Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival.

PubMed

Echenique-Rivera, Hebert; Muzzi, Alessandro; Del Tordello, Elena; Seib, Kate L; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

2011-05-01

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism.

Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

PubMed Central

Del Tordello, Elena; Seib, Kate L.; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

2011-01-01

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism. PMID:21589640

Effect of antibiotic prophylaxis on Coagulase-negative Staphylococcus virulence factor profiles in patients undergoing cataract surgery.

PubMed

López, Yolanda; Samudio, Margarita; Fariña, Norma; Castillo, Verónica; Abente, Sonia; Nentwich, Martin M; González-Britez, Nilsa; Laspina, Florentina; Carron, Agustín; Cibils, Diógenes; de Kaspar, Herminia Miño

2017-08-01

In this prospective study, multiplex polymerase chain reaction (PCR) was used to identify genes encoding virulence factors (ica, atlE and mecA) in Coagulase-negative Staphylococcus (CNS) isolates from the ocular microbiota of patients undergoing cataract surgery and to investigate possible changes in the CNS profile due to antibiotic prophylaxis. Between 09/2011 and 08/2013, patients undergoing cataract surgery were recruited at the Department of Ophthalmology, National University of Asuncion, Paraguay. In the eye to be operated on, patients received moxifloxacin 0.5 % eye drops four times at the day before surgery and a last drop 1 hour before surgery (T1). The other eye remained as control (T0). Conjunctival swabs were taken from both eyes 1 hour after the last drop. The presence of genes encoding biofilm formation (ica and atlE) and methicillin resistance (mecA) was detected by a multiplex PCR. Of the 162 patients (162 study eyes, 162 fellow eye as control group), 87 (53.7 %) eyes were positive for CNS at T0 yielding 96 CNS isolates; 70 eyes (43.2 %) were positive at T1 yielding 77 CNS isolates. For this study, 43 CNS isolates (44.8 %) from T0 and 45 (64.3 %) from T1 were used. Of the total isolates, 81.8 % (72/88) had at least one virulence factor gene (37/43 from T0 and 35/45 from T1) (p = 0.314). Simultaneous detection of ica and atlE genes was higher in T0 (58.0 %) than T1 (46.7 %), but the difference was not significant (p = 0.28). A high frequency of genes encoding virulence factors was observed in the coagulase-negative Staphylococcus isolates. The use of moxifloxacin did not significantly modify the CNS virulence factor profiles.

FNR Regulates the Expression of Important Virulence Factors Contributing to the Pathogenicity of Avian Pathogenic Escherichia coli

PubMed Central

Barbieri, Nicolle L.; Vande Vorde, Jessica A.; Baker, Alison R.; Horn, Fabiana; Li, Ganwu; Logue, Catherine M.; Nolan, Lisa K.

2017-01-01

Avian pathogenic Escherichia coli (APEC) is the etiologic agent of colibacillosis, an important cause of morbidity and mortality in poultry. Though, many virulence factors associated with APEC pathogenicity are known, their regulation remains unclear. FNR (fumarate and nitrate reduction) is a well-known global regulator that works as an oxygen sensor and has previously been described as a virulence regulator in bacterial pathogens. The goal of this study was to examine the role of FNR in the regulation of APEC virulence factors, such as Type I fimbriae, and processes such as adherence and invasion, type VI secretion, survival during oxidative stress, and growth in iron-restricted environments. To accomplish this goal, APEC O1, a well-characterized, highly virulent, and fully sequenced strain of APEC harboring multiple virulence mechanisms, some of which are plasmid-linked, was compared to its FNR mutant for expression of various virulence traits. Deletion of FNR was found to affect APEC O1's adherence, invasion and expression of ompT, a plasmid-encoded outer membrane protein, type I fimbriae, and aatA, encoding an autotransporter. Indeed, the fnr− mutant showed an 8-fold reduction in expression of type I fimbriae and a highly significant (P < 0.0001) reduction in expression of fimA, ompT (plasmid-borne), and aatA. FNR was also found to regulate expression of the type VI secretion system, affecting the expression of vgrG. Further, FNR was found to be important to APEC O1's growth in iron-deficient media and survival during oxidative stress with the mutant showing a 4-fold decrease in tolerance to oxidative stress, as compared to the wild type. Thus, our results suggest that FNR functions as an important regulator of APEC virulence. PMID:28690981

Ciprofloxacin and Trimethoprim Cause Phage Induction and Virulence Modulation in Staphylococcus aureus

PubMed Central

Goerke, Christiane; Köller, Johanna; Wolz, Christiane

2006-01-01

In Staphylococcus aureus strains of human origin, phages which integrate into the chromosomal gene coding for β-hemolysin (hlb) are widely distributed. Most of them encode accessory virulence determinants such as staphylokinase (sak) or enterotoxins. Here, we analyzed the effects of ciprofloxacin and trimethoprim on phage induction and expression of phage-encoded virulence factors by using isolates from patients with cystic fibrosis for which the induction of hlb-converting phages was demonstrated in vivo (C. Goerke, S. Matias y Papenberg, S. Dasbach, K. Dietz, R. Ziebach, B. C. Kahl, and C. Wolz, J. Infect. Dis. 189:724-734, 2004) as well as a φ13 lysogen of phage-cured strain 8325-4. Treatment of lysogens with subinhibitory concentrations of either antibiotic resulted in (i) delysogenization of strains resembling the isolates picked up after chronic lung infection and (ii) replication of phages in the bacterial host in a dose-dependent manner. Ciprofloxacin treatment resulted in enhanced recA transcription, indicating involvement of the SOS response in phage mobilization. Induction of φ13 was linked to elevated expression of the phage-encoded virulence gene sak, chiefly due to the activation of latent phage promoters. In summary, we could show the induction of hlb-converting phages and a subsequent virulence modulation of the host bacterium by ciprofloxacin and trimethoprim. PMID:16377683

The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence

PubMed Central

2010-01-01

Background Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated. Results Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence. Conclusion The functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host. PMID:21192786

Phenotypic, antimicrobial susceptibility profile and virulence factors of Klebsiella pneumoniae isolated from buffalo and cow mastitic milk.

PubMed

Osman, Kamelia M; Hassan, Hany M; Orabi, Ahmed; Abdelhafez, Ahmed S T

2014-06-01

Studies on the prevalence and virulence genes of Klebsiella mastitis pathogens in a buffalo population are undocumented. Also, the association of rmpA kfu, uge, magA, Aerobactin, K1 and K2 virulent factors with K. pneumoniae buffalo, and cow mastitis is unreported. The virulence of K. pneumoniae was evaluated through both phenotypic and molecular assays. In vivo virulence was assessed by the Vero cell cytotoxicity, suckling mouse assay and mice lethality test. Antimicrobial susceptibility was tested by disk diffusion method. The 45 K. pneumoniae isolates from buffalo (n = 10/232) and cow (n = 35/293) milk were isolated (45/525; 8.6%) and screened via PCR for seven virulence genes encoding uridine diphosphate galactose 4 epimerase encoding gene responsible for capsule and smooth lipopolysaccharide synthesis (uge), siderophores (kfu and aerobactin), protectines or invasins (rmpA and magA), and the capsule and hypermucoviscosity (K1 and K2). The most common virulence genes were rmpA, kfu, uge, and magA (77.8% each). Aerobactin and K1 genes were found at medium rates of 66.7% each and K2 (55.6%). The Vero cell cytotoxicity and LD (50) in mice were found in 100% of isolates. A multidrug resistance pattern was observed for 40% of the antimicrobials. The distribution of virulence profiles indicate a role of rmpA, kfu, uge, magA, Aerobactin, and K1 and K2 in pathogenicity of K. pneumoniae in udder infections and invasiveness, and constitutes a threat for vulnerable animals, even more if they are in combination with antibiotic resistance.

Escherichia coli O104:H4 Pathogenesis: an Enteroaggregative E. coli/Shiga Toxin-Producing E. coli Explosive Cocktail of High Virulence.

PubMed

Navarro-Garcia, Fernando

2014-12-01

A major outbreak caused by Escherichia coli of serotype O104:H4 spread throughout Europe in 2011. This large outbreak was caused by an unusual strain that is most similar to enteroaggregative E. coli (EAEC) of serotype O104:H4. A significant difference, however, is the presence of a prophage encoding the Shiga toxin, which is characteristic of enterohemorrhagic E. coli (EHEC) strains. This combination of genomic features, associating characteristics from both EAEC and EHEC, represents a new pathotype. The 2011 E. coli O104:H4 outbreak of hemorrhagic diarrhea in Germany is an example of the explosive cocktail of high virulence and resistance that can emerge in this species. A total of 46 deaths, 782 cases of hemolytic-uremic syndrome, and 3,128 cases of acute gastroenteritis were attributed to this new clone of EAEC/EHEC. In addition, recent identification in France of similar O104:H4 clones exhibiting the same virulence factors suggests that the EHEC O104:H4 pathogen has become endemically established in Europe after the end of the outbreak. EAEC strains of serotype O104:H4 contain a large set of virulence-associated genes regulated by the AggR transcription factor. They include, among other factors, the pAA plasmid genes encoding the aggregative adherence fimbriae, which anchor the bacterium to the intestinal mucosa (stacked-brick adherence pattern on epithelial cells). Furthermore, sequencing studies showed that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga toxin-producing EAEC O104:H4 strain that caused the German outbreak. This article discusses the role these virulence factors could have in EAEC/EHEC O104:H4 pathogenesis.

Virulence potential of Staphylococcus aureus isolates from Buruli ulcer patients.

PubMed

Amissah, Nana Ama; Chlebowicz, Monika A; Ablordey, Anthony; Tetteh, Caitlin S; Prah, Isaac; van der Werf, Tjip S; Friedrich, Alex W; van Dijl, Jan Maarten; Stienstra, Ymkje; Rossen, John W

2017-06-01

Buruli ulcer (BU) is a necrotizing infection of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU wounds may also be colonized with other microorganisms including Staphylococcus aureus. This study aimed to characterize the virulence factors of S. aureus isolated from BU patients. Previously sequenced genomes of 21 S. aureus isolates from BU patients were screened for the presence of virulence genes. The results show that all S. aureus isolates harbored on their core genomes genes for known virulence factors like α-hemolysin, and the α- and β-phenol soluble modulins. Besides the core genome virulence genes, mobile genetic elements (MGEs), i.e. prophages, genomic islands, pathogenicity islands and a Staphylococcal cassette chromosome (SCC) were found to carry different combinations of virulence factors, among them genes that are known to encode factors that promote immune evasion, superantigens and Panton-Valentine Leucocidin. The present observations imply that the S. aureus isolates from BU patients harbor a diverse repertoire of virulence genes that may enhance bacterial survival and persistence in the wound environment and potentially contribute to delayed wound healing. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Regulation of virulence by a two-component system in group B streptococcus.

PubMed

Jiang, Sheng-Mei; Cieslewicz, Michael J; Kasper, Dennis L; Wessels, Michael R

2005-02-01

Group B Streptococcus (GBS) is frequently carried in the gastrointestinal or genitourinary tract as a commensal organism, yet it has the potential to cause life-threatening infection in newborn infants, pregnant women, and individuals with chronic illness. Regulation of virulence factor expression may affect whether GBS behaves as an asymptomatic colonizer or an invasive pathogen, but little is known about how such factors are controlled in GBS. We now report the characterization of a GBS locus that encodes a two-component regulatory system similar to CsrRS (or CovRS) in Streptococcus pyogenes. Inactivation of csrR, encoding the putative response regulator, in two unrelated wild-type strains of GBS resulted in a marked increase in production of beta-hemolysin/cytolysin and a striking decrease in production of CAMP factor, an unrelated cytolytic toxin. Quantitative RNA hybridization experiments revealed that these two phenotypes were associated with a marked increase and decrease in expression of the corresponding genes, cylE and cfb, respectively. The CsrR mutant strains also displayed increased expression of scpB encoding C5a peptidase. Similar, but less marked, changes in gene expression were observed in CsrS (putative sensor component) mutants, evidence that CsrR and CsrS constitute a functional two-component system. Experimental infection studies in mice demonstrated reduced virulence of both CsrR and CsrS mutant strains relative to the wild type. Together, these results indicate that CsrRS regulates expression of multiple GBS virulence determinants and is likely to play an important role in GBS pathogenesis.

A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans.

PubMed

Eshghi, Azad; Becam, Jérôme; Lambert, Ambroise; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Wunder, Elsio A; Ko, Albert I; Coppee, Jean-Yves; Goarant, Cyrille; Picardeau, Mathieu

2014-06-01

Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139(-) mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

LEE-encoded regulator (Ler) mutants elicit serotype-specific protection, but not cross protection, against attaching and effacing E. coli strains.

PubMed

Zhu, C; Feng, S; Yang, Z; Davis, K; Rios, H; Kaper, J B; Boedeker, E C

2007-02-26

We previously showed that single dose orogastric immunization with an attenuated regulatory Lee-encoded regulator (ler) mutant of the rabbit enteropathogenic Escherichia coli (REPEC) strain E22 (O103:H2) protected rabbits from fatal infection with the highly virulent parent strain. In the current study we assessed the degree of homologous (serotype-specific) and heterologous (cross-serotype) protection induced by immunization with REPEC ler mutant strains of differing serotypes, or with a prototype strain RDEC-1 (O15:H-) which expresses a full array of ler up-regulated proteins. We constructed an additional ler mutant using RDEC-1 thus, permitting immunization with a ler mutant of either serotype, O15 or O103, followed by challenge with a virulent REPEC strain of the same or different serotypes. Consistent with our previous data, the current study demonstrated that rabbits immunized with a RDEC-1 ler mutant were protected from challenge with virulent RDEC-H19A (RDEC-1 transduced with Shiga toxin-producing phage H19A) of the same serotype. Rabbits immunized with RDEC-1 or E22 derivative ler mutants demonstrated significant increase in serum antibody titers to the respective whole bacterial cells expressing O antigen but not to the LEE-encoded proteins. However, immunization with the ler mutants of either E22 or RDEC-1 failed to protect rabbits from infections with virulent organisms belonging to different serotypes. In contrast, rabbits immunized with the prototype RDEC-1 were cross protected against challenge with the heterologous E22 strain as shown by normal weight gain, and the absence of clinical signs of disease or characteristic attaching and effacing (A/E) lesions. Immunization with RDEC-1 induced significantly elevated serum IgG titers to LEE-encoded proteins. We thus, demonstrated homologous protection induced by the REPEC ler mutants and heterologous protection by RDEC-1. The observed correlation between elevated immune responses to the LEE-encoded proteins and the protection against challenge with heterologous virulent REPEC strain suggests that serotype-non-specific cross protection requires the expression of, and induction of antibody to, LEE-encoded virulence factors.

Comparative Transcriptome Analysis of Vibrio splendidus JZ6 Reveals the Mechanism of Its Pathogenicity at Low Temperatures

PubMed Central

Liu, Rui; Chen, Hao; Zhang, Ran; Zhou, Zhi; Hou, Zhanhui; Gao, Dahai; Zhang, Huan; Wang, Lingling

2016-01-01

Yesso scallop-pathogenic Vibrio splendidus strain JZ6 was found to have the highest virulence at 10°C, while its pathogenicity was significantly reduced with increased temperature and completely incapacitated at 28°C. In the present study, comparative transcriptome analyses of JZ6 and another nonpathogenic V. splendidus strain, TZ19, were conducted at two crucial culture temperatures (10°C and 28°C) in order to determine the possible mechanism of temperature regulation of virulence. Comparisons among four libraries, constructed from JZ6 and TZ19 cultured at 10°C and 28°C (designated JZ6_10, JZ6_28, TZ19_10, and TZ19_28), revealed that 241 genes were possibly related to the increased virulence of JZ6 at 10°C. There were 10 genes, including 2 encoding Flp pilus assembly proteins (FlhG and VS_2437), 6 encoding proteins of the “Vibrio cholerae pathogenic cycle” (ToxS, CqsA, CqsS, RpoS, HapR, and Vsm), and 2 encoding proteins in the Sec-dependent pathway (SecE and FtsY), that were significantly upregulated in JZ6_10 (P < 0.05) compared to those in JZ6_28, TZ19_10, and TZ19_28, which were supposed to be responsible for adhesion, quorum sensing, virulence, and protein secretion of V. splendidus. When cultured at 10°C, JZ6 cells were larger and tended to aggregate more than those cultured at 28°C. The virulence factor (extracellular metalloprotease) was also found to be highly expressed in the extracellular product (ECP) of JZ6 at 10°C, and this ECP exhibited obvious cytotoxicity to oyster primary hemocytes, A549 cells, and L929 cells. These results indicated that low temperatures (10°C) could enhance adhesion, activate the quorum sensing systems, upregulate virulence factor synthesis and secretion, and, lastly, increase the pathogenicity of JZ6. PMID:26801576

The Chromobacterium violaceum type III effector CopE, a guanine nucleotide exchange factor for Rac1 and Cdc42, is involved in bacterial invasion of epithelial cells and pathogenesis.

PubMed

Miki, Tsuyoshi; Akiba, Kinari; Iguchi, Mirei; Danbara, Hirofumi; Okada, Nobuhiko

2011-06-01

The type III secretion system (T3SS) encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a) is critical for Chromobacterium violaceum pathogenesis. T3SS-dependent virulence is commonly characterized by type III effector virulence function, but the full repertoire of the effector proteins of Cpi-1/-1a T3SS is unknown. In this study, we showed that expression of Cpi-1/-1a T3SS is controlled by the master regulator CilA. We used transcriptional profiling with DNA microarrays to define CilA regulon and identified genes encoding T3SS effectors whose translocation into host cells was dependent on Cpi-1/-1a T3SS. From these effectors, we found that CopE (CV0296) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases in its C-terminal portion. The N-terminal portions (1-81 amino acids) of CopE and a CivB as a putative chaperone were required for its translocation. CopE specifically activates Rac1 and Cdc42 followed by the induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades human epithelial HeLa cells in a Cpi-1/-1a-encoded T3SS- and CopE-dependent manner. Finally, C. violaceum strains lacking copE and expressing a CopE-G168V deficient in GEF activity were attenuated for virulence in mice, suggesting that CopE contributes to the virulence of this pathogen. © 2011 Blackwell Publishing Ltd.

Nitrogen Metabolite Repression of Metabolism and Virulence in the Human Fungal Pathogen Cryptococcus neoformans

PubMed Central

Lee, I. Russel; Chow, Eve W. L.; Morrow, Carl A.; Djordjevic, Julianne T.; Fraser, James A.

2011-01-01

Proper regulation of metabolism is essential to maximizing fitness of organisms in their chosen environmental niche. Nitrogen metabolite repression is an example of a regulatory mechanism in fungi that enables preferential utilization of easily assimilated nitrogen sources, such as ammonium, to conserve resources. Here we provide genetic, transcriptional, and phenotypic evidence of nitrogen metabolite repression in the human pathogen Cryptococcus neoformans. In addition to loss of transcriptional activation of catabolic enzyme-encoding genes of the uric acid and proline assimilation pathways in the presence of ammonium, nitrogen metabolite repression also regulates the production of the virulence determinants capsule and melanin. Since GATA transcription factors are known to play a key role in nitrogen metabolite repression, bioinformatic analyses of the C. neoformans genome were undertaken and seven predicted GATA-type genes were identified. A screen of these deletion mutants revealed GAT1, encoding the only global transcription factor essential for utilization of a wide range of nitrogen sources, including uric acid, urea, and creatinine—three predominant nitrogen constituents found in the C. neoformans ecological niche. In addition to its evolutionarily conserved role in mediating nitrogen metabolite repression and controlling the expression of catabolic enzyme and permease-encoding genes, Gat1 also negatively regulates virulence traits, including infectious basidiospore production, melanin formation, and growth at high body temperature (39°–40°). Conversely, Gat1 positively regulates capsule production. A murine inhalation model of cryptococcosis revealed that the gat1Δ mutant is slightly more virulent than wild type, indicating that Gat1 plays a complex regulatory role during infection. PMID:21441208

Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

PubMed Central

Tabata, Atsushi; Nakano, Kota; Ohkura, Kazuto; Tomoyasu, Toshifumi; Kikuchi, Ken; Whiley, Robert A.

2013-01-01

Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci. PMID:23292771

The Catabolite Control Protein E (CcpE) Affects Virulence Determinant Production and Pathogenesis of Staphylococcus aureus*

PubMed Central

Hartmann, Torsten; Baronian, Grégory; Nippe, Nadine; Voss, Meike; Schulthess, Bettina; Wolz, Christiane; Eisenbeis, Janina; Schmidt-Hohagen, Kerstin; Gaupp, Rosmarie; Sunderkötter, Cord; Beisswenger, Christoph; Bals, Robert; Somerville, Greg A.; Herrmann, Mathias; Molle, Virginie; Bischoff, Markus

2014-01-01

Carbon metabolism and virulence determinant production are often linked in pathogenic bacteria, and several regulatory elements have been reported to mediate this linkage in Staphylococcus aureus. Previously, we described a novel protein, catabolite control protein E (CcpE) that functions as a regulator of the tricarboxylic acid cycle. Here we demonstrate that CcpE also regulates virulence determinant biosynthesis and pathogenesis. Specifically, deletion of ccpE in S. aureus strain Newman revealed that CcpE affects transcription of virulence factors such as capA, the first gene in the capsule biosynthetic operon; hla, encoding α-toxin; and psmα, encoding the phenol-soluble modulin cluster α. Electrophoretic mobility shift assays demonstrated that CcpE binds to the hla promoter. Mice challenged with S. aureus strain Newman or its isogenic ΔccpE derivative revealed increased disease severity in the ΔccpE mutant using two animal models; an acute lung infection model and a skin infection model. Complementation of the mutant with the ccpE wild-type allele restored all phenotypes, demonstrating that CcpE is negative regulator of virulence in S. aureus. PMID:25193664

A Unique cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner.

PubMed

Oliva, Giulia; Sahr, Tobias; Rolando, Monica; Knoth, Maike; Buchrieser, Carmen

2017-01-10

Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires' disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5' untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and adaptation traits is crucial. Yet, the regulatory circuits governing the life cycle of L. pneumophila from replicating to virulent bacteria are only partly uncovered. Here we show that the life cycle-dependent regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded antisense to the hfq-encoding gene through a base pairing mechanism. Furthermore, Hfq regulates its own expression in an autoregulatory loop. The discovery of this RNA regulatory mechanism in L. pneumophila is an important step forward in the understanding of how the switch from inoffensive, replicating to highly virulent, transmissive L. pneumophila is regulated. Copyright © 2017 Oliva et al.

How Do the Virulence Factors of Shigella Work Together to Cause Disease?

PubMed

Mattock, Emily; Blocker, Ariel J

2017-01-01

Shigella is the major cause of bacillary dysentery world-wide. It is divided into four species, named S. flexneri, S. sonnei, S. dysenteriae , and S. boydii , which are distinct genomically and in their ability to cause disease. Shigellosis, the clinical presentation of Shigella infection, is characterized by watery diarrhea, abdominal cramps, and fever. Shigella 's ability to cause disease has been attributed to virulence factors, which are encoded on chromosomal pathogenicity islands and the virulence plasmid. However, information on these virulence factors is not often brought together to create a detailed picture of infection, and how this translates into shigellosis symptoms. Firstly, Shigella secretes virulence factors that induce severe inflammation and mediate enterotoxic effects on the colon, producing the classic watery diarrhea seen early in infection. Secondly, Shigella injects virulence effectors into epithelial cells via its Type III Secretion System to subvert the host cell structure and function. This allows invasion of epithelial cells, establishing a replicative niche, and causes erratic destruction of the colonic epithelium. Thirdly, Shigella produces effectors to down-regulate inflammation and the innate immune response. This promotes infection and limits the adaptive immune response, causing the host to remain partially susceptible to re-infection. Combinations of these virulence factors may contribute to the different symptoms and infection capabilities of the diverse Shigella species, in addition to distinct transmission patterns. Further investigation of the dominant species causing disease, using whole-genome sequencing and genotyping, will allow comparison and identification of crucial virulence factors and may contribute to the production of a pan- Shigella vaccine.

How Do the Virulence Factors of Shigella Work Together to Cause Disease?

PubMed Central

Mattock, Emily; Blocker, Ariel J.

2017-01-01

Shigella is the major cause of bacillary dysentery world-wide. It is divided into four species, named S. flexneri, S. sonnei, S. dysenteriae, and S. boydii, which are distinct genomically and in their ability to cause disease. Shigellosis, the clinical presentation of Shigella infection, is characterized by watery diarrhea, abdominal cramps, and fever. Shigella's ability to cause disease has been attributed to virulence factors, which are encoded on chromosomal pathogenicity islands and the virulence plasmid. However, information on these virulence factors is not often brought together to create a detailed picture of infection, and how this translates into shigellosis symptoms. Firstly, Shigella secretes virulence factors that induce severe inflammation and mediate enterotoxic effects on the colon, producing the classic watery diarrhea seen early in infection. Secondly, Shigella injects virulence effectors into epithelial cells via its Type III Secretion System to subvert the host cell structure and function. This allows invasion of epithelial cells, establishing a replicative niche, and causes erratic destruction of the colonic epithelium. Thirdly, Shigella produces effectors to down-regulate inflammation and the innate immune response. This promotes infection and limits the adaptive immune response, causing the host to remain partially susceptible to re-infection. Combinations of these virulence factors may contribute to the different symptoms and infection capabilities of the diverse Shigella species, in addition to distinct transmission patterns. Further investigation of the dominant species causing disease, using whole-genome sequencing and genotyping, will allow comparison and identification of crucial virulence factors and may contribute to the production of a pan-Shigella vaccine. PMID:28393050

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae.

PubMed

Mukherjee, Munmun; Kakarla, Prathusha; Kumar, Sanath; Gonzalez, Esmeralda; Floyd, Jared T; Inupakutika, Madhuri; Devireddy, Amith Reddy; Tirrell, Selena R; Bruns, Merissa; He, Guixin; Lindquist, Ingrid E; Sundararajan, Anitha; Schilkey, Faye D; Mudge, Joann; Varela, Manuel F

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae . Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence-related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations.

Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients.

PubMed

Motallebi, Mitra; Jabalameli, Fereshteh; Asadollahi, Kheirollah; Taherikalani, Morovat; Emaneini, Mohammad

2016-08-01

The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative genomic analysis and characterization of incompatibility group FIB plasmid encoded virulence factors of Salmonella enterica isolated from food sources.

PubMed

Khajanchi, Bijay K; Hasan, Nur A; Choi, Seon Young; Han, Jing; Zhao, Shaohua; Colwell, Rita R; Cerniglia, Carl E; Foley, Steven L

2017-08-02

The degree to which the chromosomal mediated iron acquisition system contributes to virulence of many bacterial pathogens is well defined. However, the functional roles of plasmid encoded iron acquisition systems, specifically Sit and aerobactin, have yet to be determined for Salmonella spp. In a recent study, Salmonella enterica strains isolated from different food sources were sequenced on the Illumina MiSeq platform and found to harbor the incompatibility group (Inc) FIB plasmid. In this study, we examined sequence diversity and the contribution of factors encoded on the IncFIB plasmid to the virulence of S. enterica. Whole genome sequences of seven S. enterica isolates were compared to genomes of serovars of S. enterica isolated from food, animal, and human sources. SeqSero analysis predicted that six strains were serovar Typhimurium and one was Heidelberg. Among the S. Typhimurium strains, single nucleotide polymorphism (SNP)-based phylogenetic analyses revealed that five of the isolates clustered as a single monophyletic S. Typhimurium subclade, while one of the other strains branched with S. Typhimurium from a bovine source. DNA sequence based phylogenetic diversity analyses showed that the IncFIB plasmid-encoded Sit and aerobactin iron acquisition systems are conserved among bacterial species including S. enterica. The IncFIB plasmid was transferred to an IncFIB plasmid deficient strain of S. enterica by conjugation. The transconjugant SE819::IncFIB persisted in human intestinal epithelial (Caco-2) cells at a higher rate than the recipient SE819. Genes of the Sit and aerobactin operons in the IncFIB plasmid were differentially expressed in iron-rich and iron-depleted growth media. Minimal sequence diversity was detected in the Sit and aerobactin operons in the IncFIB plasmids present among different bacterial species, including foodborne Salmonella strains. IncFIB plasmid encoded factors play a role during infection under low-iron conditions in host cells.

Mobile genetic element-encoded cytolysin connects virulence to methicillin resistance in MRSA.

PubMed

Queck, Shu Y; Khan, Burhan A; Wang, Rong; Bach, Thanh-Huy L; Kretschmer, Dorothee; Chen, Liang; Kreiswirth, Barry N; Peschel, Andreas; Deleo, Frank R; Otto, Michael

2009-07-01

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.

Multidrug-resistant Salmonella enterica serovar Typhimurium isolates are resistant to antibiotics that influence their swimming and swarming motility

USDA-ARS?s Scientific Manuscript database

Motile bacteria utilize one or more strategies for movement, such as darting, gliding, sliding, swarming, swimming, and twitching. The ability to move is considered a virulence factor in many pathogenic bacteria, including Salmonella. Multidrug-resistant (MDR) Salmonella encodes acquired factors t...

The Fur-Iron Complex Modulates Expression of the Quorum-Sensing Master Regulator, SmcR, To Control Expression of Virulence Factors in Vibrio vulnificus

PubMed Central

Kim, In Hwang; Wen, Yancheng; Son, Jee-Soo; Lee, Kyu-Ho

2013-01-01

The gene vvpE, encoding the virulence factor elastase, is a member of the quorum-sensing regulon in Vibrio vulnificus and displays enhanced expression at high cell density. We observed that this gene was repressed under iron-rich conditions and that the repression was due to a Fur (ferric uptake regulator)-dependent repression of smcR, a gene encoding a quorum-sensing master regulator with similarity to luxR in Vibrio harveyi. A gel mobility shift assay and a footprinting experiment demonstrated that the Fur-iron complex binds directly to two regions upstream of smcR (−82 to −36 and −2 to +27, with respect to the transcription start site) with differing affinities. However, binding of the Fur-iron complex is reversible enough to allow expression of smcR to be induced by quorum sensing at high cell density under iron-rich conditions. Under iron-limiting conditions, Fur fails to bind either region and the expression of smcR is regulated solely by quorum sensing. These results suggest that two biologically important environmental signals, iron and quorum sensing, converge to direct the expression of smcR, which then coordinates the expression of virulence factors. PMID:23716618

Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

PubMed

Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

2015-01-01

Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.

Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

PubMed Central

Shames, Stephanie R.; Liu, Luying; Havey, James C.; Schofield, Whitman B.; Goodman, Andrew L.; Roy, Craig R.

2017-01-01

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis. PMID:29133401

Genomic Diversity of Burkholderia pseudomallei Clinical Isolates: Subtractive Hybridization Reveals a Burkholderia mallei-Specific Prophage in B. pseudomallei 1026b

DTIC Science & Technology

2004-06-01

identification of several new virulence gene candidates. In particular, K96243 harbors multiple genomic islands with relatively low GC contents...differences were observed. Prophage-encoded virulence factors in other bacterial species have been described (5), and it was of interest to see if gene ... Xylella fastidiosa (11, 16, 17). The genomic sequencing results for multiple strains of Streptococcus and Xylella suggest that different disease

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae

PubMed Central

Mukherjee, Munmun; Kakarla, Prathusha; Kumar, Sanath; Gonzalez, Esmeralda; Floyd, Jared T.; Inupakutika, Madhuri; Devireddy, Amith Reddy; Tirrell, Selena R.; Bruns, Merissa; He, Guixin; Lindquist, Ingrid E.; Sundararajan, Anitha; Schilkey, Faye D.; Mudge, Joann; Varela, Manuel F.

2015-01-01

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae. Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence–related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations. PMID:25722857

Functional Analysis of the Alternative Sigma-28 Factor FliA and Its Anti-Sigma Factor FlgM of the Nonflagellated Legionella Species L. oakridgensis.

PubMed

Tlapák, Hana; Rydzewski, Kerstin; Schulz, Tino; Weschka, Dennis; Schunder, Eva; Heuner, Klaus

2017-06-01

Legionella oakridgensis causes Legionnaires' disease but is known to be less virulent than Legionella pneumophila L. oakridgensis is one of the Legionella species that is nonflagellated. The genes of the flagellar regulon are absent, except those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28 factor (FlgM). Similar to L. oakridgensis , Legionella adelaidensis and Legionella londiniensis , located in the same phylogenetic clade, have no flagellar regulon, although both are positive for fliA and flgM Here, we investigated the role and function of both genes to better understand the role of FliA, the positive regulator of flagellin expression, in nonflagellated strains. We demonstrated that the FliA gene of L. oakridgensis encodes a functional sigma-28 factor that enables the transcription start from the sigma-28-dependent promoter site. The investigations have shown that FliA is necessary for full fitness of L. oakridgensis Interestingly, expression of FliA-dependent genes depends on the growth phase and temperature, as already shown for L. pneumophila strains that are flagellated. In addition, we demonstrated that FlgM is a negative regulator of FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase- and temperature-dependent manner, instead of being exported into the medium as reported for most bacteria. The degradation of FlgM leads to an increase of FliA activity. IMPORTANCE A less virulent Legionella species, L. oakridgensis , causes Legionnaires' disease and is known to not have flagella, even though L. oakridgensis has the regulator of flagellin expression (FliA). This protein has been shown to be involved in the expression of virulence factors. Thus, the strain was chosen for use in this investigation to search for FliA target genes and to identify putative virulence factors of L. oakridgensis One of the five major target genes of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly, in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems not to be transported from the cell so that FliA gets activated. In L. oakridgensis , FlgM seems to be degraded by protease activities. Copyright © 2017 American Society for Microbiology.

CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection.

PubMed

Bikard, David; Hatoum-Aslan, Asma; Mucida, Daniel; Marraffini, Luciano A

2012-08-16

Pathogenic bacterial strains emerge largely due to transfer of virulence and antimicrobial resistance genes between bacteria, a process known as horizontal gene transfer (HGT). Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci of bacteria and archaea encode a sequence-specific defense mechanism against bacteriophages and constitute a programmable barrier to HGT. However, the impact of CRISPRs on the emergence of virulence is unknown. We programmed the human pathogen Streptococcus pneumoniae with CRISPR sequences that target capsule genes, an essential pneumococcal virulence factor, and show that CRISPR interference can prevent transformation of nonencapsulated, avirulent pneumococci into capsulated, virulent strains during infection in mice. Further, at low frequencies bacteria can lose CRISPR function, acquire capsule genes, and mount a successful infection. These results demonstrate that CRISPR interference can prevent the emergence of virulence in vivo and that strong selective pressure for virulence or antibiotic resistance can lead to CRISPR loss in bacterial pathogens. Copyright © 2012 Elsevier Inc. All rights reserved.

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments

PubMed Central

Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M

2017-01-01

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83–248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence. PMID:28915104

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments.

PubMed

Barski, Michal; Brennan, Benjamin; Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M; Schwarz-Linek, Ulrich

2017-09-15

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83-248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence.

Analysis of the hierarchy of quorum-sensing regulation in Pseudomonas aeruginosa.

PubMed

Wagner, Victoria E; Li, Luen-Luen; Isabella, Vincent M; Iglewski, Barbara H

2007-01-01

Quorum-sensing in Pseudomonas aeruginosa is known to regulate several aspects of pathogenesis, including virulence factor production, biofilm development, and antimicrobial resistance. Recent high-throughput analysis has revealed the existence of several layers of regulation within the QS-circuit. To address this complexity, mutations in genes encoding known or putative transcriptional regulators that were also identified as being regulated by the las and/or rhl QS systems were screened for their contribution in mediating several phenotypes, for example motility, secreted virulence products, and pathogenic capacity in a lettuce leaf model. These studies have further elucidated the potential contribution to virulence of these genes within the QS regulon.

Cyt toxin expression reveals an inverse regulation of insect and plant virulence factors of Dickeya dadantii.

PubMed

Costechareyre, Denis; Dridi, Bedis; Rahbé, Yvan; Condemine, Guy

2010-12-01

The plant pathogenic bacteria Dickeya dadantii is also a pathogen of the pea aphid Acyrthosiphon pisum. The genome of the bacteria contains four cyt genes, encoding homologues of Bacillus thuringiensis Cyt toxins, which are involved in its pathogenicity to insects. We show here that these genes are transcribed as an operon, and we determined the conditions necessary for their expression. Their expression is induced at high temperature and at an osmolarity equivalent to that found in the plant phloem sap. The regulators of cyt genes have also been identified: their expression is repressed by H-NS and VfmE and activated by PecS. These genes are already known to regulate plant virulence factors, but in an opposite way. When tested in a virulence assay by ingestion, the pecS mutant was almost non-pathogenic while hns and vfmE mutants behaved in the same way as the wild-type strain. Mutants of other regulators of plant virulence, GacA, OmpR and PhoP, that do not control Cyt toxin production, also showed reduced pathogenicity. In an assay by injection of bacteria, the gacA strain was less pathogenic but, surprisingly, the pecS mutant was slightly more virulent. These results show that Cyt toxins are not the only virulence factors required to kill aphids, and that these factors act at different stages of the infection. Moreover, their production is controlled by general virulence regulators known for their role in plant virulence. This integration could indicate that virulence towards insects is a normal mode of life for D. dadantii. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Highly conserved type 1 pili promote enterotoxigenic E. coli pathogen-host interactions

PubMed Central

Rashu, Rasheduzzaman; Begum, Yasmin Ara; Ciorba, Matthew A.; Hultgren, Scott J.; Qadri, Firdausi

2017-01-01

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens. PMID:28531220

A Magnaporthe grisea Cyclophilin Acts as a Virulence Determinant during Plant Infection

PubMed Central

Viaud, Muriel C.; Balhadère, Pascale V.; Talbot, Nicholas J.

2002-01-01

Cyclophilins are peptidyl prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and that are best known for being the cellular target of the immunosuppressive drug cyclosporin A (CsA). The activity of CsA is caused by the drug forming a complex with cyclophilin A and inhibiting the calmodulin-dependent phosphoprotein phosphatase calcineurin. We have investigated the role of CYP1, a cyclophilin-encoding gene in the phytopathogenic fungus Magnaporthe grisea, which is the causal agent of rice blast disease. CYP1 putatively encodes a mitochondrial and cytosolic form of cyclophilin, and targeted gene replacement has shown that CYP1 acts as a virulence determinant in rice blast. Cyp1 mutants show reduced virulence and are impaired in associated functions, such as penetration peg formation and appressorium turgor generation. CYP1 cyclophilin also is the cellular target for CsA in Magnaporthe, and CsA was found to inhibit appressorium development and hyphal growth in a CYP1-dependent manner. These data implicate cyclophilins as virulence factors in phytopathogenic fungi and also provide evidence that calcineurin signaling is required for infection structure formation by Magnaporthe. PMID:11971145

Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2.

PubMed

Sitkiewicz, Izabela; Nagiec, Michal J; Sumby, Paul; Butler, Stephanie D; Cywes-Bentley, Colette; Musser, James M

2006-10-24

The molecular basis of pathogen clone emergence is relatively poorly understood. Acquisition of a bacteriophage encoding a previously unknown secreted phospholipase A(2) (designated SlaA) has been implicated in the rapid emergence in the mid-1980s of a new hypervirulent clone of serotype M3 group A Streptococcus. Although several lines of circumstantial evidence suggest that SlaA is a virulence factor, this issue has not been addressed experimentally. We found that an isogenic DeltaslaA mutant strain was significantly impaired in ability to adhere to and kill human epithelial cells compared with the wild-type parental strain. The mutant strain was less virulent for mice than the wild-type strain, and immunization with purified SlaA significantly protected mice from invasive disease. Importantly, the mutant strain was significantly attenuated for colonization in a monkey model of pharyngitis. We conclude that transductional acquisition of the ability of a GAS strain to produce SlaA enhanced the spread and virulence of the serotype M3 precursor strain. Hence, these studies identified a crucial molecular event underlying the evolution, rapid emergence, and widespread dissemination of unusually severe human infections caused by a distinct bacterial clone.

Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences.

PubMed

Nasser, Waleed; Beres, Stephen B; Olsen, Randall J; Dean, Melissa A; Rice, Kelsey A; Long, S Wesley; Kristinsson, Karl G; Gottfredsson, Magnus; Vuopio, Jaana; Raisanen, Kati; Caugant, Dominique A; Steinbakk, Martin; Low, Donald E; McGeer, Allison; Darenberg, Jessica; Henriques-Normark, Birgitta; Van Beneden, Chris A; Hoffmann, Steen; Musser, James M

2014-04-29

We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversification of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that first contained the phage encoding an extracellular DNase virulence factor (streptococcal DNase D2, SdaD2) and subsequently acquired the phage encoding the SpeA1 variant of the streptococcal pyrogenic exotoxin A superantigen. The SpeA2 toxin variant evolved from SpeA1 by a single-nucleotide change in the M1 progenitor strain before acquisition by horizontal gene transfer of a large chromosomal region encoding secreted toxins NAD(+)-glycohydrolase and streptolysin O. Acquisition of this 36-kb region in the early 1980s into just one cell containing the phage-encoded sdaD2 and speA2 genes was the final major molecular event preceding the emergence and rapid intercontinental spread of the contemporary epidemic clone. Thus, we resolve a decades-old controversy about the type and sequence of genomic alterations that produced this explosive epidemic. Analysis of comprehensive, population-based contemporary invasive strains from seven countries identified strong patterns of temporal population structure. Compared with a preepidemic reference strain, the contemporary clone is significantly more virulent in nonhuman primate models of pharyngitis and necrotizing fasciitis. A key finding is that the molecular evolutionary events transpiring in just one bacterial cell ultimately have produced millions of human infections worldwide.

Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences

PubMed Central

Nasser, Waleed; Beres, Stephen B.; Olsen, Randall J.; Dean, Melissa A.; Rice, Kelsey A.; Long, S. Wesley; Kristinsson, Karl G.; Gottfredsson, Magnus; Vuopio, Jaana; Raisanen, Kati; Caugant, Dominique A.; Steinbakk, Martin; Low, Donald E.; McGeer, Allison; Darenberg, Jessica; Henriques-Normark, Birgitta; Van Beneden, Chris A.; Hoffmann, Steen; Musser, James M.

2014-01-01

We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversification of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that first contained the phage encoding an extracellular DNase virulence factor (streptococcal DNase D2, SdaD2) and subsequently acquired the phage encoding the SpeA1 variant of the streptococcal pyrogenic exotoxin A superantigen. The SpeA2 toxin variant evolved from SpeA1 by a single-nucleotide change in the M1 progenitor strain before acquisition by horizontal gene transfer of a large chromosomal region encoding secreted toxins NAD+-glycohydrolase and streptolysin O. Acquisition of this 36-kb region in the early 1980s into just one cell containing the phage-encoded sdaD2 and speA2 genes was the final major molecular event preceding the emergence and rapid intercontinental spread of the contemporary epidemic clone. Thus, we resolve a decades-old controversy about the type and sequence of genomic alterations that produced this explosive epidemic. Analysis of comprehensive, population-based contemporary invasive strains from seven countries identified strong patterns of temporal population structure. Compared with a preepidemic reference strain, the contemporary clone is significantly more virulent in nonhuman primate models of pharyngitis and necrotizing fasciitis. A key finding is that the molecular evolutionary events transpiring in just one bacterial cell ultimately have produced millions of human infections worldwide. PMID:24733896

Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence.

PubMed

Beres, Stephen B; Sylva, Gail L; Barbian, Kent D; Lei, Benfang; Hoff, Jessica S; Mammarella, Nicole D; Liu, Meng-Yao; Smoot, James C; Porcella, Stephen F; Parkins, Larye D; Campbell, David S; Smith, Todd M; McCormick, John K; Leung, Donald Y M; Schlievert, Patrick M; Musser, James M

2002-07-23

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares approximately 1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A(2) (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.

The FUN of identifying gene function in bacterial pathogens; insights from Salmonella functional genomics.

PubMed

Hammarlöf, Disa L; Canals, Rocío; Hinton, Jay C D

2013-10-01

The availability of thousands of genome sequences of bacterial pathogens poses a particular challenge because each genome contains hundreds of genes of unknown function (FUN). How can we easily discover which FUN genes encode important virulence factors? One solution is to combine two different functional genomic approaches. First, transcriptomics identifies bacterial FUN genes that show differential expression during the process of mammalian infection. Second, global mutagenesis identifies individual FUN genes that the pathogen requires to cause disease. The intersection of these datasets can reveal a small set of candidate genes most likely to encode novel virulence attributes. We demonstrate this approach with the Salmonella infection model, and propose that a similar strategy could be used for other bacterial pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Gene expression patterns and dynamics of the colonization of common bean (Phaseolus vulgaris L.) by highly virulent and weakly virulent strains of Fusarium oxysporum

PubMed Central

Niño-Sánchez, Jonathan; Tello, Vega; Casado-del Castillo, Virginia; Thon, Michael R.; Benito, Ernesto P.; Díaz-Mínguez, José María

2015-01-01

The dynamics of root and hypocotyl colonization, and the gene expression patterns of several fungal virulence factors and plant defense factors have been analyzed and compared in the interaction of two Fusarium oxysporum f. sp. phaseoli strains displaying clear differences in virulence, with a susceptible common bean cultivar. The growth of the two strains on the root surface and the colonization of the root was quantitatively similar although the highly virulent (HV) strain was more efficient reaching the central root cylinder. The main differences between both strains were found in the temporal and spatial dynamics of crown root and hypocotyl colonization. The increase of fungal biomass in the crown root was considerably larger for the HV strain, which, after an initial stage of global colonization of both the vascular cylinder and the parenchymal cells, restricted its growth to the newly differentiated xylem vessels. The weakly virulent (WV) strain was a much slower and less efficient colonizer of the xylem vessels, showing also growth in the intercellular spaces of the parenchyma. Most of the virulence genes analyzed showed similar expression patterns in both strains, except SIX1, SIX6 and the gene encoding the transcription factor FTF1, which were highly upregulated in root crown and hypocotyl. The response induced in the infected plant showed interesting differences for both strains. The WV strain induced an early and strong transcription of the PR1 gene, involved in SAR response, while the HV strain preferentially induced the early expression of the ethylene responsive factor ERF2. PMID:25883592

Virulence properties of methicillin-susceptible Staphylococcus aureus food isolates encoding Panton-Valentine Leukocidin gene.

PubMed

Sudagidan, Mert; Aydin, Ali

2010-04-15

In this study, three Panton-Valentine Leukocidin gene carrying methicillin-susceptible Staphylococcus aureus (MSSA) strains (M1-AAG42B, PY30C-b and YF1B-b) were isolated from different food samples in Kesan-Edirne, Turkey. These strains were characterized on the basis of MLST type, spa type, virulence factor gene contents, antibiotic susceptibilities against 21 antibiotics and biofilm formation. The genetic relatedness of the strains was determined by PFGE. In addition, the complete gene sequences of lukS-PV and lukF-PV were also investigated. All strains were found to be susceptible to tested antibiotics and they were mecA negative. Three strains showed the same PFGE band pattern, ST152 clonal type and t355 spa type. In the detection of virulence factor genes, sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, seu, eta, etb, set1, geh and tst genes were not detected. All strains showed the positive results for alpha- and beta-haemolysin genes (hla and hlb), protease encoding genes (sspA, sspB and aur), lukE and lukD leukocidin genes (lukED). The strains were found to be non-biofilm formers. By this study, the virulence properties of the strains were described and this is one of the first reports regarding PVL-positive MSSA strains from food. (c) 2010 Elsevier B.V. All rights reserved.

Inactivation of DNA-Binding Response Regulator Sak189 Abrogates β-Antigen Expression and Affects Virulence of Streptococcus agalactiae

PubMed Central

Rozhdestvenskaya, Anastasia S.; Totolian, Artem A.; Dmitriev, Alexander V.

2010-01-01

Background Streptococcus agalactiae is able to colonize numerous tissues employing different mechanisms of gene regulation, particularly via two-component regulatory systems. These systems sense the environmental stimuli and regulate expression of the genes including virulence genes. Recently, the novel two-component regulatory system Sak188/Sak189 was identified. In S. agalactiae genome, it was adjacent to the bac gene encoding for β-antigen, an important virulence factor. Methodology/Principal Findings In this study, the sak188 and sak189 genes were inactivated, and the functional role of Sak188/Sak189 two-component system in regulation of the β-antigen expression was investigated. It was demonstrated that both transcription of bac gene and expression of encoded β-antigen were controlled by Sak189 response regulator, but not Sak188 histidine kinase. It was also found that the regulation occurred at transcriptional level. Finally, insertional inactivation of sak189 gene, but not sak188 gene, significantly affected virulent properties of S. agalactiae. Conclusions/Significance Sak189 response regulator is necessary for activation of bac gene transcription. It also controls the virulent properties of S. agalactiae. Given that the primary functional role of Sak188/Sak189 two-component systems is a control of bac gene transcription, this system can be annotated as BgrR/S (bac gene regulatory system). PMID:20419089

Detection of Streptococcus pyogenes virulence genes in Streptococcus dysgalactiae subsp. equisimilis from Vellore, India.

PubMed

Babbar, Anshu; Itzek, Andreas; Pieper, Dietmar H; Nitsche-Schmitz, D Patric

2018-03-12

Streptococcus dysgalactiae subsp. equisimilis (SDSE), belonging to the group C and G streptococci, are human pathogens reported to cause clinical manifestations similar to infections caused by Streptococcus pyogenes. To scrutinize the distribution of gene coding for S. pyogenes virulence factors in SDSE, 255 isolates were collected from humans infected with SDSE in Vellore, a region in southern India, with high incidence of SDSE infections. Initial evaluation indicated SDSE isolates comprising of 82.35% group G and 17.64% group C. A multiplex PCR system was used to detect 21 gene encoding virulence-associated factors of S. pyogenes, like superantigens, DNases, proteinases, and other immune modulatory toxins. As validated by DNA sequencing of the PCR products, sequences homologous to speC, speG, speH, speI, speL, ssa and smeZ of the family of superantigen coding genes and for DNases like sdaD and sdc were detected in the SDSE collection. Furthermore, there was high abundance (48.12% in group G and 86.6% in group C SDSE) of scpA, the gene coding for C5a peptidase in these isolates. Higher abundance of S. pyogenes virulence factor genes was observed in SDSE of Lancefield group C as compared to group G, even though the incidence rates in former were lower. This study not only substantiates detection of S. pyogenes virulence factor genes in whole genome sequenced SDSE but also makes significant contribution towards the understanding of SDSE and its increasing virulence potential.

The PE/PPE multigene family codes for virulence factors and is a possible source of mycobacterial antigenic variation: perhaps more?

PubMed

Akhter, Yusuf; Ehebauer, Matthias T; Mukhopadhyay, Sangita; Hasnain, Seyed E

2012-01-01

The PE/PPE multigene family codes for approximately 10% of the Mycobacterium tuberculosis proteome and is encoded by 176 open reading frames. These proteins possess, and have been named after, the conserved proline-glutamate (PE) or proline-proline-glutamate (PPE) motifs at their N-terminus. Their genes have a conserved structure and repeat motifs that could be a potential source of antigenic variation in M. tuberculosis. PE/PPE genes are scattered throughout the genome and PE/PPE pairs are usually encoded in bicistronic operons although this is not universally so. This gene family has evolved by specific gene duplication events. PE/PPE proteins are either secreted or localized to the cell surface. Several are thought to be virulence factors, which participate in evasion of the host immune response. This review summarizes the current knowledge about the gene family in order to better understand its biological function. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Both msa genes in Renibacterium salmoninarum are needed for full virulence in bacterial kidney disease

USGS Publications Warehouse

Coady, A.M.; Murray, A.L.; Elliott, D.G.; Rhodes, L.D.

2006-01-01

Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile Chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression. Copyright ?? 2006, American Society for Microbiology. All Rights Reserved.

Staphylococcus aureus nasal carriage in Ukraine: antibacterial resistance and virulence factor encoding genes.

PubMed

Netsvyetayeva, Irina; Fraczek, Mariusz; Piskorska, Katarzyna; Golas, Marlena; Sikora, Magdalena; Mlynarczyk, Andrzej; Swoboda-Kopec, Ewa; Marusza, Wojciech; Palmieri, Beniamino; Iannitti, Tommaso

2014-03-05

The number of studies regarding the incidence of multidrug resistant strains and distribution of genes encoding virulence factors, which have colonized the post-Soviet states, is considerably limited. The aim of the study was (1) to assess the Staphylococcus (S.) aureus nasal carriage rate, including Methicillin Resistant S. aureus (MRSA) strains in adult Ukrainian population, (2) to determine antibiotic resistant pattern and (3) the occurrence of Panton Valentine Leukocidine (PVL)-, Fibronectin-Binding Protein A (FnBPA)- and Exfoliative Toxin (ET)-encoding genes. Nasal samples for S. aureus culture were obtained from 245 adults. The susceptibility pattern for several classes of antibiotics was determined by disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The virulence factor encoding genes, mecA, lukS-lukF, eta, etb, etd, fnbA, were detected by Polymerase Chain Reaction (PCR). The S. aureus nasal carriage rate was 40%. The prevalence of nasal MRSA carriage in adults was 3.7%. LukS-lukF genes were detected in over 58% of the strains. ET-encoding genes were detected in over 39% of the strains and the most prevalent was etd. The fnbA gene was detected in over 59% of the strains. All MRSA isolates tested were positive for the mecA gene. LukS-lukF genes and the etd gene were commonly co-present in MRSA, while lukS-lukF genes and the fnbA gene were commonly co-present in Methicillin Sensitive S. aureus (MSSA) isolates. No significant difference was detected between the occurrence of lukS-lukF genes (P > 0.05) and the etd gene (P > 0.05) when comparing MRSA and MSSA. The occurrence of the fnbA gene was significantly more frequent in MSSA strains (P < 0.05). In Ukraine, S. aureus is a common cause of infection. The prevalence of S. aureus nasal carriage in our cohort of patients from Ukraine was 40.4%. We found that 9.1% of the strains were classified as MRSA and all MRSA isolates tested positive for the mecA gene. We also observed a high prevalence of PVL- and ET- encoding genes among S. aureus nasal carriage strains. A systematic surveillance system can help prevent transmission and spread of drug resistant toxin producing S. aureus strains.

Modulation of innate immune responses by Yersinia type III secretion system translocators and effectors.

PubMed

Bliska, James B; Wang, Xiaoying; Viboud, Gloria I; Brodsky, Igor E

2013-10-01

The innate immune system of mammals responds to microbial infection through detection of conserved molecular determinants called 'pathogen-associated molecular patterns' (PAMPs). Pathogens use virulence factors to counteract PAMP-directed responses. The innate immune system can in turn recognize signals generated by virulence factors, allowing for a heightened response to dangerous pathogens. Many Gram-negative bacterial pathogens encode type III secretion systems (T3SSs) that translocate effector proteins, subvert PAMP-directed responses and are critical for infection. A plasmid-encoded T3SS in the human-pathogenic Yersinia species translocates seven effectors into infected host cells. Delivery of effectors by the T3SS requires plasma membrane insertion of two translocators, which are thought to form a channel called a translocon. Studies of the Yersinia T3SS have provided key advances in our understanding of how innate immune responses are generated by perturbations in plasma membrane and other signals that result from translocon insertion. Additionally, studies in this system revealed that effectors function to inhibit innateimmune responses resulting from insertion of translocons into plasma membrane. Here, we review these advances with the goal of providing insight into how a T3SS can activate and inhibit innate immune responses, allowing a virulent pathogen to bypass host defences. © 2013 John Wiley & Sons Ltd.

Virulence gene regulation by CvfA, a putative RNase: the CvfA-enolase complex in Streptococcus pyogenes links nutritional stress, growth-phase control, and virulence gene expression.

PubMed

Kang, Song Ok; Caparon, Michael G; Cho, Kyu Hong

2010-06-01

Streptococcus pyogenes, a multiple-auxotrophic human pathogen, regulates virulence gene expression according to nutritional availability during various stages in the infection process or in different infection sites. We discovered that CvfA influenced the expression of virulence genes according to growth phase and nutritional status. The influence of CvfA in C medium, rich in peptides and poor in carbohydrates, was most pronounced at the stationary phase. Under these conditions, up to 30% of the transcriptome exhibited altered expression; the levels of expression of multiple virulence genes were altered, including the genes encoding streptokinase, CAMP factor, streptolysin O, M protein (more abundant in the CvfA(-) mutant), SpeB, mitogenic factor, and streptolysin S (less abundant). The increase of carbohydrates or peptides in media restored the levels of expression of the virulence genes in the CvfA(-) mutant to wild-type levels (emm, ska, and cfa by carbohydrates; speB by peptides). Even though the regulation of gene expression dependent on nutritional stress is commonly linked to the stringent response, the levels of ppGpp were not altered by deletion of cvfA. Instead, CvfA interacted with enolase, implying that CvfA, a putative RNase, controls the transcript decay rates of virulence factors or their regulators according to nutritional status. The virulence of CvfA(-) mutants was highly attenuated in murine models, indicating that CvfA-mediated gene regulation is necessary for the pathogenesis of S. pyogenes. Taken together, the CvfA-enolase complex in S. pyogenes is involved in the regulation of virulence gene expression by controlling RNA degradation according to nutritional stress.

A Commensal Gone Bad: Complete Genome Sequence of the Prototypical Enterotoxigenic Escherichia coli Strain H10407

DTIC Science & Technology

2010-11-01

and Escherichia ferguso- . TABLE 2. General characteristics of the plasm ids from ETEC strains H10407 and E1392/75 Value in E. c·oli: Characteristic...0352). consetved proteins with unknown func- tions (CDSs 0673 to 0678), a flavoprotein electron transfer system (CDSs 1730 to 1734), the colanic...mediating diarrhea are not chromosomally encoded. indicating that the essential virulence factors are encoded on the plasm ids (61 ). Potentia l

The Transcription Factor BcLTF1 Regulates Virulence and Light Responses in the Necrotrophic Plant Pathogen Botrytis cinerea

PubMed Central

Schumacher, Julia; Simon, Adeline; Cohrs, Kim Christopher; Viaud, Muriel; Tudzynski, Paul

2014-01-01

Botrytis cinerea is the causal agent of gray mold diseases in a range of dicotyledonous plant species. The fungus can reproduce asexually by forming macroconidia for dispersal and sclerotia for survival; the latter also participate in sexual reproduction by bearing the apothecia after fertilization by microconidia. Light induces the differentiation of conidia and apothecia, while sclerotia are exclusively formed in the absence of light. The relevance of light for virulence of the fungus is not obvious, but infections are observed under natural illumination as well as in constant darkness. By a random mutagenesis approach, we identified a novel virulence-related gene encoding a GATA transcription factor (BcLTF1 for light-responsive TF1) with characterized homologues in Aspergillus nidulans (NsdD) and Neurospora crassa (SUB-1). By deletion and over-expression of bcltf1, we confirmed the predicted role of the transcription factor in virulence, and discovered furthermore its functions in regulation of light-dependent differentiation, the equilibrium between production and scavenging of reactive oxygen species (ROS), and secondary metabolism. Microarray analyses revealed 293 light-responsive genes, and that the expression levels of the majority of these genes (66%) are modulated by BcLTF1. In addition, the deletion of bcltf1 affects the expression of 1,539 genes irrespective of the light conditions, including the overexpression of known and so far uncharacterized secondary metabolism-related genes. Increased expression of genes encoding alternative respiration enzymes, such as the alternative oxidase (AOX), suggest a mitochondrial dysfunction in the absence of bcltf1. The hypersensitivity of Δbctlf1 mutants to exogenously applied oxidative stress - even in the absence of light - and the restoration of virulence and growth rates in continuous light by antioxidants, indicate that BcLTF1 is required to cope with oxidative stress that is caused either by exposure to light or arising during host infection. PMID:24415947

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection

PubMed Central

De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain

2016-01-01

ABSTRACT Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae. Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum. IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete’s foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae. Comparing gene expression during infection on guinea pigs with keratin degradation in vitro, which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo, encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates. PMID:27822542

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection.

PubMed

Tran, Van Du T; De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain; Pagni, Marco; Monod, Michel

2016-01-01

Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae . Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum . IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete's foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae . Comparing gene expression during infection on guinea pigs with keratin degradation in vitro , which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo , encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates.

RegA, an AraC-like protein, is a global transcriptional regulator that controls virulence gene expression in Citrobacter rodentium.

PubMed

Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J; Frankel, Gad; Hartland, Elizabeth L; Robins-Browne, Roy M

2008-11-01

Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5alpha, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.

RegA, an AraC-Like Protein, Is a Global Transcriptional Regulator That Controls Virulence Gene Expression in Citrobacter rodentium▿

PubMed Central

Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J.; Frankel, Gad; Hartland, Elizabeth L.; Robins-Browne, Roy M.

2008-01-01

Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5α, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization. PMID:18765720

THE WIDESPREAD OCCURRENCE OF THE ENTEROHEMOLYSIN GENE EHLYA AMONG ENVIRONMENTAL STRAINS OF ESCHERICHIA COLI

EPA Science Inventory

The putative virulence factor enterohemolysin, encoded for by the ehlyA gene, has been closely associated with the pathogenic enterohemorrhagic Escherichia coli (EHEC) group. E. coli isolates from effluents from seven geographically dispersed municipal ...

EBR1 genomic expansion and its role in virulence of Fusarium species

USDA-ARS?s Scientific Manuscript database

Genome sequencing of Fusarium oxysporum revealed that pathogenic forms of this fungus harbor supernumerary chromosomes with a wide variety of genes, many of which likely encode traits required for pathogenicity or niche specialization. Specific transcription factor (TF) gene families are expanded on...

Comparative transcriptional profiling reveals differential expression of pathways directly and indirectly influencing biofilm formation in Escherichia coli O157:H7

USDA-ARS?s Scientific Manuscript database

Introduction: Escherichia coli O157:H7 (O157) is a frequent cause of foodborne disease outbreaks. O157 encodes virulence factors for colonizing and survival in reservoir animals and the environment. For example, genetic factors promoting biofilm formation are linked to survival of O157 in and outsid...

Assessment of virulence factors, antibiotic resistance and amino-decarboxylase activity in Enterococcus faecium MXVK29 isolated from Mexican chorizo.

PubMed

Alvarez-Cisneros, Y M; Fernández, F J; Sainz-Espuñez, T; Ponce-Alquicira, E

2017-02-01

Enterococcus faecium MXVK29 has the ability to produce an antimicrobial compound that belongs to Class IIa of the Klaenhammer classification, and could be used as part of a biopreservation technology through direct inoculation of the strain as a starter or protective culture. However, Enterococcus is considered as an opportunistic pathogen, hence, the purpose of this work was to study the food safety determinants of E. faecium MXVK29. The strain was sensitive to all of the antibiotics tested (penicillin, tetracycline, vancomycin, erythromycin, chloramphenicol, gentamicin, neomycin, kanamycin and netilmicin) and did not demonstrate histamine, cadaverine or putrescine formation. Furthermore, tyrosine-decarboxylase activity was detected by qualitative assays and PCR. Among the virulence factors analysed for the strain, only the genes encoding the sexual pheromone cCF10 precursor lipoprotein (ccf) and cell-wall adhesion (efaA fm ) were amplified. The presence of these genes has low impact on pathogenesis, as there are no other genes encoding for virulence factors, such as aggregation proteins. Therefore, Enterococcus faecium could be employed as part of a bioconservation method, because it does not produce risk factors for consumer's health; in addition, it could be used as part of the hurdle technology in foods. The use of molecular techniques has allowed, in recent years, to detect pathogenicity genes present in the genome of starter cultures used in food processing and preservation. The presence of these genes is undesirable, because horizontal transfer may occur with the natural biota of consumers. For this reason, it is important to analyse the presence of pathogenicity genes in such cultures. In this work, virulence factors and antibiotic resistance of Enterococcus faecium strain MXVK29, producing an antimicrobial compound with high antilisterial activity, were analysed. The results indicate that the strain is safe to be used in food processing as starter culture. © 2016 The Society for Applied Microbiology.

ArgO145, a Stx2a prophage of a bovine O145:H- STEC strain, is closely related to phages of virulent human strains.

PubMed

Krüger, A; Burgán, J; Friedrich, A W; Rossen, J W A; Lucchesi, P M A

2018-06-01

Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both Stx prophages and Stx. In particular, the toxin subtype Stx2a is associated with high virulence and HUS. Here, we report the genome of ArgO145, an inducible Stx2a prophage identified in a bovine O145:H- strain which produced high levels of Shiga toxin and Stx phage particles. The ArgO145 genome shared lambda phage organization, with recombination, regulation, replication, lysis, and head and tail structural gene regions, although some lambda genes encoding regulatory proteins could not be identified. Remarkably, some Stx2a phages of strains isolated from patients in other countries showed high similarity to ArgO145. Copyright © 2018 Elsevier B.V. All rights reserved.

Plasmid-Encoded MCP Is Involved in Virulence, Motility, and Biofilm Formation of Cronobacter sakazakii ATCC 29544

PubMed Central

Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun

2014-01-01

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage. PMID:25332122

Plasmid-encoded MCP is involved in virulence, motility, and biofilm formation of Cronobacter sakazakii ATCC 29544.

PubMed

Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun; Ryu, Sangryeol

2015-01-01

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Virulence Factors of Erwinia amylovora: A Review.

PubMed

Piqué, Núria; Miñana-Galbis, David; Merino, Susana; Tomás, Juan M

2015-06-05

Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3'-5')-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.

Environmental signals modulate ToxT-dependent virulence factor expression in Vibrio cholerae.

PubMed

Schuhmacher, D A; Klose, K E

1999-03-01

The regulatory protein ToxT directly activates the transcription of virulence factors in Vibrio cholerae, including cholera toxin (CT) and the toxin-coregulated pilus (TCP). Specific environmental signals stimulate virulence factor expression by inducing the transcription of toxT. We demonstrate that transcriptional activation by the ToxT protein is also modulated by environmental signals. ToxT expressed from an inducible promoter activated high-level expression of CT and TCP in V. cholerae at 30 degrees C, but expression of CT and TCP was significantly decreased or abolished by the addition of 0.4% bile to the medium and/or an increase of the temperature to 37 degrees C. Also, expression of six ToxT-dependent TnphoA fusions was modulated by temperature and bile. Measurement of ToxT-dependent transcription of genes encoding CT and TCP by ctxAp- and tcpAp-luciferase fusions confirmed that negative regulation by 37 degrees C or bile occurs at the transcriptional level in V. cholerae. Interestingly, ToxT-dependent transcription of these same promoters in Salmonella typhimurium was relatively insensitive to regulation by temperature or bile. These data are consistent with ToxT transcriptional activity being modulated by environmental signals in V. cholerae and demonstrate an additional level of complexity governing the expression of virulence factors in this pathogen. We propose that negative regulation of ToxT-dependent transcription by environmental signals prevents the incorrect temporal and spatial expression of virulence factors during cholera pathogenesis.

Increased virulence of Mycobacterium tuberculosis H37Rv overexpressing LipY in a murine model.

PubMed

Singh, Vipul K; Srivastava, Mrigank; Dasgupta, Arunava; Singh, Mohan P; Srivastava, Ranjana; Srivastava, Brahm S

2014-05-01

We have investigated the role of Rv3097c-encoded lipase (LipY) on the virulence of Mycobacterium tuberculosis. It has been shown that the overexpression of LipY in strain H37Rv induced increase in virulence of recombinant H37Rv::LipY strain. Compared to H37Rv, infection with H37Rv::LipY caused enhanced mortality, weight loss, bacterial load in lungs, splenomegaly, worsening lung morphology and pathology. Mice immunized with recombinant LipY antigen were protected against challenge with H37Rv::LipY, which correlated with enhanced survival of challenged mice and striking decrease in pathological features observed in unimmunized mice. To probe the cause of increase in virulence of H37Rv::LipY, the immune status of the host infected with H37Rv and H37Rv::LipY was compared. It was found that overexpression of LipY compromised immune responses resulting in attenuation of Th1 and Th17 responses, significant increase in IL-10, decrease in number of macrophages and T cells, and increase in numbers of Treg, and DCs in the lungs whereas in mice immunized with LipY an increased pool of T cells and DCs was observed. This led us to conclude that the increase in the virulence of H37Rv::LipY was due to downregulation of the host's protective immunity and the Rv3097c encoded LipY lipase is a virulence factor of M. tuberculosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

How to kill the honey bee larva: genomic potential and virulence mechanisms of Paenibacillus larvae.

PubMed

Djukic, Marvin; Brzuszkiewicz, Elzbieta; Fünfhaus, Anne; Voss, Jörn; Gollnow, Kathleen; Poppinga, Lena; Liesegang, Heiko; Garcia-Gonzalez, Eva; Genersch, Elke; Daniel, Rolf

2014-01-01

Paenibacillus larvae, a Gram positive bacterial pathogen, causes American Foulbrood (AFB), which is the most serious infectious disease of honey bees. In order to investigate the genomic potential of P. larvae, two strains belonging to two different genotypes were sequenced and used for comparative genome analysis. The complete genome sequence of P. larvae strain DSM 25430 (genotype ERIC II) consisted of 4,056,006 bp and harbored 3,928 predicted protein-encoding genes. The draft genome sequence of P. larvae strain DSM 25719 (genotype ERIC I) comprised 4,579,589 bp and contained 4,868 protein-encoding genes. Both strains harbored a 9.7 kb plasmid and encoded a large number of virulence-associated proteins such as toxins and collagenases. In addition, genes encoding large multimodular enzymes producing nonribosomally peptides or polyketides were identified. In the genome of strain DSM 25719 seven toxin associated loci were identified and analyzed. Five of them encoded putatively functional toxins. The genome of strain DSM 25430 harbored several toxin loci that showed similarity to corresponding loci in the genome of strain DSM 25719, but were non-functional due to point mutations or disruption by transposases. Although both strains cause AFB, significant differences between the genomes were observed including genome size, number and composition of transposases, insertion elements, predicted phage regions, and strain-specific island-like regions. Transposases, integrases and recombinases are important drivers for genome plasticity. A total of 390 and 273 mobile elements were found in strain DSM 25430 and strain DSM 25719, respectively. Comparative genomics of both strains revealed acquisition of virulence factors by horizontal gene transfer and provided insights into evolution and pathogenicity.

Evaluating the pathogenic potential of environmental Escherichia coli by using the Caenorhabditis elegans infection model.

PubMed

Merkx-Jacques, Alexandra; Coors, Anja; Brousseau, Roland; Masson, Luke; Mazza, Alberto; Tien, Yuan-Ching; Topp, Edward

2013-04-01

The detection and abundance of Escherichia coli in water is used to monitor and mandate the quality of drinking and recreational water. Distinguishing commensal waterborne E. coli isolates from those that cause diarrhea or extraintestinal disease in humans is important for quantifying human health risk. A DNA microarray was used to evaluate the distribution of virulence genes in 148 E. coli environmental isolates from a watershed in eastern Ontario, Canada, and in eight clinical isolates. Their pathogenic potential was evaluated with Caenorhabditis elegans, and the concordance between the bioassay result and the pathotype deduced by genotyping was explored. Isolates identified as potentially pathogenic on the basis of their complement of virulence genes were significantly more likely to be pathogenic to C. elegans than those determined to be potentially nonpathogenic. A number of isolates that were identified as nonpathogenic on the basis of genotyping were pathogenic in the infection assay, suggesting that genotyping did not capture all potentially pathogenic types. The detection of the adhesin-encoding genes sfaD, focA, and focG, which encode adhesins; of iroN2, which encodes a siderophore receptor; of pic, which encodes an autotransporter protein; and of b1432, which encodes a putative transposase, was significantly associated with pathogenicity in the infection assay. Overall, E. coli isolates predicted to be pathogenic on the basis of genotyping were indeed so in the C. elegans infection assay. Furthermore, the detection of C. elegans-infective environmental isolates predicted to be nonpathogenic on the basis of genotyping suggests that there are hitherto-unrecognized virulence factors or combinations thereof that are important in the establishment of infection.

A secretome view of colonisation factors in Shiga toxin-encoding Escherichia coli (STEC): from enterohaemorrhagic E. coli (EHEC) to related enteropathotypes.

PubMed

Monteiro, Ricardo; Ageorges, Valentin; Rojas-Lopez, Maricarmen; Schmidt, Herbert; Weiss, Agnes; Bertin, Yolande; Forano, Evelyne; Jubelin, Grégory; Henderson, Ian R; Livrelli, Valérie; Gobert, Alain P; Rosini, Roberto; Soriani, Marco; Desvaux, Mickaël

2016-08-01

Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E. coli, enterohaemorrhagic E. coli (EHEC) constitute the major subgroup of virulent STEC. EHEC cause serious human disease such as haemorrhagic colitis and haemolytic-uremic syndrome. While EHEC have evolved from enteropathogenic E. coli, hybrids with enteroaggregative E. coli have recently emerged. Of note, some enteroinvasive E. coli also belong to the STEC group. While the LEE (locus of enterocyte effacement) is a key and prominent molecular determinant in the pathogenicity, neither all EHEC nor STEC contain the LEE, suggesting that they possess additional virulence and colonisation factors. Currently, nine protein secretion systems have been described in diderm-lipopolysaccharide bacteria (archetypal Gram-negative) and can be involved in the secretion of extracellular effectors, cell-surface proteins or assembly of cell-surface organelles, such as flagella or pili. In this review, we focus on the secretome of STEC and related enteropathotypes, which are relevant to the colonisation of biotic and abiotic surfaces. Considering the wealth of potential protein trafficking mechanisms, the different combinations of colonisation factors and modulation of their expression is further emphasised with regard to the ecophysiology of STEC. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Countermeasure development for Rift Valley fever: deletion, modification or targeting of major virulence factor NSs.

PubMed

Lihoradova, Olga; Ikegami, Tetsuro

2014-01-01

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease characterized by a high rate of abortion in ruminants, and febrile illness, hemorrhagic fever, retinitis and encephalitis in humans. RVF is caused by the RVF virus (RVFV), belonging to the genus Phlebovirus of the family Bunyaviridae . RVFV encodes a major virulence factor, NSs , which is dispensable for viral replication, yet required for evasion of host innate immune responses. RVFV NSs inhibits host gene upregulation at the transcriptional level, while promoting viral translation in the cytoplasm. In this article, we summarize the virology and pathology of RVF, and countermeasure development for RVF, with emphasis on NSs function and applications.

Countermeasure development for Rift Valley fever: deletion, modification or targeting of major virulence factor NSs

PubMed Central

Lihoradova, Olga; Ikegami, Tetsuro

2014-01-01

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease characterized by a high rate of abortion in ruminants, and febrile illness, hemorrhagic fever, retinitis and encephalitis in humans. RVF is caused by the RVF virus (RVFV), belonging to the genus Phlebovirus of the family Bunyaviridae. RVFV encodes a major virulence factor, NSs, which is dispensable for viral replication, yet required for evasion of host innate immune responses. RVFV NSs inhibits host gene upregulation at the transcriptional level, while promoting viral translation in the cytoplasm. In this article, we summarize the virology and pathology of RVF, and countermeasure development for RVF, with emphasis on NSs function and applications. PMID:24910709

AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000

USDA-ARS?s Scientific Manuscript database

Plant pathogenic bacteria are able to integrate information about their environment and adjust gene expression to provide adaptive functions. AlgU, an ECF sigma factor encoded by Pseudomonas syringae, controls expression of genes for alginate biosynthesis and is active while the bacteria are associa...

Vibrio cholerae ToxR downregulates virulence factor production in response to cyclo(Phe-Pro).

PubMed

Bina, X Renee; Taylor, Dawn L; Vikram, Amit; Ante, Vanessa M; Bina, James E

2013-08-27

Vibrio cholerae is an aquatic organism that causes the severe acute diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the production of two critical virulence determinants, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The expression of the genes that encode for CT and TCP production is under the control of a hierarchical regulatory system called the ToxR regulon, which functions to activate virulence gene expression in response to in vivo stimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown. V. cholerae has been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibited V. cholerae virulence factor production. For this study, we report on the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We have demonstrated that exogenous cyclo(Phe-Pro) activated the expression of leuO, a LysR-family regulator that had not been previously associated with V. cholerae virulence. Increased leuO expression repressed aphA transcription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction of leuO expression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affect toxR transcription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription of leuO. These results have identified leuO as a new component of the ToxR regulon and demonstrate for the first time that ToxR is capable of downregulating virulence gene expression in response to an environmental cue. The ToxR regulon has been a focus of cholera research for more than three decades. During this time, a model has emerged wherein ToxR functions to activate the expression of Vibrio cholerae virulence factors upon host entry. V. cholerae and other enteric bacteria produce cyclo(Phe-Pro), a cyclic dipeptide that we identified as an inhibitor of V. cholerae virulence factor production. This finding suggested that cyclo(Phe-Pro) was a negative effector of virulence factor production and represented a molecule that could potentially be exploited for therapeutic development. In this work, we investigated the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We found that cyclo(Phe-Pro) signaled through ToxR to activate the expression of leuO, a new virulence regulator that functioned to repress virulence factor production. Our results have identified a new arm of the ToxR regulon and suggest that ToxR may play a broader role in pathogenesis than previously known.

Unprecedented Abundance of Protein Tyrosine Phosphorylation Modulates Shigella flexneri Virulence.

PubMed

Standish, Alistair James; Teh, Min Yan; Tran, Elizabeth Ngoc Hoa; Doyle, Matthew Thomas; Baker, Paul J; Morona, Renato

2016-10-09

Evidence is accumulating that protein tyrosine phosphorylation plays a crucial role in the ability of important human bacterial pathogens to cause disease. While most works have concentrated on its role in the regulation of a major bacterial virulence factor, the polysaccharide capsule, recent studies have suggested a much broader role for this post-translational modification. This prompted us to investigate protein tyrosine phosphorylation in the human pathogen Shigella flexneri. We first completed a tyrosine phosphoproteome, identifying 905 unique tyrosine phosphorylation sites on at least 573 proteins (approximately 15% of all proteins). This is the most tyrosine-phosphorylated sites and proteins in a single bacterium identified to date, substantially more than the level seen in eukaryotic cells. Most had not previously been identified and included proteins encoded by the virulence plasmid, which is essential for S. flexneri to invade cells and cause disease. In order to investigate the function of these phosphorylation sites in important virulence factors, phosphomimetic and ablative mutations were constructed in the type 3 secretion system ATPase Spa47 and the master virulence regulator VirB. This revealed that tyrosine residues phosphorylated in our study are critical for Spa47 and VirB activity, and tyrosine phosphorylation likely regulates their functional activity and subsequently the virulence of this major human pathogen. This study suggests that tyrosine phosphorylation plays a critical role in regulating a wide variety of virulence factors in the human pathogen S. flexneri and serves as a base for future studies defining its complete role. Copyright © 2016 Elsevier Ltd. All rights reserved.

Variola virus immune evasion proteins.

PubMed

Dunlop, Lance R; Oehlberg, Katherine A; Reid, Jeremy J; Avci, Dilek; Rosengard, Ariella M

2003-09-01

Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.

The Genotoxin Colibactin Is a Determinant of Virulence in Escherichia coli K1 Experimental Neonatal Systemic Infection.

PubMed

McCarthy, Alex J; Martin, Patricia; Cloup, Emilie; Stabler, Richard A; Oswald, Eric; Taylor, Peter W

2015-09-01

Escherichia coli strains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34 E. coli K1 neonatal isolates carried clbA and clbP, genes contained within the pks pathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation of clbA and clbP in E. coli A192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation of clbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation of clbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity of E. coli K1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Genotoxin Colibactin Is a Determinant of Virulence in Escherichia coli K1 Experimental Neonatal Systemic Infection

PubMed Central

McCarthy, Alex J.; Martin, Patricia; Cloup, Emilie; Stabler, Richard A.

2015-01-01

Escherichia coli strains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34 E. coli K1 neonatal isolates carried clbA and clbP, genes contained within the pks pathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation of clbA and clbP in E. coli A192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation of clbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation of clbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity of E. coli K1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate. PMID:26150540

Gene Overexpression/Suppression Analysis of Candidate Virulence Factors of Candida albicans▿

PubMed Central

Fu, Yue; Luo, Guanpingsheng; Spellberg, Brad J.; Edwards, John E.; Ibrahim, Ashraf S.

2008-01-01

We developed a conditional overexpression/suppression genetic strategy in Candida albicans to enable simultaneous testing of gain or loss of function in order to identify new virulence factors. The strategy involved insertion of a strong, tetracycline-regulated promoter in front of the gene of interest. To validate the strategy, a library of genes encoding glycosylphosphatidylinositol (GPI)-anchored surface proteins was screened for virulence phenotypes in vitro. During the screening, overexpression of IFF4 was found to increase the adherence of C. albicans to plastic and to human epithelial cells, but not endothelial cells. Consistent with the in vitro results, IFF4 overexpression modestly increased the tissue fungal burden during murine vaginal candidiasis. In addition to the in vitro screening tests, IFF4 overexpression was found to increase C. albicans susceptibility to neutrophil-mediated killing. Furthermore, IFF4 overexpression decreased the severity of hematogenously disseminated candidiasis in normal mice, but not in neutropenic mice, again consistent with the in vitro phenotype. Overexpression of 12 other GPI proteins did not affect normal GPI protein cell surface accumulation, demonstrating that the overexpression strategy did not affect the cell capacity for making such proteins. These data indicate that the same gene can increase or decrease candidal virulence in distinct models of infection, emphasizing the importance of studying virulence genes in different anatomical contexts. Finally, these data validate the use of a conditional overexpression/suppression genetic strategy to identify candidal virulence factors. PMID:18178776

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

PubMed Central

Brüssow, Harald; Canchaya, Carlos; Hardt, Wolf-Dietrich

2004-01-01

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework. PMID:15353570

The Streptococcus sanguinis competence regulon is not required for infective endocarditis virulence in a rabbit model.

PubMed

Callahan, Jill E; Munro, Cindy L; Kitten, Todd

2011-01-01

Streptococcus sanguinis is an important component of dental plaque and a leading cause of infective endocarditis. Genetic competence in S. sanguinis requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternative sigma factor, ComX. Previous studies of Streptococcus pneumoniae and Streptococcus mutans have identified functions for the >100-gene com regulon in addition to DNA uptake, including virulence. We investigated this possibility in S. sanguinis. Strains deleted for the comCDE or comX master regulatory genes were created. Using a rabbit endocarditis model in conjunction with a variety of virulence assays, we determined that both mutants possessed infectivity equivalent to that of a virulent control strain, and that measures of disease were similar in rabbits infected with each strain. These results suggest that the com regulon is not required for S. sanguinis infective endocarditis virulence in this model. We propose that the different roles of the S. sanguinis, S. pneumoniae, and S. mutans com regulons in virulence can be understood in relation to the pathogenic mechanisms employed by each species.

Season-Specific Occurrence of Potentially Pathogenic Vibrio spp. on the Southern Coast of South Korea.

PubMed

Di, Doris Y W; Lee, Anna; Jang, Jeonghwan; Han, Dukki; Hur, Hor-Gil

2017-02-01

Vibrio species are widely distributed in warm estuarine and coastal environments, and they can infect humans through the consumption of raw and mishandled contaminated seafood. In this study, we aimed to isolate and observe the distribution of enteropathogenic Vibrio spp. from environments of the southern coast of South Korea over a season cycle. A total of 10,983 isolates of Vibrio spp. were obtained from tidal water and mud samples over a 1-year period from five sampling sites along the southwest coast of South Korea. We found that Vibrio alginolyticus (n = 6,262) and Vibrio parahaemolyticus (n = 1,757) were ubiquitous in both tidal water and mud year round, whereas Vibrio cholerae (n = 24) and Vibrio vulnificus (n = 130) were seasonally specific to summer. While all V. cholerae isolates were nontoxigenic (non-O1 and non-O139), more than 88% of V. vulnificus isolates possessed the virulence factor elastolytic protease (encoded by vvp). Interestingly, V. parahaemolyticus, which was omnipresent in all seasons, contained the virulence factors thermostable direct hemolysin (encoded by tdh) and thermostable direct hemolysin-related hemolysin (encoded by trh) in larger amounts in June (29 trh-positive strains) and September (14 tdh-, 36 trh-, and 12 tdh- and trh-positive strains) than in December (4 trh-positive strains) and February (3 tdh-positive strains), and virulence factors were absent from isolates detected in April. To understand why virulence factors were detected only in the warm season and were absent in the cold season although the locations are static, long-term monitoring and particularly seasonal study are necessary. The presence of enteropathogenic Vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus), which cause acute diarrheal infection, septicemia, and wound infections upon ingestion through food and water, is usually associated with temperature. The World Health Organization (WHO) has estimated that there are 1.4 to 4.3 million cases and 28,000 to 142,000 deaths per year worldwide caused by cholera disease. In South Korea alone, consumption is as much as 52.4 kg of fish and shellfish per year per capita. Our findings suggested that seasonally specific acceleration of these possible pathogenic Vibrio spp. may threaten seafood safety and increase the risk of illness in South Korea, where local people consume raw fish during warmer months. Copyright © 2017 American Society for Microbiology.

Season-Specific Occurrence of Potentially Pathogenic Vibrio spp. on the Southern Coast of South Korea

PubMed Central

Di, Doris Y. W.; Lee, Anna; Jang, Jeonghwan; Han, Dukki

2016-01-01

ABSTRACT Vibrio species are widely distributed in warm estuarine and coastal environments, and they can infect humans through the consumption of raw and mishandled contaminated seafood. In this study, we aimed to isolate and observe the distribution of enteropathogenic Vibrio spp. from environments of the southern coast of South Korea over a season cycle. A total of 10,983 isolates of Vibrio spp. were obtained from tidal water and mud samples over a 1-year period from five sampling sites along the southwest coast of South Korea. We found that Vibrio alginolyticus (n = 6,262) and Vibrio parahaemolyticus (n = 1,757) were ubiquitous in both tidal water and mud year round, whereas Vibrio cholerae (n = 24) and Vibrio vulnificus (n = 130) were seasonally specific to summer. While all V. cholerae isolates were nontoxigenic (non-O1 and non-O139), more than 88% of V. vulnificus isolates possessed the virulence factor elastolytic protease (encoded by vvp). Interestingly, V. parahaemolyticus, which was omnipresent in all seasons, contained the virulence factors thermostable direct hemolysin (encoded by tdh) and thermostable direct hemolysin-related hemolysin (encoded by trh) in larger amounts in June (29 trh-positive strains) and September (14 tdh-, 36 trh-, and 12 tdh- and trh-positive strains) than in December (4 trh-positive strains) and February (3 tdh-positive strains), and virulence factors were absent from isolates detected in April. To understand why virulence factors were detected only in the warm season and were absent in the cold season although the locations are static, long-term monitoring and particularly seasonal study are necessary. IMPORTANCE The presence of enteropathogenic Vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus), which cause acute diarrheal infection, septicemia, and wound infections upon ingestion through food and water, is usually associated with temperature. The World Health Organization (WHO) has estimated that there are 1.4 to 4.3 million cases and 28,000 to 142,000 deaths per year worldwide caused by cholera disease. In South Korea alone, consumption is as much as 52.4 kg of fish and shellfish per year per capita. Our findings suggested that seasonally specific acceleration of these possible pathogenic Vibrio spp. may threaten seafood safety and increase the risk of illness in South Korea, where local people consume raw fish during warmer months. PMID:27836844

The prrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

PubMed Central

Reinhart, Alexandria A.; Powell, Daniel A.; Nguyen, Angela T.; O'Neill, Maura; Djapgne, Louise; Wilks, Angela; Ernst, Robert K.

2014-01-01

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence. PMID:25510881

A Family of Indoles Regulate Virulence and Shiga Toxin Production in Pathogenic E. coli

PubMed Central

Izrayelit, Yevgeniy; Bhatt, Shantanu; Cartwright, Emily; Wang, Wei; Swimm, Alyson I.; Benian, Guy M.; Schroeder, Frank C.; Kalman, Daniel

2013-01-01

Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC) are intestinal pathogens that cause food and water-borne disease in humans. Using biochemical methods and NMR-based comparative metabolomics in conjunction with the nematode Caenorhabditis elegans, we developed a bioassay to identify secreted small molecules produced by these pathogens. We identified indole, indole-3-carboxaldehyde (ICA), and indole-3-acetic acid (IAA), as factors that only in combination are sufficient to kill C. elegans. Importantly, although lethal to C. elegans, these molecules downregulate several bacterial processes important for pathogenesis in mammals. These include motility, biofilm formation and production of Shiga toxins. Some pathogenic E. coli strains are known to contain a Locus of Enterocyte Effacement (LEE), which encodes virulence factors that cause “attaching and effacing” (A/E) lesions in mammals, including formation of actin pedestals. We found that these indole derivatives also downregulate production of LEE virulence factors and inhibit pedestal formation on mammalian cells. Finally, upon oral administration, ICA inhibited virulence and promoted survival in a lethal mouse infection model. In summary, the C. elegans model in conjunction with metabolomics has facilitated identification of a family of indole derivatives that broadly regulate physiology in E. coli, and virulence in pathogenic strains. These molecules may enable development of new therapeutics that interfere with bacterial small-molecule signaling. PMID:23372726

Mobile genetic elements of Staphylococcus aureus.

PubMed

Malachowa, Natalia; DeLeo, Frank R

2010-09-01

Bacteria such as Staphylococcus aureus are successful as commensal organisms or pathogens in part because they adapt rapidly to selective pressures imparted by the human host. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information (DNA) among and within bacterial species. Importantly, MGEs encode putative virulence factors and molecules that confer resistance to antibiotics, including the gene that confers resistance to beta-lactam antibiotics in methicillin-resistant S. aureus (MRSA). Inasmuch as MRSA infections are a significant problem worldwide and continue to emerge in epidemic waves, there has been significant effort to improve diagnostic assays and to develop new antimicrobial agents for treatment of disease. Our understanding of S. aureus MGEs and the molecules they encode has played an important role toward these ends and has provided detailed insight into the evolution of antimicrobial resistance mechanisms and virulence.

Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence.

PubMed

Davies, Bryan W; Bogard, Ryan W; Young, Travis S; Mekalanos, John J

2012-04-13

The function of the Vibrio 7(th) pandemic island-1 (VSP-1) in cholera pathogenesis has remained obscure. Utilizing chromatin immunoprecipitation sequencing and RNA sequencing to map the regulon of the master virulence regulator ToxT, we identify a TCP island-encoded small RNA that reduces the expression of a previously unrecognized VSP-1-encoded transcription factor termed VspR. VspR modulates the expression of several VSP-1 genes including one that encodes a novel class of di-nucleotide cyclase (DncV), which preferentially synthesizes a previously undescribed hybrid cyclic AMP-GMP molecule. We show that DncV is required for efficient intestinal colonization and downregulates V. cholerae chemotaxis, a phenotype previously associated with hyperinfectivity. This pathway couples the actions of previously disparate genomic islands, defines VSP-1 as a pathogenicity island in V. cholerae, and implicates its occurrence in 7(th) pandemic strains as a benefit for host adaptation through the production of a regulatory cyclic di-nucleotide. Copyright © 2012 Elsevier Inc. All rights reserved.

Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix.

PubMed

Smith, Nicola L; Taylor, Edward J; Lindsay, Anna-Marie; Charnock, Simon J; Turkenburg, Johan P; Dodson, Eleanor J; Davies, Gideon J; Black, Gary W

2005-12-06

Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyogenes is frequently mediated by bacteriophage infection. One of the key bacteriophage-encoded virulence factors is a putative "hyaluronidase," HylP1, a phage tail-fiber protein responsible for the digestion of the S. pyogenes hyaluronan capsule during phage infection. Here we demonstrate that HylP1 is a hyaluronate lyase. The 3D structure, at 1.8-angstroms resolution, reveals an unusual triple-stranded beta-helical structure and provides insight into the structural basis for phage tail assembly and the role of phage tail proteins in virulence. Unlike the triple-stranded beta-helix assemblies of the bacteriophage T4 injection machinery and the tailspike endosialidase of the Escherichia coli K1 bacteriophage K1F, HylP1 possesses three copies of the active center on the triple-helical fiber itself without the need for an accessory catalytic domain. The triple-stranded beta-helix is not simply a structural scaffold, as previously envisaged; it is harnessed to provide a 200-angstroms-long substrate-binding groove for the optimal reduction in hyaluronan viscosity to aid phage penetration of the capsule.

Natural variation in the VELVET gene bcvel1 affects virulence and light-dependent differentiation in Botrytis cinerea.

PubMed

Schumacher, Julia; Pradier, Jean-Marc; Simon, Adeline; Traeger, Stefanie; Moraga, Javier; Collado, Isidro González; Viaud, Muriel; Tudzynski, Bettina

2012-01-01

Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the full-length protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Δbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Δbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Δbcvel1 mutants to nutrient starvation conditions.

Natural Variation in the VELVET Gene bcvel1 Affects Virulence and Light-Dependent Differentiation in Botrytis cinerea

PubMed Central

Schumacher, Julia; Pradier, Jean-Marc; Simon, Adeline; Traeger, Stefanie; Moraga, Javier; Collado, Isidro González; Viaud, Muriel; Tudzynski, Bettina

2012-01-01

Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the full-length protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Δbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Δbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Δbcvel1 mutants to nutrient starvation conditions. PMID:23118899

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali.

PubMed

Song, Na; Dai, Qingqing; Zhu, Baitao; Wu, Yuxing; Xu, Ming; Voegele, Ralf Thomas; Gao, Xiaoning; Kang, Zhensheng; Huang, Lili

2017-01-01

In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. 'Fuji' leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes-important virulence factors in V. mali-are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.

Regulation of Yersina pestis Virulence by AI-2 Mediated Quorum Sensing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Segelke, B; Hok, S; Lao, V

The proposed research was motivated by an interest in understanding Y. pestis virulence mechanisms and bacteria cell-cell communication. It is expected that a greater understanding of virulence mechanisms will ultimately lead to biothreat countermeasures and novel therapeutics. Y. pestis is the etiological agent of plague, the most devastating disease in human history. Y. pestis infection has a high mortality rate and a short incubation before mortality. There is no widely available and effective vaccine for Y. pestis and multi-drug resistant strains are emerging. Y. pestis is a recognized biothreat agent based on the wide distribution of the bacteria in researchmore » laboratories around the world and on the knowledge that methods exist to produce and aerosolize large amounts of bacteria. We hypothesized that cell-cell communication via signaling molecules, or quorum sensing, by Y. pestis is important for the regulation of virulence factor gene expression during host invasion, though a causative link had never been established. Quorum sensing is a mode of intercellular communication which enables orchestration of gene expression for many bacteria as a function of population density and available evidence suggests there may be a link between quorum sensing and regulation of Y. pesits virulence. Several pathogenic bacteria have been shown to regulate expression of virulence factor genes, including genes encoding type III secretion, via quorum sensing. The Y. pestis genome encodes several cell-cell signaling pathways and the interaction of at least three of these are thought to be involved in one or more modes of host invasion. Furthermore, Y. pestis gene expression array studies carried out at LLNL have established a correlation between expression of known virulence factors and genes involved in processing of the AI-2 quorum sensing signal. This was a basic research project that was intended to provide new insights into bacterial intercellular communication and how it is used to regulate virulence in Y. pestis. It is known that many bacteria use intercellular signaling molecules to orchestrate gene expression and cellular function. A fair amount is known about production and uptake of signaling molecules, but very little is known about how intercellular signaling regulates other pathways. Although several studies demonstrate that intercellular signaling plays a role in regulating virulence in other pathogens, the link between signaling and regulation of virulence has not been established. Very little work had been done directly with Y. pestis intercellular signaling apart from the work carried out at LLNL. The research we proposed was intended to both establish a causative link between AI-2 intercellular signaling and regulation of virulence in Y. pestis and elucidate the fate of the AI-2 signaling molecule after it is taken up and processed by Y. pestis. Elucidating the fate of AI-2 was expected to lead directly to the understanding of how AI-2 signal processing regulates other pathways as well as provide new insights in this direction.« less

Genome Sequences for Five Strains of the Emerging Pathogen Haemophilus haemolyticus

PubMed Central

Jordan, I. King; Conley, Andrew B.; Antonov, Ivan V.; Arthur, Robert A.; Cook, Erin D.; Cooper, Guy P.; Jones, Bernard L.; Knipe, Kristen M.; Lee, Kevin J.; Liu, Xing; Mitchell, Gabriel J.; Pande, Pushkar R.; Petit, Robert A.; Qin, Shaopu; Rajan, Vani N.; Sarda, Shruti; Sebastian, Aswathy; Tang, Shiyuyun; Thapliyal, Racchit; Varghese, Neha J.; Ye, Tianjun; Katz, Lee S.; Wang, Xin; Rowe, Lori; Frace, Michael; Mayer, Leonard W.

2011-01-01

We report the first whole-genome sequences for five strains, two carried and three pathogenic, of the emerging pathogen Haemophilus haemolyticus. Preliminary analyses indicate that these genome sequences encode markers that distinguish H. haemolyticus from its closest Haemophilus relatives and provide clues to the identity of its virulence factors. PMID:21952546

Conjugative type IVb pilus recognizes lipopolysaccharide of recipient cells to initiate PAPI-1 pathogenicity island transfer in Pseudomonas aeruginosa

USDA-ARS?s Scientific Manuscript database

Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1) is one of the largest genomic islands of this important opportunistic human pathogen. Previous studies have shown that PAPI-1 encodes several putative virulence factors, a major regulator of biofilm formation, and antibiotic-resistance traits, a...

A chromosomally encoded virulence factor protects the Lyme disease pathogen against host-adaptive immunity.

PubMed

Yang, Xiuli; Coleman, Adam S; Anguita, Juan; Pal, Utpal

2009-03-01

Borrelia burgdorferi, the bacterial pathogen of Lyme borreliosis, differentially expresses select genes in vivo, likely contributing to microbial persistence and disease. Expression analysis of spirochete genes encoding potential membrane proteins showed that surface-located membrane protein 1 (lmp1) transcripts were expressed at high levels in the infected murine heart, especially during early stages of infection. Mice and humans with diagnosed Lyme borreliosis also developed antibodies against Lmp1. Deletion of lmp1 severely impaired the pathogen's ability to persist in diverse murine tissues including the heart, and to induce disease, which was restored upon chromosomal complementation of the mutant with the lmp1 gene. Lmp1 performs an immune-related rather than a metabolic function, as its deletion did not affect microbial persistence in immunodeficient mice, but significantly decreased spirochete resistance to the borreliacidal effects of anti-B. burgdorferi sera in a complement-independent manner. These data demonstrate the existence of a virulence factor that helps the pathogen evade host-acquired immune defense and establish persistent infection in mammals.

Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

PubMed

Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

2013-07-01

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

Virulence Factors of Erwinia amylovora: A Review

PubMed Central

Piqué, Núria; Miñana-Galbis, David; Merino, Susana; Tomás, Juan M.

2015-01-01

Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3′-5′)-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them. PMID:26057748

Distribution of virulence determinants among antimicrobial-resistant and antimicrobial-susceptible Escherichia coli implicated in urinary tract infections.

PubMed

Stephenson, Sam; Brown, P D

2016-01-01

Uropathogenic Escherichia coli (UPEC) rely on the correlation of virulence expression with antimicrobial resistance to persist and cause severe urinary tract infections (UTIs). We assessed the virulence pattern and prevalence among UPEC strains susceptible and resistant to multiple antimicrobial classes. A total of 174 non-duplicate UPEC strains from patients with clinically significant UTIs were analysed for susceptibility to aminoglycoside, antifolate, cephalosporin, nitrofuran and quinolone antibiotics for the production of extended-spectrum β-lactamases and for the presence of six virulence determinants encoding adhesins (afimbrial, Type 1 fimbriae, P and S-fimbriae) and toxins (cytotoxic necrotising factor and haemolysin). Relatively high resistance rates to nalidixic acid, ciprofloxacin, cephalothin and trimethoprim-sulfamethoxazole (82%, 78%, 62% and 59%, respectively) were observed. Fourteen distinct patterns were identified for the virulence determinants such as afaBC, cnfI, fimH, hylA, papEF and sfaDE. The toxin gene, cnfI (75.3%), was the second most prevalent marker to the adhesin, fimH (97.1%). The significant association of sfaDE/hylA (P < 0.01) among antimicrobial resistant and susceptible strains was also observed notwithstanding an overall greater occurrence of virulence factors among the latter. This study provides a snapshot of UPEC complexity in Jamaica and highlights the significant clonal heterogeneity among strains. Such outcomes emphasise the need for evidence-based strategies in the effective management and control of UTIs.

Route of infection alters virulence of neonatal septicemia Escherichia coli clinical isolates

PubMed Central

Cole, Bryan K.; Scott, Edgar; Ilikj, Marko; Bard, David; Akins, Darrin R.; Dyer, David W.

2017-01-01

Escherichia coli is the leading cause of Gram-negative neonatal septicemia in the United States. Invasion and passage across the neonatal gut after ingestion of maternal E. coli strains produce bacteremia. In this study, we compared the virulence properties of the neonatal E. coli bacteremia clinical isolate SCB34 with the archetypal neonatal E. coli meningitis strain RS218. Whole-genome sequencing data was used to compare the protein coding sequences among these clinical isolates and 33 other representative E. coli strains. Oral inoculation of newborn animals with either strain produced septicemia, whereas intraperitoneal injection caused septicemia only in pups infected with RS218 but not in those injected with SCB34. In addition to being virulent only through the oral route, SCB34 demonstrated significantly greater invasion and transcytosis of polarized intestinal epithelial cells in vitro as compared to RS218. Protein coding sequences comparisons highlighted the presence of known virulence factors that are shared among several of these isolates, and revealed the existence of proteins exclusively encoded in SCB34, many of which remain uncharacterized. Our study demonstrates that oral acquisition is crucial for the virulence properties of the neonatal bacteremia clinical isolate SCB34. This characteristic, along with its enhanced ability to invade and transcytose intestinal epithelium are likely determined by the specific virulence factors that predominate in this strain. PMID:29236742

A Yersinia pestis YscN ATPase mutant functions as a live attenuated vaccine against bubonic plague in mice.

PubMed

Bozue, Joel; Cote, Christopher K; Webster, Wendy; Bassett, Anthony; Tobery, Steven; Little, Stephen; Swietnicki, Wieslaw

2012-07-01

Yersinia pestis is the causative agent responsible for bubonic and pneumonic plague. The bacterium uses the pLcr plasmid-encoded type III secretion system to deliver virulence factors into host cells. Delivery requires ATP hydrolysis by the YscN ATPase encoded by the yscN gene also on pLcr. A yscN mutant was constructed in the fully virulent CO92 strain containing a nonpolar, in-frame internal deletion within the gene. We demonstrate that CO92 with a yscN mutation was not able to secrete the LcrV protein (V-Antigen) and attenuated in a subcutaneous model of plague demonstrating that the YscN ATPase was essential for virulence. However, if the yscN mutant was complemented with a functional yscN gene in trans, virulence was restored. To evaluate the mutant as a live vaccine, Swiss-Webster mice were vaccinated twice with the ΔyscN mutant at varying doses and were protected against bubonic plague in a dose-dependent manner. Antibodies to F1 capsule but not to LcrV were detected in sera from the vaccinated mice. These preliminary results suggest a proof-of-concept for an attenuated, genetically engineered, live vaccine effective against bubonic plague. Published 2012. This article is a US Government work and is in the public domain in the USA.

Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression.

PubMed

Kasper, Stephen H; Bonocora, Richard P; Wade, Joseph T; Musah, Rabi Ann; Cady, Nathaniel C

2016-04-15

The opportunistic pathogen Pseudomonas aeruginosa utilizes multiple quorum sensing (QS) pathways to coordinate an arsenal of virulence factors. We previously identified several cysteine-based compounds inspired by natural products from the plant Petiveria alliacea which are capable of antagonizing multiple QS circuits as well as reducing P. aeruginosa biofilm formation. To understand the global effects of such compounds on virulence factor production and elucidate their mechanism of action, RNA-seq transcriptomic analysis was performed on P. aeruginosa PAO1 exposed to S-phenyl-l-cysteine sulfoxide, the most potent inhibitor from the prior study. Exposure to this inhibitor down-regulated expression of several QS-regulated virulence operons (e.g., phenazine biosynthesis, type VI secretion systems). Interestingly, many genes that were differentially regulated pertain to the related metabolic pathways that yield precursors of pyochelin, tricarboxylic acid cycle intermediates, phenazines, and Pseudomonas quinolone signal (PQS). Activation of the MexT-regulon was also indicated, including the multidrug efflux pump encoded by mexEF-oprN, which has previously been shown to inhibit QS and pathogenicity. Deeper investigation of the metabolites involved in these systems revealed that S-phenyl-l-cysteine sulfoxide has structural similarity to kynurenine, a precursor of anthranilate, which is critical for P. aeruginosa virulence. By supplementing exogenous anthranilate, the QS-inhibitory effect was reversed. Finally, it was shown that S-phenyl-l-cysteine sulfoxide competitively inhibits P. aeruginosa kynureninase (KynU) activity in vitro and reduces PQS production in vivo. The kynurenine pathway has been implicated in P. aeruginosa QS and virulence factor expression; however, this is the first study to show that targeted inhibition of KynU affects P. aeruginosa gene expression and QS, suggesting a potential antivirulence strategy.

Trigger factor of Streptococcus suis is involved in stress tolerance and virulence.

PubMed

Wu, Tao; Zhao, Zhanqin; Zhang, Lin; Ma, Hongwei; Lu, Ka; Ren, Wen; Liu, Zhengya; Chang, Haitao; Bei, Weicheng; Qiu, Yinsheng; Chen, Huanchun

2011-01-01

Streptococcus suis serotype 2 is an important zoonotic pathogen that causes serious diseases such as meningitis, septicemia, endocarditis, arthritis and septic shock in pigs and humans. Little is known about the regulation of virulence gene expression in S. suis serotype 2. In this study, we cloned and deleted the entire tig gene from the chromosome of S. suis serotype 2 SC21 strain, and constructed a mutant strain (Δtig) and a complementation strain (CΔtig). The results demonstrated that the tig gene, encoding trigger factor from S. suis serotype 2 SC21, affects the stress tolerance and the expression of a few virulence genes of S. suis serotype 2. Deletion of the tig gene of S. suis serotype 2 resulted in mutant strain, ΔTig, which exhibited a significant decrease in adherence to cell line HEp-2, and lacked hemolytic activity. Tig deficiency diminishes stresses tolerance of S. suis serotype 2 such as survive thermal, oxidative and acid stresses. Quantification of expression levels of known S. suis serotype 2 SC21 virulence genes by real-time polymerase chain reaction in vitro revealed that trigger factor influences the expression of epf, cps, adh, rpob, fbps, hyl, sly, mrp and hrcA virulence-associated genes. ΔTig was shown to be attenuated in a LD50 assay and bacteriology, indicating that trigger factor plays an important part in the pathogenesis and stress tolerance of. S. suis serotype 2 infection. Mutant ΔTig was 100% defective in virulence in CD1 mice at up to 107 CFU, and provided 100% protection when challenged with 107 CFU of the SC21 strain. Copyright © 2010. Published by Elsevier India Pvt Ltd.

Probiotic properties of lactic acid bacteria isolated from water-buffalo mozzarella cheese.

PubMed

Jeronymo-Ceneviva, Ana Beatriz; de Paula, Aline Teodoro; Silva, Luana Faria; Todorov, Svetoslav Dimitrov; Franco, Bernadette Dora G Mello; Penna, Ana Lúcia B

2014-12-01

This study evaluated the probiotic properties (stability at different pH values and bile salt concentration, auto-aggregation and co-aggregation, survival in the presence of antibiotics and commercial drugs, study of β-galactosidase production, evaluation of the presence of genes encoding MapA and Mub adhesion proteins and EF-Tu elongation factor, and the presence of genes encoding virulence factor) of four LAB strains (Lactobacillus casei SJRP35, Leuconostoc citreum SJRP44, Lactobacillus delbrueckii subsp. bulgaricus SJRP57 and Leuconostoc mesenteroides subsp. mesenteroides SJRP58) which produced antimicrobial substances (antimicrobial peptides). The strains survived the simulated GIT modeled in MRS broth, whole and skim milk. In addition, auto-aggregation and the cell surface hydrophobicity of all strains were high, and various degrees of co-aggregation were observed with indicator strains. All strains presented low resistance to several antibiotics and survived in the presence of commercial drugs. Only the strain SJRP44 did not produce the β-galactosidase enzyme. Moreover, the strain SJRP57 did not show the presence of any genes encoding virulence factors; however, the strain SJRP35 presented vancomycin resistance and adhesion of collagen genes, the strain SJRP44 harbored the ornithine decarboxylase gene and the strain SJRP58 generated positive results for aggregation substance and histidine decarboxylase genes. In conclusion, the strain SJRP57 was considered the best candidate as probiotic cultures for further in vivo studies and functional food products development.

PecS is an important player in the regulatory network governing the coordinated expression of virulence genes during the interaction between Dickeya dadantii 3937 and plants.

PubMed

Mhedbi-Hajri, Nadia; Malfatti, Pierrette; Pédron, Jacques; Gaubert, Stéphane; Reverchon, Sylvie; Van Gijsegem, Frédérique

2011-11-01

Successful infection of a pathogen relies on the coordinated expression of numerous virulence factor-encoding genes. In plant-bacteria interactions, this control is very often achieved through the integration of several regulatory circuits controlling cell-cell communication or sensing environmental conditions. Dickeya dadantii (formerly Erwinia chrysanthemi), the causal agent of soft rot on many crops and ornamentals, provokes maceration of infected plants mainly by producing and secreting a battery of plant cell wall-degrading enzymes. However, several other virulence factors have also been characterized. During Arabidopsis infection, most D. dadantii virulence gene transcripts accumulated in a coordinated manner during infection. This activation requires a functional GacA-GacS two-component regulatory system but the Gac system is not involved in the growth phase dependence of virulence gene expression. Here we show that, contrary to Pectobacterium, the AHL-mediated ExpIR quorum-sensing system does not play a major role in the growth phase-dependent control of D. dadantii virulence genes. On the other hand, the global regulator PecS participates in this coordinated expression since, in a pecS mutant, an early activation of virulence genes is observed both in vitro and in planta. This correlated with the known hypervirulence phenotype of the pecS mutant. Analysis of the relationship between the regulatory circuits governed by the PecS and GacA global regulators indicates that these two regulators act independently. PecS prevents a premature expression of virulence genes in the first stages of colonization whereas GacA, presumably in conjunction with other regulators, is required for the activation of virulence genes at the onset of symptom occurrence. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Context-Dependent Requirements for FimH and Other Canonical Virulence Factors in Gut Colonization by Extraintestinal Pathogenic Escherichia coli

PubMed Central

Russell, Colin W.; Fleming, Brittany A.; Jost, Courtney A.; Tran, Alexander; Stenquist, Alan T.; Wambaugh, Morgan A.; Bronner, Mary P.

2018-01-01

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) acts as a commensal within the mammalian gut but can induce pathology upon dissemination to other host environments such as the urinary tract and bloodstream. ExPEC genomes are likely shaped by evolutionary forces encountered within the gut, where the bacteria spend much of their time, provoking the question of how their extraintestinal virulence traits arose. The principle of coincidental evolution, in which a gene that evolved in one niche happens to be advantageous in another, has been used to argue that ExPEC virulence factors originated in response to selective pressures within the gut ecosystem. As a test of this hypothesis, the fitness of ExPEC mutants lacking canonical virulence factors was assessed within the intact murine gut in the absence of antibiotic treatment. We found that most of the tested factors, including cytotoxic necrotizing factor type 1 (CNF1), Usp, colibactin, flagella, and plasmid pUTI89, were dispensable for gut colonization. The deletion of genes encoding the adhesin PapG or the toxin HlyA had transient effects but did not interfere with longer-term persistence. In contrast, a mutant missing the type 1 pilus-associated adhesin FimH displayed somewhat reduced persistence within the gut. However, this phenotype varied dependent on the presence of specific competing strains and was partially attributable to aberrant flagellin expression in the absence of fimH. These data indicate that FimH and other key ExPEC-associated factors are not strictly required for gut colonization, suggesting that the development of extraintestinal virulence traits is not driven solely by selective pressures within the gut. PMID:29311232

Contemporary Avian Influenza A Virus Subtype H1, H6, H7, H10, and H15 Hemagglutinin Genes Encode a Mammalian Virulence Factor Similar to the 1918 Pandemic Virus H1 Hemagglutinin

PubMed Central

Qi, Li; Pujanauski, Lindsey M.; Davis, A. Sally; Schwartzman, Louis M.; Chertow, Daniel S.; Baxter, David; Scherler, Kelsey; Hartshorn, Kevan L.; Slemons, Richard D.; Walters, Kathie-Anne; Kash, John C.

2014-01-01

ABSTRACT Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. PMID:25406382

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

PubMed

Yassin, Atteyet F; Langenberg, Stefan; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Mukherjee, Supratim; Reddy, T B K; Daum, Chris; Shapiro, Nicole; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos C

2017-01-01

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Evaluation of approaches to monitor Staphylococcus aureus virulence factor expression during human disease.

PubMed

Rozemeijer, Wouter; Fink, Pamela; Rojas, Eduardo; Jones, C Hal; Pavliakova, Danka; Giardina, Peter; Murphy, Ellen; Liberator, Paul; Jiang, Qin; Girgenti, Douglas; Peters, Remco P H; Savelkoul, Paul H M; Jansen, Kathrin U; Anderson, Annaliesa S; Kluytmans, Jan

2015-01-01

Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag) vaccine comprising capsular polysaccharide (types 5 and 8) conjugates, clumping factor A (ClfA) and manganese transporter C (MntC) is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27) or bloodstream (24) infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR) analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine.

Pleiotropic Regulation of Virulence Genes in Streptococcus mutans by the Conserved Small Protein SprV

PubMed Central

Shankar, Manoharan; Hossain, Mohammad S.

2017-01-01

ABSTRACT Streptococcus mutans, an oral pathogen associated with dental caries, colonizes tooth surfaces as polymicrobial biofilms known as dental plaque. S. mutans expresses several virulence factors that allow the organism to tolerate environmental fluctuations and compete with other microorganisms. We recently identified a small hypothetical protein (90 amino acids) essential for the normal growth of the bacterium. Inactivation of the gene, SMU.2137, encoding this protein caused a significant growth defect and loss of various virulence-associated functions. An S. mutans strain lacking this gene was more sensitive to acid, temperature, osmotic, oxidative, and DNA damage-inducing stresses. In addition, we observed an altered protein profile and defects in biofilm formation, bacteriocin production, and natural competence development, possibly due to the fitness defect associated with SMU.2137 deletion. Transcriptome sequencing revealed that nearly 20% of the S. mutans genes were differentially expressed upon SMU.2137 deletion, thereby suggesting a pleiotropic effect. Therefore, we have renamed this hitherto uncharacterized gene as sprV (streptococcal pleiotropic regulator of virulence). The transcript levels of several relevant genes in the sprV mutant corroborated the phenotypes observed upon sprV deletion. Owing to its highly conserved nature, inactivation of the sprV ortholog in Streptococcus gordonii also resulted in poor growth and defective UV tolerance and competence development as in the case of S. mutans. Our experiments suggest that SprV is functionally distinct from its homologs identified by structure and sequence homology. Nonetheless, our current work is aimed at understanding the importance of SprV in the S. mutans biology. IMPORTANCE Streptococcus mutans employs several virulence factors and stress resistance mechanisms to colonize tooth surfaces and cause dental caries. Bacterial pathogenesis is generally controlled by regulators of fitness that are critical for successful disease establishment. Sometimes these regulators, which are potential targets for antimicrobials, are lost in the genomic context due to the lack of annotated homologs. This work outlines the regulatory impact of a small, highly conserved hypothetical protein, SprV, encoded by S. mutans. We show that SprV affects the transcript levels of various virulence factors required for normal growth, biofilm formation, stress tolerance, genetic competence, and bacteriocin production. PMID:28167518

FbpA, a novel multifunctional Listeria monocytogenes virulence factor.

PubMed

Dramsi, S; Bourdichon, F; Cabanes, D; Lecuit, M; Fsihi, H; Cossart, P

2004-07-01

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.

Genomes and Virulence Factors of Novel Bacterial Pathogens Causing Bleaching Disease in the Marine Red Alga Delisea pulchra

PubMed Central

Fernandes, Neil; Case, Rebecca J.; Longford, Sharon R.; Seyedsayamdost, Mohammad R.; Steinberg, Peter D.; Kjelleberg, Staffan; Thomas, Torsten

2011-01-01

Nautella sp. R11, a member of the marine Roseobacter clade, causes a bleaching disease in the temperate-marine red macroalga, Delisea pulchra. To begin to elucidate the molecular mechanisms underpinning the ability of Nautella sp. R11 to colonize, invade and induce bleaching of D. pulchra, we sequenced and analyzed its genome. The genome encodes several factors such as adhesion mechanisms, systems for the transport of algal metabolites, enzymes that confer resistance to oxidative stress, cytolysins, and global regulatory mechanisms that may allow for the switch of Nautella sp. R11 to a pathogenic lifestyle. Many virulence effectors common in phytopathogenic bacteria are also found in the R11 genome, such as the plant hormone indole acetic acid, cellulose fibrils, succinoglycan and nodulation protein L. Comparative genomics with non-pathogenic Roseobacter strains and a newly identified pathogen, Phaeobacter sp. LSS9, revealed a patchy distribution of putative virulence factors in all genomes, but also led to the identification of a quorum sensing (QS) dependent transcriptional regulator that was unique to pathogenic Roseobacter strains. This observation supports the model that a combination of virulence factors and QS-dependent regulatory mechanisms enables indigenous members of the host alga's epiphytic microbial community to switch to a pathogenic lifestyle, especially under environmental conditions when innate host defence mechanisms are compromised. PMID:22162749

Key role of an ADP - ribose - dependent transcriptional regulator of NAD metabolism for fitness and virulence of Pseudomonas aeruginosa.

PubMed

Okon, Elza; Dethlefsen, Sarah; Pelnikevich, Anna; Barneveld, Andrea van; Munder, Antje; Tümmler, Burkhard

2017-01-01

NAD is an essential co-factor of redox reactions and metabolic conversions of NAD-dependent enzymes. NAD biosynthesis in the opportunistic pathogen Pseudomonas aeruginosa has yet not been experimentally explored. The in silico search for orthologs in the P. aeruginosa PAO1 genome identified the operon pncA - pncB1-nadE (PA4918-PA4920) to encode the nicotinamidase, nicotinate phosporibosyltransferase and Nad synthase of salvage pathway I. The functional role of the preceding genes PA4917 and PA4916 was resolved by the characterization of recombinant protein. PA4917 turned out to encode the nicotinate mononucleotide adenylyltransferase NadD2 and PA4916 was determined to encode the transcriptional repressor NrtR that binds to an intergenic sequence between nadD2 and pncA. Complex formation between the catalytically inactive Nudix protein NrtR and its DNA binding site was suppressed by the antirepressor ADP-ribose. NrtR plasposon mutagenesis abrogated virulence of P. aeruginosa TBCF10839 in a murine acute airway infection model and constrained its metabolite profile. When grown together with other isogenic plasposon mutants, the nrtR knock-out was most compromised in competitive fitness to persist in nutrient-rich medium in vitro or murine airways in vivo. This example demonstrates how tightly metabolism and virulence can be intertwined by key elements of metabolic control. Copyright © 2016 Elsevier GmbH. All rights reserved.

Regulation of the yjjQ-bglJ Operon, Encoding LuxR-Type Transcription Factors, and the Divergent yjjP Gene by H-NS and LeuO▿ †

PubMed Central

Stratmann, Thomas; Madhusudan, S.; Schnetz, Karin

2008-01-01

The yjjQ and bglJ genes encode LuxR-type transcription factors conserved in several enterobacterial species. YjjQ is a potential virulence factor in avian pathogenic Escherichia coli. BglJ counteracts the silencing of the bgl (β-glucoside) operon by H-NS in E. coli K-12. Here we show that yjjQ and bglJ form an operon carried by E. coli K-12, whose expression is repressed by the histone-like nucleoid structuring (H-NS) protein. The LysR-type transcription factor LeuO counteracts this repression. Furthermore, the yjjP gene, encoding a membrane protein of unknown function and located upstream in divergent orientation to the yjjQ-bglJ operon, is likewise repressed by H-NS. Mapping of the promoters as well as the H-NS and LeuO binding sites within the 555-bp intergenic region revealed that H-NS binds to the center of the AT-rich regulatory region and distal to the divergent promoters. LeuO sites map to the center and to positions distal to the yjjQ promoters, while one LeuO binding site overlaps with the divergent yjjP promoter. This latter LeuO site is required for full derepression of the yjjQ promoters. The arrangement of regulatory sites suggests that LeuO restructures the nucleoprotein complex formed by H-NS. Furthermore, the data support the conclusion that LeuO, whose expression is likewise repressed by H-NS and which is a virulence factor in Salmonella enterica, is a master regulator that among other loci, also controls the yjjQ-bglJ operon and thus indirectly the presumptive targets of YjjQ and BglJ. PMID:18055596

Concerted Action of Sphingomyelinase and Non-Hemolytic Enterotoxin in Pathogenic Bacillus cereus

PubMed Central

Doll, Viktoria M.

2013-01-01

Bacillus cereus causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe), which is present in most B. cereus strains, is considered to be one of the main virulence factors. However, a B. cereus ΔnheBC mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an in vitro model for polarized colon epithelial cells we identified B. cereus sphingomyelinase (SMase) as a strong inducer of epithelial cell death. Using single and double deletion mutants of sph, the gene encoding for SMase, and nheBC in B. cereus we demonstrated that SMase is an important factor for B. cereus cytotoxicity in vitro and pathogenicity in vivo. SMase substantially complemented Nhe induced cytotoxicity in vitro. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae in vivo in the insect model Galleria mellonella. Our study suggests that the role of B. cereus SMase as a secreted virulence factor for in vivo pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full B. cereus virulence hence disease formation. PMID:23613846

The Regulatory Small RNA MarS Supports Virulence of Streptococcus pyogenes.

PubMed

Pappesch, Roberto; Warnke, Philipp; Mikkat, Stefan; Normann, Jana; Wisniewska-Kucper, Aleksandra; Huschka, Franziska; Wittmann, Maja; Khani, Afsaneh; Schwengers, Oliver; Oehmcke-Hecht, Sonja; Hain, Torsten; Kreikemeyer, Bernd; Patenge, Nadja

2017-09-25

Small regulatory RNAs (sRNAs) play a role in the control of bacterial virulence gene expression. In this study, we investigated an sRNA that was identified in Streptococcus pyogenes (group A Streptococcus, GAS) but is conserved throughout various streptococci. In a deletion strain, expression of mga, the gene encoding the multiple virulence gene regulator, was reduced. Accordingly, transcript and proteome analyses revealed decreased expression of several Mga-activated genes. Therefore, and because the sRNA was shown to interact with the 5' UTR of the mga transcript in a gel-shift assay, we designated it MarS for m ga-activating regulatory sRNA. Down-regulation of important virulence factors, including the antiphagocytic M-protein, led to increased susceptibility of the deletion strain to phagocytosis and reduced adherence to human keratinocytes. In a mouse infection model, the marS deletion mutant showed reduced dissemination to the liver, kidney, and spleen. Additionally, deletion of marS led to increased tolerance towards oxidative stress. Our in vitro and in vivo results indicate a modulating effect of MarS on virulence gene expression and on the pathogenic potential of GAS.

The Streptococcus sanguinis Competence Regulon Is Not Required for Infective Endocarditis Virulence in a Rabbit Model

PubMed Central

Callahan, Jill E.; Munro, Cindy L.; Kitten, Todd

2011-01-01

Streptococcus sanguinis is an important component of dental plaque and a leading cause of infective endocarditis. Genetic competence in S. sanguinis requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternative sigma factor, ComX. Previous studies of Streptococcus pneumoniae and Streptococcus mutans have identified functions for the >100-gene com regulon in addition to DNA uptake, including virulence. We investigated this possibility in S. sanguinis. Strains deleted for the comCDE or comX master regulatory genes were created. Using a rabbit endocarditis model in conjunction with a variety of virulence assays, we determined that both mutants possessed infectivity equivalent to that of a virulent control strain, and that measures of disease were similar in rabbits infected with each strain. These results suggest that the com regulon is not required for S. sanguinis infective endocarditis virulence in this model. We propose that the different roles of the S. sanguinis, S. pneumoniae, and S. mutans com regulons in virulence can be understood in relation to the pathogenic mechanisms employed by each species. PMID:22039480

Yersinia pestis YopJ suppresses tumor necrosis factor alpha induction and contributes to apoptosis of immune cells in the lymph node but is not required for virulence in a rat model of bubonic plague.

PubMed

Lemaître, Nadine; Sebbane, Florent; Long, Daniel; Hinnebusch, B Joseph

2006-09-01

The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system that transfers six Yop effector proteins into host cells. One of these proteins, YopJ, has been shown to disrupt host cell signaling pathways involved in proinflammatory cytokine production and to induce macrophage apoptosis in vitro. YopJ-dependent apoptosis in mesenteric lymph nodes has also been demonstrated in a mouse model of Yersinia pseudotuberculosis infection. These results suggest that YopJ attenuates the host innate and adaptive immune response during infection, but the role of YopJ during bubonic plague has not been completely established. We evaluated the role of Yersinia pestis YopJ in a rat model of bubonic plague following intradermal infection with a fully virulent Y. pestis strain and an isogenic yopJ mutant. Deletion of yopJ resulted in a twofold decrease in the number of apoptotic immune cells in the bubo and a threefold increase in serum tumor necrosis factor alpha levels but did not result in decreased virulence, systemic spread, or colonization levels in the spleen and blood. Our results indicate that YopJ is not essential for bubonic plague pathogenesis, even after peripheral inoculation of low doses of Y. pestis. Instead, the effects of YopJ appear to overlap and augment the immunomodulatory effects of other Y. pestis virulence factors.

Mutations in the Gene Encoding the Ancillary Pilin Subunit of the Streptococcus suis srtF Cluster Result in Pili Formed by the Major Subunit Only

PubMed Central

Fittipaldi, Nahuel; Takamatsu, Daisuke; Domínguez-Punaro, María de la Cruz; Lecours, Marie-Pier; Montpetit, Diane; Osaki, Makoto; Sekizaki, Tsutomu; Gottschalk, Marcelo

2010-01-01

Pili have been shown to contribute to the virulence of different Gram-positive pathogenic species. Among other critical steps of bacterial pathogenesis, these structures participate in adherence to host cells, colonization and systemic virulence. Recently, the presence of at least four discrete gene clusters encoding putative pili has been revealed in the major swine pathogen and emerging zoonotic agent Streptococcus suis. However, pili production by this species has not yet been demonstrated. In this study, we investigated the functionality of one of these pili clusters, known as the srtF pilus cluster, by the construction of mutant strains for each of the four genes of the cluster as well as by the generation of antibodies against the putative pilin subunits. Results revealed that the S. suis serotype 2 strain P1/7, as well as several other highly virulent invasive S. suis serotype 2 isolates express pili from this cluster. However, in most cases tested, and as a result of nonsense mutations at the 5′ end of the gene encoding the minor pilin subunit (a putative adhesin), pili were formed by the major pilin subunit only. We then evaluated the role these pili play in S. suis virulence. Abolishment of the expression of srtF cluster-encoded pili did not result in impaired interactions of S. suis with porcine brain microvascular endothelial cells. Furthermore, non-piliated mutants were as virulent as the wild type strain when evaluated in a murine model of S. suis sepsis. Our results show that srtF cluster-encoded, S. suis pili are atypical compared to other Gram-positive pili. In addition, since the highly virulent strains under investigation are unlikely to produce other pili, our results suggest that pili might be dispensable for critical steps of the S. suis pathogenesis of infection. PMID:20052283

Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus

PubMed Central

Raghunath, Pendru

2015-01-01

Vibrio parahaemolyticus is the leading cause of seafood borne bacterial gastroenteritis in the world, often associated with the consumption of raw or undercooked seafood. However, not all strains of V. parahaemolyticus are pathogenic. The thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH) encoded by tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus. However, about 10% of clinical strains do not contain tdh and/or trh. Environmental isolates of V. parahaemolyticus lacking tdh and/or trh are also highly cytotoxic to human gastrointestinal cells. Even in the absence of these hemolysins, V. parahaemolyticus remains pathogenic indicating other virulence factors exist. This mini review aims at discussing the possible roles of tdh and trh genes in clinical and environmental isolates of V. parahaemolyticus. PMID:25657643

Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus.

PubMed

Raghunath, Pendru

2014-01-01

Vibrio parahaemolyticus is the leading cause of seafood borne bacterial gastroenteritis in the world, often associated with the consumption of raw or undercooked seafood. However, not all strains of V. parahaemolyticus are pathogenic. The thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH) encoded by tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus. However, about 10% of clinical strains do not contain tdh and/or trh. Environmental isolates of V. parahaemolyticus lacking tdh and/or trh are also highly cytotoxic to human gastrointestinal cells. Even in the absence of these hemolysins, V. parahaemolyticus remains pathogenic indicating other virulence factors exist. This mini review aims at discussing the possible roles of tdh and trh genes in clinical and environmental isolates of V. parahaemolyticus.

The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae.

PubMed

Gross, Roy; Guzman, Carlos A; Sebaihia, Mohammed; dos Santos, Vítor A P Martins; Pieper, Dietmar H; Koebnik, Ralf; Lechner, Melanie; Bartels, Daniela; Buhrmester, Jens; Choudhuri, Jomuna V; Ebensen, Thomas; Gaigalat, Lars; Herrmann, Stefanie; Khachane, Amit N; Larisch, Christof; Link, Stefanie; Linke, Burkhard; Meyer, Folker; Mormann, Sascha; Nakunst, Diana; Rückert, Christian; Schneiker-Bekel, Susanne; Schulze, Kai; Vorhölter, Frank-Jörg; Yevsa, Tetyana; Engle, Jacquelyn T; Goldman, William E; Pühler, Alfred; Göbel, Ulf B; Goesmann, Alexander; Blöcker, Helmut; Kaiser, Olaf; Martinez-Arias, Rosa

2008-09-30

Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches.

The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae

PubMed Central

Gross, Roy; Guzman, Carlos A; Sebaihia, Mohammed; Martins dos Santos, Vítor AP; Pieper, Dietmar H; Koebnik, Ralf; Lechner, Melanie; Bartels, Daniela; Buhrmester, Jens; Choudhuri, Jomuna V; Ebensen, Thomas; Gaigalat, Lars; Herrmann, Stefanie; Khachane, Amit N; Larisch, Christof; Link, Stefanie; Linke, Burkhard; Meyer, Folker; Mormann, Sascha; Nakunst, Diana; Rückert, Christian; Schneiker-Bekel, Susanne; Schulze, Kai; Vorhölter, Frank-Jörg; Yevsa, Tetyana; Engle, Jacquelyn T; Goldman, William E; Pühler, Alfred; Göbel, Ulf B; Goesmann, Alexander; Blöcker, Helmut; Kaiser, Olaf; Martinez-Arias, Rosa

2008-01-01

Background Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. Results In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. Conclusion The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches. PMID:18826580

Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis.

PubMed

Paik, Sehmi; Senty, Lauren; Das, Sankar; Noe, Jody C; Munro, Cindy L; Kitten, Todd

2005-09-01

Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.

Identification of Virulence Determinants for Endocarditis in Streptococcus sanguinis by Signature-Tagged Mutagenesis†

PubMed Central

Paik, Sehmi; Senty, Lauren; Das, Sankar; Noe, Jody C.; Munro, Cindy L.; Kitten, Todd

2005-01-01

Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis. PMID:16113327

Anthrax Pathogenesis.

PubMed

Moayeri, Mahtab; Leppla, Stephen H; Vrentas, Catherine; Pomerantsev, Andrei P; Liu, Shihui

2015-01-01

Anthrax is caused by the spore-forming, gram-positive bacterium Bacillus anthracis. The bacterium's major virulence factors are (a) the anthrax toxins and (b) an antiphagocytic polyglutamic capsule. These are encoded by two large plasmids, the former by pXO1 and the latter by pXO2. The expression of both is controlled by the bicarbonate-responsive transcriptional regulator, AtxA. The anthrax toxins are three polypeptides-protective antigen (PA), lethal factor (LF), and edema factor (EF)-that come together in binary combinations to form lethal toxin and edema toxin. PA binds to cellular receptors to translocate LF (a protease) and EF (an adenylate cyclase) into cells. The toxins alter cell signaling pathways in the host to interfere with innate immune responses in early stages of infection and to induce vascular collapse at late stages. This review focuses on the role of anthrax toxins in pathogenesis. Other virulence determinants, as well as vaccines and therapeutics, are briefly discussed.

Genome Sequence of Enterohemorrhagic Escherichia coli NCCP15658

PubMed Central

Song, Ju Yeon; Yoo, Ran Hee; Jang, Song Yee; Seong, Won-Keun; Kim, Seon-Young; Jeong, Haeyoung; Kang, Sung Gyun; Kim, Byung Kwon; Kwon, Soon-Kyeong; Lee, Choong Hoon; Yu, Dong Su; Park, Mi-Sun

2012-01-01

Enterohemorrhagic Escherichia coli causes severe food-borne disease in the guts of humans and animals. Here, we report the high-quality draft genome sequence of E. coli NCCP15658 isolated from a patient in the Republic of Korea. Its genome size was determined to be 5.46 Mb, and its genomic features, including genes encoding virulence factors, were analyzed. PMID:22740673

The Posttranslocation Chaperone PrsA2 Contributes to Multiple Facets of Listeria monocytogenes Pathogenesis▿ †

PubMed Central

Alonzo, Francis; Port, Gary C.; Cao, Min; Freitag, Nancy E.

2009-01-01

Listeria monocytogenes is an intracellular bacterial pathogen whose virulence depends on the regulated expression of numerous secreted bacterial factors. As for other gram-positive bacteria, many proteins secreted by L. monocytogenes are translocated across the bacterial membrane in an unfolded state to the compartment existing between the membrane and the cell wall. This compartment presents a challenging environment for protein folding due to its high density of negative charge, high concentrations of cations, and low pH. We recently identified PrsA2 as a gene product required for L. monocytogenes virulence. PrsA2 was identified based on its increased secretion by strains containing a mutationally activated form of prfA, the key regulator of L. monocytogenes virulence gene expression. The prsA2 gene product is one of at least two predicted peptidyl-prolyl cis/trans-isomerases encoded by L. monocytogenes; these proteins function as posttranslocation protein chaperones and/or foldases. In this study, we demonstrate that PrsA2 plays a unique and important role in L. monocytogenes pathogenesis by promoting the activity and stability of at least two critical secreted virulence factors: listeriolysin O (LLO) and a broad-specificity phospholipase. Loss of PrsA2 activity severely attenuated virulence in mice and impaired bacterial cell-to-cell spread in host cells. In contrast, mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth and cell-to-cell spread as well as virulence in mice. PrsA2 is thus distinct from PrsA1 in its unique requirement for the stability and full activity of L. monocytogenes-secreted factors that contribute to host infection. PMID:19451247

The Pseudomonas aeruginosa Periplasmic Protease CtpA Can Affect Systems That Impact Its Ability To Mount Both Acute and Chronic Infections

PubMed Central

Seo, Jin

2013-01-01

Proteases play important roles in the virulence of Pseudomonas aeruginosa. Some are exported to act on host targets and facilitate tissue destruction and bacterial dissemination. Others work within the bacterial cell to process virulence factors and regulate virulence gene expression. Relatively little is known about the role of one class of bacterial serine proteases known as the carboxyl-terminal processing proteases (CTPs). The P. aeruginosa genome encodes two CTPs annotated as PA3257/Prc and PA5134/CtpA in strain PAO1. Prc degrades mutant forms of the anti-sigma factor MucA to promote mucoidy in some cystic fibrosis lung isolates. However, nothing is known about the role or importance of CtpA. We have now found that endogenous CtpA is a soluble periplasmic protein and that a ctpA null mutant has specific phenotypes consistent with an altered cell envelope. Although a ctpA null mutation has no major effect on bacterial growth in the laboratory, CtpA is essential for the normal function of the type 3 secretion system (T3SS), for cytotoxicity toward host cells, and for virulence in a mouse model of acute pneumonia. Conversely, increasing the amount of CtpA above its endogenous level induces an uncharacterized extracytoplasmic function sigma factor regulon, an event that has been reported to attenuate P. aeruginosa in a rat model of chronic lung infection. Therefore, a normal level of CtpA activity is critical for T3SS function and acute virulence, whereas too much activity can trigger an apparent stress response that is detrimental to chronic virulence. PMID:24082078

Computational Analysis Reveals a Key Regulator of Cryptococcal Virulence and Determinant of Host Response

PubMed Central

Gish, Stacey R.; Maier, Ezekiel J.; Haynes, Brian C.; Santiago-Tirado, Felipe H.; Srikanta, Deepa L.; Ma, Cynthia Z.; Li, Lucy X.; Williams, Matthew; Crouch, Erika C.; Khader, Shabaana A.

2016-01-01

ABSTRACT Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills over 600,000 people annually. Here, we report integrated computational and experimental investigations of the role and mechanisms of transcriptional regulation in cryptococcal infection. Major cryptococcal virulence traits include melanin production and the development of a large polysaccharide capsule upon host entry; shed capsule polysaccharides also impair host defenses. We found that both transcription and translation are required for capsule growth and that Usv101 is a master regulator of pathogenesis, regulating melanin production, capsule growth, and capsule shedding. It does this by directly regulating genes encoding glycoactive enzymes and genes encoding three other transcription factors that are essential for capsule growth: GAT201, RIM101, and SP1. Murine infection with cryptococci lacking Usv101 significantly alters the kinetics and pathogenesis of disease, with extended survival and, unexpectedly, death by pneumonia rather than meningitis. Our approaches and findings will inform studies of other pathogenic microbes. PMID:27094327

Virulence gene content in Escherichia coli isolates from poultry flocks with clinical signs of colibacillosis in Brazil.

PubMed

De Carli, Silvia; Ikuta, Nilo; Lehmann, Fernanda Kieling Moreira; da Silveira, Vinicius Proença; de Melo Predebon, Gabriela; Fonseca, André Salvador Kazantzi; Lunge, Vagner Ricardo

2015-11-01

Escherichia coli is a commensal bacterium of the bird's intestinal tract, but it can invade different tissues resulting in systemic symptoms (colibacillosis). This disease occurs only when the E. coli infecting strain presents virulence factors (encoded by specific genes) that enable the adhesion and proliferation in the host organism. Thus, it is important to differentiate pathogenic (APEC, avian pathogenic E. coli) and non-pathogenic or fecal (AFEC, avian fecal E. coli) isolates. Previous studies analyzed the occurrence of virulence factors in E. coli strains isolated from birds with colibacillosis, demonstrating a high frequency of the bacterial genes cvaC, iroN, iss, iutA, sitA, tsh, fyuA, irp-2, ompT and hlyF in pathogenic strains. The aim of the present study was to evaluate the occurrence and frequency of these virulence genes in E. coli isolated from poultry flocks in Brazil. A total of 138 isolates of E. coli was obtained from samples of different tissues and/or organs (spleen, liver, kidney, trachea, lungs, skin, ovary, oviduct, intestine, cloaca) and environmental swabs collected from chicken and turkey flocks suspected to have colibacillosis in farms from the main Brazilian producing regions. Total DNA was extracted and the 10 virulence genes were detected by traditional and/or real-time PCR. At least 11 samples of each gene were sequenced and compared to reference strains. All 10 virulence factors were detected in Brazilian E. coli isolates, with frequencies ranging from 39.9% (irp-2) to 68.8% (hlyF and sitA). Moreover, a high nucleotide similarity (over 99%) was observed between gene sequences of Brazilian isolates and reference strains. Seventy-nine isolates were defined as pathogenic (APEC) and 59 as fecal (AFEC) based on previously described criteria. In conclusion, the main virulence genes of the reference E. coli strains are also present in isolates associated with colibacillosis in Brazil. The analysis of this set of virulence factors can be used to differentiate between APEC and AFEC isolates in Brazil. © 2015 Poultry Science Association Inc.

Modulation of Gene Expression in Actinobacillus pleuropneumoniae Exposed to Bronchoalveolar Fluid

PubMed Central

Lone, Abdul G.; Deslandes, Vincent; Nash, John H. E.; Jacques, Mario; MacInnes, Janet I.

2009-01-01

Background Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia, is an important pathogen of swine throughout the world. It must rapidly overcome the innate pulmonary immune defenses of the pig to cause disease. To better understand this process, the objective of this study was to identify genes that are differentially expressed in a medium that mimics the lung environment early in the infection process. Methods and Principal Findings Since bronchoalveolar lavage fluid (BALF) contains innate immune and other components found in the lungs, we examined gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after a 30 min exposure to BALF, using DNA microarrays and real-time PCR. The functional classes of genes found to be up-regulated most often in BALF were those encoding proteins involved in energy metabolism, especially anaerobic metabolism, and in cell envelope, DNA, and protein biosynthesis. Transcription of a number of known virulence genes including apxIVA and the gene for SapF, a protein which is involved in resistance to antimicrobial peptides, was also up-regulated in BALF. Seventy-nine percent of the genes that were up-regulated in BALF encoded a known protein product, and of these, 44% had been reported to be either expressed in vivo and/or involved in virulence. Conclusions The results of this study suggest that in early stages of infection, A. pleuropneumoniae may modulate expression of genes involved in anaerobic energy generation and in the synthesis of proteins involved in cell wall biogenesis, as well as established virulence factors. Given that many of these genes are thought to be expressed in vivo or involved in virulence, incubation in BALF appears, at least partially, to simulate in vivo conditions and may provide a useful medium for the discovery of novel vaccine or therapeutic targets. PMID:19578537

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

PubMed

Mattow, J; Jungblut, P R; Schaible, U E; Mollenkopf, H J; Lamer, S; Zimny-Arndt, U; Hagens, K; Müller, E C; Kaufmann, S H

2001-08-01

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

LOV-domain photoreceptor, encoded in a genomic island, attenuates the virulence of Pseudomonas syringae in light-exposed Arabidopsis leaves.

PubMed

Moriconi, Victoria; Sellaro, Romina; Ayub, Nicolás; Soto, Gabriela; Rugnone, Matías; Shah, Rashmi; Pathak, Gopal P; Gärtner, Wolfgang; Casal, Jorge J

2013-10-01

In Arabidopsis thaliana, light signals modulate the defences against bacteria. Here we show that light perceived by the LOV domain-regulated two-component system (Pst-Lov) of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) modulates virulence against A. thaliana. Bioinformatic analysis and the existence of an episomal circular intermediate indicate that the locus encoding Pst-Lov is present in an active genomic island acquired by horizontal transfer. Strains mutated at Pst-Lov showed enhanced growth on minimal medium and in leaves of A. thaliana exposed to light, but not in leaves incubated in darkness or buried in the soil. Pst-Lov repressed the expression of principal and alternative sigma factor genes and their downstream targets linked to bacterial growth, virulence and quorum sensing, in a strictly light-dependent manner. We propose that the function of Pst-Lov is to distinguish between soil (dark) and leaf (light) environments, attenuating the damage caused to host tissues while releasing growth out of the host. Therefore, in addition to its direct actions via photosynthesis and plant sensory receptors, light may affect plants indirectly via the sensory receptors of bacterial pathogens. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

LitR of Vibrio salmonicida Is a Salinity-Sensitive Quorum-Sensing Regulator of Phenotypes Involved in Host Interactions and Virulence

PubMed Central

Bjelland, Ane Mohn; Sørum, Henning; Tegegne, Daget Ayana; Winther-Larsen, Hanne C.; Willassen, Nils Peder

2012-01-01

Vibrio (Aliivibrio) salmonicida is the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth of V. salmonicida in the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome of V. salmonicida LFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS in V. fischeri, was deleted. Compared to the parental strain, the litR mutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, the litR mutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with the litR mutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host. PMID:22371373

Diversity, virulence, and antimicrobial resistance of the KPC-producing Klebsiella pneumoniae ST307 clone.

PubMed

Villa, Laura; Feudi, Claudia; Fortini, Daniela; Brisse, Sylvain; Passet, Virginie; Bonura, Celestino; Endimiani, Andrea; Mammina, Caterina; Ocampo, Ana Maria; Jimenez, Judy Natalia; Doumith, Michel; Woodford, Neil; Hopkins, Katie; Carattoli, Alessandra

2017-04-01

The global spread of Klebsiella pneumoniae producing Klebsiella pneumoniae carbapenemase (KPC) has been mainly associated with the dissemination of high-risk clones. In the last decade, hospital outbreaks involving KPC-producing K. pneumoniae have been predominantly attributed to isolates belonging to clonal group (CG) 258. However, results of recent epidemiological analysis indicate that KPC-producing sequence type (ST) 307, is emerging in different parts of the world and is a candidate to become a prevalent high-risk clone in the near future. Here we show that the ST307 genome encodes genetic features that may provide an advantage in adaptation to the hospital environment and the human host. Sequence analysis revealed novel plasmid-located virulence factors, including a cluster for glycogen synthesis. Glycogen production is considered to be one of the possible adaptive responses to long-term survival and growth in environments outside the host. Chromosomally-encoded virulence traits in the clone comprised fimbriae, an integrative conjugative element carrying the yersiniabactin siderophore, and two different capsular loci. Compared with the ST258 clone, capsulated ST307 isolates showed higher resistance to complement-mediated killing. The acquired genetic features identified in the genome of this new emerging clone may contribute to increased persistence of ST307 in the hospital environment and shed light on its potential epidemiological success.

First insights into the pleiotropic role of vrf (yedF), a newly characterized gene of Salmonella Typhimurium.

PubMed

Ballesté-Delpierre, Clara; Fernandez-Orth, Dietmar; Ferrer-Navarro, Mario; Díaz-Peña, Ramón; Odena-Caballol, Antonia; Oliveira, Eliandre; Fàbrega, Anna; Vila, Jordi

2017-11-10

Salmonella possesses virulence determinants that allow replication under extreme conditions and invasion of host cells, causing disease. Here, we examined four putative genes predicted to encode membrane proteins (ydiY, ybdJ, STM1441 and ynaJ) and a putative transcriptional factor (yedF). These genes were identified in a previous study of a S. Typhimurium clinical isolate and its multidrug-resistant counterpart. For STM1441 and yedF a reduced ability to interact with HeLa cells was observed in the knock-out mutants, but an increase in this ability was absent when these genes were overexpressed, except for yedF which phenotype was rescued when yedF was restored. In the absence of yedF, decreased expression was seen for: i) virulence-related genes involved in motility, chemotaxis, attachment and survival inside the host cell; ii) global regulators of the invasion process (hilA, hilC and hilD); and iii) factors involved in LPS biosynthesis. In contrast, an increased expression was observed for anaerobic metabolism genes. We propose yedF is involved in the regulation of Salmonella pathogenesis and contributes to the activation of the virulence machinery. Moreover, we propose that, when oxygen is available, yedF contributes sustained repression of the anaerobic pathway. Therefore, we recommend this gene be named vrf, for virulence-related factor.

Streptococcal collagen-like protein A and general stress protein 24 are immunomodulating virulence factors of group A Streptococcus.

PubMed

Tsatsaronis, James A; Hollands, Andrew; Cole, Jason N; Maamary, Peter G; Gillen, Christine M; Ben Zakour, Nouri L; Kotb, Malak; Nizet, Victor; Beatson, Scott A; Walker, Mark J; Sanderson-Smith, Martina L

2013-07-01

In Western countries, invasive infections caused by M1T1 serotype group A Streptococcus (GAS) are epidemiologically linked to mutations in the control of virulence regulatory 2-component operon (covRS). In indigenous communities and developing countries, severe GAS disease is associated with genetically diverse non-M1T1 GAS serotypes. Hypervirulent M1T1 covRS mutant strains arise through selection by human polymorphonuclear cells for increased expression of GAS virulence factors such as the DNase Sda1, which promotes neutrophil resistance. The GAS bacteremia isolate NS88.2 (emm 98.1) is a covS mutant that exhibits a hypervirulent phenotype and neutrophil resistance yet lacks the phage-encoded Sda1. Here, we have employed a comprehensive systems biology (genomic, transcriptomic, and proteomic) approach to identify NS88.2 virulence determinants that enhance neutrophil resistance in the non-M1T1 GAS genetic background. Using this approach, we have identified streptococcal collagen-like protein A and general stress protein 24 proteins as NS88.2 determinants that contribute to survival in whole blood and neutrophil resistance in non-M1T1 GAS. This study has revealed new factors that contribute to GAS pathogenicity that may play important roles in resisting innate immune defenses and the development of human invasive infections.

The response regulator expM is essential for the virulence of Erwinia carotovora subsp. carotovora and acts negatively on the sigma factor RpoS (sigma s).

PubMed

Andersson, R A; Palva, E T; Pirhonen, M

1999-07-01

The main virulence factors of Erwinia carotovora subsp. carotovora, the secreted, extracellular cell-wall-degrading enzymes, are controlled by several regulatory mechanisms. We have isolated transposon mutants with reduced virulence on tobacco. One of these mutants, with a mutation in a gene designated expM, was characterized in this study. This mutant produces slightly reduced amounts of extracellular enzymes in vitro and the secretion of the enzymes is also affected. The expM wild-type allele was cloned together with an upstream gene, designated expL, that has an unknown function. The expM gene was sequenced and found to encode a protein with similarity to the RssB/SprE protein of Escherichia coli and the MviA protein of Salmonella typhimurium. These proteins belong to a new type of two-component response regulators that negatively regulate the stability of the Sigma factor RpoS (sigma s) at the protein level. The results of this study suggest that ExpM has a similar function in E. carotovora subsp. carotovora. We also provide evidence that the overproduction of RpoS in the expM mutant is an important factor for the reduced virulence phenotype and that it partly causes the observed phenotype seen in vitro. However, an expM/rpoS double mutant is still affected in secretion of extracellular enzymes, suggesting that ExpM in addition to RpoS also acts on other targets.

Bacteriophages encode factors required for protection in a symbiotic mutualism.

PubMed

Oliver, Kerry M; Degnan, Patrick H; Hunter, Martha S; Moran, Nancy A

2009-08-21

Bacteriophages are known to carry key virulence factors for pathogenic bacteria, but their roles in symbiotic bacteria are less well understood. The heritable symbiont Hamiltonella defensa protects the aphid Acyrthosiphon pisum from attack by the parasitoid Aphidius ervi by killing developing wasp larvae. In a controlled genetic background, we show that a toxin-encoding bacteriophage is required to produce the protective phenotype. Phage loss occurs repeatedly in laboratory-held H. defensa-infected aphid clonal lines, resulting in increased susceptibility to parasitism in each instance. Our results show that these mobile genetic elements can endow a bacterial symbiont with benefits that extend to the animal host. Thus, phages vector ecologically important traits, such as defense against parasitoids, within and among symbiont and animal host lineages.

Role and Regulation of the Flp/Tad Pilus in the Virulence of Pectobacterium atrosepticum SCRI1043 and Pectobacterium wasabiae SCC3193

PubMed Central

Nykyri, Johanna; Mattinen, Laura; Niemi, Outi; Adhikari, Satish; Kõiv, Viia; Somervuo, Panu; Fang, Xin; Auvinen, Petri; Mäe, Andres; Palva, E. Tapio; Pirhonen, Minna

2013-01-01

In this study, we characterized a putative Flp/Tad pilus-encoding gene cluster, and we examined its regulation at the transcriptional level and its role in the virulence of potato pathogenic enterobacteria of the genus Pectobacterium. The Flp/Tad pilus-encoding gene clusters in Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium aroidearum were compared to previously characterized flp/tad gene clusters, including that of the well-studied Flp/Tad pilus model organism Aggregatibacter actinomycetemcomitans, in which this pilus is a major virulence determinant. Comparative analyses revealed substantial protein sequence similarity and open reading frame synteny between the previously characterized flp/tad gene clusters and the cluster in Pectobacterium, suggesting that the predicted flp/tad gene cluster in Pectobacterium encodes a Flp/Tad pilus-like structure. We detected genes for a novel two-component system adjacent to the flp/tad gene cluster in Pectobacterium, and mutant analysis demonstrated that this system has a positive effect on the transcription of selected Flp/Tad pilus biogenesis genes, suggesting that this response regulator regulate the flp/tad gene cluster. Mutagenesis of either the predicted regulator gene or selected Flp/Tad pilus biogenesis genes had a significant impact on the maceration ability of the bacterial strains in potato tubers, indicating that the Flp/Tad pilus-encoding gene cluster represents a novel virulence determinant in Pectobacterium. Soft-rot enterobacteria in the genera Pectobacterium and Dickeya are of great agricultural importance, and an investigation of the virulence of these pathogens could facilitate improvements in agricultural practices, thus benefiting farmers, the potato industry and consumers. PMID:24040039

Role and regulation of the Flp/Tad pilus in the virulence of Pectobacterium atrosepticum SCRI1043 and Pectobacterium wasabiae SCC3193.

PubMed

Nykyri, Johanna; Mattinen, Laura; Niemi, Outi; Adhikari, Satish; Kõiv, Viia; Somervuo, Panu; Fang, Xin; Auvinen, Petri; Mäe, Andres; Palva, E Tapio; Pirhonen, Minna

2013-01-01

In this study, we characterized a putative Flp/Tad pilus-encoding gene cluster, and we examined its regulation at the transcriptional level and its role in the virulence of potato pathogenic enterobacteria of the genus Pectobacterium. The Flp/Tad pilus-encoding gene clusters in Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium aroidearum were compared to previously characterized flp/tad gene clusters, including that of the well-studied Flp/Tad pilus model organism Aggregatibacter actinomycetemcomitans, in which this pilus is a major virulence determinant. Comparative analyses revealed substantial protein sequence similarity and open reading frame synteny between the previously characterized flp/tad gene clusters and the cluster in Pectobacterium, suggesting that the predicted flp/tad gene cluster in Pectobacterium encodes a Flp/Tad pilus-like structure. We detected genes for a novel two-component system adjacent to the flp/tad gene cluster in Pectobacterium, and mutant analysis demonstrated that this system has a positive effect on the transcription of selected Flp/Tad pilus biogenesis genes, suggesting that this response regulator regulate the flp/tad gene cluster. Mutagenesis of either the predicted regulator gene or selected Flp/Tad pilus biogenesis genes had a significant impact on the maceration ability of the bacterial strains in potato tubers, indicating that the Flp/Tad pilus-encoding gene cluster represents a novel virulence determinant in Pectobacterium. Soft-rot enterobacteria in the genera Pectobacterium and Dickeya are of great agricultural importance, and an investigation of the virulence of these pathogens could facilitate improvements in agricultural practices, thus benefiting farmers, the potato industry and consumers.

Novel mutants of Erwinia carotovora subsp. carotovora defective in the production of plant cell wall degrading enzymes generated by Mu transpososome-mediated insertion mutagenesis.

PubMed

Laasik, Eve; Ojarand, Merli; Pajunen, Maria; Savilahti, Harri; Mäe, Andres

2005-02-01

As in Erwinia carotovora subsp. carotovora the regulation details of the main virulence factors, encoding extracellular enzymes that degrade the plant cell wall, is only rudimentally understood, we performed a genetic screen to identify novel candidate genes involved in the process. Initially, we used Mu transpososome-mediated mutagenesis approach to generate a comprehensive transposon insertion mutant library of ca. 10000 clones and screened the clones for the loss of extracellular enzyme production. Extracellular enzymes production was abolished by mutations in the chromosomal helEcc, trkAEcc yheLEcc, glsEcc, igaAEcc and cysQEcc genes. The findings reported here demonstrate that we have isolated six new representatives that belong to the pool of genes modulating the production of virulence factors in E. carotovora.

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

PubMed Central

Pavlova, Olga; Ieva, Raffaele; Bernstein, Harris D

2013-01-01

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 Å. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry. PMID:24378574

MRSA virulence and spread

PubMed Central

Otto, Michael

2012-01-01

Summary Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of hospital- and community-associated infections. Resistance to the entire class of β-lactam antibiotics, such as methicillin and penicillin, makes MRSA infections difficult to treat. Hospital-associated MRSA strains are often multi-drug resistant, leaving only lower efficiency drugs such as vancomycin as treatments options. Like many other S. aureus strains, MRSA strains produce a series of virulence factors, such as toxins and adhesion proteins. Recent findings have shed some new light on the molecular events that underlie MRSA epidemic waves. Newly emerging MRSA clones appear to have acquired phenotypic traits that render them more virulent or able to colonize better, either via mobile genetic elements or adaptation of gene expression. Acquisition of Panton-Valentine leukocidin genes and increased expression of core genome-encoded toxins are being discussed as potentially contributing to the success of the recently emerged community-associated MRSA strains. However, the molecular factors underlying the spread of hospital- and community-associated MRSA strains are still far from being completely understood, a situation calling for enhanced research efforts in that area. PMID:22747834

Antimicrobial resistance, serotypes, and virulence factors of Streptococcus suis isolates from diseased pigs.

PubMed

Li, Lu-Lu; Liao, Xiao-Ping; Sun, Jian; Yang, Yu-Rong; Liu, Bao-Tao; Yang, Shou-Shen; Zhao, Dong-Hao; Liu, Ya-Hong

2012-07-01

Streptococcus suis isolates from diseased pigs were examined for susceptibility to nine antimicrobials, possession of virulence-associated factors (VFs), and distribution of serotypes. The association between antimicrobial resistance (AMR) and serotypes as well as VFs was subsequently assessed. Among the isolates investigated, serotype 2 (66.04%) was mostly prevalent, followed by serotypes 1 (23.27%), 9 (1.26%), and 7 (0.63%), whereas 14 isolates were untypable by the polymerase chain reaction typing method used. Analysis with pulsed-field gel electrophoresis revealed the isolates had diverse DNA macrorestriction patterns. The frequency of antimicrobial resistance among the S. suis isolates was higher than that reported from other countries. It is notable that multiple antimicrobial resistance (three or more antimicrobials) was observed with 98.73% of the S. suis isolates, and the dominant resistance phenotype was erythromycin-tilmicosin-clindamycin-chloramphenicol-levofloxacin-ceftiofur-kanamycin-tetracycline-penicillin (35.85%). The most prevalent VFs were those encoded by muramidase-released protein (61.64%), followed by suilysin (56.60%) and extracellular factor (46.54%). Presence of VFs and the possession of certain AMR phenotypes were significantly associated as determined by statistical analysis. Together, these findings indicate that the clinical S. suis isolates obtained from diseased pigs in China are genetically diverse, are resistant to multiple antibiotics of clinical importance, and carry known virulence factors.

A peptide factor secreted by Staphylococcus pseudintermedius exhibits properties of both bacteriocins and virulence factors.

PubMed

Wladyka, Benedykt; Piejko, Marcin; Bzowska, Monika; Pieta, Piotr; Krzysik, Monika; Mazurek, Łukasz; Guevara-Lora, Ibeth; Bukowski, Michał; Sabat, Artur J; Friedrich, Alexander W; Bonar, Emilia; Międzobrodzki, Jacek; Dubin, Adam; Mak, Paweł

2015-09-28

Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups.

A peptide factor secreted by Staphylococcus pseudintermedius exhibits properties of both bacteriocins and virulence factors

PubMed Central

Wladyka, Benedykt; Piejko, Marcin; Bzowska, Monika; Pieta, Piotr; Krzysik, Monika; Mazurek, Łukasz; Guevara-Lora, Ibeth; Bukowski, Michał; Sabat, Artur J.; Friedrich, Alexander W.; Bonar, Emilia; Międzobrodzki, Jacek; Dubin, Adam; Mak, Paweł

2015-01-01

Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups. PMID:26411997

EcpA, an extracellular protease, is a specific virulence factor required by Xanthomonas oryzae pv. oryzicola but not by X. oryzae pv. oryzae in rice

USDA-ARS?s Scientific Manuscript database

Previously, twelve protease-deficient mutants of Xanthomonas oryzae pv. oryzicola (Xoc) RS105 strain were recovered from a Tn5-tagged mutant library. In the current study, the Tn5 insertion site in each mutant was mapped. Mutations in genes encoding components of the type II secretion apparatus, cAM...

Detection of virulence and β-lactamase encoding genes in Enterobacter aerogenes and Enterobacter cloacae clinical isolates from Brazil.

PubMed

Azevedo, Paola Aparecida Alves; Furlan, João Pedro Rueda; Oliveira-Silva, Mariana; Nakamura-Silva, Rafael; Gomes, Carolina Nogueira; Costa, Karen Regina Carim; Stehling, Eliana Guedes; Pitondo-Silva, André

2018-05-21

Enterobacter cloacae and E. aerogenes have been increasingly reported as important opportunistic pathogens. In this study, a high prevalence of multi-drug resistant isolates from Brazil, harboring several β-lactamase encoding genes was found. Several virulence genes were observed in E. aerogenes, contrasting with the E. cloacae isolates which presented none. Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

PubMed

Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

2013-03-01

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

Pleiotropic Regulation of Virulence Genes in Streptococcus mutans by the Conserved Small Protein SprV.

PubMed

Shankar, Manoharan; Hossain, Mohammad S; Biswas, Indranil

2017-04-15

Streptococcus mutans , an oral pathogen associated with dental caries, colonizes tooth surfaces as polymicrobial biofilms known as dental plaque. S. mutans expresses several virulence factors that allow the organism to tolerate environmental fluctuations and compete with other microorganisms. We recently identified a small hypothetical protein (90 amino acids) essential for the normal growth of the bacterium. Inactivation of the gene, SMU.2137, encoding this protein caused a significant growth defect and loss of various virulence-associated functions. An S. mutans strain lacking this gene was more sensitive to acid, temperature, osmotic, oxidative, and DNA damage-inducing stresses. In addition, we observed an altered protein profile and defects in biofilm formation, bacteriocin production, and natural competence development, possibly due to the fitness defect associated with SMU.2137 deletion. Transcriptome sequencing revealed that nearly 20% of the S. mutans genes were differentially expressed upon SMU.2137 deletion, thereby suggesting a pleiotropic effect. Therefore, we have renamed this hitherto uncharacterized gene as sprV ( s treptococcal p leiotropic r egulator of v irulence). The transcript levels of several relevant genes in the sprV mutant corroborated the phenotypes observed upon sprV deletion. Owing to its highly conserved nature, inactivation of the sprV ortholog in Streptococcus gordonii also resulted in poor growth and defective UV tolerance and competence development as in the case of S. mutans Our experiments suggest that SprV is functionally distinct from its homologs identified by structure and sequence homology. Nonetheless, our current work is aimed at understanding the importance of SprV in the S. mutans biology. IMPORTANCE Streptococcus mutans employs several virulence factors and stress resistance mechanisms to colonize tooth surfaces and cause dental caries. Bacterial pathogenesis is generally controlled by regulators of fitness that are critical for successful disease establishment. Sometimes these regulators, which are potential targets for antimicrobials, are lost in the genomic context due to the lack of annotated homologs. This work outlines the regulatory impact of a small, highly conserved hypothetical protein, SprV, encoded by S. mutans We show that SprV affects the transcript levels of various virulence factors required for normal growth, biofilm formation, stress tolerance, genetic competence, and bacteriocin production. Copyright © 2017 American Society for Microbiology.

Host-Associated Genomic Features of the Novel Uncultured Intracellular Pathogen Ca. Ichthyocystis Revealed by Direct Sequencing of Epitheliocysts

PubMed Central

Qi, Weihong; Vaughan, Lloyd; Katharios, Pantelis; Schlapbach, Ralph; Seth-Smith, Helena M.B.

2016-01-01

Advances in single-cell and mini-metagenome sequencing have enabled important investigations into uncultured bacteria. In this study, we applied the mini-metagenome sequencing method to assemble genome drafts of the uncultured causative agents of epitheliocystis, an emerging infectious disease in the Mediterranean aquaculture species gilthead seabream. We sequenced multiple cyst samples and constructed 11 genome drafts from a novel beta-proteobacterial lineage, Candidatus Ichthyocystis. The draft genomes demonstrate features typical of pathogenic bacteria with an obligate intracellular lifestyle: a reduced genome of up to 2.6 Mb, reduced G + C content, and reduced metabolic capacity. Reconstruction of metabolic pathways reveals that Ca. Ichthyocystis genomes lack all amino acid synthesis pathways, compelling them to scavenge from the fish host. All genomes encode type II, III, and IV secretion systems, a large repertoire of predicted effectors, and a type IV pilus. These are all considered to be virulence factors, required for adherence, invasion, and host manipulation. However, no evidence of lipopolysaccharide synthesis could be found. Beyond the core functions shared within the genus, alignments showed distinction into different species, characterized by alternative large gene families. These comprise up to a third of each genome, appear to have arisen through duplication and diversification, encode many effector proteins, and are seemingly critical for virulence. Thus, Ca. Ichthyocystis represents a novel obligatory intracellular pathogenic beta-proteobacterial lineage. The methods used: mini-metagenome analysis and manual annotation, have generated important insights into the lifestyle and evolution of the novel, uncultured pathogens, elucidating many putative virulence factors including an unprecedented array of novel gene families. PMID:27190004

Discovery of Novel Secreted Virulence Factors from Salmonella enterica Serovar Typhimurium by Proteomic Analysis of Culture Supernatants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niemann, George; Brown, Roslyn N.; Gustin, Jean K.

The intracellular pathogen Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis in the world. This pathogen has two type-III secretion systems (TTSS) necessary for virulence that are encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) and are expressed during extracellular or intracellular infectious states, respectively, to deliver virulence factors (effectors) to the host cell cytoplasm. While many have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this mass spectrometry-based proteomics study, we identified effector proteins secreted under minimal acidic medium growth conditions that induced themore » SPI-2 TTSS and its effectors, and compared the secretome from the parent strain to the secretome from strains missing either essential (SsaK) or regulatory components (SsaL) of the SPI-2 secretion apparatus. We identified 75% of the known TTSS effector repertoire. Excluding translocon components, 95% of the known effectors were biased for identification in the ssaL mutant background, which demonstrated that SsaL regulates SPI-2 type III secretion. To confirm secretion to animal cells, we made translational fusions of several of the best candidates to the calmodulin-dependent adenylate cyclase of Bordetella pertussis and assayed cAMP levels of infected J774 macrophage-like cells. From these infected cells we identified six new TTSS effectors and two others that are secreted independent of TTSS. Our results substantiate reports of additional secretion systems encoded by Salmonella other than TTSS.« less

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

PubMed

Reid, S D; Green, N M; Buss, J K; Lei, B; Musser, J M

2001-06-19

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

Development of a Novel Method for Analyzing Pseudomonas aeruginosa Twitching Motility and Its Application to Define the AmrZ Regulon

PubMed Central

Xu, Binjie; Wozniak, Daniel J.

2015-01-01

Twitching motility is an important migration mechanism for the Gram-negative bacterium Pseudomonas aeruginosa. In the commonly used subsurface twitching assay, the sub-population of P. aeruginosa with active twitching motility is difficult to harvest for high-throughput studies. Here we describe the development of a novel method that allows efficient isolation of bacterial sub-populations conducting highly active twitching motility. The transcription factor AmrZ regulates multiple P. aeruginosa virulence factors including twitching motility, yet the mechanism of this activation remains unclear. We therefore set out to understand this mechanism by defining the AmrZ regulon using DNA microarrays in combination with the newly developed twitching motility method. We discovered 112 genes in the AmrZ regulon and many encode virulence factors. One gene of interest and the subsequent focus was lecB, which encodes a fucose-binding lectin. DNA binding assays revealed that AmrZ activates lecB transcription by directly binding to its promoter. The lecB gene was previously shown to be required for twitching motility in P. aeruginosa strain PAK; however, our lecB deletion had no effect on twitching motility in strain PAO1. Collectively, in this study a novel condition was developed for quantitative studies of twitching motility, under which the AmrZ regulon was defined. PMID:26309248

Virulence and extended-spectrum β-lactamase encoding genes in Escherichia coli recovered from chicken meat intended for hospitalized human consumption.

PubMed

Younis, Gamal A; Elkenany, Rasha M; Fouda, Mohamed A; Mostafa, Noura F

2017-10-01

This study describes the prevalence of Escherichia coli in frozen chicken meat intended for human consumption with emphasis on their virulence determinants through detection of the virulence genes and recognition of the extended-spectrum β-lactamase (ESBL) encoding genes ( bla OXA and bla TEM genes). A total of 120 frozen chicken meat samples were investigated for isolation of E. coli . All isolates were subjected to biochemical and serological tests. Eight serotypes isolated from samples were analyzed for the presence of various virulence genes ( stx1, stx2 , and eae A genes) using multiplex polymerase chain reaction (PCR) technique. Moreover, the strains were evaluated for the ESBL encoding genes ( bla TEM and bla OXA ). Overall, 11.66% (14/120) chicken meat samples carried E. coli according to cultural and biochemical properties. The most predominant serotypes were O78 and O128: H2 (21.5%, each), followed by O121: H7 and O44: H18. Molecular method detected that 2 strains (25%) harbored stx1 , 3 strains (37.5%) stx2 , and 3 strains (37.5%) both stx1 and stx2 , while 1 (12.5%) strain carried eae A gene. Particularly, only O26 serotype had all tested virulence genes ( stx1, stx2, and eae A ). The results revealed that all examined 8 serotypes were Shiga toxin-producing E. coli (STEC). The ESBL encoding genes ( bla TEM and bla OXA ) of STEC were detected in 4 (50%) isolates by multiplex PCR. The overall incidence of bla TEM and bla OXA genes was 3 (37.5%) and 2 (25%) isolates. The present study indicates the prevalence of virulent and ESBL-producing E. coli in frozen chicken meat intended for hospitalized human consumption due to poor hygienic measures and irregular use of antibiotics. Therefore, the basic instructions regarding good hygienic measures should be adapted to limit public health hazard.

Sporangiospore size dimorphism is linked to virulence of Mucor circinelloides.

PubMed

Li, Charles H; Cervantes, Maria; Springer, Deborah J; Boekhout, Teun; Ruiz-Vazquez, Rosa M; Torres-Martinez, Santiago R; Heitman, Joseph; Lee, Soo Chan

2011-06-01

Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (-) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (-) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex.

Sporangiospore Size Dimorphism Is Linked to Virulence of Mucor circinelloides

PubMed Central

Li, Charles H.; Cervantes, Maria; Springer, Deborah J.; Boekhout, Teun; Ruiz-Vazquez, Rosa M.; Torres-Martinez, Santiago R.; Heitman, Joseph; Lee, Soo Chan

2011-01-01

Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (−) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (−) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex. PMID:21698218

Large subunit of the ribonucleotide reductase gene is a virulent factor and plays a critical role in Marek's disease virus pathogenesis

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) encodes a ribonucleotide reductase (RR) gene consisting of two subunits UL39 (RR1) and UL40 (RR2). Both RR1 and RR2 form an active holoenzyme and are necessary for enzyme activity. This gene was indentified by monoclonal antibody T81 in a gt11 MDV expression library and f...

Leptospiral outer membrane protein LipL41 is not essential for acute leptospirosis but requires a small chaperone protein, lep, for stable expression.

PubMed

King, Amy M; Bartpho, Thanatchaporn; Sermswan, Rasana W; Bulach, Dieter M; Eshghi, Azad; Picardeau, Mathieu; Adler, Ben; Murray, Gerald L

2013-08-01

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp., but knowledge of leptospiral pathogenesis remains limited. However, the development of mutagenesis systems has allowed the investigation of putative virulence factors and their involvement in leptospirosis. LipL41 is the third most abundant lipoprotein found in the outer membranes of pathogenic leptospires and has been considered a putative virulence factor. LipL41 is encoded on the large chromosome 28 bp upstream of a small open reading frame encoding a hypothetical protein of unknown function. This gene was named lep, for LipL41 expression partner. In this study, lipL41 was found to be cotranscribed with lep. Two transposon mutants were characterized: a lipL41 mutant and a lep mutant. In the lep mutant, LipL41 protein levels were reduced by approximately 90%. Lep was shown through cross-linking and coexpression experiments to bind to LipL41. Lep is proposed to be a molecular chaperone essential for the stable expression of LipL41. The roles of LipL41 and Lep in the pathogenesis of Leptospira interrogans were investigated; surprisingly, neither of these two unique proteins was essential for acute leptospirosis.

Detection of tlh and tdh genes in Vibrio Parahaemolyticus inhabiting farmed water ecosystem used for L. Vannamei aquaculture

NASA Astrophysics Data System (ADS)

Nawan Hasrimi, Adila; Budiharjo, Anto; Nur Jannah, Siti

2018-05-01

Vibrio parahaemolyticus is hallophilic gram-negative bacteria that live as natural inhabitant in aquatic environment. All Vibrio parahaemolyticus strain known to have thermolabile hemolysin encoded by tlh gene as species marker. Thermostable direct hemolysin encoded by tdh gene is responsible for regulating virulence factor in Vibrio parhaemolyticus. Aim of this research is to detect tlh and tdh gene from water of L. vannamei aquaculture in Rembang regency. Colonies of green-blueish bacteria grew from isolation of L. vannamei aquaculture water in CD-VP media which was identified as Vibrio parahaemolyticus. Colonies of V. parahaemolyticus grew to be small and green-blueish bacteria colonies in TCBS agar. Result of molecular analysis showed that bacteria isolated from water sample are specifically identified as Vibrio parahaemolyticus bacteria by the detection of tlh gene. Vibrio parahaemolyticus isolated from water of L. vannamei aquaculture detected as tdh negative that indicates tdh gene is not present in isolated bacteria. Vibrio parahaemolyticus isolate were cultured in Wagatsuma agar for tdh gene confirmation test that showed Kanagawa negative result, which indicated that V. parahaemolyticus did not produce thermostable direct hemolysin. These results showed that Vibrio parahaemolyticus isolated from aquatic environment of L. vannamei aquaculture in Rembang regency did not show virulence factors.

Diversity, virulence, and antimicrobial resistance of the KPC-producing Klebsiella pneumoniae ST307 clone

PubMed Central

Villa, Laura; Feudi, Claudia; Fortini, Daniela; Brisse, Sylvain; Passet, Virginie; Bonura, Celestino; Endimiani, Andrea; Mammina, Caterina; Ocampo, Ana Maria; Jimenez, Judy Natalia; Doumith, Michel; Woodford, Neil; Hopkins, Katie

2017-01-01

The global spread of Klebsiella pneumoniae producing Klebsiella pneumoniae carbapenemase (KPC) has been mainly associated with the dissemination of high-risk clones. In the last decade, hospital outbreaks involving KPC-producing K. pneumoniae have been predominantly attributed to isolates belonging to clonal group (CG) 258. However, results of recent epidemiological analysis indicate that KPC-producing sequence type (ST) 307, is emerging in different parts of the world and is a candidate to become a prevalent high-risk clone in the near future. Here we show that the ST307 genome encodes genetic features that may provide an advantage in adaptation to the hospital environment and the human host. Sequence analysis revealed novel plasmid-located virulence factors, including a cluster for glycogen synthesis. Glycogen production is considered to be one of the possible adaptive responses to long-term survival and growth in environments outside the host. Chromosomally-encoded virulence traits in the clone comprised fimbriae, an integrative conjugative element carrying the yersiniabactin siderophore, and two different capsular loci. Compared with the ST258 clone, capsulated ST307 isolates showed higher resistance to complement-mediated killing. The acquired genetic features identified in the genome of this new emerging clone may contribute to increased persistence of ST307 in the hospital environment and shed light on its potential epidemiological success. PMID:28785421

Prevalence of genes encoding virulence factors among Escherichia coli with K1 antigen and non-K1 E. coli strains.

PubMed

Kaczmarek, Agnieszka; Budzynska, Anna; Gospodarek, Eugenia

2012-10-01

Multiplex PCR was used to detect genes encoding selected virulence determinants associated with strains of Escherichia coli with K1 antigen (K1(+)) and non-K1 E. coli (K1(-)). The prevalence of the fimA, fimH, sfa/foc, ibeA, iutA and hlyF genes was studied for 134 (67 K1(+) and 67 K1(-)) E. coli strains isolated from pregnant women and neonates. The fimA gene was present in 83.6 % of E. coli K1(+) and in 86.6 % of E. coli K1(-) strains. The fimH gene was present in all tested E. coli K1(+) strains and in 97.0 % of non-K1 strains. E. coli K1(+) strains were significantly more likely to possess the following genes than E. coli K1(-) strains: sfa/foc (37.3 vs 16.4 %, P = 0.006), ibeA (35.8 vs 4.5 %, P<0.001), iutA (82.1 vs 35.8 %, P<0.001) and hlyF (28.4 vs 6.0 %, P<0.001). In conclusion, E. coli K1(+) seems to be more virulent than E. coli K1(-) strains in developing severe infections, thereby increasing possible sepsis or neonatal bacterial meningitis.

Adaptation of Escherichia coli Traversing From the Faecal Environment to the Urinary Tract

PubMed Central

Nielsen, Karen L.; Stegger, Marc; Godfrey, Paul A.; Feldgarden, Michael; Andersen, Paal S.; Frimodt-Møller, Niels

2016-01-01

The majority of extraintestinal pathogenic Escherichia coli (ExPEC) causing urinary tract infections (UTI) are found in the patient's own gut flora, but only limited knowledge is available on the potential adaptation that may occur in the bacteria for them to traverse the perineum and successfully infect the urinary tract. Here, matching faecal and UTI isolates from 42 patients were compared pairwise using in-depth whole-genome sequencing to investigate whether genetic changes were evident for successful colonization in these two different environments. The identified non-synonymous mutations (0-12 substitutions in each pair) were primarily associated to genes encoding virulence factors and nutrient metabolism; and indications of parallel evolution were observed in genes encoding the major phase-variable protein antigen 43, a toxin/antitoxin locus and haemolysin B. No differences in virulence potential were observed in a mouse UTI model for five matching faecal and UTI isolates with or without mutations in antigen 43 and haemolysin B. Variations in plasmid content were observed in only four of the 42 pairs. Although, we observed mutations in known UTI virulence genes for a few pairs, the majority showed no detectable differences in mutations or mobilome changes when compared to their faecal counterpart. The results show that UPECs are successful in colonizing both the bladder and gut without adaptation. PMID:27825516

Adaptation of Escherichia coli traversing from the faecal environment to the urinary tract.

PubMed

Nielsen, Karen L; Stegger, Marc; Godfrey, Paul A; Feldgarden, Michael; Andersen, Paal S; Frimodt-Møller, Niels

2016-12-01

The majority of extraintestinal pathogenic Escherichia coli (ExPEC) causing urinary tract infections (UTI) are found in the patient's own gut flora, but only limited knowledge is available on the potential adaptation that may occur in the bacteria in order to traverse the perineum and successfully infect the urinary tract. Here, matching pairs of faecal and UTI isolates from 42 patients were compared pairwise using in-depth whole-genome sequencing to investigate whether genetic changes were evident for successful colonization in these two different environments. The identified non-synonymous mutations (0-12 substitutions in each pair) were primarily associated to genes encoding virulence factors and nutrient metabolism; and indications of parallel evolution were observed in genes encoding the major phase-variable protein antigen 43, a toxin/antitoxin locus and haemolysin B. No differences in virulence potential were observed in a mouse UTI model for five matching faecal and UTI isolates with or without mutations in antigen 43 and haemolysin B. Variations in plasmid content were observed in only four of the 42 pairs. Although, we observed mutations in known UTI virulence genes for a few pairs, the majority showed no detectable differences with respect to mutations or mobilome when compared to their faecal counterpart. The results show that UPECs are successful in colonizing both the bladder and gut without adaptation. Copyright © 2016 Elsevier GmbH. All rights reserved.

Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence.

PubMed

Brown, Darby G; Swanson, Jill K; Allen, Caitilyn

2007-05-01

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10(7) CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds.

Transcriptome analysis of trichothecene-induced gene expression in barley.

PubMed

Boddu, Jayanand; Cho, Seungho; Muehlbauer, Gary J

2007-11-01

Fusarium head blight, caused primarily by Fusarium graminearum, is a major disease problem on barley (Hordeum vulgare L.). Trichothecene mycotoxins produced by the fungus during infection increase the aggressiveness of the fungus and promote infection in wheat (Triticum aestivum L.). Loss-of-function mutations in the TRI5 gene in F. graminearum result in the inability to synthesize trichothecenes and in reduced virulence on wheat. We examined the impact of pathogen-derived trichothecenes on virulence and the transcriptional differences in barley spikes infected with a trichothecene-producing wild-type strain and a loss-of-function tri5 trichothecene nonproducing mutant. Disease severity, fungal biomass, and floret necrosis and bleaching were reduced in spikes inoculated with the tri5 mutant strain compared with the wild-type strain, indicating that the inability to synthesize trichothecenes results in reduced virulence in barley. We detected 63 transcripts that were induced during trichothecene accumulation, including genes encoding putative trichothecene detoxification and transport proteins, ubiquitination-related proteins, programmed cell death-related proteins, transcription factors, and cytochrome P450s. We also detected 414 gene transcripts that were designated as basal defense response genes largely independent of trichothecene accumulation. Our results show that barley exhibits a specific response to trichothecene accumulation that can be separated from the basal defense response. We propose that barley responds to trichothecene accumulation by inducing at least two general responses. One response is the induction of genes encoding trichothecene detoxification and transport activities that may reduce the impact of trichothecenes. The other response is to induce genes encoding proteins associated with ubiquitination and cell death which may promote successful establishment of the disease.

Draft genome sequence of a multidrug-resistant KPC-2-producing Enterobacter aerogenes isolated from a hospitalised patient in Brazil.

PubMed

Moura, Quézia; Fernandes, Miriam R; Cerdeira, Louise; Nhambe, Lúcia F; Ienne, Susan; Souza, Tiago A; Lincopan, Nilton

2017-09-01

Multidrug-resistant (MDR) Enterobacter aerogenes strains are frequently associated with nosocomial infections and high mortality rates, representing a serious public health problem. The aim of this study was to present the draft genome sequence of a MDR KPC-2-producing E. aerogenes isolated from a perineal swab of a hospitalised patient in Brazil. Genomic DNA was sequenced using an Illumina MiSeq platform. De novo genome assembly was carried out using the A5-Miseq pipeline, and whole-genome sequence analysis was performed using tools from the Center for Genomic Epidemiology. The strain harboured resistance genes to β-lactams, aminoglycosides, sulphonamides and trimethoprim in addition to genes encoding multidrug efflux system proteins, a quaternary ammonium transporter and heavy metal efflux system proteins. In addition, the strain harboured genes encoding diverse virulence factors. These data might allow a better understanding of the genetic basis of antimicrobial resistance and virulence in E. aerogenes strains. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Emerging Functions for the Staphylococcus aureus RNome

PubMed Central

Felden, Brice

2013-01-01

Staphylococcus aureus is a leading pathogen for animals and humans, not only being one of the most frequently isolated bacteria in hospital-associated infections but also causing diseases in the community. To coordinate the expression of its numerous virulence genes for growth and survival, S. aureus uses various signalling pathways that include two-component regulatory systems, transcription factors, and also around 250 regulatory RNAs. Biological roles have only been determined for a handful of these sRNAs, including cis, trans, and cis-trans acting RNAs, some internally encoding small, functional peptides and others possessing dual or multiple functions. Here we put forward an inventory of these fascinating sRNAs; the proteins involved in their activities; and those involved in stress response, metabolisms, and virulence. PMID:24348246

Genetic and Biochemical Characterization of a Gene Operon for trans-Aconitic Acid, a Novel Nematicide from Bacillus thuringiensis.

PubMed

Du, Cuiying; Cao, Shiyun; Shi, Xiangyu; Nie, Xiangtao; Zheng, Jinshui; Deng, Yun; Ruan, Lifang; Peng, Donghai; Sun, Ming

2017-02-24

trans -Aconitic acid (TAA) is an isomer of cis -aconitic acid (CAA), an intermediate of the tricarboxylic acid cycle that is synthesized by aconitase. Although TAA production has been detected in bacteria and plants for many years and is known to be a potent inhibitor of aconitase, its biosynthetic origins and the physiological relevance of its activity have remained unclear. We have serendipitously uncovered key information relevant to both of these questions. Specifically, in a search for novel nematicidal factors from Bacillus thuringiensis , a significant nematode pathogen harboring many protein virulence factors, we discovered a high yielding component that showed activity against the plant-parasitic nematode Meloidogyne incognita and surprisingly identified it as TAA. Comparison with CAA, which displayed a much weaker nematicidal effect, suggested that TAA is specifically synthesized by B. thuringiensis as a virulence factor. Analysis of mutants deficient in plasmids that were anticipated to encode virulence factors allowed us to isolate a TAA biosynthesis-related ( tbr ) operon consisting of two genes, tbrA and tbrB We expressed the corresponding proteins, TbrA and TbrB, and characterized them as an aconitate isomerase and TAA transporter, respectively. Bioinformatics analysis of the TAA biosynthetic gene cluster revealed the association of the TAA genes with transposable elements relevant for horizontal gene transfer as well as a distribution across B. cereus bacteria and other B. thuringiensis strains, suggesting a general role for TAA in the interactions of B. cereus group bacteria with nematode hosts in the soil environment. This study reveals new bioactivity for TAA and the TAA biosynthetic pathway, improving our understanding of virulence factors employed by B. thuringiensis pathogenesis and providing potential implications for nematode management applications. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.

PubMed

Lüneberg, E; Mayer, B; Daryab, N; Kooistra, O; Zähringer, U; Rohde, M; Swanson, J; Frosch, M

2001-03-01

We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.

Antimicrobial medium- and long-chain free fatty acids prevent PrfA-dependent activation of virulence genes in Listeria monocytogenes.

PubMed

Sternkopf Lillebæk, Eva Maria; Lambert Nielsen, Stine; Scheel Thomasen, Rikke; Færgeman, Nils J; Kallipolitis, Birgitte H

The foodborne pathogen Listeria monocytogenes is the causative agent of the invasive disease listeriosis. Infection by L. monocytogenes involves bacterial crossing of the intestinal barrier and intracellular replication in a variety of host cells. The PrfA protein is the master regulator of virulence factors required for bacterial entry, intracellular replication and cell-to-cell spread. PrfA-dependent activation of virulence genes occurs primarily in the blood and during intracellular infection. In contrast, PrfA does not play a significant role in regulation of virulence gene expression in the intestinal environment. In the gastrointestinal phase of infection, the bacterium encounters a variety of antimicrobial agents, including medium- and long-chain free fatty acids that are commonly found in our diet and as active components of bile. Here we show that subinhibitory concentrations of specific antimicrobial free fatty acids act to downregulate transcription of PrfA-activated virulence genes. Interestingly, the inhibitory effect is also evident in cells encoding a constitutively active variant of PrfA. Collectively, our data suggest that antimicrobial medium- and long-chain free fatty acids may act as signals to prevent PrfA-mediated activation of virulence genes in environments where PrfA activation is not required, such as in food and the gastrointestinal tract. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Genomic Epidemiology of Hypervirulent Serogroup W, ST-11 Neisseria meningitidis

PubMed Central

Mustapha, Mustapha M.; Marsh, Jane W.; Krauland, Mary G.; Fernandez, Jorge O.; de Lemos, Ana Paula S.; Dunning Hotopp, Julie C.; Wang, Xin; Mayer, Leonard W.; Lawrence, Jeffrey G.; Hiller, N. Luisa; Harrison, Lee H.

2015-01-01

Neisseria meningitidis is a leading bacterial cause of sepsis and meningitis globally with dynamic strain distribution over time. Beginning with an epidemic among Hajj pilgrims in 2000, serogroup W (W) sequence type (ST) 11 emerged as a leading cause of epidemic meningitis in the African ‘meningitis belt’ and endemic cases in South America, Europe, Middle East and China. Previous genotyping studies were unable to reliably discriminate sporadic W ST-11 strains in circulation since 1970 from the Hajj outbreak strain (Hajj clone). It is also unclear what proportion of more recent W ST-11 disease clusters are caused by direct descendants of the Hajj clone. Whole genome sequences of 270 meningococcal strains isolated from patients with invasive meningococcal disease globally from 1970 to 2013 were compared using whole genome phylogenetic and major antigen-encoding gene sequence analyses. We found that all W ST-11 strains were descendants of an ancestral strain that had undergone unique capsular switching events. The Hajj clone and its descendants were distinct from other W ST-11 strains in that they shared a common antigen gene profile and had undergone recombination involving virulence genes encoding factor H binding protein, nitric oxide reductase, and nitrite reductase. These data demonstrate that recent acquisition of a distinct antigen-encoding gene profile and variations in meningococcal virulence genes was associated with the emergence of the Hajj clone. Importantly, W ST-11 strains unrelated to the Hajj outbreak contribute a significant proportion of W ST-11 cases globally. This study helps illuminate genomic factors associated with meningococcal strain emergence and evolution. PMID:26629539

Distribution of genes encoding virulence factors and molecular analysis of Shigella spp. isolated from patients with diarrhea in Kerman, Iran.

PubMed

Hosseini Nave, Hossein; Mansouri, Shahla; Emaneini, Mohammad; Moradi, Mohammad

2016-03-01

Shigella is one of the important causes of diarrhea worldwide. Shigella has several virulence factors contributing in colonization and invasion of epithelial cells and eventually death of host cells. The present study was performed in order to investigate the distribution of virulence factors genes in Shigella spp. isolated from patients with acute diarrhea in Kerman, Iran as well as the genetic relationship of these isolates. A total of 56 isolates including 31 S. flexneri, 18 S. sonnei and 7 S. boydii were evaluated by polymerase chain reaction (PCR) for the presence of 11 virulence genes (ipaH, ial, set1A, set1B, sen, virF, invE, sat, sigA, pic and sepA). Then, the clonal relationship of these strains was analyzed by multilocus variable-number tandem repeat analysis (MLVA) method. All isolates were positive for ipaH gene. The other genes include ial, invE and virF were found in 80.4%, 60.7% and 67.9% of the isolates, respectively. Both set1A and set1B were detected in 32.3% of S. flexneri isolates, whereas 66.1% of the isolates belonging to different serogroup carried sen gene. The sat gene was present in all S. flexneri isolates, but not in the S. sonnei and S. boydii isolates. The result showed, 30.4% of isolates were simultaneously positive and the rest of the isolates were negative for sepA and pic genes. The Shigella isolates were divided into 29 MLVA types. This study, for the first time, investigated distribution of 11 virulence genes in Shigella spp. Our results revealed heterogeneity of virulence genes in different Shigella serogroups. Furthermore, the strains belonging to the same species had little diversity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Two-component regulators involved in the global control of virulence in Erwinia carotovora subsp. carotovora.

PubMed

Eriksson, A R; Andersson, R A; Pirhonen, M; Palva, E T

1998-08-01

Production of extracellular, plant cell wall degrading enzymes, the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, is coordinately controlled by a complex regulatory network. Insertion mutants in the exp (extracellular enzyme production) loci exhibit pleiotropic defects in virulence and the growth-phase-dependent transcriptional activation of genes encoding extracellular enzymes. Two new exp mutations, designated expA and expS, were characterized. Introduction of the corresponding wild-type alleles to the mutants complemented both the lack of virulence and the impaired production of plant cell wall degrading enzymes. The expA gene was shown to encode a 24-kDa polypeptide that is structurally and functionally related to the uvrY gene product of Escherichia coli and the GacA response regulator of Pseudomonas fluorescens. Functional similarity of expA and uvrY was demonstrated by genetic complementation. The expA gene is organized in an operon together with a uvrC-like gene, identical to the organization of uvrY and uvrC in E. coli. The unlinked expS gene encodes a putative sensor kinase that shows 92% identity to the recently described rpfA gene product from another E. carotovora subsp. carotovora strain. Our data suggest that ExpS and ExpA are members of two-component sensor kinase and response regulator families, respectively. These two proteins might interact in controlling virulence gene expression in E. carotovora subsp. carotovora.

Molecular Characterization of Salmonella from Human and Animal Origins in Uganda

PubMed Central

Kagirita, Atek Atwiine; Owalla, Tonny Jimmy; Majalija, Samuel

2017-01-01

Sporadic Salmonella outbreaks with varying clinical presentations have been on the rise in various parts of Uganda. The sources of outbreaks and factors underlying the different clinical manifestation are curtailed by paucity of information on Salmonella genotypes and the associated virulence genes. This study reports molecular diversity of Salmonella enterica and their genetic virulence profiles among human and animal isolates. Characterization was done using Kauffman-White classification scheme and virulence genes analysis using multiplex PCR. Overall, 52% of the isolates belonged to serogroup D, 16% to serogroup E, 15% to poly F, H-S, and 12% to serogroup B. Serogroups A, C1, and C2 each consisted of only one isolate representing 5%. Virulence genes located on SPI-1 [spaN and sipB] and on SPI-2 [spiA] in addition to pagC and msgA were equally distributed in isolates obtained from all sources. Plasmid encoded virulence gene spvB was found in <5% of isolates from both human epidemic and animal origins whereas it occurred in 80% of clinical isolates. This study reveals that serogroup D is the predominant Salmonella serogroup in circulation and it is widely shared among animals and humans and calls for joint and coordinated surveillance for one health implementation in Uganda. PMID:28634597

Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes.

PubMed

Zhu, Luchang; Olsen, Randall J; Horstmann, Nicola; Shelburne, Samuel A; Fan, Jia; Hu, Ye; Musser, James M

2016-07-01

Variable-number tandem-repeat (VNTR) polymorphisms are ubiquitous in bacteria. However, only a small fraction of them has been functionally studied. Here, we report an intergenic VNTR polymorphism that confers an altered level of toxin production and increased virulence in Streptococcus pyogenes The nature of the polymorphism is a one-unit deletion in a three-tandem-repeat locus upstream of the rocA gene encoding a sensor kinase. S. pyogenes strains with this type of polymorphism cause human infection and produce significantly larger amounts of the secreted cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO). Using isogenic mutant strains, we demonstrate that deleting one or more units of the tandem repeats abolished RocA production, reduced CovR phosphorylation, derepressed multiple CovR-regulated virulence factors (such as SPN and SLO), and increased virulence in a mouse model of necrotizing fasciitis. The phenotypic effect of the VNTR polymorphism was nearly the same as that of inactivating the rocA gene. In summary, we identified and characterized an intergenic VNTR polymorphism in S. pyogenes that affects toxin production and virulence. These new findings enhance understanding of rocA biology and the function of VNTR polymorphisms in S. pyogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Molecular and phenotypic characteristics of methicillin-resistant Staphylococcus aureus isolated from hospitalized patients.

PubMed

de Oliveira, Caio Ferreira; Morey, Alexandre Tadachi; Santos, Jussevania Pereira; Gomes, Ludmila Vilela Pereira; Cardoso, Juscélio Donizete; Pinge-Filho, Phileno; Perugini, Márcia Regina Eches; Yamauchi, Lucy Megumi; Yamada-Ogatta, Sueli Fumie

2015-07-30

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of infections acquired in both community and hospital settings. In this study, MRSA isolated from different sources of hospitalized patients was characterized by molecular and phenotypic methods. A total of 123 S. aureus isolates were characterized according to their genetic relatedness by repetitive element sequence based-PCR (REP-PCR), in vitro antimicrobial susceptibility profile, SCCmec typing and presence of seven virulence factor-encoding genes. REP-PCR fingerprinting showed low relatedness between the isolates, and the predominance of one specific lineage or clonal group was not observed. All isolates were susceptible to teicoplanin and linezolide. All isolates were resistant to cefoxitin and penicillin, and the majority were also resistant to one or more other antimicrobials. Fifty isolates (41.7%) were intermediately resistant to vancomycin. Most isolates harbored SCCmec type II (53.7%), followed by type I (22.8%), type IV (8.1%) and type III (1.6%). All isolates harbored at least two virulence factor-encoding genes, and the prevalence was as follows: coa, 100%; icaA, 100%; hla, 13.0%; hlb, 91.1%, hld, 91.1%; lukS-PV and lukF-PV, 2.4%; and tst, 34.1%. A positive association with the presence of hla and SCCmec type II, and tst and SCCmec type I was observed. This study showed the high virulence potential of multidrug-resistant MRSA circulating in a teaching hospital. A high prevalence of MRSA showing intermediate vancomycin resistance was also observed, indicating the urgent need to improve strategies for controlling the use of antimicrobials for appropriate management of S. aureus infections.

Probing Genomic Aspects of the Multi-Host Pathogen Clostridium perfringens Reveals Significant Pangenome Diversity, and a Diverse Array of Virulence Factors

PubMed Central

Kiu, Raymond; Caim, Shabhonam; Alexander, Sarah; Pachori, Purnima; Hall, Lindsay J.

2017-01-01

Clostridium perfringens is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and characterise the genomic variation, pangenomic diversity, and key virulence traits of 56 C. perfringens strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that C. perfringens has an “open” pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined C. perfringens phylogeny (16S rRNA gene) in relation to some 25 Clostridium species, with C. baratii and C. sardiniense determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised C. perfringens-associated exotoxins genes including α-toxin (plc), enterotoxin (cpe), and Perfringolysin O (pfo or pfoA), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes (tet) and anti-defensins genes (mprF) were consistently detected in silico (tet: 75%; mprF: 100%). However, pre-antibiotic era strain genomes did not encode for tet, thus implying antimicrobial selective pressures in C. perfringens evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen. PMID:29312194

Probing Genomic Aspects of the Multi-Host Pathogen Clostridium perfringens Reveals Significant Pangenome Diversity, and a Diverse Array of Virulence Factors.

PubMed

Kiu, Raymond; Caim, Shabhonam; Alexander, Sarah; Pachori, Purnima; Hall, Lindsay J

2017-01-01

Clostridium perfringens is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and characterise the genomic variation, pangenomic diversity, and key virulence traits of 56 C. perfringens strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that C. perfringens has an "open" pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined C. perfringens phylogeny (16S rRNA gene) in relation to some 25 Clostridium species, with C. baratii and C. sardiniense determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised C. perfringens -associated exotoxins genes including α-toxin ( plc ), enterotoxin ( cpe ), and Perfringolysin O ( pfo or pfoA ), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes ( tet ) and anti-defensins genes ( mprF ) were consistently detected in silico ( tet : 75%; mprF : 100%). However, pre-antibiotic era strain genomes did not encode for tet , thus implying antimicrobial selective pressures in C. perfringens evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen.

Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whittingham, Jean L.; Blagova, Elena V.; Finn, Ciaran E.

2014-08-01

VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded β-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vapmore » proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.« less

Multiple Functional Domains of Enterococcus faecalis Aggregation Substance Asc10 Contribute to Endocarditis Virulence ▿ †

PubMed Central

Chuang, Olivia N.; Schlievert, Patrick M.; Wells, Carol L.; Manias, Dawn A.; Tripp, Timothy J.; Dunny, Gary M.

2009-01-01

Aggregation substance proteins encoded by sex pheromone plasmids increase the virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis models. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation substance Asc10, encoded by plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the infected tissue was the best indicator of virulence. Isogenic strains carrying either no plasmid, wild-type pCF10, a pCF10 derivative with an in-frame deletion of the prgB gene encoding Asc10, or pCF10 derivatives expressing other alleles of prgB were examined in this model. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative in which glycine residues in two RGD motifs were changed to alanine residues showed the greatest reduction in virulence. Remarkably, this strain and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo. PMID:18955479

aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species.

PubMed

Lescat, Mathilde; Hoede, Claire; Clermont, Olivier; Garry, Louis; Darlu, Pierre; Tuffery, Pierre; Denamur, Erick; Picard, Bertrand

2009-12-29

Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B2 variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B1 variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene. We identified the gene encoding esterase B as the acetyl-esterase gene (aes) using gene disruption. The analysis of aes nucleotide sequences in a panel of 78 reference strains, including the E. coli reference (ECOR) strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from aes sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B1 and B2 by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between aes and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of aes to be associated with virulence. Our findings suggest that aes does not play a direct role in the virulence of E. coli extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.

The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence.

PubMed

Ren, Jun; Prescott, John F

2004-11-15

An 81 kb virulence plasmid containing a pathogenicity island (PI) plays a crucial role in the pathogenesis of Rhodococcus equi pneumonia in foals but its specific function in virulence and regulation of plasmid-encoded virulence genes is unclear. Using a LacZ selection marker developed for R. equi in this study, in combination with an apramycin resistance gene, an efficient two-stage homologous recombination targeted gene mutation procedure was used to mutate three virulence plasmid genes, a LysR regulatory gene homologue (ORF4), a ResD-like two-component response regulator homologue (ORF8), and a gene (ORF10) of unknown function that is highly expressed by R. equi inside macrophages, as well as the chromosomal gene operon, phoPR. Virulence testing by liver clearance after intravenous injection in mice showed that the ORF4 and ORF8 mutants were fully attenuated, that the phoPR mutant was hypervirulent, and that virulence of the ORF10 mutant remained unchanged. A virulence plasmid DNA microarray was used to compare the plasmid gene expression profile of each of the four gene-targeted mutants against the parental R. equi strain. Changes were limited to PI genes and gene induction was observed for all mutants, suggesting that expression of virulence plasmid genes is dominated by a negative regulatory network. The finding of attenuation of ORF4 and ORF8 mutants despite enhanced transcription of vapA suggests that factors other than VapA are important for full expression of virulence. ORF1, a putative Lsr antigen gene, was strongly and similarly induced in all mutants, implying a common regulatory pathway affecting this gene for all four mutated genes. ORF8 is apparently the centre of this common pathway. Two distinct highly correlated gene induction patterns were observed, that of the ORF4 and ORF8 mutants, and that of the ORF10 and phoPR mutants. The gene induction pattern distinguishing these two groups paralleled their virulence in mice.

Disruption of the Aspergillus fumigatus ECM33 homologue results in rapid conidial germination, antifungal resistance and hypervirulence.

PubMed

Romano, Jacob; Nimrod, Guy; Ben-Tal, Nir; Shadkchan, Yona; Baruch, Koti; Sharon, Haim; Osherov, Nir

2006-07-01

The ECM33/SPS2 family of glycosylphosphatidylinositol-anchored proteins plays an important role in maintaining fungal cell wall integrity and virulence. However, the precise molecular role of these proteins is unknown. In this work, AfuEcm33, the gene encoding the ECM33 homologue in the important pathogenic fungus Aspergillus fumigatus, has been cloned and its function analysed. It is shown that disruption of AfuEcm33 results in rapid conidial germination, increased cell-cell adhesion, resistance to the antifungal agent caspofungin and increased virulence in an immunocompromised mouse model for disseminated aspergillosis. These results suggest that the protein encoded by AfuEcm33 is involved in key aspects of cell wall morphogenesis and plays an important role in A. fumigatus virulence.

Mechanosensing regulates virulence in Escherichia coli O157:H7.

PubMed

Islam, Md Shahidul; Krachler, Anne Marie

2016-01-01

Enterohemorrhagic Escherichia coli O157:H7 is a food-borne pathogen transmitted via the fecal-oral route, and can cause bloody diarrhea and hemolytic uremic syndrome (HUS) in the human host. Although a range of colonization factors, Shiga toxins and a type III secretion system (T3SS) all contribute to disease development, the locus of enterocyte effacement (LEE) encoded T3SS is responsible for the formation of lesions in the intestinal tract. While a variety of chemical cues in the host environment are known to up-regulate LEE expression, we recently demonstrated that changes in physical forces at the site of attachment are required for localized, full induction of the system and thus spatial regulation of virulence in the intestinal tract. Here, we discuss our findings in the light of other recent studies describing mechanosensing of the host and force-dependent induction of virulence mechanisms. We discuss potential mechanisms of mechanosensing and mechanotransduction, and the level of conservation across bacterial species.

Proteomics Analysis of the Causative Agent of Typhoid Fever

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ansong, Charles; Yoon, Hyunjin; Norbeck, Angela D.

2008-02-01

Typhoid fever is a potentially fatal disease caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. typhi). S. typhi infection is a complex process that involves numerous bacterially-encoded virulence determinants, and these are thought to confer both stringent human host specificity and a high mortality rate. In the present study we used a liquid chromatography-mass spectrometry (LC-MS) based proteomics strategy to investigate the proteome of logarithmic, stationary phase, and low pH/low magnesium (MgM) S. typhi cultures. This represents the first large scale comprehensive characterization of the S. typhi proteome. Our analysis identified a total of 2066 S. typhi proteins.more » In an effort to identify putative S. typhi-specific virulence factors, we then compared our S. typhi results to those obtained in a previously published study of the S. typhimurium proteome under similar conditions (Adkins J.N. et al (2006) Mol Cell Prot). Comparative proteomic analysis of S. typhi (strain Ty2) and S. typhimurium (strains LT2 and 14028) revealed a subset of highly expressed proteins unique to S. typhi that were exclusively detected under conditions that mimic the infective state in macrophage cells. These proteins included CdtB, HlyE, and a conserved protein encoded by t1476. The differential expression of selected proteins was confirmed by Western blot analysis. Taken together with the current literature, our observations suggest that this subset of proteins may play a role in S. typhi pathogenesis and human host specificity. In addition, we observed products of the biotin (bio) operon displayed a higher abundance in the more virulent strains S. typhi-Ty2 and S. typhimurium-14028 compared to the virulence attenuated S. typhimurium strain LT2, suggesting bio proteins may contribute to Salmonella pathogenesis.« less

Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori.

PubMed

Bury-Moné, Stéphanie; Thiberge, Jean-Michel; Contreras, Monica; Maitournam, Aboubakar; Labigne, Agnès; De Reuse, Hilde

2004-07-01

The virulence of pathogenic bacteria is dependent on their adaptation to and survival in the stressful conditions encountered in their hosts. Helicobacter pylori exclusively colonizes the acid stomach of primates, making it an ideal study model. Little is known about how H. pylori responds to the moderately acidic conditions encountered at its colonization site, the gastric mucus layer. Thus, we compared gene expression profiles of H. pylori 26695 grown at neutral and acidic pH, and validated the data for a selection of genes by real-time polymerase chain reaction, dot-blots or enzymatic assays. During growth in acidic conditions, 56 genes were upregulated and 45 genes downregulated. We found that acidity is a signal modulating the expression of several virulence factors. Regulation of genes related to metal ion homeostasis suggests protective mechanisms involving diminished transport and enhanced storage. Genes encoding subunits of the F0F1 ATPase and of a newly identified Na+/H+ antiporter (NhaC-HP0946) were downregulated, revealing that this bacterium uses original mechanisms to control proton entry. Five of the upregulated genes encoded proteins controlling intracellular ammonia synthesis, including urease, amidase and formamidase, underlining the major role of this buffering compound in the protection against acidity in H. pylori. Regulatory networks and transcriptome analysis as well as enzymatic assays implicated two metal-responsive transcriptional regulators (NikR and Fur) and an essential two-component response regulator (HP0166, OmpR-like) as effectors of the H. pylori acid response. Finally, a nikR-fur mutant is attenuated in the mouse model, emphasizing the link between response to acidity, metal metabolism and virulence in this gastric pathogen.

AmrZ Beta-Sheet Residues Are Essential for DNA Binding and Transcriptional Control of Pseudomonas aeruginosa Virulence Genes ▿ †

PubMed Central

Waligora, Elizabeth A.; Ramsey, Deborah M.; Pryor, Edward E.; Lu, Haiping; Hollis, Thomas; Sloan, Gina P.; Deora, Rajendar; Wozniak, Daniel J.

2010-01-01

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection. PMID:20709902

Gene expansion shapes genome architecture in the human pathogen Lichtheimia corymbifera: an evolutionary genomics analysis in the ancient terrestrial mucorales (Mucoromycotina).

PubMed

Schwartze, Volker U; Winter, Sascha; Shelest, Ekaterina; Marcet-Houben, Marina; Horn, Fabian; Wehner, Stefanie; Linde, Jörg; Valiante, Vito; Sammeth, Michael; Riege, Konstantin; Nowrousian, Minou; Kaerger, Kerstin; Jacobsen, Ilse D; Marz, Manja; Brakhage, Axel A; Gabaldón, Toni; Böcker, Sebastian; Voigt, Kerstin

2014-08-01

Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1-4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparison to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae.

Gene Expansion Shapes Genome Architecture in the Human Pathogen Lichtheimia corymbifera: An Evolutionary Genomics Analysis in the Ancient Terrestrial Mucorales (Mucoromycotina)

PubMed Central

Wehner, Stefanie; Linde, Jörg; Valiante, Vito; Sammeth, Michael; Riege, Konstantin; Nowrousian, Minou; Kaerger, Kerstin; Jacobsen, Ilse D.; Marz, Manja; Brakhage, Axel A.; Gabaldón, Toni; Böcker, Sebastian; Voigt, Kerstin

2014-01-01

Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1–4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparision to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae. PMID:25121733

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity.

PubMed

Marko, Victoria A; Kilmury, Sara L N; MacNeil, Lesley T; Burrows, Lori L

2018-05-18

Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 was proposed to be a mechanosensory component that-in conjunction with minor pilins-triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins/PilY1 and virulence. pilW, pilX, and pilY1 mutants had reduced virulence towards Caenorhabditis elegans relative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the AlgR regulon and delaying C. elegans killing. Reporter assays confirmed that FimS-AlgR were required for increased expression of the minor pilin operon upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via a phosphomimetic mutation reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants required FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR that suppresses expression of acute-phase virulence factors and delays killing. This mechanism could contribute to adaptation of P. aeruginosa in chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors-such as alginate-that are characteristic of such infections.

Cross-Talk between Staphylococcus aureus and Other Staphylococcal Species via the agr Quorum Sensing System

PubMed Central

Canovas, Jaime; Baldry, Mara; Bojer, Martin S.; Andersen, Paal S.; Gless, Bengt H.; Grzeskowiak, Piotr K.; Stegger, Marc; Damborg, Peter; Olsen, Christian A.; Ingmer, Hanne

2016-01-01

Staphylococci are associated with both humans and animals. While most are non-pathogenic colonizers, Staphylococcus aureus is an opportunistic pathogen capable of causing severe infections. S. aureus virulence is controlled by the agr quorum sensing system responding to secreted auto-inducing peptides (AIPs) sensed by AgrC, a two component histidine kinase. agr loci are found also in other staphylococcal species and for Staphylococcus epidermidis, the encoded AIP represses expression of agr regulated virulence genes in S. aureus. In this study we aimed to better understand the interaction between staphylococci and S. aureus, and show that this interaction may eventually lead to the identification of new anti-virulence candidates to target S. aureus infections. Here we show that culture supernatants of 37 out of 52 staphylococcal isolates representing 17 different species inhibit S. aureus agr. The dog pathogen, Staphylococcus schleiferi, expressed the most potent inhibitory activity and was active against all four agr classes found in S. aureus. By employing a S. aureus strain encoding a constitutively active AIP receptor we show that the activity is mediated via agr. Subsequent cloning and heterologous expression of the S. schleiferi AIP in S. aureus demonstrated that this molecule was likely responsible for the inhibitory activity, and further proof was provided when pure synthetic S. schleiferi AIP was able to completely abolish agr induction of an S. aureus reporter strain. To assess impact on S. aureus virulence, we co-inoculated S. aureus and S. schleiferi in vivo in the Galleria mellonella wax moth larva, and found that expression of key S. aureus virulence factors was abrogated. Our data show that the S. aureus agr locus is highly responsive to other staphylococcal species suggesting that agr is an inter-species communication system. Based on these results we speculate that interactions between S. aureus and other colonizing staphylococci will significantly influence the ability of S. aureus to cause infection, and we propose that other staphylococci are potential sources of compounds that can be applied as anti-virulence therapy for combating S. aureus infections. PMID:27877157

Virulence Factor Genes in Staphylococcus aureus Isolated From Diabetic Foot Soft Tissue and Bone Infections.

PubMed

Víquez-Molina, Gerardo; Aragón-Sánchez, Javier; Pérez-Corrales, Cristian; Murillo-Vargas, Christian; López-Valverde, María Eugenia; Lipsky, Benjamin A

2018-03-01

The aim of this study is to describe the presence of genes encoding for 4 virulence factors (pvl, eta, etb, and tsst), as well as the mecA gene conferring resistance to beta-lactam antibiotics, in patients with diabetes and a staphylococcal foot infection. We have also analyzed whether isolates of Staphylococcus aureus from bone infections have a different profile for these genes compared with those from exclusively soft tissue infections. In this cross-sectional study of a prospectively recruited series of patients admitted to the Diabetic Foot Unit, San Juan de Dios Hospital, San José, Costa Rica with a moderate or severe diabetic foot infection (DFI), we collected samples from infected soft tissue and from bone during debridement. During the study period (June 1, 2014 to May 31, 2016), we treated 379 patients for a DFI. S aureus was isolated from 101 wound samples, of which 43 were polymicrobial infections; we only included the 58 infections that were monomicrobial S aureus for this study. Infections were exclusively soft tissue in 17 patients (29.3%) while 41 (70.7%) had bone involvement (osteomyelitis). The mecA gene was detected in 35 cases (60.3%), pvl gene in 4 cases (6.9%), and tsst gene in 3 (5.2%). We did not detect etA and etB in any of the cases. There were no differences in the profile of S aureus genes encoding for virulence factors (pvl, etA, etB, and tsst) recovered from DFIs between those with just soft tissue compared to those with osteomyelitis. However, we found a significantly higher prevalence of pvl+ strains of S aureus associated with soft tissue compared with bone infections. Furthermore, we observed a significantly longer time to healing among patients infected with mecA+ (methicillin-resistant) S aureus (MRSA).

Contemporary avian influenza A virus subtype H1, H6, H7, H10, and H15 hemagglutinin genes encode a mammalian virulence factor similar to the 1918 pandemic virus H1 hemagglutinin.

PubMed

Qi, Li; Pujanauski, Lindsey M; Davis, A Sally; Schwartzman, Louis M; Chertow, Daniel S; Baxter, David; Scherler, Kelsey; Hartshorn, Kevan L; Slemons, Richard D; Walters, Kathie-Anne; Kash, John C; Taubenberger, Jeffery K

2014-11-18

Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections with these subtypes may be important in controlling future outbreaks. Copyright © 2014 Qi et al.

Giardia secretome highlights secreted tenascins as a key component of pathogenesis

PubMed Central

Dubourg, Audrey; Xia, Dong; Winpenny, John P; Al Naimi, Suha; Bouzid, Maha; Sexton, Darren W; Wastling, Jonathan M; Hunter, Paul R; Tyler, Kevin M

2018-01-01

Abstract Background Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood. Results The genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites. Conclusions We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators. PMID:29385462

Giardia secretome highlights secreted tenascins as a key component of pathogenesis.

PubMed

Dubourg, Audrey; Xia, Dong; Winpenny, John P; Al Naimi, Suha; Bouzid, Maha; Sexton, Darren W; Wastling, Jonathan M; Hunter, Paul R; Tyler, Kevin M

2018-03-01

Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood. The genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites. We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators.

The yersiniabactin transport system is critical for the pathogenesis of bubonic and pneumonic plague.

PubMed

Fetherston, Jacqueline D; Kirillina, Olga; Bobrov, Alexander G; Paulley, James T; Perry, Robert D

2010-05-01

Iron acquisition from the host is an important step in the pathogenic process. While Yersinia pestis has multiple iron transporters, the yersiniabactin (Ybt) siderophore-dependent system plays a major role in iron acquisition in vitro and in vivo. In this study, we determined that the Ybt system is required for the use of iron bound by transferrin and lactoferrin and examined the importance of the Ybt system for virulence in mouse models of bubonic and pneumonic plague. Y. pestis mutants unable to either transport Ybt or synthesize the siderophore were both essentially avirulent via subcutaneous injection (bubonic plague model). Surprisingly, via intranasal instillation (pneumonic plague model), we saw a difference in the virulence of Ybt biosynthetic and transport mutants. Ybt biosynthetic mutants displayed an approximately 24-fold-higher 50% lethal dose (LD(50)) than transport mutants. In contrast, under iron-restricted conditions in vitro, a Ybt transport mutant had a more severe growth defect than the Ybt biosynthetic mutant. Finally, a Delta pgm mutant had a greater loss of virulence than the Ybt biosynthetic mutant, indicating that the 102-kb pgm locus encodes a virulence factor, in addition to Ybt, that plays a role in the pathogenesis of pneumonic plague.

Haemophilus ducreyi Hfq contributes to virulence gene regulation as cells enter stationary phase.

PubMed

Gangaiah, Dharanesh; Labandeira-Rey, Maria; Zhang, Xinjun; Fortney, Kate R; Ellinger, Sheila; Zwickl, Beth; Baker, Beth; Liu, Yunlong; Janowicz, Diane M; Katz, Barry P; Brautigam, Chad A; Munson, Robert S; Hansen, Eric J; Spinola, Stanley M

2014-02-11

To adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes of Haemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog of hfq. Insertional inactivation of hfq altered the expression of ~16% of the H. ducreyi genes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in the hfq inactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation of hfq downregulated genes in the flp-tad and lspB-lspA2 operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarked hfq deletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion of hfq downregulated Flp1 and impaired the ability of H. ducreyi to form microcolonies, downregulated DsrA and rendered H. ducreyi serum susceptible, and downregulated LspB and LspA2, which allow H. ducreyi to resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor of H. ducreyi stationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog. Pathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, including Haemophilus ducreyi, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required for H. ducreyi to infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.

Molecular genetics of Erwinia amylovora involved in the development of fire blight.

PubMed

Oh, Chang-Sik; Beer, Steven V

2005-12-15

The bacterial plant pathogen, Erwinia amylovora, causes the devastating disease known as fire blight in some Rosaceous plants like apple, pear, quince, raspberry and several ornamentals. Knowledge of the factors affecting the development of fire blight has mushroomed in the last quarter century. On the molecular level, genes encoding a Hrp type III secretion system, genes encoding enzymes involved in synthesis of extracellular polysaccharides and genes facilitating the growth of E. amylovora in its host plants have been characterized. The Hrp pathogenicity island, delimited by genes suggesting horizontal gene transfer, is composed of four distinct regions, the hrp/hrc region, the HEE (Hrp effectors and elicitors) region, the HAE (Hrp-associated enzymes) region, and the IT (Island transfer) region. The Hrp pathogenicity island encodes a Hrp type III secretion system (TTSS), which delivers several proteins from bacteria to plant apoplasts or cytoplasm. E. amylovora produces two exopolysaccharides, amylovoran and levan, which cause the characteristic fire blight wilting symptom in host plants. In addition, other genes, and their encoded proteins, have been characterized as virulence factors of E. amylovora that encode enzymes facilitating sorbitol metabolism, proteolytic activity and iron harvesting. This review summarizes our understanding of the genes and gene products of E. amylovora that are involved in the development of the fire blight disease.

Inhibition of Virulence Gene Expression in Staphylococcus aureus by Novel Depsipeptides from a Marine Photobacterium

PubMed Central

Mansson, Maria; Nielsen, Anita; Kjærulff, Louise; Gotfredsen, Charlotte H.; Wietz, Matthias; Ingmer, Hanne; Gram, Lone; Larsen, Thomas O.

2011-01-01

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium. PMID:22363239

Identification of New Virulence Factors and Vaccine Candidates for Yersinia pestis

PubMed Central

Andersson, Jourdan A.; Sha, Jian; Erova, Tatiana E.; Fitts, Eric C.; Ponnusamy, Duraisamy; Kozlova, Elena V.; Kirtley, Michelle L.; Chopra, Ashok K.

2017-01-01

Earlier, we reported the identification of new virulence factors/mechanisms of Yersinia pestis using an in vivo signature-tagged mutagenesis (STM) screening approach. From this screen, the role of rbsA, which encodes an ATP-binding protein of ribose transport system, and vasK, an essential component of the type VI secretion system (T6SS), were evaluated in mouse models of plague and confirmed to be important during Y. pestis infection. However, many of the identified genes from the screen remained uncharacterized. In this study, in-frame deletion mutants of ypo0815, ypo2884, ypo3614-3168 (cyoABCDE), and ypo1119-1120, identified from the STM screen, were generated. While ypo0815 codes for a general secretion pathway protein E (GspE) of the T2SS, the ypo2884-encoded protein has homology to the βγ crystallin superfamily, cyoABCDE codes for the cytochrome o oxidase operon, and the ypo1119-1120 genes are within the Tol-Pal system which has multiple functions. Additionally, as our STM screen identified three T6SS-associated genes, and, based on in silico analysis, six T6SS clusters and multiple homologs of the T6SS effector hemolysin-coregulated protein (Hcp) exist in Y. pestis CO92, we also targeted these T6SS clusters and effectors for generating deletion mutants. These deletion mutant strains exhibited varying levels of attenuation (up to 100%), in bubonic or pneumonic murine infection models. The attenuation could be further augmented by generation of combinatorial deletion mutants, namely ΔlppΔypo0815, ΔlppΔypo2884, ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3. We earlier showed that deletion of the lpp gene, which encodes Braun lipoprotein (Lpp) and activates Toll-like receptor-2, reduced virulence of Y. pestis CO92 in murine models of bubonic and pneumonic plague. The surviving mice infected with ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3 mutant strains were 55–100% protected upon subsequent re-challenge with wild-type CO92 in a pneumonic model. Further, evaluation of the attenuated T6SS mutant strains in vitro revealed significant alterations in phagocytosis, intracellular survival in murine macrophages, and their ability to induce cytotoxic effects on macrophages. The results reported here provide further evidence of the utility of the STM screening approach for the identification of novel virulence factors and to possibly target such genes for the development of novel live-attenuated vaccine candidates for plague. PMID:29090192

Identification of New Virulence Factors and Vaccine Candidates for Yersinia pestis.

PubMed

Andersson, Jourdan A; Sha, Jian; Erova, Tatiana E; Fitts, Eric C; Ponnusamy, Duraisamy; Kozlova, Elena V; Kirtley, Michelle L; Chopra, Ashok K

2017-01-01

Earlier, we reported the identification of new virulence factors/mechanisms of Yersinia pestis using an in vivo signature-tagged mutagenesis (STM) screening approach. From this screen, the role of rbsA , which encodes an ATP-binding protein of ribose transport system, and vasK , an essential component of the type VI secretion system (T6SS), were evaluated in mouse models of plague and confirmed to be important during Y. pestis infection. However, many of the identified genes from the screen remained uncharacterized. In this study, in-frame deletion mutants of ypo0815, ypo2884, ypo3614-3168 (cyoABCDE) , and ypo1119-1120 , identified from the STM screen, were generated. While ypo0815 codes for a general secretion pathway protein E (GspE) of the T2SS, the ypo2884 -encoded protein has homology to the βγ crystallin superfamily, cyoABCDE codes for the cytochrome o oxidase operon, and the ypo1119-1120 genes are within the Tol-Pal system which has multiple functions. Additionally, as our STM screen identified three T6SS-associated genes, and, based on in silico analysis, six T6SS clusters and multiple homologs of the T6SS effector hemolysin-coregulated protein (Hcp) exist in Y. pestis CO92, we also targeted these T6SS clusters and effectors for generating deletion mutants. These deletion mutant strains exhibited varying levels of attenuation (up to 100%), in bubonic or pneumonic murine infection models. The attenuation could be further augmented by generation of combinatorial deletion mutants, namely Δ lpp Δ ypo0815 , Δ lpp Δ ypo2884 , Δ lpp Δ cyoABCDE , Δ vasK Δ hcp6 , and Δ ypo2720-2733 Δ hcp3 . We earlier showed that deletion of the lpp gene, which encodes Braun lipoprotein (Lpp) and activates Toll-like receptor-2, reduced virulence of Y. pestis CO92 in murine models of bubonic and pneumonic plague. The surviving mice infected with Δ lpp Δ cyoABCDE , Δ vasK Δ hcp6 , and Δ ypo2720-2733 Δ hcp3 mutant strains were 55-100% protected upon subsequent re-challenge with wild-type CO92 in a pneumonic model. Further, evaluation of the attenuated T6SS mutant strains in vitro revealed significant alterations in phagocytosis, intracellular survival in murine macrophages, and their ability to induce cytotoxic effects on macrophages. The results reported here provide further evidence of the utility of the STM screening approach for the identification of novel virulence factors and to possibly target such genes for the development of novel live-attenuated vaccine candidates for plague.

Effects of the HN gene c-terminal extensions on the Newcastle disease virus virulence

USDA-ARS?s Scientific Manuscript database

The hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is a multifunctional protein that has receptor recognition, neuraminidase and fusion promotion activities. Sequence analysis revealed that the HN gene of many extremely low virulence NDV strains encodes a larger open reading frame...

A Novel ESAT-6 Secretion System-Secreted Protein EsxX of Community-Associated Staphylococcus aureus Lineage ST398 Contributes to Immune Evasion and Virulence.

PubMed

Dai, Yingxin; Wang, Yanan; Liu, Qian; Gao, Qianqian; Lu, Huiying; Meng, Hongwei; Qin, Juanxiu; Hu, Mo; Li, Min

2017-01-01

The ESAT-6 secretion system (ESS) has been reported to contribute to the virulence and pathogenicity of several Staphylococcus aureus strains such as USA300 and Newman. However, the role of the ESS in community-associated S. aureus (CA-SA) lineage ST398 in China is not well understood. By comparing the ess locus of ST398 with the published S. aureus sequence in the NCBI database, we found one gene in the ess locus encoding a novel WXG superfamily protein that is highly conserved only in ST398. LC-MS/MS and Western blot analysis revealed that this protein is a novel secreted protein controlled by the ST398 ESS, and we named the protein EsxX. Although EsxX was not under the control of the accessory gene regulator like many other virulence factors and had no influence on several phenotypes of ST398, such as growth, hemolysis, and biofilm formation, it showed important impacts on immune evasion and virulence in ST398. An esxX deletion mutant led to significantly reduced resistance to neutrophil killing and decreased virulence in murine skin and blood infection models, indicating its essential contribution to the evasion of innate host defense and virulence to support the pathogenesis of ST398 infections. The function of this novel secreted protein EsxX might help us better understand the role of the ESS in the virulence and epidemic success of the CA-SA lineage ST398.

Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice (Oryza sativa) varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae.

PubMed

Verdier, Valérie; Triplett, Lindsay R; Hummel, Aaron W; Corral, Rene; Cernadas, R Andres; Schmidt, Clarice L; Bogdanove, Adam J; Leach, Jan E

2012-12-01

Genomes of the rice (Oryza sativa) xylem and mesophyll pathogens Xanthomonas oryzae pv. oryzae (Xoo) and pv. oryzicola (Xoc) encode numerous secreted transcription factors called transcription activator-like (TAL) effectors. In a few studied rice varieties, some of these contribute to virulence by activating corresponding host susceptibility genes. Some activate disease resistance genes. The roles of X. oryzae TAL effectors in diverse rice backgrounds, however, are poorly understood. Xoo TAL effectors that promote infection by activating SWEET sucrose transporter genes were expressed in TAL effector-deficient X. oryzae strain X11-5A, and assessed in 21 rice varieties. Some were also tested in Xoc on variety Nipponbare. Several Xoc TAL effectors were tested in X11-5A on four rice varieties. Xoo TAL effectors enhanced X11-5A virulence on most varieties, but to varying extents depending on the effector and variety. SWEET genes were activated in all tested varieties, but increased virulence did not correlate with activation level. SWEET activators also enhanced Xoc virulence on Nipponbare. Xoc TAL effectors did not alter X11-5A virulence. SWEET-targeting TAL effectors contribute broadly and non-tissue-specifically to virulence in rice, and their function is affected by host differences besides target sequences. Further, the utility of X11-5A for characterizing individual TAL effectors in rice was established. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Porphyromonas gingivalis and Treponema denticola Exhibit Metabolic Symbioses

PubMed Central

Mitchell, Helen L.; Pyke, James S.; Meuric, Vincent; Slakeski, Nada; Cleal, Steven M.; Chambers, Jenny L.; McConville, Malcolm J.; Reynolds, Eric C.

2014-01-01

Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using 13C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections. PMID:24603978

Molecular characterization of the 2011 Hong Kong scarlet fever outbreak.

PubMed

Tse, Herman; Bao, Jessie Y J; Davies, Mark R; Maamary, Peter; Tsoi, Hoi-Wah; Tong, Amy H Y; Ho, Tom C C; Lin, Chi-Ho; Gillen, Christine M; Barnett, Timothy C; Chen, Jonathan H K; Lee, Mianne; Yam, Wing-Cheong; Wong, Chi-Kin; Ong, Cheryl-Lynn Y; Chan, Yee-Wai; Wu, Cheng-Wei; Ng, Tony; Lim, Wilina W L; Tsang, Thomas H F; Tse, Cindy W S; Dougan, Gordon; Walker, Mark J; Lok, Si; Yuen, Kwok-Yung

2012-08-01

A scarlet fever outbreak occurred in Hong Kong in 2011. The majority of cases resulted in the isolation of Streptococcus pyogenes emm12 with multiple antibiotic resistances. Phylogenetic analysis of 22 emm12 scarlet fever outbreak isolates, 7 temporally and geographically matched emm12 non-scarlet fever isolates, and 18 emm12 strains isolated during 2005-2010 indicated the outbreak was multiclonal. Genome sequencing of 2 nonclonal scarlet fever isolates (HKU16 and HKU30), coupled with diagnostic polymerase chain reaction assays, identified 2 mobile genetic elements distributed across the major lineages: a 64.9-kb integrative and conjugative element encoding tetracycline and macrolide resistance and a 46.4-kb prophage encoding superantigens SSA and SpeC and the DNase Spd1. Phenotypic comparison of HKU16 and HKU30 with the S. pyogenes M1T1 strain 5448 revealed that HKU16 displays increased adherence to HEp-2 human epithelial cells, whereas HKU16, HKU30, and 5448 exhibit equivalent resistance to neutrophils and virulence in a humanized plasminogen murine model. However, in contrast to M1T1, the virulence of HKU16 and HKU30 was not associated with covRS mutation. The multiclonal nature of the emm12 scarlet fever isolates suggests that factors such as mobile genetic elements, environmental factors, and host immune status may have contributed to the 2011 scarlet fever outbreak.

Molecular Characterization of the 2011 Hong Kong Scarlet Fever Outbreak

PubMed Central

Tse, Herman; Bao, Jessie Y. J.; Davies, Mark R.; Maamary, Peter; Tsoi, Hoi-Wah; Tong, Amy H. Y.; Ho, Tom C. C.; Lin, Chi-Ho; Gillen, Christine M.; Barnett, Timothy C.; Chen, Jonathan H. K.; Lee, Mianne; Yam, Wing-Cheong; Wong, Chi-Kin; Ong, Cheryl-lynn Y.; Chan, Yee-Wai; Wu, Cheng-Wei; Ng, Tony; Lim, Wilina W. L.; Tsang, Thomas H. F.; Tse, Cindy W. S.; Dougan, Gordon; Walker, Mark J.; Lok, Si; Yuen, Kwok-Yung

2012-01-01

A scarlet fever outbreak occurred in Hong Kong in 2011. The majority of cases resulted in the isolation of Streptococcus pyogenes emm12 with multiple antibiotic resistances. Phylogenetic analysis of 22 emm12 scarlet fever outbreak isolates, 7 temporally and geographically matched emm12 non–scarlet fever isolates, and 18 emm12 strains isolated during 2005–2010 indicated the outbreak was multiclonal. Genome sequencing of 2 nonclonal scarlet fever isolates (HKU16 and HKU30), coupled with diagnostic polymerase chain reaction assays, identified 2 mobile genetic elements distributed across the major lineages: a 64.9-kb integrative and conjugative element encoding tetracycline and macrolide resistance and a 46.4-kb prophage encoding superantigens SSA and SpeC and the DNase Spd1. Phenotypic comparison of HKU16 and HKU30 with the S. pyogenes M1T1 strain 5448 revealed that HKU16 displays increased adherence to HEp-2 human epithelial cells, whereas HKU16, HKU30, and 5448 exhibit equivalent resistance to neutrophils and virulence in a humanized plasminogen murine model. However, in contrast to M1T1, the virulence of HKU16 and HKU30 was not associated with covRS mutation. The multiclonal nature of the emm12 scarlet fever isolates suggests that factors such as mobile genetic elements, environmental factors, and host immune status may have contributed to the 2011 scarlet fever outbreak. PMID:22615319

Genome-wide transcriptome analysis of Clavibacter michiganensis subsp. michiganensis grown in xylem mimicking medium.

PubMed

Hiery, Eva; Adam, Susanne; Reid, Stephen; Hofmann, Jörg; Sonnewald, Sophia; Burkovski, Andreas

2013-12-01

The interaction between Clavibacter michiganensis subsp. michiganensis with its host, the tomato plant (Solanum lycopersicum), is poorly understood and only few virulence factors are known. While studying of the bacteria in planta is time-consuming and difficult, the analysis in vitro would facilitate research. Therefore, a xylem mimicking medium (XMM) for C. michiganensis subsp. michiganensis was established in this study based on an apoplast medium for Xanthomonas campestris pv. vesicatoria. In contrast to the apoplast medium, XMM contains no sugars, but amino acids which serve as nitrogen and carbon source. As a result, growth in XMM induced transcriptional changes of genes encoding putative sugar, amino acid and iron uptake systems. In summary, mRNA levels of about 8% of all C. michiganensis subsp. michiganensis genes were changed when XMM-grown bacteria were compared to M9 minimal medium-grown cells. Almost no transcriptional changes of genes encoding hydrolytic enzymes were detected, leading to the idea that XMM reflects the situation in the beginning of infection and therefore allows the characterization of virulence factors in this early stage of infection. The addition of the plant wound substance acetosyringone to the XMM medium led to a change in transcript amount, including genes coding for proteins involved in protein transport, iron uptake and regulation processes. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains.

PubMed

Johnson, Timothy J; Siek, Kylie E; Johnson, Sara J; Nolan, Lisa K

2006-01-01

ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains.

DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains

PubMed Central

Johnson, Timothy J.; Siek, Kylie E.; Johnson, Sara J.; Nolan, Lisa K.

2006-01-01

ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains. PMID:16385064

In vitro and intra-macrophage gene expression by Rhodococcus equi strain 103.

PubMed

Rahman, Md Tanvir; Parreira, Valeria; Prescott, John F

2005-09-30

Rhodococcus equi is a facultative intracellular respiratory pathogen of foals that persists and multiplies within macrophages. In foals, virulence is associated with 80-90 kb plasmids, which include a pathogenicity island (PI) containing the virulence-associated protein (vap) gene family, but detailed understanding of the basis of virulence is still poor. A 60 spot-based DNA microarray was developed containing eight PI genes and 42 chromosomal putative virulence or virulence-associated genes selected from a recent partial genome sequence in order to study transcription of these genes by R. equi grown inside macrophages and under in vitro conditions thought to simulate those of macrophages. In addition to seven PI genes, nine chromosomal genes involved in fatty acid and lipid metabolism (choD, fadD13, fbpB), heme biosynthesis (hemE), iron utilization (mbtF), heat shock resistance and genes encoding chaperones (clpB, groEL), a sigma factor (sigK), and a transcriptional regulator (moxR) were significantly induced in R. equi growing inside macrophages. The pattern of R. equi chromosomal genes significantly transcribed inside macrophages largely differed from those transcribed under in vitro conditions (37 degrees C, pH 5.0 or 50mM H2O2 for 30 min). This study has identified genes, other than those of the virulence plasmid, the transcription of which is enhanced within equine macrophages. These genes should be investigated further to improve understanding of how this organism survives intracellularly.

Pleiotropic effects of temperature-regulated 2-OH-lauroytransferase (PA0011) on Pseudomonas aeruginosa antibiotic resistance, virulence and type III secretion system.

PubMed

Wang, Bobo; Li, Bo; Liang, Ying; Li, Jing; Gao, Lang; Chen, Lin; Duan, Kangmin; Shen, Lixin

2016-02-01

Pseudomonas aeruginosa is an important human pathogen which adapts to changing environment, such as temperature variations and entering host by regulating their gene expression. Here, we report that gene PA0011 in P. aeruginosa PAO1, which encodes a 2-OH-lauroytransferase participating in lipid A biosynthesis, is involved in carbapenem resistance and virulence in a temperature-regulated manner in PAO1. The expression of PA0011 was higher at an environment temperature (21 °C) than that at a body temperature (37 °C). The inactivation of PA0011 rendered increased antibiotic susceptibility and decreased virulence both in vivo and in vitro. The impaired integrity and the decreased stability of the outer membrane were the cause of the increased susceptibility of PAO1(Δ0011) to carbapenem and many other common antibiotics. The reduced endotoxic activity of lipopolysaccharide (LPS) contributed to the decreased virulence both at 21 °C and 37 °C in PAO1 (Δ0011). In addition, we have found that PA0011 repressed the expression of TTSS virulence factors both at transcriptional and translational levels, similar to the effect of O antigen of LPS but unlike any effect of its homologue reported in other bacteria. The effect of PA0011 on resistance to many antibiotics including carbapenem and virulence in P. aeruginosa makes it a target for novel antimicrobial therapies. Copyright © 2015 Elsevier Ltd. All rights reserved.

The mazEF toxin-antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis.

PubMed

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

2015-01-01

The toxin-antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains.

The mazEF toxin–antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis

PubMed Central

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

2015-01-01

The toxin–antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains. PMID:26005332

Klebsiella pneumoniae: Going on the Offense with a Strong Defense

PubMed Central

Paczosa, Michelle K.

2016-01-01

SUMMARY Klebsiella pneumoniae causes a wide range of infections, including pneumonias, urinary tract infections, bacteremias, and liver abscesses. Historically, K. pneumoniae has caused serious infection primarily in immunocompromised individuals, but the recent emergence and spread of hypervirulent strains have broadened the number of people susceptible to infections to include those who are healthy and immunosufficient. Furthermore, K. pneumoniae strains have become increasingly resistant to antibiotics, rendering infection by these strains very challenging to treat. The emergence of hypervirulent and antibiotic-resistant strains has driven a number of recent studies. Work has described the worldwide spread of one drug-resistant strain and a host defense axis, interleukin-17 (IL-17), that is important for controlling infection. Four factors, capsule, lipopolysaccharide, fimbriae, and siderophores, have been well studied and are important for virulence in at least one infection model. Several other factors have been less well characterized but are also important in at least one infection model. However, there is a significant amount of heterogeneity in K. pneumoniae strains, and not every factor plays the same critical role in all virulent Klebsiella strains. Recent studies have identified additional K. pneumoniae virulence factors and led to more insights about factors important for the growth of this pathogen at a variety of tissue sites. Many of these genes encode proteins that function in metabolism and the regulation of transcription. However, much work is left to be done in characterizing these newly discovered factors, understanding how infections differ between healthy and immunocompromised patients, and identifying attractive bacterial or host targets for treating these infections. PMID:27307579

Type IV Pili in Francisella tularensis: Roles of pilF and pilT in Fiber Assembly, Host Cell Adherence, and Virulence ▿

PubMed Central

Chakraborty, Subhra; Monfett, Michael; Maier, Tamara M.; Benach, Jorge L.; Frank, Dara W.; Thanassi, David G.

2008-01-01

Francisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensis subspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pil genes. We show here that deletion of the pilF putative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilT putative disassembly ATPase also caused a complete loss of pili, indicating that pilT functions differently in F. tularensis than in model Tfp systems such as those found in Pseudomonas aeruginosa and Neisseria spp. The LVS pilF and pilT mutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilF and pilT mutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pil genes and provides evidence that the Francisella pili contribute to host cell adhesion and virulence. PMID:18426883

Chromobacterium pathogenicity island 1 type III secretion system is a major virulence determinant for Chromobacterium violaceum-induced cell death in hepatocytes.

PubMed

Miki, Tsuyoshi; Iguchi, Mirei; Akiba, Kinari; Hosono, Masato; Sobue, Tomoyoshi; Danbara, Hirofumi; Okada, Nobuhiko

2010-08-01

Chromobacterium violaceum is a Gram-negative bacterium that causes fatal septicaemia in humans and animals. C. violaceum ATCC 12472 possesses genes associated with two distinct type III secretion systems (T3SSs). One of these systems is encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a), another is encoded by Chromobacterium pathogenicity island 2 (Cpi-2). Here we show that C. violaceum causes fulminant hepatitis in a mouse infection model, and Cpi-1/-1a-encoded T3SS is required for its virulence. In addition, using C. violaceum strains with defined mutations in the genes that encode the Cpi-1/-1a or Cpi-2 locus in combination with cultured mammalian cell lines, we found that C. violaceum is able to induce cytotoxicity in a Cpi-1/-1a-dependent manner. Characterization of Chromobacterium-induced cytotoxicity revealed that cell lysis by C. violaceum infection involves the formation of pore structures on the host cell membrane, as demonstrated by protection by cytotoxicity in the presence of osmoprotectants. Finally, we demonstrated that CipB, a Cpi-1/-1a effector, is implicated in translocator-mediated pore formation and the ability of CipB to form a pore is essential for Chromobacterium-induced cytotoxicity. These results strongly suggest that Cpi-1/-1a-encoded T3SS is a virulence determinant that causes fatal infection by the induction of cell death in hepatocytes. © 2010 Blackwell Publishing Ltd.

Toxic shock syndrome toxin-1, not α-toxin, mediated Bundaberg fatalities.

PubMed

Mueller, Elizabeth A; Merriman, Joseph A; Schlievert, Patrick M

2015-12-01

The 1928 Bundaberg disaster is one of the greatest vaccine tragedies in history. Of 21 children immunized with a diphtheria toxin-antitoxin preparation contaminated with Staphylococcus aureus, 18 developed life-threatening disease and 12 died within 48  h. Historically, the deaths have been attributed to α-toxin, a secreted cytotoxin produced by most S. aureus strains, yet the ability of the Bundaberg contaminant microbe to produce the toxin has never been verified. For the first time, the ability of the original strain to produce α-toxin and other virulence factors is investigated. The study investigates the genetic and regulatory loci mediating α-toxin expression by PCR and assesses production of the cytotoxin in vitro using an erythrocyte haemolysis assay. This analysis is extended to other secreted virulence factors produced by the strain, and their sufficiency to cause lethality in New Zealand white rabbits is determined. Although the strain possesses a wild-type allele for α-toxin, it must have a defective regulatory system, which is responsible for the strain's minimal α-toxin production. The strain encodes and produces staphylococcal superantigens, including toxic shock syndrome toxin-1 (TSST-1), which is sufficient to cause lethality in patients. The findings cast doubt on the belief that α-toxin is the major virulence factor responsible for the Bundaberg fatalities and point to the superantigen TSST-1 as the cause of the disaster.

Clavibacter michiganensis ssp. michiganensis: bacterial canker of tomato, molecular interactions and disease management.

PubMed

Nandi, Munmun; Macdonald, Jacqueline; Liu, Peng; Weselowski, Brian; Yuan, Ze-Chun

2018-03-12

Bacterial canker disease is considered to be one of the most destructive diseases of tomato (Solanum lycopersicum), and is caused by the seed-borne Gram-positive bacterium Clavibacter michiganensis ssp. michiganensis (Cmm). This vascular pathogen generally invades and proliferates in the xylem through natural openings or wounds, causing wilt and canker symptoms. The incidence of symptomless latent infections and the invasion of tomato seeds by Cmm are widespread. Pathogenicity is mediated by virulence factors and transcriptional regulators encoded by the chromosome and two natural plasmids. The virulence factors include serine proteases, cell wall-degrading enzymes (cellulases, xylanases, pectinases) and others. Mutational analyses of these genes and gene expression profiling (via quantitative reverse transcription-polymerase chain reaction, transcriptomics and proteomics) have begun to shed light on their roles in colonization and virulence, whereas the expression of tomato genes in response to Cmm infection suggests plant factors involved in the defence response. These findings may aid in the generation of target-specific bactericides or new resistant varieties of tomato. Meanwhile, various chemical and biological controls have been researched to control Cmm. This review presents a detailed investigation regarding the pathogen Cmm, bacterial canker infection, molecular interactions between Cmm and tomato, and current perspectives on improved disease management. © 2018 AGRICULTURE AND AGRI-FOOD CANADA. MOLECULAR PLANT PATHOLOGY © 2018 JOHN WILEY & SONS LTD.

Characterization of EhaJ, a New Autotransporter Protein from Enterohemorrhagic and Enteropathogenic Escherichia coli

PubMed Central

Easton, Donna M.; Totsika, Makrina; Allsopp, Luke P.; Phan, Minh-Duy; Idris, Adi; Wurpel, Daniël J.; Sherlock, Orla; Zhang, Bing; Venturini, Carola; Beatson, Scott A.; Mahony, Timothy J.; Cobbold, Rowland N.; Schembri, Mark A.

2011-01-01

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms have been extensively studied, the factors that mediate host colonization remain to be properly defined. Previously, we identified four genes (ehaA, ehaB, ehaC, and ehaD) from the prototypic EHEC strain EDL933 that encode for proteins that belong to the autotransporter (AT) family. Here we have examined the prevalence of these genes, as well as several other AT-encoding genes, in a collection of EHEC and EPEC strains. We show that the complement of AT-encoding genes in EHEC and EPEC strains is variable, with some AT-encoding genes being highly prevalent. One previously uncharacterized AT-encoding gene, which we have termed ehaJ, was identified in 12/44 (27%) of EHEC and 2/20 (10%) of EPEC strains. The ehaJ gene lies immediately adjacent to a gene encoding a putative glycosyltransferase (referred to as egtA). Western blot analysis using an EhaJ-specific antibody indicated that EhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealed EhaJ is located at the cell surface and in the presence of the egtA glycosyltransferase gene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ also mediated adherence to a range of extracellular matrix proteins, however this occurred independent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-type EPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alter its adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC. Further studies are required to elucidate the function of EhaJ in colonization and virulence. PMID:21687429

Regulation of host-pathogen interactions via the post-transcriptional Csr/Rsm system.

PubMed

Kusmierek, Maria; Dersch, Petra

2018-02-01

A successful colonization of specific hosts requires a rapid and efficient adaptation of the virulence-relevant gene expression program by bacterial pathogens. An important element in this endeavor is the Csr/Rsm system. This multi-component, post-transcriptional control system forms a central hub within complex regulatory networks and coordinately adjusts virulence properties with metabolic and physiological attributes of the pathogen. A key function is elicited by the RNA-binding protein CsrA/RsmA. CsrA/RsmA interacts with numerous target mRNAs, many of which encode crucial virulence factors, and alters their translation, stability or elongation of transcription. Recent studies highlighted that important colonization factors, toxins, and bacterial secretion systems are under CsrA/RsmA control. CsrA/RsmA deficiency impairs host colonization and attenuates virulence, making this post-transcriptional regulator a suitable drug target. The CsrA/RsmA protein can be inactivated through sequestration by non-coding RNAs, or via binding to specific highly abundant mRNAs and interacting proteins. The wide range of interaction partners and RNA targets, as well as the overarching, interlinked genetic control circuits illustrate the complexity of this regulatory system in the different pathogens. Future work addressing spatio-temporal changes of Csr/Rsm-mediated control during the course of an infection will help us to understand how bacteria reprogram their expression profile to cope with continuous changes experienced in colonized niches. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of an essential virulence gene of cyprinid herpesvirus 3.

PubMed

Boutier, Maxime; Gao, Yuan; Vancsok, Catherine; Suárez, Nicolás M; Davison, Andrew J; Vanderplasschen, Alain

2017-09-01

The genus Cyprinivirus consists of a growing list of phylogenetically related viruses, some of which cause severe economic losses to the aquaculture industry. The archetypal member, cyprinid herpesvirus 3 (CyHV-3) causes mass mortalities worldwide in koi and common carp. A CyHV-3 mutant was described previously that is attenuated in vivo by a deletion affecting two genes (ORF56 and ORF57). The relative contributions of ORF56 and ORF57 to the safety and efficacy profile of this vaccine candidate have now been assessed by analysing viruses individually deleted for ORF56 or ORF57. Inoculation of these viruses into carp demonstrated that the absence of ORF56 did not affect virulence, whereas the absence of ORF57 led to an attenuation comparable to, though slightly less than, that of the doubly deleted virus. To demonstrate further the role of ORF57 as a key virulence factor, a mutant retaining the ORF57 region but unable to express the ORF57 protein was produced by inserting multiple in-frame stop codons into the coding region. Analysis of this virus in vivo revealed a safety and efficacy profile comparable to that of the doubly deleted virus. These findings show that ORF57 encodes an essential CyHV-3 virulence factor. They also indicate that ORF57 orthologues in other cypriniviruses may offer promising targets for the rational design of attenuated recombinant vaccines. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

An emerging mycoplasma associated with trichomoniasis, vaginal infection and disease.

PubMed

Fettweis, Jennifer M; Serrano, Myrna G; Huang, Bernice; Brooks, J Paul; Glascock, Abigail L; Sheth, Nihar U; Strauss, Jerome F; Jefferson, Kimberly K; Buck, Gregory A

2014-01-01

Humans are colonized by thousands of bacterial species, but it is difficult to assess the metabolic and pathogenic potential of the majority of these because they have yet to be cultured. Here, we characterize an uncultivated vaginal mycoplasma tightly associated with trichomoniasis that was previously known by its 16S rRNA sequence as "Mnola." In this study, the mycoplasma was found almost exclusively in women infected with the sexually transmitted pathogen Trichomonas vaginalis, but rarely observed in women with no diagnosed disease. The genomes of four strains of this species were reconstructed using metagenome sequencing and assembly of DNA from four discrete mid-vaginal samples, one of which was obtained from a pregnant woman with trichomoniasis who delivered prematurely. These bacteria harbor several putative virulence factors and display unique metabolic strategies. Genes encoding proteins with high similarity to potential virulence factors include two collagenases, a hemolysin, an O-sialoglycoprotein endopeptidase and a feoB-type ferrous iron transport system. We propose the name "Candidatus Mycoplasma girerdii" for this potential new pathogen.

Myxoma virus M130R is a novel virulence factor required for lethal myxomatosis in rabbits.

PubMed

Barrett, John W; Werden, Steven J; Wang, Fuan; McKillop, William M; Jimenez, June; Villeneuve, Danielle; McFadden, Grant; Dekaban, Gregory A

2009-09-01

Myxoma virus (MV) is a highly lethal, rabbit-specific poxvirus that induces a disease called myxomatosis in European rabbits. In an effort to understand the function of predicted immunomodulatory genes we have deleted various viral genes from MV and tested the ability of these knockout viruses to induce lethal myxomatosis. MV encodes a unique 15 kD cytoplasmic protein (M130R) that is expressed late (12h post infection) during infection. M130R is a non-essential gene for MV replication in rabbit, monkey or human cell lines. Construction of a targeted gene knockout virus (vMyx130KO) and infection of susceptible rabbits demonstrate that the M130R knockout virus is attenuated and that loss of M130R expression allows the rabbit host immune system to effectively respond to and control the lethal effects of MV. M130R expression is a bona fide poxviral virulence factor necessary for full and lethal development of myxomatosis.

Identification of Genetic Bases of Vibrio fluvialis Species-Specific Biochemical Pathways and Potential Virulence Factors by Comparative Genomic Analysis

PubMed Central

Lu, Xin; Liang, Weili; Wang, Yunduan; Xu, Jialiang

2014-01-01

Vibrio fluvialis is an important food-borne pathogen that causes diarrheal illness and sometimes extraintestinal infections in humans. In this study, we sequenced the genome of a clinical V. fluvialis strain and determined its phylogenetic relationships with other Vibrio species by comparative genomic analysis. We found that the closest relationship was between V. fluvialis and V. furnissii, followed by those with V. cholerae and V. mimicus. Moreover, based on genome comparisons and gene complementation experiments, we revealed genetic mechanisms of the biochemical tests that differentiate V. fluvialis from closely related species. Importantly, we identified a variety of genes encoding potential virulence factors, including multiple hemolysins, transcriptional regulators, and environmental survival and adaptation apparatuses, and the type VI secretion system, which is indicative of complex regulatory pathways modulating pathogenesis in this organism. The availability of V. fluvialis genome sequences may promote our understanding of pathogenic mechanisms for this emerging pathogen. PMID:24441165

Three regulators of G protein signaling differentially affect mating, morphology and virulence in the smut fungus Ustilago maydis.

PubMed

Moretti, Marino; Wang, Lei; Grognet, Pierre; Lanver, Daniel; Link, Hannes; Kahmann, Regine

2017-09-01

Regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling negatively. To broaden an understanding of the roles of RGS proteins in fungal pathogens, we functionally characterized the three RGS protein-encoding genes (rgs1, rgs2 and rgs3) in the phytopathogenic fungus Ustilago maydis. It was found that RGS proteins played distinct roles in the regulation of development and virulence. rgs1 had a minor role in virulence when deleted in a solopathogenic strain. In crosses, rgs1 was dispensable for mating and filamentation, but was required for teliospore production. Haploid rgs2 mutants were affected in cell morphology, growth, mating and were unable to cause disease symptoms in crosses. However, virulence was unaffected when rgs2 was deleted in a solopathogenic strain, suggesting an exclusive involvement in pre-fusion events. These rgs2 phenotypes are likely connected to elevated intracellular cAMP levels. rgs3 mutants were severely attenuated in mating, in their response to pheromone, virulence and formation of mature teliospores. The mating defect could be traced back to reduced expression of the transcription factor rop1. It was speculated that the distinct roles of the three U. maydis RGS proteins were achieved by direct modulation of the Gα subunit-activated signaling pathways as well as through Gα-independent functions. © 2017 John Wiley & Sons Ltd.

Knocking out Bcsas1 in Botrytis cinerea impacts growth, development, and secretion of extracellular proteins, which decreases virulence.

PubMed

Zhang, Zhanquan; Qin, Guozheng; Li, Boqiang; Tian, Shiping

2014-06-01

Pathogenic fungi usually secrete a series of virulence factors to the extracellular environment to facilitate infection. Rab GTPases play a central role in the secretory pathway. To explore the function of Rab/GTPase in filamentous fungi, we knocked out a Rab/GTPase family gene, Bcsas1, in Botrytis cinerea, an aggressive fungal pathogen that infects more than 200 plant species. A detailed analysis was conducted on the virulence and the secretory capability of the mutants. The results indicated that knockout of Bcsas1 inhibited hyphal development and reduced sporulation of B. cinerea on potato dextrose agar plates resulting in reduced virulence on various fruit hosts. Knocking out the Bcsas1 gene led to an accumulation of transport vesicles at the hyphal tip, significantly reduced extracellular protein content, and lowered the activity of polygalacturonase and xylanase in the extracellular medium. However, mutation of Bcsas1 did not affect the expression of genes encoding polygalacturonase and xylanase, suggesting the secretion of these two family enzymes was suppressed in the mutant. Moreover, a comparative analysis of the secretome provided further evidence that the disruption of Bcsas1 in mutant strains significantly depressed the secretion of polysaccharide hydrolases and proteases. The results indicate that Bcsas1, the Rab8/SEC4-like gene, plays a crucial role in development, protein secretion, and virulence of B. cinerea.

Inheritance of the lysozyme inhibitor Ivy was an important evolutionary step by Yersinia pestis to avoid the host innate immune response.

PubMed

Derbise, Anne; Pierre, François; Merchez, Maud; Pradel, Elizabeth; Laouami, Sabrina; Ricard, Isabelle; Sirard, Jean-Claude; Fritz, Jill; Lemaître, Nadine; Akinbi, Henry; Boneca, Ivo G; Sebbane, Florent

2013-05-15

Yersinia pestis (the plague bacillus) and its ancestor, Yersinia pseudotuberculosis (which causes self-limited bowel disease), encode putative homologues of the periplasmic lysozyme inhibitor Ivy and the membrane-bound lysozyme inhibitor MliC. The involvement of both inhibitors in virulence remains subject to debate. Mutants lacking ivy and/or mliC were generated. We evaluated the mutants' ability to counter lysozyme, grow in serum, and/or counter leukocytes; to produce disease in wild-type, neutropenic, or lysozyme-deficient rodents; and to induce host inflammation. MliC was not required for lysozyme resistance and the development of plague. Deletion of ivy decreased Y. pestis' ability to counter lysozyme and polymorphonuclear neutrophils, but it did not affect the bacterium's ability to grow in serum or resist macrophages. Y. pestis lacking Ivy had attenuated virulence, unless animals were neutropenic or lysozyme deficient. The Ivy mutant induced inflammation to a degree similar to that of the parental strain. Last, Y. pseudotuberculosis did not require Ivy to counter lysozyme and for virulence. Ivy is required to counter lysozyme during infection, but its role as a virulence factor is species dependent. Our study also shows that a gene that is not necessary for the virulence of an ancestral bacterium may become essential in the emergence of a new pathogen.

Deletion analysis of Streptococcus pneumoniae late competence genes distinguishes virulence determinants that are dependent or independent of competence induction

PubMed Central

Zhu, Luchang; Lin, Jingjun; Kuang, Zhizhou; Vidal, Jorge E.; Lau, Gee W.

2015-01-01

Summary The competence regulon of Streptococcus pneumoniae (pneumococcus) is crucial for genetic transformation. During competence development, the alternative sigma factor ComX is activated, which in turn, initiates transcription of 80 “late” competence genes. Interestingly, only 16 late genes are essential for genetic transformation. We hypothesized that these late genes that are dispensable for competence are beneficial to pneumococcal fitness during infection. These late genes were systematically deleted, and the resulting mutants were examined for their fitness during mouse models of bacteremia and acute pneumonia. Among these, 14 late genes were important for fitness in mice. Significantly, deletion of some late genes attenuated pneumococcal fitness to the same level in both wild-type and ComX-null genetic backgrounds, suggesting that the constitutive baseline expression of these genes was important for bacterial fitness. In contrast, some mutants were attenuated only in the wild-type genetic background but not in the ComX-null background, suggesting that specific expression of these genes during competence state contributed to pneumococcal fitness. Increased virulence during competence state was partially caused by the induction of allolytic enzymes that enhanced pneumolysin release. These results distinguish the role of basal expression versus competence induction in virulence functions encoded by ComX-regulated late competence genes. Graphical abstract During genetic transformation of pneumococcus, the alternative sigma factor ComX regulates expression of 14 late competence genes important for virulence. The constitutive baseline expression of some of these genes is important for bacteremia and acute pneumonia infections. In contrast, elevated expression of DprA, CbpD, CibAB, and Cinbox are dependent on competence development, enhancing the release of pneumolysin. These results distinguish the role of basal expression versus competence induction in virulence determinants regulated by ComX. PMID:25846124

Inhibition of IRF-3 activation by VP35 is critical for the high level of virulence of ebola virus.

PubMed

Hartman, Amy L; Bird, Brian H; Towner, Jonathan S; Antoniadou, Zoi-Anna; Zaki, Sherif R; Nichol, Stuart T

2008-03-01

Zaire ebolavirus causes a rapidly progressing hemorrhagic disease with high mortality. Identification of the viral virulence factors that contribute to the severity of disease induced by Ebola virus is critical for the design of therapeutics and vaccines against the disease. Given the rapidity of disease progression, virus interaction with the innate immune system early in the course of infection likely plays an important role in determining the outcome of the disease. The Ebola virus VP35 protein inhibits the activation of IRF-3, a critical transcription factor for the induction of early antiviral immunity. Previous studies revealed that a single amino acid change (R312A) in VP35 renders the protein unable to inhibit IRF-3 activation. A reverse-genetics-generated, mouse-adapted, recombinant Ebola virus that encodes the R312A mutation in VP35 was produced. We found that relative to the case for wild-type virus containing the authentic VP35 sequence, this single amino acid change in VP35 renders the virus completely attenuated in mice. Given that these viruses differ by only a single amino acid in the IRF-3 inhibitory domain of VP35, the level of alteration of virulence is remarkable and highlights the importance of VP35 for the pathogenesis of Ebola virus.

Common duckweed (Lemna minor) is a versatile high-throughput infection model for the Burkholderia cepacia complex and other pathogenic bacteria.

PubMed

Thomson, Euan L S; Dennis, Jonathan J

2013-01-01

Members of the Burkholderia cepacia complex (Bcc) have emerged in recent decades as problematic pulmonary pathogens of cystic fibrosis (CF) patients, with severe infections progressing to acute necrotizing pneumonia and sepsis. This study presents evidence that Lemna minor (Common duckweed) is useful as a plant model for the Bcc infectious process, and has potential as a model system for bacterial pathogenesis in general. To investigate the relationship between Bcc virulence in duckweed and Galleria mellonella (Greater wax moth) larvae, a previously established Bcc infection model, a duckweed survival assay was developed and used to determine LD50 values. A strong correlation (R(2) = 0.81) was found between the strains' virulence ranks in the two infection models, suggesting conserved pathways in these vastly different hosts. To broaden the application of the duckweed model, enteropathogenic Escherichia coli (EPEC) and five isogenic mutants with previously established LD50 values in the larval model were tested against duckweed, and a strong correlation (R(2) = 0.93) was found between their raw LD50 values. Potential virulence factors in B. cenocepacia K56-2 were identified using a high-throughput screen against single duckweed plants. In addition to the previously characterized antifungal compound (AFC) cluster genes, several uncharacterized genes were discovered including a novel lysR regulator, a histidine biosynthesis gene hisG, and a gene located near the gene encoding the recently characterized virulence factor SuhB(Bc). Finally, to demonstrate the utility of this model in therapeutic applications, duckweed was rescued from Bcc infection by treating with bacteriophage at 6-h intervals. It was observed that phage application became ineffective at a timepoint that coincided with a sharp increase in bacterial invasion of plant tissue. These results indicate that common duckweed can serve as an effective infection model for the investigation of bacterial virulence factors and therapeutic strategies to combat them.

Differences in Virulence Markers between Helicobacter pylori Strains from Iraq and Those from Iran: Potential Importance of Regional Differences in H. pylori-Associated Disease▿

PubMed Central

Hussein, Nawfal R.; Mohammadi, Marjan; Talebkhan, Yeganeh; Doraghi, Masoumeh; Letley, Darren P.; Muhammad, Merdan K.; Argent, Richard H.; Atherton, John C.

2008-01-01

Helicobacter pylori causes peptic ulceration and gastric adenocarcinoma; the latter is common in Iran but not in Iraq. We hypothesized that more virulent H. pylori strains may be found in Iran than in Iraq and so compared established and newly described virulence factors in strains from these countries. We studied 59 unselected dyspeptic patients from Iran and 49 from Iraq. cagA was found in similar proportions of strains from both countries (76% in Iran versus 71% in Iraq) and was significantly associated with peptic ulcer disease in Iraq (P ≤ 0.01) but not in Iran. cagA alleles encoding four or more tyrosine phosphorylation motifs were found in 12% of the Iranian strains but none of the Iraqi strains (P = 0.02). There were no significant differences in the vacA signal-, middle-, or intermediate-region types between Iranian and Iraqi strains. Among the strains from Iran, vacA genotypes showed no specific peptic ulcer associations, but among the strains from Iraq, vacA i1 strains were associated with gastric ulcer (P ≤ 0.02), mimicking their previously demonstrated association with gastric cancer in Iran. dupA was found in similar proportions of Iranian and Iraqi strains (38% and 32%, respectively) and was associated with peptic ulceration in Iraqi patients (P ≤ 0.01) but not Iranian patients. H. pylori strains from Iraq and Iran possess virulence factors similar to those in Western countries. The presence of cagA with more phosphorylation motifs in Iranian strains may contribute to the higher incidence of gastric cancer. However, the association between strain virulence markers and disease in Iraq but not Iran suggests that other host and environmental factors may be more important in the disease-prone Iranian population. PMID:18353934

Differences in virulence markers between Helicobacter pylori strains from Iraq and those from Iran: potential importance of regional differences in H. pylori-associated disease.

PubMed

Hussein, Nawfal R; Mohammadi, Marjan; Talebkhan, Yeganeh; Doraghi, Masoumeh; Letley, Darren P; Muhammad, Merdan K; Argent, Richard H; Atherton, John C

2008-05-01

Helicobacter pylori causes peptic ulceration and gastric adenocarcinoma; the latter is common in Iran but not in Iraq. We hypothesized that more virulent H. pylori strains may be found in Iran than in Iraq and so compared established and newly described virulence factors in strains from these countries. We studied 59 unselected dyspeptic patients from Iran and 49 from Iraq. cagA was found in similar proportions of strains from both countries (76% in Iran versus 71% in Iraq) and was significantly associated with peptic ulcer disease in Iraq (P

Common Duckweed (Lemna minor) Is a Versatile High-Throughput Infection Model For the Burkholderia cepacia Complex and Other Pathogenic Bacteria

PubMed Central

Thomson, Euan L. S.; Dennis, Jonathan J.

2013-01-01

Members of the Burkholderia cepacia complex (Bcc) have emerged in recent decades as problematic pulmonary pathogens of cystic fibrosis (CF) patients, with severe infections progressing to acute necrotizing pneumonia and sepsis. This study presents evidence that Lemna minor (Common duckweed) is useful as a plant model for the Bcc infectious process, and has potential as a model system for bacterial pathogenesis in general. To investigate the relationship between Bcc virulence in duckweed and Galleria mellonella (Greater wax moth) larvae, a previously established Bcc infection model, a duckweed survival assay was developed and used to determine LD50 values. A strong correlation (R2 = 0.81) was found between the strains’ virulence ranks in the two infection models, suggesting conserved pathways in these vastly different hosts. To broaden the application of the duckweed model, enteropathogenic Escherichia coli (EPEC) and five isogenic mutants with previously established LD50 values in the larval model were tested against duckweed, and a strong correlation (R2 = 0.93) was found between their raw LD50 values. Potential virulence factors in B. cenocepacia K56-2 were identified using a high-throughput screen against single duckweed plants. In addition to the previously characterized antifungal compound (AFC) cluster genes, several uncharacterized genes were discovered including a novel lysR regulator, a histidine biosynthesis gene hisG, and a gene located near the gene encoding the recently characterized virulence factor SuhBBc. Finally, to demonstrate the utility of this model in therapeutic applications, duckweed was rescued from Bcc infection by treating with bacteriophage at 6-h intervals. It was observed that phage application became ineffective at a timepoint that coincided with a sharp increase in bacterial invasion of plant tissue. These results indicate that common duckweed can serve as an effective infection model for the investigation of bacterial virulence factors and therapeutic strategies to combat them. PMID:24223216

Ape parasite origins of human malaria virulence genes

PubMed Central

Larremore, Daniel B.; Sundararaman, Sesh A.; Liu, Weimin; Proto, William R.; Clauset, Aaron; Loy, Dorothy E.; Speede, Sheri; Plenderleith, Lindsey J.; Sharp, Paul M.; Hahn, Beatrice H.; Rayner, Julian C.; Buckee, Caroline O.

2015-01-01

Antigens encoded by the var gene family are major virulence factors of the human malaria parasite Plasmodium falciparum, exhibiting enormous intra- and interstrain diversity. Here we use network analysis to show that var architecture and mosaicism are conserved at multiple levels across the Laverania subgenus, based on var-like sequences from eight single-species and three multi-species Plasmodium infections of wild-living or sanctuary African apes. Using select whole-genome amplification, we also find evidence of multi-domain var structure and synteny in Plasmodium gaboni, one of the ape Laverania species most distantly related to P. falciparum, as well as a new class of Duffy-binding-like domains. These findings indicate that the modular genetic architecture and sequence diversity underlying var-mediated host-parasite interactions evolved before the radiation of the Laverania subgenus, long before the emergence of P. falciparum. PMID:26456841

The effect of deletion of the edema factor on Bacillus anthracis pathogenicity in guinea pigs and rabbits.

PubMed

Levy, Haim; Weiss, Shay; Altboum, Zeev; Schlomovitz, Josef; Rothschild, Nili; Glinert, Itai; Sittner, Assa; Kobiler, David

2012-01-01

Bacillus anthracis secretes three major components, which assemble into two bipartite toxins: lethal toxin (LT), composed of lethal factor (LF) and protective antigen (PA) and edema toxin (ET), composed of edema factor (EF) and PA. EF is a potent calmodulin-dependent adenylate cyclase, which is internalized into the target cell following PA binding. Once inside the cell, EF elevates cAMP levels, interrupting intracellular signaling. Effects of ET were demonstrated on monocytes, neutrophils and T-cells. In an earlier work we demonstrated that a deletion of LF in a fully virulent strain had no effect in guinea pigs and a significant, but not major, effect in the rabbit model. These results suggested that EF might play an important role in the development of infection and mortality following exposure to B. anthracis spores. To evaluate the role of EF in B. anthracis pathogenicity we deleted the cya gene, which encodes the EF protein, in the fully virulent Vollum strain. The Δcya mutant was fully virulent in the guinea pig model as determined by LD(50) experiments. In the rabbit model, when infected subcutaneously, the absence of EF had no effect on the virulence of the mutant. However an increase of two orders of magnitude in the LD(50) was demonstrated when the rabbits were infected by intranasal instillation accompanied with partial mortality and increased mean time to death. These results argue that in the guinea pig model the presence of one of the toxins, ET or LT is sufficient for the development of the infection. In the rabbit model ET plays a role in respiratory infection, most probably mediating the early steps of host colonization. Copyright © 2011 Elsevier Ltd. All rights reserved.

Epidemic Population Structure of Pseudomonas aeruginosa: Evidence for a Clone That Is Pathogenic to the Eye and That Has a Distinct Combination of Virulence Factors

PubMed Central

Lomholt, Jeanet A.; Poulsen, Knud; Kilian, Mogens

2001-01-01

The genetic structure of a population of Pseudomonas aeruginosa, isolated from patients with keratitis, endophthalmitis, and contact lens-associated red eye, contact lens storage cases, urine, ear, blood, lungs, wounds, feces, and the environment was determined by multilocus enzyme electrophoresis. The presence and characteristics of virulence factors were determined by restriction fragment length polymorphism analysis with DNA probes for lasA, lasB, aprA, exoS, exoT, exoU, and ctx and by zymography of staphylolysin, elastase, and alkaline protease. These analyses revealed an epidemic population structure of P. aeruginosa, characterized by frequent recombination in which a particular successful clone may increase, predominate for a time, and then disasappear as a result of recombination. Epidemic clones were found among isolates from patients with keratitis. They were characterized by high activity of a hitherto-unrecognized size variant of elastase, high alkaline protease activity, and possession of the exoU gene encoding the cytotoxic exoenzyme U. These virulence determinants are not exclusive traits in strains causing keratitis, as strains with other properties may cause keratitis in the presence of predisposing conditions. There were no uniform patterns of characteristics of isolates from other types of infection; however, all strains from urinary tract infections possessed the exoS gene, all strains from environment and feces and the major part of keratitis and wound isolates exhibited high elastase and alkaline protease activity, and all strains from feces showed high staphylolysin activity, indicating that these virulence factors may be important in the pathogenesis of these infectious diseases. PMID:11553572

LMOf2365_0442 Encoding for a Fructose Specific PTS Permease IIA May Be Required for Virulence in L. monocytogenes Strain F2365

PubMed Central

Liu, Yanhong; Yoo, Brian B.; Hwang, Cheng-An; Suo, Yujuan; Sheen, Shiowshuh; Khosravi, Parvaneh; Huang, Lihan

2017-01-01

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, which is a major public health concern due to the high fatality rate. LMOf2365_0442, 0443, and 0444 encode for fructose-specific EIIABC components of phosphotransferase transport system (PTS) permease that is responsible for sugar transport. In previous studies, in-frame deletion mutants of a putative fructose-specific PTS permease (LMOf2365_0442, 0443, and 0444) were constructed and analyzed. However, the virulence potential of these deletion mutants has not been studied. In this study, two in vitro methods were used to analyze the virulence potential of these L. monocytogenes deletion mutants. First, invasion assays were used to measure the invasion efficiencies to host cells using the human HT-29 cell line. Second, plaque forming assays were used to measure cell-to-cell spread in host cells. Our results showed that the deletion mutant ΔLMOf2365_0442 had reduced invasion and cell-to-cell spread efficiencies in human cell line compared to the parental strain LMOf2365, indicating that LMOf2365_0442 encoding for a fructose specific PTS permease IIA may be required for virulence in L. monocytogenes strain F2365. In addition, the gene expression levels of 15 virulence and stress-related genes were analyzed in the stationary phase cells of the deletion mutants using RT-PCR assays. Virulence-related gene expression levels were elevated in the deletion mutants ΔLMOf2365_0442-0444 compared to the wild type parental strain LMOf2365, indicating the down-regulation of virulence genes by this PTS permease in L. monocytogenes. Finally, stress-related gene clpC expression levels were also increased in all of the deletion mutants, suggesting the involvement of this PTS permease in stress response. Furthermore, these deletion mutants displayed the same pressure tolerance and the same capacity for biofilm formation compared to the wild-type parental strain LMOf2365. In summary, our findings suggest that the LMOf2365_0442 gene can be used as a potential target to develop inhibitors for new therapeutic and pathogen control strategies for public health. PMID:28900418

Ulcerogenic Helicobacter pylori Strains Isolated from Children: A Contribution to Get Insight into the Virulence of the Bacteria

PubMed Central

Vitoriano, Inês; Saraiva-Pava, Kathy D.; Rocha-Gonçalves, Alexandra; Santos, Andrea; Lopes, Ana I.; Oleastro, Mónica; Roxo-Rosa, Mónica

2011-01-01

Infection with Helicobacter pylori is the major cause for the development of peptic ulcer disease (PUD). In children, with no other etiology for the disease, this rare event occurs shortly after infection. In these young patients, habits of smoking, diet, consumption of alcohol and non-steroid anti-inflammatory drugs and stress, in addition to the genetic susceptibility of the patient, represent a minor influence. Accordingly, the virulence of the implicated H. pylori strain should play a crucial role in the development of PUD. Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins. To uncover virulence features, we compared the proteome of these two groups of H. pylori strains. Two-dimensional gel electrophoresis followed by mass-spectrometry allowed us to detect 27 differentially expressed proteins between them. In addition to the presence of genes encoding well established virulence factors, namely cagA, vacAs1, oipA “on” status, homB and jhp562 genes, the pediatric ulcerogenic strains shared a proteome profile characterized by changes in the abundance of: motility-associated proteins, accounting for higher motility; antioxidant proteins, which may confer increased resistance to inflammation; and enzymes involved in key steps in the metabolism of glucose, amino acids and urea, which may be advantageous to face fluctuations of nutrients. In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors. PMID:22039453

Clavibacter michiganensis subsp. michiganensis: first steps in the understanding of virulence of a Gram-positive phytopathogenic bacterium.

PubMed

Gartemann, Karl-Heinz; Kirchner, Oliver; Engemann, Jutta; Gräfen, Ines; Eichenlaub, Rudolf; Burger, Annette

2003-12-19

Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete. It infects tomato, spreads through the xylem and causes bacterial wilt and canker. The wild-type strain NCPPB382 carries two plasmids, pCM1 and pCM2. The cured plasmid-free derivative CMM100 is still able to colonize tomato, but no disease symptoms develop indicating that all genes required for successful infection, establishment and growth in the plant reside on the chromosome. Both plasmids carry one virulence factor, a gene encoding a cellulase, CelA in case of pCM1 and a putative serine protease Pat-1 on pCM2. These genes can independently convert the non-virulent strain CMM100 into a pathogen causing wilt on tomatoes. Currently, genome projects for Cmm and the closely related potato-pathogen C. michiganensis subsp. sepedonicus have been initiated. The data from the genome project shall give clues on further genes involved in plant-microbe interaction that can be tested experimentally. Especially, identification of genes related to host-specificity through genome comparison of the two subspecies might be possible.

VirF-Independent Regulation of Shigella virB Transcription is Mediated by the Small RNA RyhB

PubMed Central

Broach, William H.; Egan, Nicholas; Wing, Helen J.; Payne, Shelley M.; Murphy, Erin R.

2012-01-01

Infection of the human host by Shigella species requires the coordinated production of specific Shigella virulence factors, a process mediated largely by the VirF/VirB regulatory cascade. VirF promotes the transcription of virB, a gene encoding the transcriptional activator of several virulence-associated genes. This study reveals that transcription of virB is also regulated by the small RNA RyhB, and importantly, that this regulation is not achieved indirectly via modulation of VirF activity. These data are the first to demonstrate that the regulation of virB transcription can be uncoupled from the master regulator VirF. It is also established that efficient RyhB-dependent regulation of transcription is facilitated by specific nucleic acid sequences within virB. This study not only reveals RyhB-dependent regulation of virB transcription as a novel point of control in the central regulatory circuit modulating Shigella virulence, but also highlights the versatility of RyhB in controlling bacterial gene expression. PMID:22701677

A Plasmodium yoelii HECT-like E3 ubiquitin ligase regulates parasite growth and virulence.

PubMed

Nair, Sethu C; Xu, Ruixue; Pattaradilokrat, Sittiporn; Wu, Jian; Qi, Yanwei; Zilversmit, Martine; Ganesan, Sundar; Nagarajan, Vijayaraj; Eastman, Richard T; Orandle, Marlene S; Tan, John C; Myers, Timothy G; Liu, Shengfa; Long, Carole A; Li, Jian; Su, Xin-Zhuan

2017-08-09

Infection of mice with strains of Plasmodium yoelii parasites can result in different pathology, but molecular mechanisms to explain this variation are unclear. Here we show that a P. yoelii gene encoding a HECT-like E3 ubiquitin ligase (Pyheul) influences parasitemia and host mortality. We genetically cross two lethal parasites with distinct disease phenotypes, and identify 43 genetically diverse progeny by typing with microsatellites and 9230 single-nucleotide polymorphisms. A genome-wide quantitative trait loci scan links parasite growth and host mortality to two major loci on chromosomes 1 and 7 with LOD (logarithm of the odds) scores = 6.1 and 8.1, respectively. Allelic exchange of partial sequences of Pyheul in the chromosome 7 locus and modification of the gene expression alter parasite growth and host mortality. This study identifies a gene that may have a function in parasite growth, virulence, and host-parasite interaction, and therefore could be a target for drug or vaccine development.Many strains of Plasmodium differ in virulence, but factors that control these distinctions are not known. Here the authors comparatively map virulence loci using the offspring from a P. yoelii YM and N67 genetic cross, and identify a putative HECT E3 ubiquitin ligase that may explain the variance.

Association of plasmid-mediated quinolone resistance and virulence markers in Escherichia coli isolated from water.

PubMed

Mendonça, Nuno; Ramalho, Joana; Vieira, Pedro; Da Silva, Gabriela Jorge

2012-06-01

This work aimed to investigate the association of the carriage of plasmid-mediated quinolone resistance (PMQR) genes, the virulence potential encoded in pathogenicity islands (PAIs) and the phylogenetic background in Escherichia coli strains isolated from waters of diverse origin. Antimicrobial susceptibilities were determined by the disc diffusion method. Screening for PMQR (qnr, aac(6')-Ib-variant and qepA) genes, PAIs and the determination of phylogroup was performed by PCR. Nineteen percent of strains were resistant to nalidixic acid, 11% to ciprofloxacin and 5% to gentamicin. qnrA was the only PMQR detected in 16% of strains, susceptible to quinolones and grouped in phylogenetic lineage B1. Sixty-seven percent of the isolates were assigned to the less-virulent groups A and B1. PAIs IV(536) and II(CFT073) were detected in 16 and 3% of the isolates, respectively. All PAIs were detected in the phylogroups D and B1. The presence of PAIs in isolates from waters may represent an increased risk for public health, as they were isolated from samples collected from surface and drinking waters. As E. coli is an important indicator of microbiological water quality, and also a potential pathogen, routine analysis for its detection could be complemented by screening for virulence factors and antimicrobial genes.

The Genome Sequence of Mannheimia haemolytica A1: Insights into Virulence, Natural Competence, and Pasteurellaceae Phylogeny†

PubMed Central

Gioia, Jason; Qin, Xiang; Jiang, Huaiyang; Clinkenbeard, Kenneth; Lo, Reggie; Liu, Yamei; Fox, George E.; Yerrapragada, Shailaja; McLeod, Michael P.; McNeill, Thomas Z.; Hemphill, Lisa; Sodergren, Erica; Wang, Qiaoyan; Muzny, Donna M.; Homsi, Farah J.; Weinstock, George M.; Highlander, Sarah K.

2006-01-01

The draft genome sequence of Mannheimia haemolytica A1, the causative agent of bovine respiratory disease complex (BRDC), is presented. Strain ATCC BAA-410, isolated from the lung of a calf with BRDC, was the DNA source. The annotated genome includes 2,839 coding sequences, 1,966 of which were assigned a function and 436 of which are unique to M. haemolytica. Through genome annotation many features of interest were identified, including bacteriophages and genes related to virulence, natural competence, and transcriptional regulation. In addition to previously described virulence factors, M. haemolytica encodes adhesins, including the filamentous hemagglutinin FhaB and two trimeric autotransporter adhesins. Two dual-function immunoglobulin-protease/adhesins are also present, as is a third immunoglobulin protease. Genes related to iron acquisition and drug resistance were identified and are likely important for survival in the host and virulence. Analysis of the genome indicates that M. haemolytica is naturally competent, as genes for natural competence and DNA uptake signal sequences (USS) are present. Comparison of competence loci and USS in other species in the family Pasteurellaceae indicates that M. haemolytica, Actinobacillus pleuropneumoniae, and Haemophilus ducreyi form a lineage distinct from other Pasteurellaceae. This observation was supported by a phylogenetic analysis using sequences of predicted housekeeping genes. PMID:17015664

The Yersiniabactin Transport System Is Critical for the Pathogenesis of Bubonic and Pneumonic Plague▿

PubMed Central

Fetherston, Jacqueline D.; Kirillina, Olga; Bobrov, Alexander G.; Paulley, James T.; Perry, Robert D.

2010-01-01

Iron acquisition from the host is an important step in the pathogenic process. While Yersinia pestis has multiple iron transporters, the yersiniabactin (Ybt) siderophore-dependent system plays a major role in iron acquisition in vitro and in vivo. In this study, we determined that the Ybt system is required for the use of iron bound by transferrin and lactoferrin and examined the importance of the Ybt system for virulence in mouse models of bubonic and pneumonic plague. Y. pestis mutants unable to either transport Ybt or synthesize the siderophore were both essentially avirulent via subcutaneous injection (bubonic plague model). Surprisingly, via intranasal instillation (pneumonic plague model), we saw a difference in the virulence of Ybt biosynthetic and transport mutants. Ybt biosynthetic mutants displayed an ∼24-fold-higher 50% lethal dose (LD50) than transport mutants. In contrast, under iron-restricted conditions in vitro, a Ybt transport mutant had a more severe growth defect than the Ybt biosynthetic mutant. Finally, a Δpgm mutant had a greater loss of virulence than the Ybt biosynthetic mutant, indicating that the 102-kb pgm locus encodes a virulence factor, in addition to Ybt, that plays a role in the pathogenesis of pneumonic plague. PMID:20160020

Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

PubMed Central

Ogier, Jean-Claude; Pagès, Sylvie; Bisch, Gaëlle; Chiapello, Hélène; Médigue, Claudine; Rouy, Zoé; Teyssier, Corinne; Vincent, Stéphanie; Tailliez, Patrick; Givaudan, Alain; Gaudriault, Sophie

2014-01-01

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500–700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin–antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host. PMID:24904010

Development of genetic tools for in vivo virulence analysis of Streptococcus sanguinis.

PubMed

Turner, Lauren Senty; Das, Sankar; Kanamoto, Taisei; Munro, Cindy L; Kitten, Todd

2009-08-01

Completion of the genome sequence of Streptococcus sanguinis SK36 necessitates tools for further characterization of this species. It is often desirable to insert antibiotic resistance markers and other exogenous genes into the chromosome; therefore, we sought to identify a chromosomal site for ectopic expression of foreign genes, and to verify that insertion into this site did not affect important cellular phenotypes. We designed three plasmid constructs for insertion of erm, aad9 or tetM resistance determinants into a genomic region encoding only a small (65 aa) hypothetical protein. To determine whether this insertion affected important cellular properties, SK36 and its erythromycin-resistant derivative, JFP36, were compared for: (i) growth in vitro, (ii) genetic competence, (iii) biofilm formation and (iv) virulence for endocarditis in the rabbit model of infective endocarditis (IE). The spectinomycin-resistant strain, JFP56, and tetracycline-resistant strain, JFP76, were also tested for virulence in vivo. Insertion of erm did not affect growth, competence or biofilm development of JFP36. Recovery of bacteria from heart valves of co-inoculated rabbits was similar to wild-type for JFP36, JFP56 and JFP76, indicating that IE virulence was not significantly affected. The capacity for mutant complementation in vivo was explored in an avirulent ssaB mutant background. Expression of ssaB from its predicted promoter in the target region restored IE virulence. Thus, the chromosomal site utilized is a good candidate for further manipulations of S. sanguinis. In addition, the resistant strains developed may be further applied as controls to facilitate screening for virulence factors in vivo.

Development of genetic tools for in vivo virulence analysis of Streptococcus sanguinis

PubMed Central

Senty Turner, Lauren; Das, Sankar; Kanamoto, Taisei; Munro, Cindy L.; Kitten, Todd

2009-01-01

Completion of the genome sequence of Streptococcus sanguinis SK36 necessitates tools for further characterization of this species. It is often desirable to insert antibiotic resistance markers and other exogenous genes into the chromosome; therefore, we sought to identify a chromosomal site for ectopic expression of foreign genes, and to verify that insertion into this site did not affect important cellular phenotypes. We designed three plasmid constructs for insertion of erm, aad9 or tetM resistance determinants into a genomic region encoding only a small (65 aa) hypothetical protein. To determine whether this insertion affected important cellular properties, SK36 and its erythromycin-resistant derivative, JFP36, were compared for: (i) growth in vitro, (ii) genetic competence, (iii) biofilm formation and (iv) virulence for endocarditis in the rabbit model of infective endocarditis (IE). The spectinomycin-resistant strain, JFP56, and tetracycline-resistant strain, JFP76, were also tested for virulence in vivo. Insertion of erm did not affect growth, competence or biofilm development of JFP36. Recovery of bacteria from heart valves of co-inoculated rabbits was similar to wild-type for JFP36, JFP56 and JFP76, indicating that IE virulence was not significantly affected. The capacity for mutant complementation in vivo was explored in an avirulent ssaB mutant background. Expression of ssaB from its predicted promoter in the target region restored IE virulence. Thus, the chromosomal site utilized is a good candidate for further manipulations of S. sanguinis. In addition, the resistant strains developed may be further applied as controls to facilitate screening for virulence factors in vivo. PMID:19423626

Photobacterium damselae subsp. damselae, an Emerging Fish Pathogen in the Black Sea: Evidence of a Multiclonal Origin

PubMed Central

Terceti, Mateus S.; Ogut, Hamdi

2016-01-01

ABSTRACT Photobacterium damselae subsp. damselae is considered to be an emerging pathogen of marine fish of importance in aquaculture, with a notable increase in its geographical distribution during the last several years. In this study, we carried out for the first time to our knowledge a genetic and pathobiological characterization of 14 strains isolated from sea bass (Dicentrarchus labrax) reared in the Southeastern Black Sea, where high mortalities were observed at two aquaculture farms during the summer and autumn of 2011. Heterogeneity was evidenced among strains in phenotypical traits, such as sucrose fermentation, motility, and hemolysis. Although 11 of 14 isolates were hemolytic, we found that all of the isolates lacked the pPHDD1 virulence plasmid that encodes the phospholipase-D damselysin (Dly) and the pore-forming toxin PhlyP, two hemolysins previously reported to constitute major virulence factors for turbot. Subsequent PCR and sequencing analyses demonstrated that the 11 hemolytic isolates harbored a complete hlyAch gene, a chromosome I-borne gene that encodes HlyAch hemolysin, whereas the three nonhemolytic isolates contained hlyAch pseudogenes caused by insertion sequence elements. Virulence challenges with two representative strains revealed that, albeit less virulent than the pPHDD1-harboring strain RM-71, the plasmidless hlyAch-positive and hlyAch-negative Black Sea isolates were pathogenic for sea bass. A phylogenetic analysis based on the toxR gene sequence uncovered a greater diversity in the isolates, indicating that the presence of this pathogen in the Black Sea was not caused by the introduction and spread of a single virulent clone but by the proliferation of different clones. IMPORTANCE The geographical distribution of marine bacterial pathogens is undergoing a worldwide increase. In particular, bacteria of the group vibrios are increasingly being isolated as the causative agents of disease in novel species of cultivated fish in areas where they had not been previously reported. Here we characterize for the first time to our knowledge a collection of isolates of the fish and human pathogen Photobacterium damselae subsp. damselae from diseased sea bass reared in the Black Sea. We uncovered great genetic diversity in the Black Sea isolates of this pathogen, suggesting a multiclonal origin. We also demonstrate for the first time that these isolates bear pathogenic potential for sea bass cultures by virulence challenges. PMID:27084008

An Enterotoxin-Bearing Pathogenicity Island in Staphylococcus epidermidis▿†

PubMed Central

Madhusoodanan, Jyoti; Seo, Keun Seok; Remortel, Brian; Park, Joo Youn; Hwang, Sun Young; Fox, Lawrence K.; Park, Yong Ho; Deobald, Claudia F.; Wang, Dan; Liu, Song; Daugherty, Sean C.; Gill, Ann Lindley; Bohach, Gregory A.; Gill, Steven R.

2011-01-01

Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage. PMID:21317317

Development of a miniaturized DNA microarray for identification of 66 virulence genes of Legionella pneumophila.

PubMed

Żak, Mariusz; Zaborowski, Piotr; Baczewska-Rej, Milena; Zasada, Aleksandra A; Matuszewska, Renata; Krogulska, Bożena

2011-12-20

For the last five years, Legionella sp. infections and legionnaire's disease in Poland have been receiving a lot of attention, because of the new regulations concerning microbiological quality of drinking water. This was the inspiration to search for and develop a new assay to identify many virulence genes of Legionella pneumophila to better understand their distribution in environmental and clinical strains. The method might be an invaluable help in infection risk assessment and in epidemiological investigations. The microarray is based on Array Tube technology. It contains 3 positive and 1 negative control. Target genes encode structural elements of T4SS, effector proteins and factors not related to T4SS. Probes were designed using OligoWiz software and data analyzed using IconoClust software. To isolate environmental and clinical strains, BAL samples and samples of hot water from different and independent hot water distribution systems of public utility buildings were collected. We have developed a miniaturized DNA microarray for identification of 66 virulence genes of L. pneumophila. The assay is specific to L. pneumophila sg 1 with sensitivity sufficient to perform the assay using DNA isolated from a single L. pneumophila colony. Seven environmental strains were analyzed. Two exhibited a hybridization pattern distinct from the reference strain. The method is time- and cost-effective. Initial studies have shown that genes encoding effector proteins may vary among environmental strains. Further studies might help to identify set of genes increasing the risk of clinical disease and to determine the pathogenic potential of environmental strains.

Binding of NAD+-Glycohydrolase to Streptolysin O Stabilizes Both Toxins and Promotes Virulence of Group A Streptococcus

PubMed Central

Velarde, Jorge J.; O’Seaghdha, Maghnus; Baddal, Buket; Bastiat-Sempe, Benedicte

2017-01-01

ABSTRACT The globally dominant, invasive M1T1 strain of group A Streptococcus (GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD+-glycohydrolase (NADase), resulting in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. DNA sequencing of such strains has revealed that reduced or absent enzymatic activity can be associated with a variety of point mutations in nga, the gene encoding NADase; a commonly observed polymorphism associated with near-complete abrogation of activity is a substitution of aspartic acid for glycine at position 330 (G330D). However, nga has not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. It has been suggested that NADase that lacks NAD-glycohydrolase activity retains an as-yet-unidentified inherent cytotoxicity to mammalian cells and thus is still a potent virulence factor. We now show that expression of NADase, either enzymatically active or inactive, augments SLO-mediated toxicity for keratinocytes. In culture supernatants, SLO and NADase are mutually interdependent for protein stability. We demonstrate that the two proteins interact in solution and that both the translocation domain and catalytic domain of NADase are required for maximal binding between the two toxins. We conclude that binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS virulence. PMID:28900022

Comparative Transcriptome Analysis of Salivary Glands of Two Populations of Rice Brown Planthopper, Nilaparvata lugens, That Differ in Virulence

PubMed Central

Chen, Hongdan; Ye, Wenfeng; Li, Shaohui; Lou, Yonggen

2013-01-01

Background The brown planthopper (BPH), Nilaparvata lugens (Stål), a destructive rice pest in Asia, can quickly overcome rice resistance by evolving new virulent populations. Herbivore saliva plays an important role in plant–herbivore interactions, including in plant defense and herbivore virulence. However, thus far little is known about BPH saliva at the molecular level, especially its role in virulence and BPH–rice interaction. Methodology/Principal Findings Using cDNA amplification in combination with Illumina short-read sequencing technology, we sequenced the salivary-gland transcriptomes of two BPH populations with different virulence; the populations were derived from rice variety TN1 (TN1 population) and Mudgo (M population). In total, 37,666 and 38,451 unigenes were generated from the salivary glands of these populations, respectively. When combined, a total of 43,312 unigenes were obtained, about 18 times more than the number of expressed sequence tags previously identified from these glands. Gene ontology annotations and KEGG orthology classifications indicated that genes related to metabolism, binding and transport were significantly active in the salivary glands. A total of 352 genes were predicted to encode secretory proteins, and some might play important roles in BPH feeding and BPH–rice interactions. Comparative analysis of the transcriptomes of the two populations revealed that the genes related to ‘metabolism,’ ‘digestion and absorption,’ and ‘salivary secretion’ might be associated with virulence. Moreover, 67 genes encoding putative secreted proteins were differentially expressed between the two populations, suggesting these genes may contribute to the change in virulence. Conclusions/Significance This study was the first to compare the salivary-gland transcriptomes of two BPH populations having different virulence traits and to find genes that may be related to this difference. Our data provide a rich molecular resource for future functional studies on salivary glands and will be useful for elucidating the molecular mechanisms underlying BPH feeding and virulence differences. PMID:24244529

Comparative transcriptome analysis of salivary glands of two populations of rice brown planthopper, Nilaparvata lugens, that differ in virulence.

PubMed

Ji, Rui; Yu, Haixin; Fu, Qiang; Chen, Hongdan; Ye, Wenfeng; Li, Shaohui; Lou, Yonggen

2013-01-01

The brown planthopper (BPH), Nilaparvata lugens (Stål), a destructive rice pest in Asia, can quickly overcome rice resistance by evolving new virulent populations. Herbivore saliva plays an important role in plant-herbivore interactions, including in plant defense and herbivore virulence. However, thus far little is known about BPH saliva at the molecular level, especially its role in virulence and BPH-rice interaction. Using cDNA amplification in combination with Illumina short-read sequencing technology, we sequenced the salivary-gland transcriptomes of two BPH populations with different virulence; the populations were derived from rice variety TN1 (TN1 population) and Mudgo (M population). In total, 37,666 and 38,451 unigenes were generated from the salivary glands of these populations, respectively. When combined, a total of 43,312 unigenes were obtained, about 18 times more than the number of expressed sequence tags previously identified from these glands. Gene ontology annotations and KEGG orthology classifications indicated that genes related to metabolism, binding and transport were significantly active in the salivary glands. A total of 352 genes were predicted to encode secretory proteins, and some might play important roles in BPH feeding and BPH-rice interactions. Comparative analysis of the transcriptomes of the two populations revealed that the genes related to 'metabolism,' 'digestion and absorption,' and 'salivary secretion' might be associated with virulence. Moreover, 67 genes encoding putative secreted proteins were differentially expressed between the two populations, suggesting these genes may contribute to the change in virulence. This study was the first to compare the salivary-gland transcriptomes of two BPH populations having different virulence traits and to find genes that may be related to this difference. Our data provide a rich molecular resource for future functional studies on salivary glands and will be useful for elucidating the molecular mechanisms underlying BPH feeding and virulence differences.

Complete genome sequence of Vibrio parahaemolyticus strain FORC_008, a foodborne pathogen from a flounder fish in South Korea.

PubMed

Kim, Suyeon; Chung, Han Young; Lee, Dong-Hoon; Lim, Jong Gyu; Kim, Se Keun; Ku, Hye-Jin; Kim, You-Tae; Kim, Heebal; Ryu, Sangryeol; Lee, Ju-Hoon; Choi, Sang Ho

2016-07-01

Vibrio parahaemolyticus is a Gram-negative, motile, nonspore-forming pathogen that causes foodborne illness associated with the consumption of contaminated seafoods. Although many cases of foodborne outbreaks caused by V. parahaemolyticus have been reported, the genomes of only five strains have been completely sequenced and analyzed using bioinformatics. In order to characterize overall virulence factors and pathogenesis of V. parahaemolyticus associated with foodborne outbreak in South Korea, a new strain FORC_008 was isolated from flounder fish and its genome was completely sequenced. The genomic analysis revealed that the genome of FORC_008 consists of two circular DNA chromosomes of 3266 132 bp (chromosome I) and 1772 036 bp (chromosome II) with a GC content of 45.36% and 45.53%, respectively. The entire genome contains 4494 predicted open reading frames, 129 tRNAs and 31 rRNA genes. While the strain FORC_008 does not have genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), its genome encodes many other virulence factors including hemolysins, pathogenesis-associated secretion systems and iron acquisition systems, suggesting that it may be a potential pathogen. This report provides an extended understanding on V. parahaemolyticus in genomic level and would be helpful for rapid detection, epidemiological investigation and prevention of foodborne outbreak in South Korea. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Decoding the genetic and functional diversity of the DSF quorum-sensing system in Stenotrophomonas maltophilia

PubMed Central

Huedo, Pol; Yero, Daniel; Martinez-Servat, Sònia; Ruyra, Àngels; Roher, Nerea; Daura, Xavier; Gibert, Isidre

2015-01-01

Stenotrophomonas maltophilia uses the Diffusible Signal Factor (DSF) quorum sensing (QS) system to mediate intra- and inter-specific signaling and regulate virulence-related processes. The components of this system are encoded by the rpf cluster, with genes rpfF and rpfC encoding for the DSF synthase RpfF and sensor RpfC, respectively. Recently, we have shown that there exist two variants of the rpf cluster (rpf-1 and rpf-2), distinguishing two groups of S. maltophilia strains. Surprisingly, only rpf-1 strains produce detectable DSF, correlating with their ability to control biofilm formation, swarming motility and virulence. The evolutive advantage of acquiring two different rpf clusters, the phylogenetic time point and mechanism of this acquisition and the conditions that activate DSF production in rpf-2 strains, are however not known. Examination of this cluster in various species suggests that its variability originated most probably by genetic exchange between rhizosphere bacteria. We propose that rpf-2 variant strains make use of a strategy recently termed as “social cheating.” Analysis of cellular and extracellular fatty acids (FAs) of strains E77 (rpf-1) and M30 (rpf-2) suggests that their RpfFs have also a thioesterase activity that facilitates the release of unspecific FAs to the medium in addition to DSF. Production of DSF in rpf-1 strains appears in fact to be modulated by some of these extracellular FAs in addition to other factors such as temperature and nutrients, while in rpf-2 strains DSF biosynthesis is derepressed only upon detection of DSF itself, suggesting that they require cohabitation with DSF-producer bacteria to activate their DSF regulatory machinery. Finally, we show that the mixed rpf-1/rpf-2 population presents synergism in DSF production and virulence capacity in an in vivo infection model. Recovery and quantification of DSF from co-infected animals correlates with the observed mortality rate. PMID:26284046

Genomics, evolution, and molecular epidemiology of the Streptococcus bovis/Streptococcus equinus complex (SBSEC).

PubMed

Jans, Christoph; Meile, Leo; Lacroix, Christophe; Stevens, Marc J A

2015-07-01

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) is a group of human and animal derived streptococci that are commensals (rumen and gastrointestinal tract), opportunistic pathogens or food fermentation associates. The classification of SBSEC has undergone massive changes and currently comprises 7 (sub)species grouped into four branches based on sequences identities: the Streptococcus gallolyticus, the Streptococcus equinus, the Streptococcus infantarius and the Streptococcus alactolyticus branch. In animals, SBSEC are causative agents for ruminal acidosis, potentially laminitis and infective endocarditis (IE). In humans, a strong association was established between bacteraemia, IE and colorectal cancer. Especially the SBSEC-species S. gallolyticus subsp. gallolyticus is an emerging pathogen for IE and prosthetic joint infections. S. gallolyticus subsp. pasteurianus and the S. infantarius branch are further associated with biliary and urinary tract infections. Knowledge on pathogenic mechanisms is so far limited to colonization factors such as pili and biofilm formation. Certain strain variants of S. gallolyticus subsp. macedonicus and S. infantarius subsp. infantarius are associated with traditional dairy and plant-based food fermentations and display traits suggesting safety. However, due to their close relationship to virulent strains, their use in food fermentation has to be critically assessed. Additionally, implementing accurate and up-to-date taxonomy is critical to enable appropriate treatment of patients and risk assessment of species and strains via recently developed multilocus sequence typing schemes to enable comparative global epidemiology. Comparative genomics revealed that SBSEC strains harbour genomics islands (GI) that seem acquired from other streptococci by horizontal gene transfer. In case of virulent strains these GI frequently encode putative virulence factors, in strains from food fermentation the GI encode functions that are pivotal for strain performance during fermentation. Comparative genomics is a powerful tool to identify acquired pathogenic functions, but there is still an urgent need for more physiological and epidemiological data to understand SBSEC-specific traits. Copyright © 2014 Elsevier B.V. All rights reserved.

Cnm is a major virulence factor of invasive Streptococcus mutans and part of a conserved three-gene locus

PubMed Central

Avilés-Reyes, A.; Miller, J.H.; Simpson-Haidaris, P.J.; Lemos, J.A.; Abranches, J.

2014-01-01

SUMMARY Cnm, a collagen- and laminin-binding protein present in a subset of Streptococcus mutans strains, mediates binding to extracellular matrices (ECM), intracellular invasion and virulence in the Galleria mellonella model. Antibodies raised against Cnm were used to confirm expression and the cell surface localization of Cnm in the highly invasive OMZ175 strain. Sequence analysis identified two additional genes (cnaB and cbpA) encoding putative surface proteins immediately upstream of cnm. Inactivation of cnaB and cbpA in OMZ175, individually or in combination, did not decrease the ability of this highly invasive and virulent strain to bind to different ECM proteins, invade human coronary artery endothelial cells (HCAEC), or kill G. mellonella. Similarly, expression of cnaB and cbpA in the cnm− strain UA159 revealed that these genes did not enhance Cnmrelated phenotypes. However, integration of cnm in the chromosome of UA159 significantly increased its ability to bind to collagen and laminin, invade HCAEC, and kill G. mellonella. Moreover, the presence of antibodies against Cnm nearly abolished the ability of OMZ175 to bind to collagen and laminin and invade HCAEC, and significantly protected G. mellonella against OMZ175 infection. We concluded that neither CnaB nor CbpA is necessary for the expression of Cnm-related traits. We also provided definitive evidence that Cnm is an important virulence factor and a suitable target for the development of novel preventive and therapeutic strategies to combat invasive S. mutans strains. PMID:24103776

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk.

PubMed

Martins, Katheryne Benini; Faccioli-Martins, Patricia Yoshida; Riboli, Danilo Flávio Moraes; Pereira, Valéria Cataneli; Fernandes, Simone; Oliveira, Aline A; Dantas, Ariane; Zafalon, Luiz Francisco; da Cunha, Maria de Lourdes Ribeiro de Souza

2015-06-01

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Detection of CDT toxin genes in Campylobacter spp. strains isolated from broiler carcasses and vegetables in São Paulo, Brazil

PubMed Central

de Carvalho, Aline Feola; da Silva, Daniela Martins; Azevedo, Sergio Santos; Piatti, Rosa Maria; Genovez, Margareth Elide; Scarcelli, Eliana

2013-01-01

Campylobacteriosis is a worldwide distributed zoonosis. One of the main virulence factors related to Campylobacter spp. in animals and humans is the cytolethal distending toxin (CDT), encoded by three adjacent genes (cdtA, cdtB, cdtC). The occurrence of Campylobacter spp. in samples of vegetables has not been reported in Brazil yet, and has seldom been described in the international literature. The detection of CDT in these strains has not been reported, either. The objectives of the present study were to determine the occurrence of Campylobacter spp. strains carrying virulence factors in samples of poultry and vegetables (lettuce and spinach) from different points of sale, thus verifying if vegetables are as an important vehicle for potentially virulent Campylobacter spp. strains as poultry. Twenty four strains were identified as Campylobacter jejuni by phenotypic and genotypic methods: 22 from broiler carcasses and two from lettuce samples. Three strains were identified as Campylobacter coli: two from broiler carcasses and one from lettuce. The presence of the cdt genes were detected in 20/24 (83.3%) C. jejuni strains, and 3/3 (100%) C. coli strains. The isolation of Campylobacter spp. strains with the cdt gene cluster in lettuce samples points to a new possible source of contamination, which could have an impact in the vegetable production chain and risk to public health. Results show that potentially virulent C. jejuni and C. coli strains remain viable in samples of broiler carcasses and vegetables at the points of sale. PMID:24516435

The stationary phase sigma factor, RpoS, regulates the production of a carbapenem antibiotic, a bioactive prodigiosin and virulence in the enterobacterial pathogen Serratia sp. ATCC 39006.

PubMed

Wilf, Nabil M; Salmond, George P C

2012-03-01

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and invertebrate animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, a prodigiosin and a carbapenem, and the exoenzymes pectate lyase and cellulase. We showed previously that deletion of the RNA chaperone Hfq abolished antibiotic production and attenuated virulence in both animal and plant hosts. Hfq and dependent small RNAs (sRNAs) are known to regulate the post-transcriptional expression of rpoS, which encodes σ(S), the stationary phase sigma factor subunit of RNA polymerase. An S39006 hfq deletion mutant showed decreased transcript levels of rpoS. Therefore, in this study we investigated whether the phenotypes regulated by Hfq were mediated through its control of rpoS. Whereas loss of Hfq abolished prodigiosin and carbapenem production and attenuated virulence in both C. elegans and potato, characterization of an S39006 rpoS mutant showed unexpectedly elevated prodigiosin and carbapenem production. Furthermore, the rpoS mutant exhibited attenuated animal pathogenesis, but not plant pathogenesis. Additionally, a homologue of the Hfq-dependent sRNA, RprA, was identified and shown to regulate prodigiosin production in a manner consistent with its role in positively regulating translation of rpoS mRNA. Combined, these results demonstrate that Hfq regulation of secondary metabolism and plant pathogenesis is independent of RpoS and establishes RpoS and RprA as regulators of antibiotic production.

Effect of the Streptococcus agalactiae Virulence Regulator CovR on the Pathogenesis of Urinary Tract Infection.

PubMed

Sullivan, Matthew J; Leclercq, Sophie Y; Ipe, Deepak S; Carey, Alison J; Smith, Joshua P; Voller, Nathan; Cripps, Allan W; Ulett, Glen C

2017-02-01

Streptococcus agalactiae can cause urinary tract infection (UTI). The role of the S. agalactiae global virulence regulator, CovR, in UTI pathogenesis is unknown. We used murine and human bladder uroepithelial cell models of UTI and S. agalactiae mutants in covR and related factors, including β-hemolysin/cytolysin (β-h/c), surface-anchored adhesin HvgA, and capsule to study the role of CovR in UTI. We found that covR-deficient serotype III S. agalactiae 874391 was significantly attenuated for colonization in mice and adhesion to uroepithelial cells. Mice infected with covR-deficient S. agalactiae produced less proinflammatory cytokines than those infected with wild-type 874391. Acute cytotoxicity in uroepithelial cells triggered by covR-deficient but not wild-type 874391 was associated with significant caspase 3 activation. Mechanistically, covR mutation significantly altered the expression of several genes in S. agalactiae 874391 that encode key virulence factors, including β-h/c and HvgA, but not capsule. Subsequent mutational analyses revealed that HvgA and capsule, but not the β-h/c, exerted significant effects on colonization of the murine urinary tract in vivo. S. agalactiae CovR promotes bladder infection and inflammation, as well as adhesion to and viability of uroepithelial cells. The pathogenesis of S. agalactiae UTI is complex, multifactorial, and influenced by virulence effects of CovR, HvgA, and capsule. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

A cell–cell signaling sensor is required for virulence and insect transmission of Xylella fastidiosa

PubMed Central

Chatterjee, Subhadeep; Wistrom, Christina; Lindow, Steven E.

2008-01-01

Cell–cell signaling in Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid Diffusible Signaling Factor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid two-component regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG, rpfB, a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF. PMID:18268318

A cell-cell signaling sensor is required for virulence and insect transmission of Xylella fastidiosa.

PubMed

Chatterjee, Subhadeep; Wistrom, Christina; Lindow, Steven E

2008-02-19

Cell-cell signaling in Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid Diffusible Signaling Factor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid two-component regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG, rpfB, a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF.

Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

PubMed

Al-Jarbou, Ahmed Nasser

2012-01-01

Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

FvSTR1, a striatin orthologue in Fusarium virguliforme, is required for asexual development and virulence.

PubMed

Islam, Kazi T; Bond, Jason P; Fakhoury, Ahmad M

2017-08-01

The soil-borne fungus Fusarium virguliforme causes sudden death syndrome (SDS), one of the most devastating diseases of soybean in North and South America. Despite the importance of SDS, a clear understanding of the fungal pathogenicity factors that affect the development of this disease is still lacking. We have identified FvSTR1, a F. virguliforme gene, which encodes a protein similar to a family of striatin proteins previously reported to regulate signalling pathways, cell differentiation, conidiation, sexual development, and virulence in filamentous fungi. Striatins are multi-domain proteins that serve as scaffolding units in the striatin-interacting phosphatase and kinase (STRIPAK) complex in fungi and animals. To address the function of a striatin homologue in F. virguliforme, FvSTR1 was disrupted and functionally characterized using a gene knock out strategy. The resulting Fvstr1 mutants were largely impaired in conidiation and pigmentation, and displayed defective conidia and conidiophore morphology compared to the wild-type and ectopic transformants. Greenhouse virulence assays revealed that the disruption of FvSTR1 resulted in complete loss of virulence in F. virguliforme. Microtome studies using fluorescence microscopy showed that the Fvstr1 mutants were defective in their ability to colonize the vascular system. The Fvstr1 mutants also showed a reduced transcript level of genes involved in asexual reproduction and in the production of secondary metabolites. These results suggest that FvSTR1 has a critical role in asexual development and virulence in F. virguliforme.

Paralogous Outer Membrane Proteins Mediate Uptake of Different Forms of Iron and Synergistically Govern Virulence in Francisella tularensis tularensis*

PubMed Central

Ramakrishnan, Girija; Sen, Bhaswati; Johnson, Richard

2012-01-01

Francisella tularensis subsp. tularensis is a highly infectious bacterium causing acute disease in mammalian hosts. Mechanisms for the acquisition of iron within the iron-limiting host environment are likely to be critical for survival of this intracellular pathogen. FslE (FTT0025) and FupA (FTT0918) are paralogous proteins that are predicted to form β-barrels in the outer membrane of virulent strain Schu S4 and are unique to Francisella species. Previous studies have implicated both FupA, initially identified as a virulence factor and FslE, encoded by the siderophore biosynthetic operon, in iron acquisition. Using single and double mutants, we demonstrated that these paralogs function in concert to promote growth under iron limitation. We used a 55Fe transport assay to demonstrate that FslE is involved in siderophore-mediated ferric iron uptake, whereas FupA facilitates high affinity ferrous iron uptake. Optimal replication within J774A.1 macrophage-like cells required at least one of these uptake systems to be functional. In a mouse model of tularemia, the ΔfupA mutant was attenuated, but the ΔfslE ΔfupA mutant was significantly more attenuated, implying that the two systems of iron acquisition function synergistically to promote virulence. These studies highlight the importance of specific iron acquisition functions, particularly that of ferrous iron, for virulence of F. tularensis in the mammalian host. PMID:22661710

Chancroid: from clinical practice to basic science.

PubMed

Lewis, D A

2000-01-01

Chancroid is a sexually transmitted disease caused by the bacterium Haemophilus ducreyi. It usually presents as a genital ulcer and may be associated with regional lymphadenopathy and bubo formation. H. ducreyi infection is predominantly seen in tropical resource-poor regions of the world where it is frequently the most common etiological cause of genital ulceration. Genital ulcer disease has been shown to be an extremely important co-factor in HIV transmission. With the advent of the AIDS epidemic, there has been increased research effort to elucidate those factors involved in the pathogenesis of chancroid. Several putative virulence factors have now been identified and isogenic H. ducreyi mutants constructed by mutagenesis of their encoding genes. This approach has facilitated investigations into the role each of these putative virulence factors may play in H. ducreyi pathogenesis through the use of in vitro and in vivo model systems. One major goal of current chancroid research is to identify antigens which are immunogenic and could form the basis of a vaccine against H. ducreyi infection. Such a vaccine, if shown to be effective in decreasing the prevalence of chancroid, could have the added benefit of slowing down the HIV incidence rates in those populations where chancroid is a major co-factor for HIV transmission.

VdCYC8, encoding CYC8 glucose repression mediator protein, is required for microsclerotia formation and full virulence in Verticillium dahliae

USDA-ARS?s Scientific Manuscript database

Verticillium dahliae is the primary causal agent for Verticillium wilt disease on a diverse array of economically important crops, including cotton. In previous research, we screened a T-DNA insertional mutant library of the highly virulent isolate Vd080 derived from cotton. In this study, the targ...

Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

PubMed

Lacroix, Benoît; Citovsky, Vitaly

2015-11-20

During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

Genome characterization and population genetic structure of the zoonotic pathogen, Streptococcus canis

PubMed Central

2012-01-01

Background Streptococcus canis is an important opportunistic pathogen of dogs and cats that can also infect a wide range of additional mammals including cows where it can cause mastitis. It is also an emerging human pathogen. Results Here we provide characterization of the first genome sequence for this species, strain FSL S3-227 (milk isolate from a cow with an intra-mammary infection). A diverse array of putative virulence factors was encoded by the S. canis FSL S3-227 genome. Approximately 75% of these gene sequences were homologous to known Streptococcal virulence factors involved in invasion, evasion, and colonization. Present in the genome are multiple potentially mobile genetic elements (MGEs) [plasmid, phage, integrative conjugative element (ICE)] and comparison to other species provided convincing evidence for lateral gene transfer (LGT) between S. canis and two additional bovine mastitis causing pathogens (Streptococcus agalactiae, and Streptococcus dysgalactiae subsp. dysgalactiae), with this transfer possibly contributing to host adaptation. Population structure among isolates obtained from Europe and USA [bovine = 56, canine = 26, and feline = 1] was explored. Ribotyping of all isolates and multi locus sequence typing (MLST) of a subset of the isolates (n = 45) detected significant differentiation between bovine and canine isolates (Fisher exact test: P = 0.0000 [ribotypes], P = 0.0030 [sequence types]), suggesting possible host adaptation of some genotypes. Concurrently, the ancestral clonal complex (54% of isolates) occurred in many tissue types, all hosts, and all geographic locations suggesting the possibility of a wide and diverse niche. Conclusion This study provides evidence highlighting the importance of LGT in the evolution of the bacteria S. canis, specifically, its possible role in host adaptation and acquisition of virulence factors. Furthermore, recent LGT detected between S. canis and human bacteria (Streptococcus urinalis) is cause for concern, as it highlights the possibility for continued acquisition of human virulence factors for this emerging zoonotic pathogen. PMID:23244770

Aspergillus Galactosaminogalactan Mediates Adherence to Host Constituents and Conceals Hyphal β-Glucan from the Immune System

PubMed Central

Liu, Hong; Lee, Mark J.; Snarr, Brendan D.; Chen, Dan; Xu, Wenjie; Kravtsov, Ilia; Hoareau, Christopher M. Q.; Vanier, Ghyslaine; Urb, Mirjam; Campoli, Paolo; Al Abdallah, Qusai; Lehoux, Melanie; Chabot, Josée C.; Ouimet, Marie-Claude; Baptista, Stefanie D.; Fritz, Jörg H.; Nierman, William C.; Latgé, Jean Paul; Mitchell, Aaron P.; Filler, Scott G.; Fontaine, Thierry; Sheppard, Donald C.

2013-01-01

Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall β-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis. PMID:23990787

Aureusimines in Staphylococcus aureus are not involved in virulence.

PubMed

Sun, Fei; Cho, Hoonsik; Jeong, Do-Won; Li, Chunling; He, Chuan; Bae, Taeok

2010-12-29

Recently, dipeptide aureusimines were reported to activate expression of staphylococcal virulence genes, such as alpha-hemolysin, and increase S. aureus virulence. Surprisingly, most of the virulence genes affected by aureusimines form part of the regulon of the SaeRS two component system (TCS), raising the possibility that SaeRS might be directly or indirectly involved in the aureusimine-dependent signaling process. Using HPLC analyses, we confirmed that a transposon mutant of ausA, the gene encoding the aureusimine dipeptide synthesis enzyme, does not produce dipeptides. However, the transposon mutant showed normal hemolysis activity and alpha-hemolysin/SaeP production. Furthermore, the P1 promoter of the sae operon, one of the targets of the SaeRS TCS, showed normal transcription activity. Moreover, in contrast to the original report, the ausA transposon mutant did not exhibit attenuated virulence in an animal infection model. DNA sequencing revealed that the ausA deletion mutant used in the original study has an 83 nt-duplication in saeS. Hemolysis activity of the original mutant was restored by a plasmid carrying the sae operon. A mutant of the sae operon showed elevated resistance to chloramphenicol and erythromycin, two antibiotics widely used during staphylococcal mutagenesis. At 43°C in the presence of erythromycin and aeration, the conditions typically employed for staphylococcal mutagenesis, an saeR transposon mutant grew much faster than a control mutant and the saeR mutant was highly enriched in a mixed culture experiment. Our results show that the previously reported roles of aureusimines in staphylococcal gene regulation and virulence were due to an unintended mutation in saeS, which was likely selected due to elevated resistance of the mutant to environmental stresses. Thus, there is no evidence indicating that the dipeptide aureusimines play a role in sae-mediated virulence factor production or contribute to staphylococcal virulence.

Molecular analysis of a novel Toll/interleukin-1 receptor (TIR)-domain containing virulence protein of Y. pseudotuberculosis among Far East scarlet-like fever serotype I strains.

PubMed

Nörenberg, Dominik; Wieser, Andreas; Magistro, Giuseppe; Hoffmann, Christiane; Meyer, Christian; Messerer, Maxim; Schubert, Sören

2013-12-01

Pathogenicity of Yersinia pseudotuberculosis is determined by an arsenal of virulence factors. Particularly, the Yersinia outer proteins (Yops) and the Type III secretion system (T3SS) encoded on the pYV virulence plasmid are required for Yersinia pathogenicity. A specific group of Y. pseudotuberculosis, responsible for the clinical syndrome described as Far East scarlet-like fever (FESLF), is known to have an altered virulence gene cluster. Far East strains cause unique clinical symptoms for which the pYV virulence plasmid plays apparently a rather secondary role. Here, we characterize a previously unknown protein of Y. pseudotuberculosis serotype I strains (TcpYI) which can be found particularly among the FESLF strain group. The TcpYI protein shares considerable sequence homology to members of the Toll/IL-1 receptor family. Bacterial TIR domain containing proteins (Tcps) interact with the innate immune system by TIR-TIR interactions and subvert host defenses via individual, multifaceted mechanisms. In terms of virulence, it appears that the TcpYI protein of Y. pseudotuberculosis displays its own virulence phenotype compared to the previously characterized bacterial Tcps. Our results clearly demonstrate that TcpYI increases the intracellular survival of the respective strains in vitro. Furthermore, we show here that the intracellular survival benefit of the wild-type strain correlates with an increase in tcpYI gene expression inside murine macrophages. In support of this, we found that TcpYI enhances the survival inside the spleens of mice in a mouse model of peritonitis. Our results may point toward involvement of the TcpYI protein in inhibition of phagocytosis, particularly in distinct Y. pseudotuberculosis strains of the FESLF strain group where the pYV virulence plasmid is absent. Copyright © 2013 Elsevier GmbH. All rights reserved.

Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene rosa in virulent and attenuated strains of Renibacterium salmoninarum

USGS Publications Warehouse

O'Farrell, C. L.; Strom, M.S.

1999-01-01

Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5, to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of rosa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization, and total p57 expression between 33209 anti MT 239 are not due to differences in rosa sequence or differences in steady state transcript levels.

Role of the Mce1 transporter in the lipid homeostasis of Mycobacterium tuberculosis.

PubMed

Forrellad, Marina Andrea; McNeil, Michael; Santangelo, María de la Paz; Blanco, Federico Carlos; García, Elizabeth; Klepp, Laura Inés; Huff, Jason; Niederweis, Michael; Jackson, Mary; Bigi, Fabiana

2014-03-01

Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the causative agent of human tuberculosis, has developed several strategies involving proteins and other compounds known collectively as virulence factors to subvert human host defences and invade the human host. The Mce proteins are among these virulence-related proteins and are encoded by the mce1, mce2, mce3 and mce4 operons in the genome of M. tuberculosis. It has been proposed that these operons encode ABC-like lipid transporters; however, the nature of their substrates has only been revealed in the case of the Mce4 proteins. Here we found that the knockout of the mce1 operon alters the lipid profile of M. tuberculosis H37Rv and the uptake of palmitic acid. Thin layer chromatography and liquid chromatography-mass spectrometry analysis showed that the mce1 mutant accumulates more mycolic acids than the wild type and complemented strains. Interestingly, this accumulation of mycolic acid is exacerbated when bacteria are cultured in the presence of palmitic acid or arachidonic acid. These results suggest that the mce1 operon may serve as a mycolic acid re-importer. Copyright © 2013 Elsevier Ltd. All rights reserved.

Staphylococcus aureus genomics and the impact of horizontal gene transfer.

PubMed

Lindsay, Jodi A

2014-03-01

Whole genome sequencing and microarrays have revealed the population structure of Staphylococcus aureus, and identified epidemiological shifts, transmission routes, and adaptation of major clones. S. aureus genomes are highly diverse. This is partly due to a population structure of conserved lineages, each with unique combinations of genes encoding surface proteins, regulators, immune evasion and virulence pathways. Even more variable are the mobile genetic elements (MGE), which encode key proteins for antibiotic resistance, virulence and host-adaptation. MGEs can transfer at high frequency between isolates of the same lineage by horizontal gene transfer (HGT). There is increasing evidence that HGT is key to bacterial adaptation and success. Recent studies have shed light on new mechanisms of DNA transfer such as transformation, the identification of receptors for transduction, on integration of DNA pathways, mechanisms blocking transfer including CRISPR and new restriction systems, strategies for evasion of restriction barriers, as well as factors influencing MGE selection and stability. These studies have also lead to new tools enabling construction of genetically modified clinical S. aureus isolates. This review will focus on HGT mechanisms and their importance in shaping the evolution of new clones adapted to antibiotic resistance, healthcare, communities and livestock. Copyright © 2013 Elsevier GmbH. All rights reserved.

Antibiotic resistance phenotypes and virulence-associated genes in Escherichia coli isolated from animals and animal food products in Tunisia.

PubMed

Badi, Souhir; Cremonesi, Paola; Abbassi, Mohamed Salah; Ibrahim, Chourouk; Snoussi, Majdi; Bignoli, Giulia; Luini, Mario; Castiglioni, Bianca; Hassen, Abdennaceur

2018-05-01

Livestock and food products of animal origin constitute important reservoirs of intestinal and extraintestinal pathogenic Escherichia coli including antibiotic-resistant E. coli isolates. To assess potential risks to public health related to E. coli strains of animal origin in Tunisia, 65 E. coli isolates recovered from healthy animals and food products of animal origin were studied. Antimicrobial susceptibility was determined according to CLSI guidelines and genes encoding antibiotic resistance as well as virulence factors were investigated by PCR. High rates of antibiotic resistance were observed to kanamycin (78.4%), gentamicin (75.3%) and streptomycin (75.3%, encoded by strA-strB (7 isolates)), amoxicillin (64.6%), amoxicillin/clavulanic acid (60%), tetracycline (44.6%; tetA (8 isolates) and tetB (7 isolates)), nalidixic acid (27.6%, qnrS (3 isolates), qnrB (2 isolates) and qnrA (one isolate)) and sulfonamides (36.9%; sul1 (1 isolate), sul2 (4 isolates), and sul3 (1 isolate)). Virulotypes classified some isolates as STEC (3%), MNEC (1.5%) and atypical EPEC (1.5%). This study demonstrated high rates of antimicrobial resistance and the presence of some pathogenic pathovars from animal origins that are a cause of concern for public health.

The well-coordinated linkage between acidogenicity and aciduricity via insoluble glucans on the surface of Streptococcus mutans

PubMed Central

Guo, Lihong; McLean, Jeffrey S.; Lux, Renate; He, Xuesong; Shi, Wenyuan

2015-01-01

Streptococcus mutans is considered the principal cariogenic bacterium for dental caries. Despite the recognition of their importance for cariogenesis, the possible coordination among S. mutans’ main virulence factors, including glucan production, acidogenicity and aciduricity, has been less well studied. In the present study, using S. mutans strains with surface-displayed pH-sensitive pHluorin, we revealed sucrose availability- and Gtf functionality-dependent proton accumulation on S. mutans surface. Consistent with this, using a pH-sensitive dye, we demonstrated that both in vivo cell-produced and in vitro enzymatically synthesized insoluble glucans displayed proton-concentrating ability. Global transcriptomics revealed proton accumulation triggers the up-regulation of genes encoding functions involved in acid tolerance response in a glucan-dependent manner. Our data suggested that this proton enrichment around S. mutans could pre-condition the bacterium for acid-stress. Consistent with this hypothesis, we found S. mutans strains defective in glucan production were more acid sensitive. Our study revealed for the first time that insoluble glucans is likely an essential factor linking acidogenicity with aciduricity. The coordination of these key virulence factors could provide new insights on how S. mutans may have become a major cariogenic pathogen. PMID:26657939

The Outer Membrane Protein A (OmpA) of Y. pestis promotes intracellular survival and virulence in mice

PubMed Central

Bartra, Sara Schesser; Gong, Xin; Lorica, Cherish D.; Jain, Chaitanya; Nair, Manoj K. M.; Schifferli, Dieter; Qian, Lianfen; Li, Zhongwei; Plano, Gregory V.; Schesser, Kurt

2011-01-01

The plague bacterium Yersinia pestis has a number of well-described strategies to protect itself from both host cells and soluble factors. In an effort to identify additional anti-host factors, we employed a transposon site hybridization (TraSH)-based approach to screen 105 Y. pestis mutants in an in vitro infection system. In addition to loci encoding various components of the well-characterized type III secretion system (T3SS), our screen unambiguously identified ompA as a pro-survival gene. We go on to show that an engineered Y. pestis ΔompA strain, as well as a ΔompA strain of the closely related pathogen Y. pseudotuberculosis, have fully functioning T3SSs but are specifically defective in surviving within macrophages. Additionally, the Y. pestis ΔompA strain was outcompeted by the wild-type strain in a mouse co-infection assay. Unlike in other bacterial pathogens in which OmpA can promote adherence, invasion, or serum resistance, the OmpA of Y. pestis is restricted to enhancing intracellular survival. Our data show that OmpA of the pathogenic Yersinia is a virulence factor on par with the T3SS. PMID:22023991

Virulence characteristics of extraintestinal pathogenic Escherichia coli deletion of gene encoding the outer membrane protein X.

PubMed

Meng, Xianrong; Liu, Xueling; Zhang, Liyuan; Hou, Bo; Li, Binyou; Tan, Chen; Li, Zili; Zhou, Rui; Li, Shaowen

2016-09-01

Outer membrane protein X (OmpX) and its homologues have been proposed to contribute to the virulence in various bacterial species. But, their role in virulence of extraintestinal pathogenic Escherichia coli (ExPEC) is yet to be determined. This study evaluates the role of OmpX in ExPEC virulence in vitro and in vivo using a clinical strain PPECC42 of porcine origin. The ompX deletion mutant exhibited increased swimming motility and decreased adhesion to, and invasion of pulmonary epithelial A549 cell, compared to the wild-type strain. A mild increase in LD50 and distinct decrease in bacterial load in such organs as heart, liver, spleen, lung and kidney were observed in mice infected with the ompX mutant. Complementation of the complete ompX gene in trans restored the virulence of mutant strain to the level of wild-type strain. Our results reveal that OmpX contributes to ExPEC virulence, but may be not an indispensable virulence determinant.

MaHog1, a Hog1-type mitogen-activated protein kinase gene, contributes to stress tolerance and virulence of the entomopathogenic fungus Metarhizium acridum.

PubMed

Jin, Kai; Ming, Yue; Xia, Yu Xian

2012-12-01

Fungal biocontrol agents have great potential in integrated pest management. However, poor efficacy and sensitivity to various adverse factors have hampered their wide application. In eukaryotic cells, Hog1 kinase plays a critical role in stress responses. In this study, MaHog1 (GenBank accession no. EFY85878), encoding a member of the Hog1/Sty1/p38 mitogen-activated protein kinase family in Metarhizium (Me.) acridum, was identified. Targeted gene disruption was used to analyse the role of MaHog1 in virulence and tolerance of adverse factors. Mutants with MaHog1 depletion showed increased sensitivity to high osmotic stress, high temperature and oxidative stress, and exhibited remarkable resistance to cell wall-disturbing agents. These results suggest that Hog1 kinase has a conserved function in regulating multistress responses among fungi, and that MaHog1 might influence cell wall biogenesis in Me. acridum. Bioassays conducted with topical inoculation and intrahaemocoel injection revealed that MaHog1 is required for both penetration and postpenetration development of Me. acridum. MaHog1 disruption resulted in a significant reduction in virulence, likely due to the combination of a decrease in conidial germination, a reduction in appressorium formation and a decline in growth rate in insect haemolymph, which might be caused by impairing fungal tolerance of various stresses during infection.

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Broussard, Tyler C.; Bose, Jeffrey L.; Rosch, Jason W.; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O.

2014-01-01

Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus. PMID:25002480

Distribution and regulation of the mobile genetic element-encoded phenol-soluble modulin PSM-mec in methicillin-resistant Staphylococcus aureus.

PubMed

Chatterjee, Som S; Chen, Liang; Joo, Hwang-Soo; Cheung, Gordon Y C; Kreiswirth, Barry N; Otto, Michael

2011-01-01

The phenol-soluble modulin PSM-mec is the only known staphylococcal toxin that is encoded on a mobile antibiotic resistance determinant, namely the staphylococcal cassette chromosome (SCC) element mec encoding resistance to methicillin. Here we show that the psm-mec gene is found frequently among methicillin-resistant Staphylococcus aureus (MRSA) strains of SCCmec types II, III, and VIII, and is a conserved part of the class A mec gene complex. Controlled expression of AgrA versus RNAIII in agr mutants of all 3 psm-mec-positive SCCmec types demonstrated that expression of psm-mec, which is highly variable, is controlled by AgrA in an RNAIII-independent manner. Furthermore, psm-mec isogenic deletion mutants showed only minor changes in PSMα peptide production and unchanged (or, as previously described, diminished) virulence compared to the corresponding wild-type strains in a mouse model of skin infection. This indicates that the recently reported regulatory impact of the psm-mec locus on MRSA virulence, which is opposite to that of the PSM-mec peptide and likely mediated by a regulatory RNA, is minor when analyzed in the original strain background. Our study gives new insight in the distribution, regulation, and role in virulence of the PSM-mec peptide and the psm-mec gene locus.

Occurrence of virulence-associated genes among Staphylococcus saprophyticus isolated from different sources.

PubMed

de Paiva-Santos, Weslley; de Sousa, Viviane Santos; Giambiagi-deMarval, Marcia

2018-03-28

Staphylococcus saprophyticus is an important pathogen responsible for community urinary tract infections (UTI). Besides composing the human microbiota, this species is widely distributed in the environment and the origins of this organism for human infection is not fully characterized. Although some virulence determinants are known, such as d-serine deaminase (DsdA), urease and cell-wall associated proteins, few studies investigated the distribution of virulence-associated genes and analyzed the pathogenic potential of S. saprophyticus strains from different sources. The aim of the present study was to detect the presence of S. saprophyticus genes encoding surface proteins UafA, Aas, Ssp, SdrI, SssF as well as the DsdA and urease enzymes. A total of 142 S. saprophyticus strains were obtained from four sources: UTI, colonization, water and food. It was found, in every tested strain, the presence of genes encoding the surface proteins UafA, Aas, Ssp and SssF and the DsdA and urease enzymes. In contrast, the gene encoding SdrI surface protein was not detected in any of the strains of S. saprophyticus. These results provide a better understanding of the characteristics of S. saprophyticus strains and suggest that isolates from non-human sources have a potential to colonize the urinary tract. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Virulence Plasmid of Yersinia, an Antihost Genome

PubMed Central

Cornelis, Guy R.; Boland, Anne; Boyd, Aoife P.; Geuijen, Cecile; Iriarte, Maite; Neyt, Cécile; Sory, Marie-Paule; Stainier, Isabelle

1998-01-01

The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds. PMID:9841674

Vaccinia virus recombinants encoding the truncated structural gene region of Venezuelan equine encephalitis virus (VEEV) give solid protection against peripheral challenge but only partial protection against airborne challenge with virulent VEEV.

PubMed

Phillpotts, R J; Lescott, T L; Jacobs, S C

2000-10-01

Vaccinia virus (VV) recombinants that contain the genes encoding the Venezuelan equine encephalitis virus (VEEV) structural gene region (C-E3-E2-6 K-E1) solidly protect mice against peripheral challenge with virulent VEEV, but provide only partial protection against airborne challenge. To improve upon these results we focussed on the principal antigens involved in protection. VV recombinants encoding the structural genes E3-E2-6 K-E1, E3-E2-6 K or 6 K-E1 were prepared and evaluated for their ability to protect Balb/c mice after a single dorsal scarification with 10(8) PFU against peripheral or airborne challenge with virulent VEEV. The antibody response was also examined. Our experiments provide new evidence that truncates of the VEEV structural region (E3-E2-6 K-E1, E3-E2-6 K), cloned and expressed in VV, protect against challenge with virulent virus. They also confirm the important role of E2 in protection. However, we were unable to improve upon previously reported levels of protection against airborne challenge. A substantial level of circulating antibodies and the presence of local IgA (not always induced by mucosal immunization) (Greenway et al., 1992) appear essential for protection against the airborne virus. Current VV-VEEV recombinants seem unable to elicit this level of immune response and further improvements are therefore required to increase the immunogenicity of VV-VEEV vaccines.

An Emerging Mycoplasma Associated with Trichomoniasis, Vaginal Infection and Disease

PubMed Central

Fettweis, Jennifer M.; Serrano, Myrna G.; Huang, Bernice; Brooks, J. Paul; Glascock, Abigail L.; Sheth, Nihar U.; Strauss, Jerome F.; Jefferson, Kimberly K.; Buck, Gregory A.

2014-01-01

Humans are colonized by thousands of bacterial species, but it is difficult to assess the metabolic and pathogenic potential of the majority of these because they have yet to be cultured. Here, we characterize an uncultivated vaginal mycoplasma tightly associated with trichomoniasis that was previously known by its 16S rRNA sequence as “Mnola.” In this study, the mycoplasma was found almost exclusively in women infected with the sexually transmitted pathogen Trichomonas vaginalis, but rarely observed in women with no diagnosed disease. The genomes of four strains of this species were reconstructed using metagenome sequencing and assembly of DNA from four discrete mid-vaginal samples, one of which was obtained from a pregnant woman with trichomoniasis who delivered prematurely. These bacteria harbor several putative virulence factors and display unique metabolic strategies. Genes encoding proteins with high similarity to potential virulence factors include two collagenases, a hemolysin, an O-sialoglycoprotein endopeptidase and a feoB-type ferrous iron transport system. We propose the name “Candidatus Mycoplasma girerdii” for this potential new pathogen. PMID:25337710

Effect of Mild Acid on Gene Expression in Staphylococcus aureus

PubMed Central

Weinrick, Brian; Dunman, Paul M.; McAleese, Fionnuala; Murphy, Ellen; Projan, Steven J.; Fang, Yuan; Novick, Richard P.

2004-01-01

During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, ∼5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus. PMID:15576791

Listeria monocytogenes Isolates Carrying Virulence-Attenuating Mutations in Internalin A Are Commonly Isolated from Ready-to-Eat Food Processing Plant and Retail Environments.

PubMed

VAN Stelten, A; Roberts, A R; Manuel, C S; Nightingale, K K

2016-10-01

Listeria monocytogenes is a human foodborne pathogen that may cause an invasive disease known as listeriosis in susceptible individuals. Internalin A (InlA; encoded by inlA) is a virulence factor that facilitates crossing of host cell barriers by L. monocytogenes . At least 19 single nucleotide polymorphisms (SNPs) in inlA that result in a premature stop codon (PMSC) have been described worldwide. SNPs leading to a PMSC in inlA have been shown to be causally associated with attenuated virulence. L. monocytogenes pathogens carrying virulence-attenuating (VA) mutations in inlA have been commonly isolated from ready-to-eat (RTE) foods but rarely have been associated with human disease. This study was conducted to determine the prevalence of VA SNPs in inlA among L. monocytogenes from environments associated with RTE food production and handling. More than 700 L. monocytogenes isolates from RTE food processing plant (n = 409) and retail (n = 319) environments were screened for the presence of VA SNPs in inlA. Overall, 26.4% of isolates from RTE food processing plant and 32.6% of isolates from retail environments carried a VA mutation in inlA. Food contact surfaces sampled at retail establishments were significantly (P < 0.0001) more likely to be contaminated by a L. monocytogenes isolate carrying a VA mutation in inlA (56% of 55 isolates) compared with nonfood contact surfaces (28% of 264 isolates). Overall, a significant proportion of L. monocytogenes isolated from RTE food production and handling environments have reduced virulence. These data will be useful in the revision of current and the development of future risk assessments that incorporate strain-specific virulence parameters.

Determination of resistance and virulence genes in Enterococcus faecalis and E. faecium strains isolated from poultry and their genotypic characterization by ADSRRS-fingerprinting.

PubMed

Nowakiewicz, A; Ziólkowska, G; Troscianczyk, A; Zieba, P; Gnat, S

2017-04-01

The aim of this study was to determine the antimicrobial resistance of E. faecalis and E. faecium strains isolated from poultry and to carry out genotypic characterization thereof with the ADSRRS-fingerprinting method (amplification of DNA fragments surrounding rare restriction sites) and analysis of the genetic relatedness between the isolates with different resistance and virulence determinants. Samples were collected from 70 4-week-old chickens and tested for Enterococcus. Minimum inhibitory concentrations of 11 antimicrobials were determined using the broth microdilution method. Detection of antibiotic resistance and virulence genes was performed using PCR, and molecular analysis was carried out using the ADSRRS-fingerprinting method. The highest percentage of strains was resistant to tetracycline (60.5%) and erythromycin (54.4%), and a large number exhibited high-level resistance to both kanamycin (42.1%) and streptomycin (34.2%). Among 8 genes encoding AME, the tested strains showed mainly the presence of [aph(3΄)-IIIa], [ant(6)-Ia], [aac(6΄)-Ie-aph(2΄΄)-Ia], and [ant(9)-Ia] genes. Phenotypic resistance to erythromycin was encoded in 98.4% strains by the ermB gene. Genotypic resistance to tetracycline in E. faecium was associated with the presence of tetM and tetL (respectively, in 95.5 and 57.7% of the isolates); in contrast, E. faecalis strains were characterized mainly by the presence of tetO (83.3%). The virulence profile was homogenous for all E. faecium strains and included only efaAfm and ccf genes. All E. faecalis strains exhibited efaAfs, gelE, and genes encoding sex pheromones. The strains tested exhibited 34 genotypic profiles. Comparative analysis of phenotypic and genotypic resistance and virulence profiles and confrontation thereof with the genotypes of the strains tested showed that strains assigned to a particular genotype have an identical phenotypic resistance profile and a panel of resistance and virulence genes. The results of this study confirm that poultry can be a reservoir of resistant E. faecium and E. faecalis strains with multiple combinations of resistance and virulence genes, whose specific panel determines not only phenotypic characteristics but also has a strong correlation with the genotypic profiles of the strains. © 2016 Poultry Science Association Inc.

Effects of Subinhibitory Concentrations of Antibiotics on Alpha-Toxin (hla) Gene Expression of Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Isolates

PubMed Central

Ohlsen, Knut; Ziebuhr, Wilma; Koller, Klaus-Peter; Hell, Wolfgang; Wichelhaus, Thomas A.; Hacker, Jörg

1998-01-01

Concentrations of antibiotics below the MIC are able to modulate the expression of virulence-associated genes. In this study, the influence of subinhibitory doses of 31 antibiotics on the expression of the gene encoding the staphylococcal alpha-toxin (hla), a major virulence factor of Staphylococcus aureus, was investigated with a novel gene fusion protocol. The most striking observation was a strong induction of hla expression by subinhibitory concentrations of β-lactams and an almost complete inhibition of alpha-toxin expression by clindamycin. Whereas glycopeptide antibiotics had no effect, the macrolide erythromycin and several aminoglycosides reduced and fluoroquinolones slightly stimulated hla expression. Furthermore, Northern blot analysis of hla mRNA and Western blot (immunoblot) analysis of culture supernatants of both methicillin-sensitive and methicillin-resistant S. aureus strains revealed that methicillin-induced alpha-toxin expression is a common phenomenon of alpha-toxin-producing strains. Some methicillin-resistant S. aureus isolates produced up to 30-fold more alpha-toxin in the presence of 10 μg of methicillin per ml than in its absence. The results indicate that the novel gene fusion technique is a useful tool for studying the modulation of virulence gene expression by antibiotics. Moreover, the results suggest that the effects of certain antibiotics on virulence properties may be relevant for the management of S. aureus infections. PMID:9797209

Identification of the anti-terminator qO111:H)- gene in Norwegian sorbitol-fermenting Escherichia coli O157:NM.

PubMed

Haugum, Kjersti; Lindstedt, Bjørn-Arne; Løbersli, Inger; Kapperud, Georg; Brandal, Lin Thorstensen

2012-04-01

Sorbitol-fermenting Escherichia coli O157:NM (SF O157) is an emerging pathogen suggested to be more virulent than nonsorbitol-fermenting Escherichia coli O157:H7 (NSF O157). Important virulence factors are the Shiga toxins (stx), encoded by stx1 and/or stx2 located within prophages integrated in the bacterial genome. The stx genes are expressed from p(R) (') as a late protein, and anti-terminator activity from the Q protein is necessary for read through of the late terminator t(R) (') and activation of p(R) (') . We investigated the regulation of stx2(EDL933) expression at the genomic level in 17 Norwegian SF O157. Sequencing of three selected SF O157 strains revealed that the anti-terminator q gene and genes upstream of stx2(EDL933) were identical or similar to the ones observed in the E. coli O111:H- strain AP010960, but different from the ones observed in the NSF O157 strain EDL933 (AE005174). This suggested divergent stx2(EDL933) -encoding bacteriophages between NSF O157 and the SF O157 strains (FR874039-41). Furthermore, different DNA structures were detected in the SF O157 strains, suggesting diversity among bacteriophages also within the SF O157 group. Further investigations are needed to elucidate whether the q(O111:H) (-) gene observed in all our SF O157 contributes to the increased virulence seen in SF O157 compared to NSF O157. An assay for detecting q(O111:H) (-) was developed. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

A Unique Chromosomal Rearrangement in the Cryptococcus neoformans var. grubii Type Strain Enhances Key Phenotypes Associated with Virulence

PubMed Central

Morrow, Carl A.; Lee, I. Russel; Chow, Eve W. L.; Ormerod, Kate L.; Goldinger, Anita; Byrnes, Edmond J.; Nielsen, Kirsten; Heitman, Joseph; Schirra, Horst Joachim; Fraser, James A.

2012-01-01

ABSTRACT The accumulation of genomic structural variation between closely related populations over time can lead to reproductive isolation and speciation. The fungal pathogen Cryptococcus is thought to have recently diversified, forming a species complex containing members with distinct morphologies, distributions, and pathologies of infection. We have investigated structural changes in genomic architecture such as inversions and translocations that distinguish the most pathogenic variety, Cryptococcus neoformans var. grubii, from the less clinically prevalent Cryptococcus neoformans var. neoformans and Cryptococcus gattii. Synteny analysis between the genomes of the three Cryptococcus species/varieties (strains H99, JEC21, and R265) reveals that C. neoformans var. grubii possesses surprisingly few unique genomic rearrangements. All but one are relatively small and are shared by all molecular subtypes of C. neoformans var. grubii. In contrast, the large translocation peculiar to the C. neoformans var. grubii type strain is found in all tested subcultures from multiple laboratories, suggesting that it has possessed this rearrangement since its isolation from a human clinical sample. Furthermore, we find that the translocation directly disrupts two genes. The first of these encodes a novel protein involved in metabolism of glucose at human body temperature and affects intracellular levels of trehalose. The second encodes a homeodomain-containing transcription factor that modulates melanin production. Both mutations would be predicted to increase pathogenicity; however, when recreated in an alternate genetic background, these mutations do not affect virulence in animal models. The type strain of C. neoformans var. grubii in which the majority of molecular studies have been performed is therefore atypical for carbon metabolism and key virulence attributes. PMID:22375073

Genome-Wide Analyses Reveal Genes Subject to Positive Selection in Pasteurella multocida

PubMed Central

Cao, Peili; Guo, Dongchun; Liu, Jiasen; Jiang, Qian; Xu, Zhuofei; Qu, Liandong

2017-01-01

Pasteurella multocida, a Gram-negative opportunistic pathogen, has led to a broad range of diseases in mammals and birds, including fowl cholera in poultry, pneumonia and atrophic rhinitis in swine and rabbit, hemorrhagic septicemia in cattle, and bite infections in humans. In order to better interpret the genetic diversity and adaptation evolution of this pathogen, seven genomes of P. multocida strains isolated from fowls, rabbit and pigs were determined by using high-throughput sequencing approach. Together with publicly available P. multocida genomes, evolutionary features were systematically analyzed in this study. Clustering of 70,565 protein-coding genes showed that the pangenome of 33 P. multocida strains was composed of 1,602 core genes, 1,364 dispensable genes, and 1,070 strain-specific genes. Of these, we identified a full spectrum of genes related to virulence factors and revealed genetic diversity of these potential virulence markers across P. multocida strains, e.g., bcbAB, fcbC, lipA, bexDCA, ctrCD, lgtA, lgtC, lic2A involved in biogenesis of surface polysaccharides, hsf encoding autotransporter adhesin, and fhaB encoding filamentous haemagglutinin. Furthermore, based on genome-wide positive selection scanning, a total of 35 genes were subject to strong selection pressure. Extensive analyses of protein subcellular location indicated that membrane-associated genes were highly abundant among all positively selected genes. The detected amino acid sites undergoing adaptive selection were preferably located in extracellular space, perhaps associated with bacterial evasion of host immune responses. Our findings shed more light on conservation and distribution of virulence-associated genes across P. multocida strains. Meanwhile, this study provides a genetic context for future researches on the mechanism of adaptive evolution in P. multocida. PMID:28611758

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

PubMed Central

Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

2005-01-01

Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

PubMed

Matsunaga, James; Barocchi, Michele A; Croda, Julio; Young, Tracy A; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A; Reis, Mitermayer G; Riley, Lee W; Haake, David A; Ko, Albert I

2003-08-01

Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.

Virulence factors in Escherichia coli urinary tract infection.

PubMed Central

Johnson, J R

1991-01-01

Uropathogenic strains of Escherichia coli are characterized by the expression of distinctive bacterial properties, products, or structures referred to as virulence factors because they help the organism overcome host defenses and colonize or invade the urinary tract. Virulence factors of recognized importance in the pathogenesis of urinary tract infection (UTI) include adhesins (P fimbriae, certain other mannose-resistant adhesins, and type 1 fimbriae), the aerobactin system, hemolysin, K capsule, and resistance to serum killing. This review summarizes the virtual explosion of information regarding the epidemiology, biochemistry, mechanisms of action, and genetic basis of these urovirulence factors that has occurred in the past decade and identifies areas in need of further study. Virulence factor expression is more common among certain genetically related groups of E. coli which constitute virulent clones within the larger E. coli population. In general, the more virulence factors a strain expresses, the more severe an infection it is able to cause. Certain virulence factors specifically favor the development of pyelonephritis, others favor cystitis, and others favor asymptomatic bacteriuria. The currently defined virulence factors clearly contribute to the virulence of wild-type strains but are usually insufficient in themselves to transform an avirulent organism into a pathogen, demonstrating that other as-yet-undefined virulence properties await discovery. Virulence factor testing is a useful epidemiological and research tool but as yet has no defined clinical role. Immunological and biochemical anti-virulence factor interventions are effective in animal models of UTI and hold promise for the prevention of UTI in humans. Images PMID:1672263

[Orthopoxvirus genes for kelch-like proteins. III. Construction of mousepox (ectromelia) virus variants with targeted gene deletions].

PubMed

Kochneva, G V; Kolosova, I V; Lupan, T A; Sivolobova, G F; Iudin, P V; Grazhdantseva, A A; Riabchikova, E I; Kandrina, N Iu; Shchelkunov, S N

2009-01-01

Mousepox (ectromelia) virus genome contains four genes encoding for kelch-like proteins EVM018, EVM027, EVM150 and EVM167. A complete set of insertion plasmids was constructed to allow the production of recombinant ectromelia viruses with targeted deletions of one to four genes of kelch family both individually (single mutants) and in different combinations (double, triple and quadruple mutants). It was shown that deletion of any of the three genes EVMO18, EVM027 or EVM167 resulted in reduction of 50% lethal dose (LD50) by five and more orders in outbred white mice infected intraperitoneally. Deletion of mousepox kelch-gene EVM150 did not influence the virus virulence. Two or more kelch-genes deletion also resulted in high level of attenuation, which could evidently be due to the lack of three genes EVM167, EVM018 and/or EVM027 identified as virulence factors. The local inflammatory process on the model of intradermal injection of mouse ear pinnae (vasodilatation level, hyperemia, cutaneous edema, arterial thrombosis) was significantly more intensive for wild type virus and virulent mutant deltaEVM150 in comparison with avirulent mutant AEVM167.

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments.

PubMed

Miller, Daniel P; Hutcherson, Justin A; Wang, Yan; Nowakowska, Zuzanna M; Potempa, Jan; Yoder-Himes, Deborah R; Scott, David A; Whiteley, Marvin; Lamont, Richard J

2017-01-01

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis , and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

Functional analysis of the cytochrome P450 monooxygenase gene bcbot1 of Botrytis cinerea indicates that botrydial is a strain-specific virulence factor.

PubMed

Siewers, Verena; Viaud, Muriel; Jimenez-Teja, Daniel; Collado, Isidro G; Gronover, Christian Schulze; Pradier, Jean-Marc; Tudzynski, Bettina; Tudzynski, Paul

2005-06-01

The micrographic phytopathogen Botrytis cinerea causes gray mold diseases in a large number of dicotyledonous crop plants and ornamentals. Colonization of host tissue is accompanied by rapid killing of plant cells ahead of the growing hyphen, probably caused by secretion of nonspecific phytotoxins, e.g., the sesquiterpene botrydial. Although all pathogenic strains tested so far had been shown to secrete botrydial and although the toxin causes comparable necrotic lesions as infection by the fungus, the role of botrydial in the infection process has not been elucidated so far. Here, we describe the functional characterization of bcbot1, encoding a P450 monooxygenase and provide evidence that it is involved in the botrydial pathway, i.e., it represents the first botrydial biosynthetic gene identified. We show that bcbot1 is expressed in planta and that expression in vitro and in planta is controlled by an alpha-subunit of a heterotrimeric GTP-binding protein, BCG1. Deletion of bcbot1 in three standard strains of B. cinerea shows that the effect on virulence (on several host plants) is strain-dependent; only deletion in one of the strains (T4) led to reduced virulence.

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

PubMed Central

Flores-Díaz, Marietta; Monturiol-Gross, Laura; Naylor, Claire

2016-01-01

SUMMARY Bacterial sphingomyelinases and phospholipases are a heterogeneous group of esterases which are usually surface associated or secreted by a wide variety of Gram-positive and Gram-negative bacteria. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, generating products identical to the ones produced by eukaryotic enzymes which play crucial roles in distinct physiological processes, including membrane dynamics, cellular signaling, migration, growth, and death. Several bacterial sphingomyelinases and phospholipases are essential for virulence of extracellular, facultative, or obligate intracellular pathogens, as these enzymes contribute to phagosomal escape or phagosomal maturation avoidance, favoring tissue colonization, infection establishment and progression, or immune response evasion. This work presents a classification proposal for bacterial sphingomyelinases and phospholipases that considers not only their enzymatic activities but also their structural aspects. An overview of the main physiopathological activities is provided for each enzyme type, as are examples in which inactivation of a sphingomyelinase- or a phospholipase-encoding gene impairs the virulence of a pathogen. The identification of sphingomyelinases and phospholipases important for bacterial pathogenesis and the development of inhibitors for these enzymes could generate candidate vaccines and therapeutic agents, which will diminish the impacts of the associated human and animal diseases. PMID:27307578

The Staphylococcus aureus leucine aminopeptidase LAP is localized to the bacterial cytosol and demonstrates a broad substrate range that extends beyond leucine

PubMed Central

Carroll, Ronan K.; Veillard, Florian; Gagne, Danielle T.; Lindenmuth, Jarrod M.; Poreba, Marcin; Drag, Marcin; Potempa, Jan; Shaw, Lindsey N.

2013-01-01

Staphylococcus aureus is a potent pathogen of humans exhibiting a broad disease range, in part, due to an extensive repertoire of secreted virulence factors, including proteases. Recently, we identified the first example of an intracellular protease (leucine aminopeptidase - LAP) that is required for virulence in S. aureus. Disruption of pepZ, the gene encoding LAP, had no affect on the growth rate of bacteria, however, in systemic and localized infection models the pepZ mutant was significantly attenuated in virulence. Recently, a contradictory report has been published, suggesting that LAP is an extracellular enzyme and it is required for growth in S. aureus. Here, we investigate these results and confirm our previous findings that LAP is localized to the bacterial cytosol and is not required for growth. In addition we conduct a biochemical investigation of purified recombinant LAP identifying optimal conditions for enzymatic activity and substrate preference for hydrolysis. Our results show that LAP has a broad substrate range, including activity against the dipeptide cysteine-glycine and that leucine is not the primary target of LAP. PMID:23241672

Identification of a novel adhesion molecule involved in the virulence of Legionella pneumophila.

PubMed

Chang, Bin; Kura, Fumiaki; Amemura-Maekawa, Junko; Koizumi, Nobuo; Watanabe, Haruo

2005-07-01

Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.

Communications of Staphylococcus aureus and non-aureus Staphylococcus species from bovine intramammary infections and teat apex colonization.

PubMed

Mahmmod, Yasser S; Klaas, Ilka Christine; Svennesen, Line; Pedersen, Karl; Ingmer, Hanne

2018-05-16

The role of non-aureus staphylococci (NAS) in the risk of acquisition of intramammary infections with Staphylococcus aureus is vague and still under debate. The objectives of this study were to (1) investigate the distribution patterns of NAS species from milk and teat skin in dairy herds with automatic milking systems, and (2) examine if the isolated NAS influences the expression of S. aureus virulence factors controlled by the accessory gene regulator (agr) quorum sensing system. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabbing and aseptic quarter foremilk samples from right hind and left front quarters. Teat skin swabs were collected using the modified wet-dry method and milk samples were taken aseptically for bacterial culture. Colonies from quarters with suspicion of having NAS in milk or teat skin samples (or both) were subjected to MALDI-TOF assay for species identification. To investigate the interaction between S. aureus and NAS, 81 isolates NAS were subjected to a qualitative β-galactosidase reporter plate assay. In total, 373 NAS isolates were identified representing 105 from milk and 268 from teat skin of 284 quarters (= 142 cows). Sixteen different NAS species were identified, 15 species from teat skin and 10 species from milk. The most prevalent NAS species identified from milk were Staphylococcus epidermidis (50%), Staphylococcus haemolyticus (15%), and Staphylococcus chromogenes (11%), accounting for 76%. Meanwhile, the most prevalent NAS species from teat skin were Staphylococcus equorum (43%), S. haemolyticus (16%), and Staphylococcus cohnii (14%), accounting for 73%. Using reporter gene fusions monitoring transcriptional activity of key virulence factors and regulators, we found that out of 81 supernatants of NAS isolates, 77% reduced expression of hla, encoding a-hemolysin, 70% reduced expression of RNAIII, the key effector molecule of agr, and 61% reduced expression of spa encoding protein A of S. aureus, respectively. Our NAS isolates showed 3 main patterns: (1) downregulation effect such as S. chromogenes (milk) and Staphylococcus xylosus (milk and teat), (2) no effect such as Staphylococcus sciuri (teat) and S. vitulinus (teat), and the third pattern (c) variable effect such as S. epidermidis (milk and teat) and S. equorum (milk and teat). The pattern of cross-talk between NAS species and S. aureus virulence genes varied according to the involved NAS species, habitat type, and herd factors. The knowledge of how NAS influences S. aureus virulence factor expression could explain the varying protective effect of NAS on S. aureus intramammary infections. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Whole Genome Sequences of Three Treponema pallidum ssp. pertenue Strains: Yaws and Syphilis Treponemes Differ in Less than 0.2% of the Genome Sequence

PubMed Central

Chen, Lei; Pospíšilová, Petra; Strouhal, Michal; Qin, Xiang; Mikalová, Lenka; Norris, Steven J.; Muzny, Donna M.; Gibbs, Richard A.; Fulton, Lucinda L.; Sodergren, Erica; Weinstock, George M.; Šmajs, David

2012-01-01

Background The yaws treponemes, Treponema pallidum ssp. pertenue (TPE) strains, are closely related to syphilis causing strains of Treponema pallidum ssp. pallidum (TPA). Both yaws and syphilis are distinguished on the basis of epidemiological characteristics, clinical symptoms, and several genetic signatures of the corresponding causative agents. Methodology/Principal Findings To precisely define genetic differences between TPA and TPE, high-quality whole genome sequences of three TPE strains (Samoa D, CDC-2, Gauthier) were determined using next-generation sequencing techniques. TPE genome sequences were compared to four genomes of TPA strains (Nichols, DAL-1, SS14, Chicago). The genome structure was identical in all three TPE strains with similar length ranging between 1,139,330 bp and 1,139,744 bp. No major genome rearrangements were found when compared to the four TPA genomes. The whole genome nucleotide divergence (dA) between TPA and TPE subspecies was 4.7 and 4.8 times higher than the observed nucleotide diversity (π) among TPA and TPE strains, respectively, corresponding to 99.8% identity between TPA and TPE genomes. A set of 97 (9.9%) TPE genes encoded proteins containing two or more amino acid replacements or other major sequence changes. The TPE divergent genes were mostly from the group encoding potential virulence factors and genes encoding proteins with unknown function. Conclusions/Significance Hypothetical genes, with genetic differences, consistently found between TPE and TPA strains are candidates for syphilitic treponemes virulence factors. Seventeen TPE genes were predicted under positive selection, and eleven of them coded either for predicted exported proteins or membrane proteins suggesting their possible association with the cell surface. Sequence changes between TPE and TPA strains and changes specific to individual strains represent suitable targets for subspecies- and strain-specific molecular diagnostics. PMID:22292095

Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria.

PubMed

Strateva, Tanya; Atanasova, Daniela; Savov, Encho; Petrova, Guergana; Mitov, Ivan

2016-01-01

To evaluate the prevalence of some virulence genes among 510 clinical Enterococcus spp. isolates and to assess the association of those genes with the species, infection site, and patient group (inpatients/outpatients). Adhesins genes (aggregation substances agg and asa1 of Enterococcus faecalis and Enterococcus faecium, respectively), enterococcal surface protein (esp), endocarditis-specific antigen A (efaA), collagen-binding proteins (ace/acm)); invasins (hyaluronidase (hyl) and gelatinase (gelE)); cytotoxines (activation of cytolysin (cylA) in E. faecalis); and modulators of the host immunity and inflammation (enhanced expression pheromone (eep) in E. faecalis) were detected by polymerase chain reaction. The overall prevalence was: esp - 44.3%, agg/asa1 - 38.4%, ace/acm - 64.3%, efaA - 85.9%, eep - 69.4%, gelE - 64.3%, hyl - 25.1%, and cylA - 47.1%. E. faecalis isolates had significantly higher frequency of adhesin genes (esp and agg/asa1) and gelatinase in comparison to E. faecium. Multiple virulence genes in E. faecalis were significantly more prevalent than in E. faecium isolates. Domination of E. faecium with or without only one gene compared to the isolates of E. faecalis were found. Enterococcus spp. isolates obtained from outpatients compared to inpatients isolates had significantly higher frequency of agg/asa1, eep, gelE and cylA. Some adhesins genes (esp, agg/asa1 and efaA) had higher prevalence among the non-invasive Enterococcus spp. isolates compared to those causing invasive bacteremia, while ace/acm revealed higher dissemination in isolates causing invasive infections compared to non-invasive isolates. Most E. faecalis attaches to abiotic surfaces in hospital environment, which correlates with higher prevalence of gene encoding for virulence factors involved in biofilm formation, such as enterococcal surface protein, aggregation substance, and gelatinase. The intestinal tract is an important reservoir for opportunistic enterococcal pathogens and allows them to access infectious sites through different virulence factors, demonstrated in outpatient isolates in this study. Copyright © 2016 Elsevier Editora Ltda. All rights reserved.

The Spl Serine Proteases Modulate Staphylococcus aureus Protein Production and Virulence in a Rabbit Model of Pneumonia

PubMed Central

Salgado-Pabon, Wilmara; Meyerholz, David K.; White, Mark J.; Schlievert, Patrick M.

2016-01-01

ABSTRACT The Spl proteases are a group of six serine proteases that are encoded on the νSaβ pathogenicity island and are unique to Staphylococcus aureus. Despite their interesting biochemistry, their biological substrates and functions in virulence have been difficult to elucidate. We found that an spl operon mutant of the community-associated methicillin-resistant S. aureus USA300 strain LAC induced localized lung damage in a rabbit model of pneumonia, characterized by bronchopneumonia observed histologically. Disease in the mutant-infected rabbits was restricted in distribution compared to that in wild-type USA300-infected rabbits. We also found that SplA is able to cleave the mucin 16 glycoprotein from the surface of the CalU-3 lung cell line, suggesting a possible mechanism for wild-type USA300 spreading pneumonia to both lungs. Investigation of the secreted and surface proteomes of wild-type USA300 and the spl mutant revealed multiple alterations in metabolic proteins and virulence factors. This study demonstrates that the Spls modulate S. aureus physiology and virulence, identifies a human target of SplA, and suggests potential S. aureus targets of the Spl proteases. IMPORTANCE Staphylococcus aureus is a versatile human pathogen that produces an array of virulence factors, including several proteases. Of these, six proteases called the Spls are the least characterized. Previous evidence suggests that the Spls are expressed during human infection; however, their function is unknown. Our study shows that the Spls are required for S. aureus to cause disseminated lung damage during pneumonia. Further, we present the first example of a human protein cut by an Spl protease. Although the Spls were predicted not to cut staphylococcal proteins, we also show that an spl mutant has altered abundance of both secreted and surface-associated proteins. This work provides novel insight into the function of Spls during infection and their potential ability to degrade both staphylococcal and human proteins. PMID:27747296

Pathogen exploitation of an abscisic acid- and jasmonate-inducible MAPK phosphatase and its interception by Arabidopsis immunity.

PubMed

Mine, Akira; Berens, Matthias L; Nobori, Tatsuya; Anver, Shajahan; Fukumoto, Kaori; Winkelmüller, Thomas M; Takeda, Atsushi; Becker, Dieter; Tsuda, Kenichi

2017-07-11

Phytopathogens promote virulence by, for example, exploiting signaling pathways mediated by phytohormones such as abscisic acid (ABA) and jasmonate (JA). Some plants can counteract pathogen virulence by invoking a potent form of immunity called effector-triggered immunity (ETI). Here, we report that ABA and JA mediate inactivation of the immune-associated MAP kinases (MAPKs), MPK3 and MPK6, in Arabidopsis thaliana ABA induced expression of genes encoding the protein phosphatases 2C (PP2Cs), HAI1 , HAI2 , and HAI3 through ABF/AREB transcription factors. These three HAI PP2Cs interacted with MPK3 and MPK6 and were required for ABA-mediated MPK3/MPK6 inactivation and immune suppression. The bacterial pathogen Pseudomonas syringae pv. tomato ( Pto ) DC3000 activates ABA signaling and produces a JA-mimicking phytotoxin, coronatine (COR), that promotes virulence. We found that Pto DC3000 induces HAI1 through COR-mediated activation of MYC2, a master transcription factor in JA signaling. HAI1 dephosphorylated MPK3 and MPK6 in vitro and was necessary for COR-mediated suppression of MPK3/MPK6 activation and immunity. Intriguingly, upon ETI activation, A. thaliana plants overcame the HAI1-dependent virulence of COR by blocking JA signaling. Finally, we showed conservation of induction of HAI PP2Cs by ABA and JA in other Brassicaceae species. Taken together, these results suggest that ABA and JA signaling pathways, which are hijacked by the bacterial pathogen, converge on the HAI PP2Cs that suppress activation of the immune-associated MAPKs. Also, our data unveil interception of JA-signaling activation as a host counterstrategy against the bacterial suppression of MAPKs during ETI.

Identification of Novel Listeria monocytogenes Secreted Virulence Factors following Mutational Activation of the Central Virulence Regulator, PrfA▿ †

PubMed Central

Port, Gary C.; Freitag, Nancy E.

2007-01-01

Upon bacterial entry into the cytosol of infected mammalian host cells, the central virulence regulator PrfA of Listeria monocytogenes becomes activated and induces the expression of numerous factors which contribute to bacterial pathogenesis. The mechanism or signal by which PrfA becomes activated during the course of infection has not yet been determined; however, several amino acid substitutions within PrfA (known as PrfA* mutations) that appear to lock the protein into a constitutively activated state have been identified. In this study, the PrfA activation statuses of several L. monocytogenes mutant strains were subjected to direct isogenic comparison and the mutant with the highest activity, the prfA(L140F) mutant, was identified. The prfA(L140F) strain was subsequently used as a tool to identify gene products secreted as a result of PrfA activation. By use of two-dimensional gel electrophoresis followed by liquid chromatography-electrospray ionization-tandem mass spectroscopy analyses, 15 proteins were identified as up-regulated in the prfA(L140F) secretome, while the secretion of two proteins was found to be reduced. Although some of the proteins identified were known to be subject to direct regulation by PrfA, the majority have not previously been associated with PrfA regulation and their expression or secretion may be influenced indirectly by a PrfA-dependent regulatory pathway. Plasmid insertion inactivation of the genes encoding four novel secreted products indicated that three of the four have significant roles in L. monocytogenes virulence. The use of mutationally activated prfA alleles therefore provides a useful approach towards identifying gene products that contribute to L. monocytogenes pathogenesis. PMID:17938228

Evidence for viral virulence as a predominant factor limiting human immunodeficiency virus vaccine efficacy.

PubMed

Mooij, P; Bogers, W M; Oostermeijer, H; Koornstra, W; Ten Haaft, P J; Verstrepen, B E; Van Der Auwera, G; Heeney, J L

2000-05-01

Current strategies in human immunodeficiency virus type 1 (HIV-1) vaccine development are often based on the production of different vaccine antigens according to particular genetic clades of HIV-1 variants. To determine if virus virulence or genetic distance had a greater impact on HIV-1 vaccine efficacy, we designed a series of heterologous chimeric simian/human immunodeficiency virus (SHIV) challenge experiments in HIV-1 subunit-vaccinated rhesus macaques. Of a total of 22 animals, 10 nonimmunized animals served as controls; the remainder were vaccinated with the CCR5 binding envelope of HIV-1(W6.1D). In the first study, heterologous challenge included two nonpathogenic SHIV chimeras encoding the envelopes of the divergent clade B HIV-1(han2) and HIV-1(sf13) strains. In the second study, all immunized animals were rechallenged with SHIV(89. 6p), a virus closely related to the vaccine strain but highly virulent. Protection from either of the divergent SHIV(sf13) or SHIV(han2) challenges was demonstrated in the majority of the vaccinated animals. In contrast, upon challenge with the more related but virulent SHIV(89.6p), protection was achieved in only one of the previously protected vaccinees. A secondary but beneficial effect of immunization on virus load and CD4(+) T-cell counts was observed despite failure to protect from infection. In addition to revealing different levels of protective immunity, these results suggest the importance of developing vaccine strategies capable of protecting from particularly virulent variants of HIV-1.

Induction of virulence gene expression in Staphylococcus aureus by pulmonary surfactant.

PubMed

Ishii, Kenichi; Adachi, Tatsuo; Yasukawa, Jyunichiro; Suzuki, Yutaka; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-04-01

We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

PubMed Central

Kretschmer, Matthias; Klose, Jana

2012-01-01

An understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogen Ustilago maydis. However, mfe2 mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in the had1 gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog, had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of an mfe2Δ mutant. We also show that short-chain fatty acids induce cell death in U. maydis and that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms by U. maydis that includes potential metabolic contributions to proliferation in planta and an effect on virulence-related morphogenesis. PMID:22707484

Contribution of lipoproteins and lipoprotein processing to endocarditis virulence in Streptococcus sanguinis.

PubMed

Das, Sankar; Kanamoto, Taisei; Ge, Xiuchun; Xu, Ping; Unoki, Takeshi; Munro, Cindy L; Kitten, Todd

2009-07-01

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [(3)H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.

Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in Streptococcus sanguinis▿ §

PubMed Central

Das, Sankar; Kanamoto, Taisei; Ge, Xiuchun; Xu, Ping; Unoki, Takeshi; Munro, Cindy L.; Kitten, Todd

2009-01-01

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [3H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence. PMID:19395487

Virulence Factors of Staphylococcus aureus Isolates in an Iranian Referral Children's Hospital.

PubMed

Sabouni, Farah; Mahmoudi, Shima; Bahador, Abbas; Pourakbari, Babak; Sadeghi, Reihaneh Hosseinpour; Ashtiani, Mohammad Taghi Haghi; Nikmanesh, Bahram; Mamishi, Setareh

2014-04-01

The clinical importance of Staphylococcus aureus (S. aureus) is attributed to notable virulence factors, surface proteins, toxins, and enzymes as well as the rapid development of drug resistance. The aim of this study was to compare the occurrence of virulence factors produced by S. aureus strains isolated from children in an Iranian referral children's hospital. The presence of genes encoding for the enterotoxins A (sea), B (seb), C (sec), D (sed), TSST-1 (tsst), exfoliative toxin A (eta), and exfoliative toxin B (etb) were detected by Multiplex polymerase chain reaction (PCR) using specific primers. In addition, the standardized Kirby-Bauer disc-diffusion method was performed on Mueller-Hinton agar. In total, 133 S. aureus isolates were obtained from different patients. Of these S. aureus isolates, 64 (48%) were methicillin-resistant S. aureus (MRSA), and all of these tested positive for the mecA gene. Regarding the classical enterotoxin genes, sea gene (40.6%) was the most prevalent followed by seb (19.6%), tsst (12.8%), eta (11.3%), etb (9%), sed (4.5%), and sec (3%). Among methicillin-susceptible S. aureus (MSSA) isolates, seb and tsst were the more prevalent toxins in comparison with MRSA isolates (p < 0.05), while the frequency of sea, sed, eta, and etb genes were higher among MRSA isolates (p > 0.05). In our study enterotoxin A was produced by 40.6% of the isolates (48% from MRSA and 33% from MSSA isolates) which was higher than in previous reports. According to our results, strict hygiene and preventative measures during food processing are highly recommended.

Generation and Surface Localization of Intact M Protein in Streptococcus pyogenes Are Dependent on sagA

PubMed Central

Biswas, Indranil; Germon, Pierre; McDade, Kathleen; Scott, June R.

2001-01-01

The M protein is an important surface-located virulence factor of Streptococcus pyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that the sag locus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription of emm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019–6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation in sagA, the first gene in the sag locus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of the emm gene was unaffected by the sagA mutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in the sagA mutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made the sagA mutant strain susceptible to phagocytosis. Thus, although sagA does not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor. PMID:11598078

Phytosynthesized silver nanoparticles as antiquorum sensing and antibiofilm agent against the nosocomial pathogen Serratia marcescens: an in vitro study.

PubMed

Ravindran, D; Ramanathan, S; Arunachalam, K; Jeyaraj, G P; Shunmugiah, K P; Arumugam, V R

2018-06-01

Serratia marcescens is an important multidrug-resistant human pathogen. The pathogenicity of S. marcescens mainly depends on the quorum sensing (QS) mechanism, which regulates the virulence factors production and biofilm formation. Hence, targeting QS mechanism in S. marcescens will ultimately pave the way to combat its pathogenicity. Thus, the present study is intended to evaluate the efficacy of Vetiveria zizanioides root extract-mediated silver nanoparticles (AgNPs) as a potent anti-QS and antibiofilm agent against S. marcescens. The AgNPs were synthesized using V. zizanioides aqueous root extract and the physiochemical properties of V. zizanioides-based AgNPs (VzAgNPs) were evaluated using analytical techniques such as ultraviolet-visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, dynamic light scattering and scanning and transmission electron microscopic techniques. VzAgNPs were found to attenuate the QS-dependent virulence factors, namely prodigiosin, protease, lipase, exopolysaccharide productions and biofilm formation of S. marcescens, without inhibiting its growth. Further, the transcriptomic analysis confirmed the down-regulation of QS-dependent genes, which encode for the production of virulence factors and biofilm formation. The current study confirms VzAgNPs as an ideal anti-QS and antibiofilm agent against S. marcescens. This is the first approach that validates the anti-QS and antibiofilm potential of phytosynthesized VzAgNPs against the nosocomial pathogen, S. marcescens. As VzAgNPs exhibits potent antivirulent activities, it could be used to treat hospital-acquired S. marcescens infections. © 2018 The Society for Applied Microbiology.

DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli.

PubMed

Fekete, Péter Z; Brzuszkiewicz, Elzbieta; Blum-Oehler, Gabriele; Olasz, Ferenc; Szabó, Mónika; Gottschalk, Gerhard; Hacker, Jörg; Nagy, Béla

2012-01-01

In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential. Copyright © 2011. Published by Elsevier GmbH.

A trans-acting leader RNA from a Salmonella virulence gene

PubMed Central

Choi, Eunna; Han, Yoontak; Cho, Yong-Joon; Nam, Daesil; Lee, Eun-Jin

2017-01-01

Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection. PMID:28874555

Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis.

PubMed

Lo, Hsueh-Hsia; Cheng, Wei-Shan

2015-01-01

Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis. Streptococcus dysgalactiae subspecies equisimilis (SDSE), the dominant human pathogenic species among group G streptococci, is the causative agent of several invasive and non-invasive diseases worldwide. However, limited information is available about the distribution of virulence factors among SDSE isolates, or their association with emm types and the isolation sites. In this study, 246 beta-hemolytic group G SDSE isolates collected in central Taiwan between February 2007 and August 2011 were under investigation. Of these, 66 isolates were obtained from normally sterile sites and 180 from non-sterile sites. emm typing revealed 32 types, with the most prevalent one being stG10.0 (39.8%), followed by stG245.0 (15.4%), stG840.0 (12.2%), stG6.1 (7.7%), and stG652.0 (4.1%). The virulence genes lmb (encoding laminin-binding protein), gapC (glyceraldehyde 3-phosphate dehydrogenase), sagA (streptolysin S), and hylB (hyaluronidase) existed in all isolates. Also, 99.2% of the isolates possessed slo (streptolysin O) and scpA (C5a peptidase) genes. In addition, 72.8%, 14.6%, 9.4%, and 2.4% of the isolates possessed the genes ska (streptokinase), cbp (putative collagen-binding protein, SDEG_1781), fbp (putative fibronectin-binding protein, SDEG_0161), and sicG (streptococcal inhibitor of complement), respectively. The only superantigen gene detected was spegg (streptococcus pyrogenic exotoxin G(dys) ), which was possessed by 74.4% of the isolates; these isolates correlated with non-sterile sites. Positive correlations were observed between the following emm types and virulence genes: stG10.0 and stG840.0 with spegg, stG6.1 and stG652.0 with ska, and stG840.0 with cbp. On the other hand, negative correlations were observed between the following: stG245.0, stG6.1, and stG652.0 types with spegg, stG10.0 with ska, and stG10.0, stG245.0, and stG6.1 types with cbp. The prevalence of emm types of SDSE in central Taiwan was investigated for the first time. Moreover, the distribution of virulence factors among beta-hemolytic group G SDSE isolates, as well as their association with emm types or isolation sites were also examined. © 2014 APMIS. Published by John Wiley & Sons Ltd.

Evaluating Different Virulence Traits of Klebsiella pneumoniae Using Dictyostelium discoideum and Zebrafish Larvae as Host Models

PubMed Central

Marcoleta, Andrés E.; Varas, Macarena A.; Ortiz-Severín, Javiera; Vásquez, Leonardo; Berríos-Pastén, Camilo; Sabag, Andrea V.; Chávez, Francisco P.; Allende, Miguel L.; Santiviago, Carlos A.; Monasterio, Octavio; Lagos, Rosalba

2018-01-01

Multiresistant and invasive hypervirulent Klebsiella pneumoniae strains have become one of the most urgent bacterial pathogen threats. Recent analyses revealed a high genomic plasticity of this species, harboring a variety of mobile genetic elements associated with virulent strains, encoding proteins of unknown function whose possible role in pathogenesis have not been addressed. K. pneumoniae virulence has been studied mainly in animal models such as mice and pigs, however, practical, financial, ethical and methodological issues limit the use of mammal hosts. Consequently, the development of simple and cost-effective experimental approaches with alternative host models is needed. In this work we described the use of both, the social amoeba and professional phagocyte Dictyostelium discoideum and the fish Danio rerio (zebrafish) as surrogate host models to study K. pneumoniae virulence. We compared three K. pneumoniae clinical isolates evaluating their resistance to phagocytosis, intracellular survival, lethality, intestinal colonization, and innate immune cells recruitment. Optical transparency of both host models permitted studying the infective process in vivo, following the Klebsiella-host interactions through live-cell imaging. We demonstrated that K. pneumoniae RYC492, but not the multiresistant strains 700603 and BAA-1705, is virulent to both host models and elicits a strong immune response. Moreover, this strain showed a high resistance to phagocytosis by D. discoideum, an increased ability to form biofilms and a more prominent and irregular capsule. Besides, the strain 700603 showed the unique ability to replicate inside amoeba cells. Genomic comparison of the K. pneumoniae strains showed that the RYC492 strain has a higher overall content of virulence factors although no specific genes could be linked to its phagocytosis resistance, nor to the intracellular survival observed for the 700603 strain. Our results indicate that both zebrafish and D. discoideum are advantageous host models to study different traits of K. pneumoniae that are associated with virulence. PMID:29479519

Genomic Analysis of Attenuation in Pandemic Vibrio parahaemolyticus

NASA Astrophysics Data System (ADS)

Pinnell, L. J.; Tallman, J. J., III; Turner, J.

2016-02-01

A critical problem in the prevention and treatment of infectious disease is the ability to differentiate virulent from avirulent bacterial strains. The distinction is commonly based on the presence or absence of specific virulence-associated genes. Alternately, serotypic or phylogenetic typing can accurately differentiate virulent from avirulent strains. When these approaches fail, more discriminatory analysis is needed. Pandemic Vibiro parahaemolyticus, distinguishable by genotyping (thermostable direct hemolysin or tdh), serotyping (O3:K6) and multilocus sequence typing (ST3), is regarded as a highly virulent clonal complex. We have previously shown, through population genetics and cytotoxicity testing, that some pandemic strains isolated from environmental sources are avirulent. To investigate the basis for attenuation, we sequenced the draft genomes of 10 pandemic V. parahaemolyticus isolates originating from environmental (N = 7) and clinical sources (N = 3). Genomic comparison of these 10 draft genomes, and the pandemic type strain (RIMD2210633), revealed a large core genome (5,158,719 bp) and a much smaller accessory genome (141,403 bp). The accessory genome was largely comprised of hypothetical proteins; however, several genes encoded phage-related proteins. Phylogenetic analysis, based on 2,902 single nucleotide polymorphisms in the core genome, did not reveal a discernable pattern. Current efforts are focused on the identification of insertions, deletions and point mutations that may alter protein expression or protein function. Preliminary results show that attenuated strains lack the virulence-associated vacB gene (VP1890). This gene encodes a 741 amino acid exoribonuclease homologous to exoribonucleases known to modulate virulence in Salmonella enterica and Helicobacter pylori. The correlation between attenuation and the absence of this gene, suggests that VP1890 plays an important role in human pathogenesis.

Systems analysis of multiple regulator perturbations allows discovery of virulence factors in Salmonella

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Hyunjin; Ansong, Charles; McDermott, Jason E.

Background: Systemic bacterial infections are highly regulated and complex processes that are orchestrated by numerous virulence factors. Genes that are coordinately controlled by the set of regulators required for systemic infection are potentially required for pathogenicity. Results: In this study we present a systems biology approach in which sample-matched multi-omic measurements of fourteen virulence-essential regulator mutants were coupled with computational network analysis to efficiently identify Salmonella virulence factors. Immunoblot experiments verified network-predicted virulence factors and a subset was determined to be secreted into the host cytoplasm, suggesting that they are virulence factors directly interacting with host cellular components. Two ofmore » these, SrfN and PagK2, were required for full mouse virulence and were shown to be translocated independent of either of the type III secretion systems in Salmonella or the type III injectisome-related flagellar mechanism. Conclusions: Integrating multi-omic datasets from Salmonella mutants lacking virulence regulators not only identified novel virulence factors but also defined a new class of translocated effectors involved in pathogenesis. The success of this strategy at discovery of known and novel virulence factors suggests that the approach may have applicability for other bacterial pathogens.« less

Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production

PubMed Central

2010-01-01

Background Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production. Results Xoo genome harbours an rpf cluster comprising rpfB, rpfF, rpfC and rpfG. The proteins encoded by these genes are highly homologous to their counterparts in X. campestris pv. campestris (Xcc), suggesting that Xcc and Xoo might use similar mechanisms for DSF biosynthesis and autoregulation. Consistent with in silico analysis, the rpfF mutant was DSF-deficient and the rpfC mutant produced about 25 times higher DSF-like activity than the wild type Xoo strain KACC10331. From the supernatants of rpfC mutant, we purified three compounds showing strong DSF-like activity. Mass spectrometry and NMR analysis revealed that two of them were the previously characterized DSF and BDSF; the third one was a novel unsaturated fatty acid with 2 double bonds and was designated as CDSF in this study. Further analysis showed that all the three DSF-family signals were synthesized via the enzyme RpfF encoded by Xoo2868. DSF and BDSF at a final concentration of 3 μM to the rpfF mutant could fully restore its extracellular xylanase activity and EPS production to the wild type level, but CDSF was less active than DSF and BDSF in induction of EPS and xylanase. DSF and CDSF shared a similar cell density-dependent production time course with the maximum production being detected at 42 h after inoculation, whereas the maximum production of BDSF was observed at 36 h after inoculation. When grown in a rich medium such as YEB, LB, PSA, and NYG, Xoo produced all the three signals with the majority being DSF. Whereas in nutritionally poor XOLN medium Xoo only produced BDSF and DSF but the majority was BDSF. Conclusions This study demonstrates that Xoo and Xcc share the conserved mechanisms for DSF biosynthesis and autoregulation. Xoo produces DSF, BDSF and CDSF signals in rich media and CDSF is a novel signal in DSF-family with two double bonds. All the three DSF-family signals promote EPS production and xylanase activity in Xoo, but CDSF is less active than its analogues DSF and BDSF. The composition and ratio of the three DSF-family signals produced by Xoo are influenced by the composition of culture media. PMID:20615263

Comparative Genomics and an Insect Model Rapidly Identify Novel Virulence Genes of Burkholderia mallei

DTIC Science & Technology

2008-04-01

IID on A pril 23, 2008 jb.asm .org D ow nloaded from metabolite-producing clusters encoding nonribosomal peptide or polyketide synthetases...BMA1848) encod- ing a subunit of acetolactate synthase III. The resultant mutant was not able to grow on minimal glucose medium and, similar to what has...caused by the wild type. BMAA1204 is a 4,200-residue CDS annotated as encoding a putative polyketide synthase (PKS) in COG family 0332. PKSs are

Involvement of the pagR gene of pXO2 in anthrax pathogenesis

PubMed Central

Liang, Xudong; Zhang, Enmin; Zhang, Huijuan; Wei, Jianchun; Li, Wei; Zhu, Jin; Wang, Bingxiang; Dong, Shulin

2016-01-01

Anthrax is a disease caused by Bacillus anthracis. Specifically, the anthrax toxins and capsules encoded by the pXO1 and pXO2 plasmids, respectively, are the major virulence factors. We previously reported that the pXO1 plasmid was retained in the attenuated strain of B. anthracis vaccine strains even after subculturing at high temperatures. In the present study, we reinvestigate the attenuation mechanism of Pasteur II. Sequencing of pXO1 and pXO2 from Pasteur II strain revealed mutations in these plasmids as compared to the reference sequences. Two deletions on these plasmids, one each on pXO1 and pXO2, were confirmed to be unique to the Pasteur II strain as compared to the wild-type strains. Gene replacement with homologous recombination revealed that the mutation in the promoter region of the pagR gene on pXO2, but not the mutation on pXO1, contributes to lethal levels of toxin production. This result was further confirmed by RT-PCR, western blot, and animal toxicity assays. Taken together, our results signify that the attenuation of the Pasteur II vaccine strain is caused by a mutation in the pagR gene on its pXO2 plasmid. Moreover, these data suggest that pXO2 plasmid encoded proteins are involved in the virulence of B. anthracis. PMID:27363681

Mapping of herpes simplex virus-1 neurovirulence to. gamma. sub 1 34. 5, a gene nonessential for growth in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, J.; Roizman, B.; Kern, E.R.

1990-11-30

The gene designated {gamma}{sub 1}34.5 maps in the inverted repeats flanking the long unique sequence of herpes simplex virus-1 (HSV-1) DNA, and therefore it is present in two copies per genome. This gene is not essential for viral growth in cell culture. Four recombinant viruses were genetically engineered to test the function of this gene. These were (i) a virus from which both copies of the gene were deleted, (ii) a virus containing a stop codon in both copies of the gene, (iii) a virus containing after the first codon an insert encoding a 16-amino acid epitope known to reactmore » with a specific monoclonal antibody, and (iv) a virus in which the deleted sequences were restored. The viruses from which the gene was deleted or which carried stop codons were avirulent on intracerebral inoculation of mice. The virus with the gene tagged by the sequence encoding the epitope was moderately virulent, whereas the restored virus reacquired the phenotype of the parent virus. Significant amounts of virus were recovered only from brains of animals inoculated with virulent viruses. Inasmuch as the product of the {gamma}{sub 1}34.5 gene extended the host range of the virus by enabling it to replicate and destroy brain cells, it is a viral neurovirulence factor.« less

Sword and shield: linked group B streptococcal beta-hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense.

PubMed

Liu, George Y; Doran, Kelly S; Lawrence, Toby; Turkson, Nicole; Puliti, Manuela; Tissi, Luciana; Nizet, Victor

2004-10-05

Group B Streptococcus (GBS) is a major cause of pneumonia, bacteremia, and meningitis in neonates and has been found to persist inside host phagocytic cells. The pore-forming GBS beta-hemolysin/cytolysin (betaH/C) encoded by cylE is an important virulence factor as demonstrated in several in vivo models. Interestingly, cylE deletion results not only in the loss of betaH/C activity, but also in the loss of a carotenoid pigment of unknown function. In this study, we sought to define the mechanism(s) by which cylE may contribute to GBS phagocyte resistance and increased virulence potential. We found that cylE-deficient GBS was more readily cleared from a mouse's bloodstream, human whole blood, and isolated macrophage and neutrophil cultures. Survival was linked to the ability of betaH/C to induce cytolysis and apoptosis of the phagocytes. At a lower bacterial inoculum, cylE also contributed to enhanced survival within phagocytes that was attributed to the ability of carotenoid to shield GBS from oxidative damage. In oxidant killing assays, cylE mutants were shown to be more susceptible to hydrogen peroxide, hypochlorite, superoxide, and singlet oxygen. Together, these data suggest a mechanism by which the linked cylE-encoded phenotypes, betaH/C (sword) and carotenoid (shield), act in partnership to thwart the immune phagocytic defenses.

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

PubMed Central

Raabe, Carsten A.; Sanchez, Cecilia P.; Randau, Gerrit; Robeck, Thomas; Skryabin, Boris V.; Chinni, Suresh V.; Kube, Michael; Reinhardt, Richard; Ng, Guey Hooi; Manickam, Ravichandran; Kuryshev, Vladimir Y.; Lanzer, Michael; Brosius, Juergen; Tang, Thean Hock; Rozhdestvensky, Timofey S.

2010-01-01

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors. PMID:19864253

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum.

PubMed

Raabe, Carsten A; Sanchez, Cecilia P; Randau, Gerrit; Robeck, Thomas; Skryabin, Boris V; Chinni, Suresh V; Kube, Michael; Reinhardt, Richard; Ng, Guey Hooi; Manickam, Ravichandran; Kuryshev, Vladimir Y; Lanzer, Michael; Brosius, Juergen; Tang, Thean Hock; Rozhdestvensky, Timofey S

2010-01-01

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense-antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors.

Antigenic profiling of Yersinia pestis infection in the Wyoming coyote (Canis latrans)

USGS Publications Warehouse

Vernati, G.; Edwards, W.H.; Rocke, T.E.; Little, S.F.; Andrews, G.P.

2011-01-01

Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.

Helicobacter pylori virulence and cancer pathogenesis

PubMed Central

Yamaoka, Yoshio; Graham, David Y

2014-01-01

Helicobacter pylori is human gastric pathogen that causes chronic and progressive gastric mucosal inflammation and is responsible for the gastric inflammation-associated diseases, gastric cancer and peptic ulcer disease. specific outcomes reflect the interplay between host-, environmental- and bacterial-specific factors. Progress in understanding putative virulence factors in disease pathogenesis has been limited and many false leads have consumed scarce resources. Few in vitro–in vivo correlations or translational applications have proved clinically relevant. Reported virulence factor-related outcomes reflect differences in relative risk of disease rather than specificity for any specific outcome. Studies of individual virulence factor associations have provided conflicting results. Since virulence factors are linked, studies of groups of putative virulence factors are needed to provide clinically useful information. Here, the authors discuss the progress made in understanding the role of H. pylori virulence factors CagA, vacuolating cytotoxin, OipA and DupA in disease pathogenesis and provide suggestions for future studies. PMID:25052757

Helicobacter pylori virulence and cancer pathogenesis.

PubMed

Yamaoka, Yoshio; Graham, David Y

2014-06-01

Helicobacter pylori is human gastric pathogen that causes chronic and progressive gastric mucosal inflammation and is responsible for the gastric inflammation-associated diseases, gastric cancer and peptic ulcer disease. Specific outcomes reflect the interplay between host-, environmental- and bacterial-specific factors. Progress in understanding putative virulence factors in disease pathogenesis has been limited and many false leads have consumed scarce resources. Few in vitro-in vivo correlations or translational applications have proved clinically relevant. Reported virulence factor-related outcomes reflect differences in relative risk of disease rather than specificity for any specific outcome. Studies of individual virulence factor associations have provided conflicting results. Since virulence factors are linked, studies of groups of putative virulence factors are needed to provide clinically useful information. Here, the authors discuss the progress made in understanding the role of H. pylori virulence factors CagA, vacuolating cytotoxin, OipA and DupA in disease pathogenesis and provide suggestions for future studies.

Clostridium botulinum: a bug with beauty and weapon.

PubMed

Shukla, H D; Sharma, S K

2005-01-01

Clostridium botulinum, a Gram-positive, anaerobic spore-forming bacteria, is distinguished by its significant clinical applications as well as its potential to be used as bioterror agent. Growing cells secrete botulinum neurotoxin (BoNT), the most poisonous of all known poisons. While BoNT is the causative agent of deadly neuroparalytic botulism, it also serves as a remarkably effective treatment for involuntary muscle disorders such as blepharospasm, strabismus, hemifacial spasm, certain types of spasticity in children, and other ailments. BoNT is also used in cosmetology for the treatment of glabellar lines, and is well-known as the active component of the anti-aging medications Botox and Dysport. In addition, recent reports show that botulinum neurotoxin can be used as a tool for pharmaceutical drug delivery. However, BoNT remains the deadliest of all toxins, and is viewed by biodefense researchers as a possible agent of bioterrorism (BT). Among seven serotypes, C. botulinum type A is responsible for the highest mortality rate in botulism, and thus has the greatest potential to act as biological weapon. Genome sequencing of C. botulinum type A Hall strain (ATCC 3502) is now complete, and has shown the genome size to be 3.89 Mb with a G+C content of approximately 28.2%. The bacterium harbors a 16.3 kb plasmid with a 26.8% G+C content--slightly lower than that of the chromosome. Most of the virulence factors in C. botulinum are chromosomally encoded; bioinformatic analysis of the genome sequence has shown that the plasmid does not harbor toxin genes or genes for related virulence factors. Interestingly, the plasmid does harbor genes essential to replication, including dnaE, which encodes the alpha subunit of DNA polymerase III which has close similarity with its counterpart in C. perfringens strain 13. The plasmid also contains similar genes to those that encode the ABC-type multidrug transport ATPase, and permease. The presence of ABC-type multidrug transport ATPase, and permease suggests putative involvement of efflux pumps in bacteriocin production, modification, and export in C. botulinum. The C. botulinum plasmid additionally harbors genes for LambdaBa04 prophage and site-specific recombinase that are similar to those found in the Ames strain of Bacillus anthracis; these genes and their products may play a role in genomic rearrangement. Completion of genome sequencing for C. botulinum will provide an opportunity to design genomic and proteomic-based systems for detecting different serotypes of C. botulinum strains in the environment. The completed sequence may also facilitate identification of potential virulence factors and drug targets, as well as help characterize neurotoxin-complexing proteins, their polycistronic expression, and phylogenetic relationships between different serotypes.

Expression of virulence factors by Staphylococcus aureus grown in serum.

PubMed

Oogai, Yuichi; Matsuo, Miki; Hashimoto, Masahito; Kato, Fuminori; Sugai, Motoyuki; Komatsuzawa, Hitoshi

2011-11-01

Staphylococcus aureus produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by in vitro experiments using bacterial medium. However, when S. aureus infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in S. aureus grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl₃ into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with agr inactivated grown in serum, the expression of RNA III, psm, and sec4 was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that S. aureus expresses virulence factors in adaptation to the host environment.

Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans

PubMed Central

Sztajer, Helena; Szafranski, Szymon P; Tomasch, Jürgen; Reck, Michael; Nimtz, Manfred; Rohde, Manfred; Wagner-Döbler, Irene

2014-01-01

Polymicrobial biofilms are of large medical importance, but relatively little is known about the role of interspecies interactions for their physiology and virulence. Here, we studied two human pathogens co-occuring in the oral cavity, the opportunistic fungus Candida albicans and the caries-promoting bacterium Streptococcus mutans. Dual-species biofilms reached higher biomass and cell numbers than mono-species biofilms, and the production of extracellular polymeric substances (EPSs) by S. mutans was strongly suppressed, which was confirmed by scanning electron microscopy, gas chromatography–mass spectrometry and transcriptome analysis. To detect interkingdom communication, C. albicans was co-cultivated with a strain of S. mutans carrying a transcriptional fusion between a green fluorescent protein-encoding gene and the promoter for sigX, the alternative sigma factor of S. mutans, which is induced by quorum sensing signals. Strong induction of sigX was observed in dual-species biofilms, but not in single-species biofilms. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion of comS encoding the synthesis of the sigX-inducing peptide precursor abolished this activity, whereas deletion of comC encoding the competence-stimulating peptide precursor had no effect. Transcriptome analysis of S. mutans confirmed induction of comS, sigX, bacteriocins and the downstream late competence genes, including fratricins, in dual-species biofilms. We show here for the first time the stimulation of the complete quorum sensing system of S. mutans by a species from another kingdom, namely the fungus C. albicans, resulting in fundamentally changed virulence properties of the caries pathogen. PMID:24824668

Two Host-Induced Ralstonia solanacearum Genes, acrA and dinF, Encode Multidrug Efflux Pumps and Contribute to Bacterial Wilt Virulence▿ †

PubMed Central

Brown, Darby G.; Swanson, Jill K.; Allen, Caitilyn

2007-01-01

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>107 CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds. PMID:17337552

Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog.

PubMed

Lo, Miranda; Murray, Gerald L; Khoo, Chen Ai; Haake, David A; Zuerner, Richard L; Adler, Ben

2010-11-01

Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

PubMed Central

Lihoradova, Olga A.; Indran, Sabarish V.; Kalveram, Birte; Lokugamage, Nandadeva; Head, Jennifer A.; Gong, Bin; Tigabu, Bersabeh; Juelich, Terry L.; Freiberg, Alexander N.; Ikegami, Tetsuro

2013-01-01

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever. PMID:23638202

Characterization of Rift Valley fever virus MP-12 strain encoding NSs of Punta Toro virus or sandfly fever Sicilian virus.

PubMed

Lihoradova, Olga A; Indran, Sabarish V; Kalveram, Birte; Lokugamage, Nandadeva; Head, Jennifer A; Gong, Bin; Tigabu, Bersabeh; Juelich, Terry L; Freiberg, Alexander N; Ikegami, Tetsuro

2013-01-01

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.

Acute Hepatopancreatic Necrosis Disease-Causing Vibrio parahaemolyticus Strains Maintain an Antibacterial Type VI Secretion System with Versatile Effector Repertoires.

PubMed

Li, Peng; Kinch, Lisa N; Ray, Ann; Dalia, Ankur B; Cong, Qian; Nunan, Linda M; Camilli, Andrew; Grishin, Nick V; Salomon, Dor; Orth, Kim

2017-07-01

Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of Vibrio parahaemolyticus that have acquired a "selfish plasmid" encoding the deadly binary toxins PirA vp /PirB vp To better understand the repertoire of virulence factors in AHPND-causing V. parahaemolyticus , we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of V. parahaemolyticus Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing V. parahaemolyticus a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control. IMPORTANCE Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Vibrio parahaemolyticus Although the plasmid-encoded binary toxins PirA vp /PirB vp have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in V. parahaemolyticus and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections. Copyright © 2017 American Society for Microbiology.

Acute Hepatopancreatic Necrosis Disease-Causing Vibrio parahaemolyticus Strains Maintain an Antibacterial Type VI Secretion System with Versatile Effector Repertoires

PubMed Central

Li, Peng; Kinch, Lisa N.; Ray, Ann; Dalia, Ankur B.; Nunan, Linda M.; Camilli, Andrew; Grishin, Nick V.

2017-01-01

ABSTRACT Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of Vibrio parahaemolyticus that have acquired a “selfish plasmid” encoding the deadly binary toxins PirAvp/PirBvp. To better understand the repertoire of virulence factors in AHPND-causing V. parahaemolyticus, we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of V. parahaemolyticus. Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing V. parahaemolyticus a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control. IMPORTANCE Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Vibrio parahaemolyticus. Although the plasmid-encoded binary toxins PirAvp/PirBvp have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in V. parahaemolyticus and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections. PMID:28432099

Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa.

PubMed

Cursino, Luciana; Galvani, Cheryl D; Athinuwat, Dusit; Zaini, Paulo A; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

2011-10-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

Comparative genomic analysis and phenazine production of Pseudomonas chlororaphis, a plant growth-promoting rhizobacterium

PubMed Central

Chen, Yawen; Shen, Xuemei; Peng, Huasong; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

2015-01-01

Pseudomonas chlororaphis HT66, a plant growth-promoting rhizobacterium that produces phenazine-1-carboxamide with high yield, was compared with three genomic sequenced P. chlororaphis strains, GP72, 30–84 and O6. The genome sizes of four strains vary from 6.66 to 7.30 Mb. Comparisons of predicted coding sequences indicated 4833 conserved genes in 5869–6455 protein-encoding genes. Phylogenetic analysis showed that the four strains are closely related to each other. Its competitive colonization indicates that P. chlororaphis can adapt well to its environment. No virulence or virulence-related factor was found in P. chlororaphis. All of the four strains could synthesize antimicrobial metabolites including different phenazines and insecticidal protein FitD. Some genes related to the regulation of phenazine biosynthesis were detected among the four strains. It was shown that P. chlororaphis is a safe PGPR in agricultural application and could also be used to produce some phenazine antibiotics with high-yield. PMID:26484173

Staphylococcus aureus Toxins and Diabetic Foot Ulcers: Role in Pathogenesis and Interest in Diagnosis

PubMed Central

Dunyach-Remy, Catherine; Ngba Essebe, Christelle; Sotto, Albert; Lavigne, Jean-Philippe

2016-01-01

Infection of foot ulcers is a common, often severe and costly complication in diabetes. Diabetic foot infections (DFI) are mainly polymicrobial, and Staphylococcus aureus is the most frequent pathogen isolated. The numerous virulence factors and toxins produced by S. aureus during an infection are well characterized. However, some particular features could be observed in DFI. The aim of this review is to describe the role of S. aureus in DFI and the implication of its toxins in the establishment of the infection. Studies on this issue have helped to distinguish two S. aureus populations in DFI: toxinogenic S. aureus strains (harboring exfoliatin-, EDIN-, PVL- or TSST-encoding genes) and non-toxinogenic strains. Toxinogenic strains are often present in infections with a more severe grade and systemic impact, whereas non-toxinogenic strains seem to remain localized in deep structures and bone involving diabetic foot osteomyelitis. Testing the virulence profile of bacteria seems to be a promising way to predict the behavior of S. aureus in the chronic wounds. PMID:27399775

Identification of a two-component signal transduction system involved in fimbriation of Porphyromonas gingivalis.

PubMed

Hayashi, J; Nishikawa, K; Hirano, R; Noguchi, T; Yoshimura, F

2000-01-01

Porphyromonas gingivalis, a periodontopathogen, is an oral anaerobic gram-negative bacterium with numerous fimbriae on the cell surface. Fimbriae have been considered to be an important virulence factor in this organism. We analyzed the genomic DNA of transposon-induced, fimbria-deficient mutants derived from ATCC 33277 and found that seven independent mutants had transposon insertions within the same restriction fragment. Cloning and sequencing of the disrupted region from one of the mutants revealed two adjacent open reading frames (ORFs) which seemed to encode a two-component signal transduction system. We also found that six of the mutants had insertions in a gene, fimS, a homologue of the genes encoding sensor kinase, and that the insertion in the remaining one disrupted the gene immediately downstream, fimR, a homologue of the response regulator genes in other bacteria. These findings suggest that this two-component regulatory system is involved in fimbriation of P. gingivalis.

Novel Antigens for enterotoxigenic Escherichia coli (ETEC) Vaccines

PubMed Central

Fleckenstein, James M.; Sheikh, Alaullah; Qadri, Firdausi

2014-01-01

Enterotoxigenic Escherichia coli (ETEC) are the most common bacterial pathogens-causing diarrhea in developing countries where they cause hundreds of thousands of deaths, mostly in children. These organisms are leading cause of diarrheal illness in travelers to endemic countries. ETEC pathogenesis, and consequently vaccine approaches, have largely focused on plasmid-encoded enterotoxins or fimbrial colonization factors. To date these approaches have not yielded a broadly protective vaccine. However, recent studies suggest that ETEC pathogenesis is more complex than previously appreciated and involves additional plasmid and chromosomally-encoded virulence molecules that can be targeted in vaccines. Here, we review recent novel antigen discovery efforts, potential contribution of these proteins to the molecular pathogenesis of ETEC and protective immunity, and the potential implications for development of next generation vaccines for important pathogens. These proteins may help to improve the effectiveness of future vaccines by making simpler and possibly broadly protective because of their conserved nature. PMID:24702311

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

PubMed

Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L

2012-01-01

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

Novel Plasmids and Resistance Phenotypes in Yersinia pestis: Unique Plasmid Inventory of Strain Java 9 Mediates High Levels of Arsenic Resistance

PubMed Central

Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L.

2012-01-01

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium. PMID:22479347

Feasibility of Genome-Wide Screening for Biosafety Assessment of Probiotics: A Case Study of Lactobacillus helveticus MTCC 5463.

PubMed

Senan, S; Prajapati, J B; Joshi, C G

2015-12-01

Recent years have witnessed an explosion in genome sequencing of probiotic strains for accurate identification and characterization. Regulatory bodies are emphasizing on the need for performing phase I safety studies for probiotics. The main hypothesis of this study was to explore the feasibility of using genome databases for safety screening of strains. In this study, we attempted to develop a framework for the safety assessment of a potential probiotic strain, Lactobacillus helveticus MTCC 5463 based on genome mining for genes associated with antibiotic resistance, production of harmful metabolites, and virulence. The sequencing of MTCC 5463 was performed using GS-FLX Titanium reagents. Genes coding for antibiotic resistance and virulence were identified using Antibiotic Resistance Genes Database and Virulence Factors Database. Results indicated that MTCC 5463 carried antibiotic resistance genes associated with beta-lactam and fluoroquinolone. There is no threat of transfer of these genes to host gut commensals because the genes are not plasmid encoded. The presence of genes for adhesion, biofilm, surface proteins, and stress-related proteins provides robustness to the strain. The presence of hemolysin gene in the genome revealed a theoretical risk of virulence. The results of in silico analysis complemented the in vitro studies and human clinical trials, confirming the safety of the probiotic strain. We propose that the safety assessment of probiotic strains administered live at high doses using a genome-wide screening could be an effective and time-saving tool for identifying prognostic biomarkers of biosafety.

The small regulatory RNA FasX enhances group A Streptococcus virulence and inhibits pilus expression via serotype-specific targets

PubMed Central

Danger, Jessica L.; Cao, Tram N.; Cao, Tran H.; Sarkar, Poulomee; Treviño, Jeanette; Pflughoeft, Kathryn J.; Sumby, Paul

2015-01-01

Summary Bacterial pathogens commonly show intra-species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T-antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT-types), and (c) the streptokinase-encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype-specific manner. Parental, fasX mutant, and complemented derivatives of serotype M1 (ska-2, FCT-2), M2 (ska-1, FCT-6), M6 (ska-2, FCT-1), and M28 (ska-1, FCT-4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT-region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen-expressing mice. Our data are the first to identify and characterize serotype-specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence. PMID:25586884

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes.

PubMed

Maury, Mylène M; Chenal-Francisque, Viviane; Bracq-Dieye, Hélène; Han, Lei; Leclercq, Alexandre; Vales, Guillaume; Moura, Alexandra; Gouin, Edith; Scortti, Mariela; Disson, Olivier; Vázquez-Boland, José A; Lecuit, Marc

2017-11-01

The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene ( hly ) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA - /LLO - ) mutants belonged to phylogenetically diverse clades of L. monocytogenes , and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA - /LLO - mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen. Copyright © 2017 Maury et al.

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes

PubMed Central

Maury, Mylène M.; Chenal-Francisque, Viviane; Bracq-Dieye, Hélène; Han, Lei; Leclercq, Alexandre; Vales, Guillaume; Moura, Alexandra; Gouin, Edith; Scortti, Mariela; Disson, Olivier

2017-01-01

ABSTRACT The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes. Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA. Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA−/LLO−) mutants belonged to phylogenetically diverse clades of L. monocytogenes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA−/LLO− mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen. PMID:28827366

Identification and quantification of virulence factors of enterotoxigenic Escherichia coli by high-resolution melting curve quantitative PCR.

PubMed

Wang, Weilan; Zijlstra, Ruurd T; Gänzle, Michael G

2017-05-15

Diagnosis of enterotoxigenic E. coli (ETEC) associated diarrhea is complicated by the diversity of E.coli virulence factors. This study developed a multiplex quantitative PCR assay based on high-resolution melting curves analysis (HRM-qPCR) to identify and quantify genes encoding five ETEC fimbriae related to diarrhea in swine, i.e. K99, F41, F18, F6 and K88. Five fimbriae expressed by ETEC were amplified in multiple HRM-qPCR reactions to allow simultaneous identification and quantification of five target genes. The assay was calibrated to allow quantification of the most abundant target gene, and validated by analysis of 30 samples obtained from piglets with diarrhea and healthy controls, and comparison to standard qPCR detection. The five amplicons with melting temperatures (Tm) ranging from 74.7 ± 0.06 to 80.5 ± 0.15 °C were well-separated by HRM-qPCR. The area of amplicons under the melting peak correlated linearly to the proportion of the template in the calibration mixture if the proportion exceeded 4.8% (K88) or <1% (all other amplicons). The suitability of the method was evaluated using 30 samples from weaned pigs aged 6-7 weeks; 14 of these animals suffered from diarrhea in consequence of poor sanitary conditions. Genes encoding fimbriae and enterotoxins were quantified by HRM-qPCR and/or qPCR. The multiplex HRM-qPCR allowed accurate analysis when the total gene copy number of targets was more than 1 × 10 5 / g wet feces and the HRM curves were able to simultaneously distinguish fimbriae genes in the fecal samples. The relative quantification of the most abundant F18 based on melting peak area was highly correlated (P < 0.001; r 2  = 0.956) with that of individual qPCR result but the correlation for less abundant fimbriae was much lower. The multiplex HRM assay identifies ETEC virulence factors specifically and efficiently. It correctly indicated the predominant fimbriae type and additionally provides information of presence/ absence of other fimbriae types and it could find broad applications for pathogen diagnosis.

Lysionotin attenuates Staphylococcus aureus pathogenicity by inhibiting α-toxin expression.

PubMed

Teng, Zihao; Shi, Dongxue; Liu, Huanyu; Shen, Ziying; Zha, Yonghong; Li, Wenhua; Deng, Xuming; Wang, Jianfeng

2017-09-01

α-Toxin, one of the best known pore-forming proteins produced by Staphylococcus aureus (S. aureus), is a critical virulence factor in multiple infections. The necessity of α-toxin for S. aureus pathogenicity suggests that this toxin is an important target for the development of a potential treatment strategy. In this study, we showed that lysionotin, a natural compound, can inhibit the hemolytic activity of culture supernatants by S. aureus by reducing α-toxin expression. Using real-time PCR analysis, we showed that transcription of hla (the gene encoding α-toxin) and agr (the locus regulating hla) was significantly inhibited by lysionotin. Lactate dehydrogenase and live/dead assays indicated that lysionotin effectively protected human alveolar epithelial cells against S. aureus, and in vivo studies also demonstrated that lysionotin can protect mice from pneumonia caused by S. aureus. These findings suggest that lysionotin is an efficient inhibitor of α-toxin expression and shows significant protection against S. aureus in vitro and in vivo. This study supports a potential strategy for the treatment of S. aureus infection by inhibiting the expression of virulence factors and indicates that lysionotin may be a potential treatment for S. aureus pneumonia.

Comparative genomic analysis of seven Mycoplasma hyosynoviae strains

PubMed Central

Bumgardner, Eric A; Kittichotirat, Weerayuth; Bumgarner, Roger E; Lawrence, Paulraj K

2015-01-01

Infection with Mycoplasma hyosynoviae can result in debilitating arthritis in pigs, particularly those aged 10 weeks or older. Strategies for controlling this pathogen are becoming increasingly important due to the rise in the number of cases of arthritis that have been attributed to infection in recent years. In order to begin to develop interventions to prevent arthritis caused by M. hyosynoviae, more information regarding the specific proteins and potential virulence factors that its genome encodes was needed. However, the genome of this emerging swine pathogen had not been sequenced previously. In this report, we present a comparative analysis of the genomes of seven strains of M. hyosynoviae isolated from different locations in North America during the years 2010 to 2013. We identified several putative virulence factors that may contribute to the ability of this pathogen to adhere to host cells. Additionally, we discovered several prophage genes present within the genomes of three strains that show significant similarity to MAV1, a phage isolated from the related species, M. arthritidis. We also identified CRISPR-Cas and type III restriction and modification systems present in two strains that may contribute to their ability to defend against phage infection. PMID:25693846

Disulfide Bond Formation and ToxR Activity in Vibrio cholerae

PubMed Central

Fengler, Vera H. I.; Boritsch, Eva C.; Tutz, Sarah; Seper, Andrea; Ebner, Hanna; Roier, Sandro; Schild, Stefan; Reidl, Joachim

2012-01-01

Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation. PMID:23144706

The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis1[OPEN

PubMed Central

Shine, M.B.; Cui, Xiaoyan; Chen, Xin; Ma, Na; Kachroo, Pradeep; Zhi, Haijan; Kachroo, Aardra

2016-01-01

The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other’s nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection. PMID:27356973

Bacterial infection as assessed by in vivo gene expression

PubMed Central

Heithoff, Douglas M.; Conner, Christopher P.; Hanna, Philip C.; Julio, Steven M.; Hentschel, Ute; Mahan, Michael J.

1997-01-01

In vivo expression technology (IVET) has been used to identify >100 Salmonella typhimurium genes that are specifically expressed during infection of BALB/c mice and/or murine cultured macrophages. Induction of these genes is shown to be required for survival in the animal under conditions of the IVET selection. One class of in vivo induced (ivi) genes, iviVI-A and iviVI-B, constitute an operon that resides in a region of the Salmonella genome with low G+C content and presumably has been acquired by horizontal transfer. These ivi genes encode predicted proteins that are similar to adhesins and invasins from prokaryotic and eukaryotic pathogens (Escherichia coli [tia], Plasmodium falciparum [PfEMP1]) and have coopted the PhoPQ regulatory circuitry of Salmonella virulence genes. Examination of the in vivo induction profile indicates (i) many ivi genes encode regulatory functions (e.g., phoPQ and pmrAB) that serve to enhance the sensitivity and amplitude of virulence gene expression (e.g., spvB); (ii) the biochemical function of many metabolic genes may not represent their sole contribution to virulence; (iii) the host ecology can be inferred from the biochemical functions of ivi genes; and (iv) nutrient limitation plays a dual signaling role in pathogenesis: to induce metabolic functions that complement host nutritional deficiencies and to induce virulence functions required for immediate survival and spread to subsequent host sites. PMID:9023360

Degradation of the benzoxazolinone class of phytoalexins is important for virulence of Fusarium pseudograminearum towards wheat.

PubMed

Kettle, Andrew J; Batley, Jacqueline; Benfield, Aurelie H; Manners, John M; Kazan, Kemal; Gardiner, Donald M

2015-12-01

Wheat, maize, rye and certain other agriculturally important species in the Poaceae family produce the benzoxazolinone class of phytoalexins on pest and pathogen attack. Benzoxazolinones can inhibit the growth of pathogens. However, certain fungi can actively detoxify these compounds. Despite this, a clear link between the ability to detoxify benzoxazolinones and pathogen virulence has not been shown. Here, through comparative genome analysis of several Fusarium species, we have identified a conserved genomic region around the FDB2 gene encoding an N-malonyltransferase enzyme known to be involved in benzoxazolinone degradation in the maize pathogen Fusarium verticillioides. Expression analyses demonstrated that a cluster of nine genes was responsive to exogenous benzoxazolinone in the important wheat pathogen Fusarium pseudograminearum. The analysis of independent F. pseudograminearum FDB2 knockouts and complementation of the knockout with FDB2 homologues from F. graminearum and F. verticillioides confirmed that the N-malonyltransferase enzyme encoded by this gene is central to the detoxification of benzoxazolinones, and that Fdb2 contributes quantitatively to virulence towards wheat in head blight inoculation assays. This contrasts with previous observations in F. verticillioides, where no effect of FDB2 mutations on pathogen virulence towards maize was observed. Overall, our results demonstrate that the detoxification of benzoxazolinones is a strategy adopted by wheat-infecting F. pseudograminearum to overcome host-derived chemical defences. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

PubMed

Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

2016-04-15

Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae. Copyright © 2016 Elsevier B.V. All rights reserved.

Type VI secretion is a major virulence determinant in Burkholderia mallei.

PubMed

Schell, Mark A; Ulrich, Ricky L; Ribot, Wilson J; Brueggemann, Ernst E; Hines, Harry B; Chen, Dan; Lipscomb, Lyla; Kim, H Stanley; Mrázek, Jan; Nierman, William C; Deshazer, David

2007-06-01

Burkholderia mallei is a host-adapted pathogen and a category B biothreat agent. Although the B. mallei VirAG two-component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown. Here we show with expression profiling that overexpression of virAG resulted in transcriptional activation of approximately 60 genes, including some involved in capsule production, actin-based intracellular motility, and type VI secretion (T6S). The 15 genes encoding the major sugar component of the homopolymeric capsule were up-expressed > 2.5-fold, but capsule was still produced in the absence of virAG. Actin tail formation required virAG as well as bimB, bimC and bimE, three previously uncharacterized genes that were activated four- to 15-fold when VirAG was overproduced. Surprisingly, actin polymerization was found to be dispensable for virulence in hamsters. In contrast, genes encoding a T6S system were up-expressed as much as 30-fold and mutations in this T6S gene cluster resulted in strains that were avirulent in hamsters. SDS-PAGE and mass spectrometry demonstrated that BMAA0742 was secreted by the T6S system when virAG was overexpressed. Purified His-tagged BMAA0742 was recognized by glanders antiserum from a horse, a human and mice, indicating that this Hcp-family protein is produced in vivo during infection.

Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.

PubMed

Yeung, Yik Andy; Foletti, Davide; Deng, Xiaodi; Abdiche, Yasmina; Strop, Pavel; Glanville, Jacob; Pitts, Steven; Lindquist, Kevin; Sundar, Purnima D; Sirota, Marina; Hasa-Moreno, Adela; Pham, Amber; Melton Witt, Jody; Ni, Irene; Pons, Jaume; Shelton, David; Rajpal, Arvind; Chaparro-Riggers, Javier

2016-11-18

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.

bdhA-patD operon as a virulence determinant, revealed by a novel large-scale approach for identification of Legionella pneumophila mutants defective for amoeba infection.

PubMed

Aurass, P; Pless, B; Rydzewski, K; Holland, G; Bannert, N; Flieger, A

2009-07-01

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular parasite of eukaryotic cells. In the environment, it colonizes amoebae. After being inhaled into the human lung, the bacteria infect and damage alveolar cells in a way that is mechanistically similar to the amoeba infection. Several L. pneumophila traits, among those the Dot/Icm type IVB protein secretion machinery, are essential for exploiting host cells. In our search for novel Legionella virulence factors, we developed an agar plate assay, designated the scatter screen, which allowed screening for mutants deficient in infecting Acanthamoeba castellanii amoebae. Likewise, an L. pneumophila clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established Legionella virulence genes, for example, dot/icm genes, were identified. Importantly, 70 novel virulence-associated genes were found. One of those is L. pneumophila bdhA, coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that bdhA is cotranscribed with patD, encoding a patatin-like protein of L. pneumophila showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, the L. pneumophila bdhA-patD mutant showed a severe replication defect in amoebae and U937 macrophages. Our data suggest that the operon is involved in poly-3-hydroxybutyrate utilization and phospholipolysis and show that the bdhA-patD operon is a virulence determinant of L. pneumophila. In summary, the screen for amoeba-sensitive Legionella clones efficiently isolated mutants that do not grow in amoebae and, in the case of the bdhA-patD mutant, also human cells.

Detection of a Bacteriophage Gene Encoding a Mu-like Portal Protein in Haemophilus parasuis Reference Strains and Field Isolates by Nested Polymerase Chain Reaction

USDA-ARS?s Scientific Manuscript database

A nested PCR assay was developed to determine the presence of a gene encoding a bacteriophage Mu-like portal protein, gp29, in 15 reference strains and 31 field isolates of Haemophilus parasuis. Specific primers, based on the gene’s sequence, were utilized. A majority of the virulent reference strai...

Crystal structure of the Vibrio cholerae colonization factor TcpF and identification of a functional immunogenic site.

PubMed

Megli, Christina J; Yuen, Alex S W; Kolappan, Subramaniapillai; Richardson, Malcolm R; Dharmasena, Madushini N; Krebs, Shelly J; Taylor, Ronald K; Craig, Lisa

2011-06-03

Vibrio cholerae relies on two main virulence factors--toxin-coregulated pilus (TCP) and cholera toxin--to cause the gastrointestinal disease cholera. TCP is a type IV pilus that mediates bacterial autoagglutination and colonization of the intestine. TCP is encoded by the tcp operon, which also encodes TcpF, a protein of unknown function that is secreted by V. cholerae in a TCP-dependent manner. Although TcpF is not required for TCP biogenesis, a tcpF mutant has a colonization defect in the infant mouse cholera model that is as severe as a pilus mutant. Furthermore, TcpF antisera protect against V. cholerae infection. TcpF has no apparent sequence homology to any known protein. Here, we report the de novo X-ray crystal structure of TcpF and the identification of an epitope that is critical for its function as a colonization factor. A monoclonal antibody recognizing this epitope is protective against V. cholerae challenge and adds to the protection provided by an anti-TcpA antibody. These data suggest that TcpF has a novel function in V. cholerae colonization and define a region crucial for this function. Copyright © 2011 Elsevier Ltd. All rights reserved.

Staphylococcus aureus clonal dynamics and virulence factors in children with atopic dermatitis.

PubMed

Lomholt, Hans; Andersen, Klaus Ejner; Kilian, Mogens

2005-11-01

A prospective cohort study was undertaken to determine the clonal dynamics of Staphylococcus aureus colonization and infection during 1 y in children with atopic dermatitis, and to correlate specific clones, accessory gene regulator (agr) groups, and production of virulence factors with eczema activity. Eleven children were examined every 6 wk with swaps taken from active eczema, anterior nose, axillae and perineum, and scoring of eczema activity by severity scoring of atopic dermatitis (SCORAD). Individual S. aureus clonal types were identified and examined for production of superantigens, toxins, and were assigned to agr groups. S. aureus colonization patterns ranged from rare colonization over transient colonization to persistent colonization by a single clone or a dynamic exchange of up to five clones. Production of no single virulence factor including superantigens and toxins was significantly associated with exacerbation of eczema. In four children there was a shift between visits in agr group of colonizing clones. These shifts were associated with an increased SCORAD value of 19 (SE = 7, p = 0.009). Change of clones belonging to the same agr group was not associated with a higher SCORAD value. In 11 of 12 cases with two different clones co-colonizing a child the clones belonged to the same agr group. In conclusion, this limited group of children with atopic dermatitis showed highly variable colonization patterns of S. aureus, and communication between strains by use of agr encoded octa peptides appeared to be active in vivo. Increased severity of eczema was related to a change in agr group and may have been because of inflammation triggered by the takeover of an antigenically different clone, as agr groups represent ancient phylogenetic lineages.

Staphylococcus aureus hyaluronidase is a CodY-regulated virulence factor.

PubMed

Ibberson, Carolyn B; Jones, Crystal L; Singh, Shweta; Wise, Matthew C; Hart, Mark E; Zurawski, Daniel V; Horswill, Alexander R

2014-10-01

Staphylococcus aureus is a Gram-positive pathogen that causes a diverse range of bacterial infections. Invasive S. aureus strains secrete an extensive arsenal of hemolysins, immunomodulators, and exoenzymes to cause disease. Our studies have focused on the secreted enzyme hyaluronidase (HysA), which cleaves the hyaluronic acid polymer at the β-1,4 glycosidic bond. In the study described in this report, we have investigated the regulation and contribution of this enzyme to S. aureus pathogenesis. Using the Nebraska Transposon Mutant Library (NTML), we identified eight insertions that modulate extracellular levels of HysA activity. Insertions in the sigB operon, as well as in genes encoding the global regulators SarA and CodY, significantly increased HysA protein levels and activity. By altering the availability of branched-chain amino acids, we further demonstrated CodY-dependent repression of HysA activity. Additionally, through mutation of the CodY binding box upstream of hysA, the repression of HysA production was lost, suggesting that CodY is a direct repressor of hysA expression. To determine whether HysA is a virulence factor, a ΔhysA mutant of a community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 strain was constructed and found to be attenuated in a neutropenic, murine model of pulmonary infection. Mice infected with this mutant strain exhibited a 4-log-unit reduction in bacterial burden in their lungs, as well as reduced lung pathology and increased levels of pulmonary hyaluronic acid, compared to mice infected with the wild-type, parent strain. Taken together, these results indicate that S. aureus hyaluronidase is a CodY-regulated virulence factor. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Identification of the Staphylococcus aureus vfrAB operon, a novel virulence factor regulatory locus.

PubMed

Bose, Jeffrey L; Daly, Seth M; Hall, Pamela R; Bayles, Kenneth W

2014-05-01

During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not been characterized previously. Interestingly, a ΔvfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrB mutant did not restore hemolytic activity in the ΔvfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary for S. aureus pathogenesis.

Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics.

PubMed

Artursson, Karin; Söderlund, Robert; Liu, Lihong; Monecke, Stefan; Schelin, Jenny

2016-09-25

Reducing the prevalence of mastitis caused by Staphylococcus aureus (S. aureus) is essential to improve animal health and reduce economic losses for farmers. The clinical outcome of acute mastitis and risk of progression to persistent mastitis can, at least to some extent, be related to genetic variants of the strain causing the infection. In the present study we have used microarrays to investigate the presence of virulence genes in S. aureus isolates from dairy cows with acute clinical mastitis (n=70) and correlated the findings to other genotypic and phenotypic characteristics. Among the most commonly found virulence factors were genes encoding several hemolysin types, leukocidins D and lukM/lukF-P83, clumping factors A and B, fibrinogen binding protein and fibronectin-binding protein A. Some virulence factors e.g. fibronectin-binding protein B and Staphylococcus aureus surface protein G were less common. Genes coding for several staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) were commonly found, especially in one major pulsotype. No beta-lactamase genes were found in any common pulsotype, while present in some rare pulsotypes, indicated to be of human origin. Production of TSST-1, enterotoxins, hemolysins and beta-lactamase could all be positively correlated to presence of the corresponding genes. This study reveals a number of genotypic differences and similarities among common and rare pulsotypes of S. aureus from cases of mastitis in Sweden. The results could help the design of diagnostic tools to guide on-farm interventions according to the expected impact on udder health from a specific S. aureus genotype. Copyright © 2016 Elsevier B.V. All rights reserved.

Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis.

PubMed

Nunvar, Jaroslav; Kalferstova, Lucie; Bloodworth, Ruhi A M; Kolar, Michal; Degrossi, Jose; Lubovich, Silvina; Cardona, Silvia T; Drevinek, Pavel

2016-01-01

Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.

Leishmania dihydroxyacetonephosphate acyltransferase LmDAT is important for ether lipid biosynthesis but not for the integrity of detergent resistant membranes.

PubMed

Zufferey, Rachel; Al-Ani, Gada K; Dunlap, Kara

2009-12-01

Glycerolipid biosynthesis in Leishmania initiates with the acylation of glycerol-3-phosphate by a single glycerol-3-phosphate acyltransferase, LmGAT, or of dihydroxyacetonephosphate by a dihydroxyacetonephosphate acyltransferase, LmDAT. We previously reported that acylation of the precursor dihydroxyacetonephosphate rather than glycerol-3-phosphate is the physiologically relevant pathway for Leishmania parasites. We demonstrated that LmDAT is important for normal growth, survival during the stationary phase, and for virulence. Here, we assessed the role of LmDAT in glycerolipid metabolism and metacyclogenesis. LmDAT was found to be implicated in the biosynthesis of ether glycerolipids, including the ether lipid derived virulence factor lipophosphoglycan and glycosylphosphatidylinositol-anchored proteins. The null mutant produced longer lipophosphoglycan molecules that were not released in the medium, and augmented levels of glycosylphosphatidylinositol-anchored proteins. In addition, the integrity of detergent resistant membranes was not affected by the absence of the LmDAT gene. Further, our genetic analyses strongly suggest that LmDAT was synthetic lethal with the glycerol-3-phosphate acyltransferase encoding gene LmGAT, implying that Leishmania expresses only two acyltransferases that initiate the biosynthesis of its cellular glycerolipids. Last, despite the fact that LmDAT is important for virulence the null mutant still exhibited the typical characteristics of metacyclics.

Comparative analysis of the in vitro and in planta secretomes from Mycosphaerella fijiensis isolates.

PubMed

Escobar-Tovar, Lina; Guzmán-Quesada, Mauricio; Sandoval-Fernández, Jorge A; Gómez-Lim, Miguel A

2015-06-01

Black Sigatoka, a devastating disease of bananas and plantains worldwide, is caused by the fungus Mycosphaerella fijiensis. Several banana cultivars such as 'Yangambi Km 5' and Calcutta IV, have been known to be resistant to the fungus, but the resistance has been broken in 'Yangambi Km 5' in Costa Rica. Since the resistance of this variety still persists in Mexico, the aim of this study was to compare the in vitro and in planta secretomes from two avirulent and virulent M. fijiensis isolates using proteomics and bioinformatics approaches. We aimed to identify differentially expressed proteins in fungal isolates that differ in pathogenicity and that might be responsible for breaking the resistance in 'Yangambi Km 5'. We were able to identify 90 protein spots in the secretomes of fungal isolates encoding 42 unique proteins and 35 differential spots between them. Proteins involved in carbohydrate transport and metabolism were more prevalent. Several proteases, pathogenicity-related, ROS detoxification and unknown proteins were also highly or specifically expressed by the virulent isolate in vitro or during in planta infection. An unknown protein representing a virulence factor candidate was also identified. These results demonstrated that the secretome reflects major differences between both M. fijiensis isolates. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments

PubMed Central

Miller, Daniel P.; Hutcherson, Justin A.; Wang, Yan; Nowakowska, Zuzanna M.; Potempa, Jan; Yoder-Himes, Deborah R.; Scott, David A.; Whiteley, Marvin; Lamont, Richard J.

2017-01-01

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism. PMID:28900609

The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.

PubMed Central

Gouin, E; Mengaud, J; Cossart, P

1994-01-01

Most known Listeria monocytogenes virulence genes cluster within a 9.6-kb chromosomal region. This region is flanked on one end by two uncharacterized open reading frames (ORF A and ORF B) and ldh, an ORF presumably encoding the L. monocytogenes lactate dehydrogenase (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). We report here that the other end is flanked by prs, and ORF homologous to phosphoribosyl PPi synthetase genes. ORF B and prs were detected in all Listeria species and thus delimit the virulence region. This virulence gene cluster was detected exclusively in hemolytic Listeria species, Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species. Images PMID:8039927

Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene-disrupted and gene-overexpressing mutant lines.

PubMed

Ben-Daniel, Bat-Hen; Bar-Zvi, Dudy; Tsror Lahkim, Leah

2012-02-01

Colletotrichum coccodes (Wallr.) S. Hughes, the causal agent of black dot on potato and anthracnose on tomato, reduces yield and crop quality. We explored the role of secreted pectate lyase (PL), a cell wall-degrading enzyme, in the aggressiveness of C. coccodes. In vitro-cultivated highly aggressive isolates secreted immunologically detectable PL levels 6 h after transfer to secondary medium versus 12 h for mildly aggressive isolates, suggesting that secreted PL is a virulence factor. The gene encoding PL, CcpelA, was cloned and used for the genetic manipulation of highly (US-41 and Si-72) and mildly (Si-60) aggressive isolates. CcpelA gene-disrupted mutants showed reduced aggressiveness towards tomato fruits and impaired PL secretion and extracellular activity. Conversely, overexpression of CcpelA in the Si-60 isolate increased its aggressiveness and PL secretion. Comparison of CcpelA cloned from isolates US-41 and Si-60 revealed that both encode identical proteins, but differ in their promoters. Bioinformatics analysis for cis-acting elements suggested that the promoters of the US-41 and Si-60 isolates contain one and no AreA-binding site (GATA box), respectively. AreA has been suggested to be involved in fungal aggressiveness; therefore, CcpelA may be a key virulence factor in C. coccodes pathogenicity, and the differences in isolate aggressiveness might result from promoter activity. Quantitative reverse transcriptase-polymerase chain reaction analyses confirmed the higher level of CcpelA transcript in isolate US-41 versus Si-60. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

Induction of Virulence Gene Expression in Staphylococcus aureus by Pulmonary Surfactant

PubMed Central

Ishii, Kenichi; Adachi, Tatsuo; Yasukawa, Jyunichiro; Suzuki, Yutaka; Hamamoto, Hiroshi

2014-01-01

We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal. PMID:24452679

OI-57, a Genomic Island of Escherichia coli O157, Is Present in Other Seropathotypes of Shiga Toxin-Producing E. coli Associated with Severe Human Disease▿

PubMed Central

Imamovic, Lejla; Tozzoli, Rosangela; Michelacci, Valeria; Minelli, Fabio; Marziano, Maria Luisa; Caprioli, Alfredo; Morabito, Stefano

2010-01-01

Strains of Shiga toxin-producing Escherichia coli (STEC) are a heterogeneous E. coli group that may cause severe disease in humans. STEC have been categorized into seropathotypes (SPTs) based on their phenotypic and molecular characteristics and the clinical features of the associated diseases. SPTs range from A to E, according to a decreasing rank of pathogenicity. To define the virulence gene asset (“virulome”) characterizing the highly pathogenic SPTs, we used microarray hybridization to compare the whole genomes of STEC belonging to SPTs B, C, and D with that of STEC O157 (SPT A). The presence of the open reading frames (ORFs) associated with SPTs A and B was subsequently investigated by PCR in a larger panel of STEC and in other E. coli strains. A genomic island termed OI-57 was present in SPTs A and B but not in the other SPTs. OI-57 harbors the putative virulence gene adfO, encoding a factor enhancing the adhesivity of STEC O157, and ckf, encoding a putative killing factor for the bacterial cell. PCR analyses showed that OI-57 was present in its entirety in the majority of the STEC genomes examined, indicating that it represents a stable acquisition of the positive clonal lineages. OI-57 was also present in a high proportion of the human enteropathogenic E. coli genomes assayed, suggesting that it could be involved in the attaching-and-effacing colonization of the intestinal mucosa. In conclusion, OI-57 appears to be part of the virulome of pathogenic STEC and further studies are needed to elucidate its role in the pathogenesis of STEC infections. PMID:20823207

Central role of a bacterial two-component gene regulatory system of previously unknown function in pathogen persistence in human saliva.

PubMed

Shelburne, Samuel A; Sumby, Paul; Sitkiewicz, Izabela; Granville, Chanel; DeLeo, Frank R; Musser, James M

2005-11-01

The molecular genetic mechanisms used by bacteria to persist in humans are poorly understood. Group A Streptococcus (GAS) causes the majority of bacterial pharyngitis cases in humans and is prone to persistently inhabit the upper respiratory tract. To gain information about how GAS survives in and infects the oropharynx, we analyzed the transcriptome of a serotype M1 strain grown in saliva. The dynamic pattern of changes in transcripts of genes [spy0874/0875, herein named sptR and sptS (sptR/S), for saliva persistence] encoding a two-component gene regulatory system of unknown function suggested that SptR/S contributed to persistence of GAS in saliva. Consistent with this idea, an isogenic nonpolar mutant strain (DeltasptR) was dramatically less able to survive in saliva compared with the parental strain. Iterative expression microarray analysis of bacteria grown in saliva revealed that transcripts of several known and putative GAS virulence factor genes were decreased significantly in the DeltasptR mutant strain. Compared with the parental strain, the isogenic mutant strain also had altered transcripts of multiple genes encoding proteins involved in complex carbohydrate acquisition and utilization pathways. Western immunoblot analysis and real-time PCR analysis of GAS in throat swabs taken from humans with pharyngitis confirmed the findings. We conclude that SptR/S optimizes persistence of GAS in human saliva, apparently by strategically influencing metabolic pathways and virulence factor production. The discovery of a genetic program that significantly increased persistence of a major human pathogen in saliva enhances understanding of how bacteria survive in the host and suggests new therapeutic strategies.

ORD/NERL CURRENT VRARS RESEARCH

EPA Science Inventory

Virulence is the degree of pathogenicity of a microorganism and virulence factors are the components of an organism that contribute to virulence. Identifying microorganisms using known virulence factors is one method used by microbiologists to distinguish pathogenic isolates fro...

Identification of putative adhesins of Actinobacillus suis and their homologues in other members of the family Pasteurellaceae.

PubMed

Bujold, Adina R; MacInnes, Janet I

2015-11-14

Actinobacillus suis disease has been reported in a wide range of vertebrate species, but is most commonly found in swine. A. suis is a commensal of the tonsils of the soft palate of swine, but in the presence of unknown stimuli it can invade the bloodstream, causing septicaemia and sequelae such as meningitis, arthritis, and death. It is genotypically and phenotypically similar to A. pleuropneumoniae, the causative agent of pleuropneumonia, and to other members of the family Pasteurellaceae that colonise tonsils. At present, very little is known about the genes involved in attachment, colonisation, and invasion by A. suis (or related members of the tonsil microbiota). Bioinformatic analyses of the A. suis H91-0380 genome were done using BASys and blastx in GenBank. Forty-seven putative adhesin-associated genes predicted to encode 24 putative adhesins were discovered. Among these are 6 autotransporters, 25 fimbriae-associated genes (encoding 3 adhesins), 12 outer membrane proteins, and 4 additional genes (encoding 3 adhesins). With the exception of 2 autotransporter-encoding genes (aidA and ycgV), both with described roles in virulence in other species, all of the putative adhesin-associated genes had homologues in A. pleuropneumoniae. However, the majority of the closest homologues of the A. suis adhesins are found in A. ureae and A. capsulatus--species not known to infect swine, but both of which can cause systemic infections. A. suis and A. pleuropneumoniae share many of the same putative adhesins, suggesting that the different diseases, tissue tropism, and host range of these pathogens are due to subtle genetic differences, or perhaps differential expression of virulence factors during infection. However, many of the putative adhesins of A. suis share even greater homology with those of other pathogens within the family Pasteurellaceae. Similar to A. suis, these pathogens (A. capsulatus and A. ureae) cause systemic infections and it is tempting to speculate that they employ similar strategies to invade the host, but more work is needed before that assertion can be made. This work begins to examine adhesin-associated factors that allow some members of the family Pasteurellaceae to invade the bloodstream while others cause a more localised infection.

Identification of VP1/2A and 2C as Virulence Genes of Hepatitis A Virus and Demonstration of Genetic Instability of 2C

PubMed Central

Emerson, Suzanne U.; Huang, Ying K.; Nguyen, Hanh; Brockington, Alicia; Govindarajan, Sugantha; St. Claire, Marisa; Shapiro, Max; Purcell, Robert H.

2002-01-01

Fourteen different chimeric virus genomes were constructed from two infectious cDNA clones encoding a virulent and an attenuated isolate, respectively, of the HM175 strain of hepatitis A virus. The ability of each recombinant virus to infect tamarins and to cause acute hepatitis was determined. Comparisons of the genotype and phenotype of each virus suggested that VP1/2A and 2C genes were responsible for virulence. The 2C gene derived from the attenuated parent virus was unstable, and one or more mutations arose in this gene during the first passage in tamarins. PMID:12163575

Candida albicans Iff11, a secreted protein required for cell wall structure and virulence.

PubMed

Bates, Steven; de la Rosa, José M; MacCallum, Donna M; Brown, Alistair J P; Gow, Neil A R; Odds, Frank C

2007-06-01

The Candida albicans cell wall is the immediate point of contact with the host and is implicated in the host-fungal interaction and virulence. To date, a number of cell wall proteins have been identified and associated with virulence. Analysis of the C. albicans genome has identified the IFF gene family as encoding the largest family of cell wall-related proteins. This family is also conserved in a range of other Candida species. Iff11 differs from other family members in lacking a GPI anchor, and we have demonstrated it to be O glycosylated and secreted in C. albicans. A null mutant lacking IFF11 was hypersensitive to cell wall-damaging agents, suggesting a role in cell wall organization. In a murine model of systemic infection the null mutant was highly attenuated in virulence, and survival-standardized infections suggest it is required to establish an infection. This work provides the first evidence of the importance of this gene family in the host-fungal interaction and virulence.

Antimicrobial resistance and virulence genes in enterococci from wild game meat in Spain.

PubMed

Guerrero-Ramos, Emilia; Cordero, Jorge; Molina-González, Diana; Poeta, Patrícia; Igrejas, Gilberto; Alonso-Calleja, Carlos; Capita, Rosa

2016-02-01

A total of 55 enterococci (45 Enterococcus faecium, 7 Enterococcus faecalis, and three Enterococcus durans) isolated from the meat of wild game animals (roe deer, boar, rabbit, pheasant, and pigeon) in North-Western Spain were tested for susceptibility to 14 antimicrobials by the disc diffusion method. All strains showed a multi-resistant phenotype (resistance to between three and 10 antimicrobials). The strains exhibited high percentages of resistance to erythromycin (89.1%), tetracycline (67.3%), ciprofloxacin (92.7%), nitrofurantoin (67.3%), and quinupristin-dalfopristin (81.8%). The lowest values (9.1%) were observed for high-level resistance to gentamicin, kanamycin, and streptomycin. The average number of resistances per strain was 5.8 for E. faecium isolates, 7.9 for E. faecalis, and 5.7 for E. durans. Genes encoding antimicrobial resistance and virulence were studied by polymerase chain reaction. A total of 15 (57.7%) of the 26 vancomycin-resistant isolates harboured the vanA gene. Other resistance genes detected included vanB, erm(B) and/or erm(C), tet(L) and/or tet(M), acc(6')-aph(2″), and aph(3')-IIIa in strains resistant to vancomycin, erythromycin, tetracycline, gentamicin, and kanamycin, respectively. Specific genes of the Tn5397 transposon were detected in 54.8% of the tet(M)-positive enterococci. Nine virulence factors (gelE, agg, ace, cpd, frs, esp, hyl, efaAfs and efaAfm) were studied. All virulence genes, with the exception of the frs gene, were found to be present in the enterococcal isolates. At least one virulence gene was detected in 20.0% of E. faecium, 71.4% of E. faecalis and 33.3% of E. durans isolates, with ace and cpd being the most frequently detected genes (6 isolates each). This suggests that wild game meat might play a role in the spreading through the food chain of enterococci with antimicrobial resistance and virulence determinants to humans. Copyright © 2015 Elsevier Ltd. All rights reserved.

NF-κB Signaling in Gastric Cancer

PubMed Central

Sokolova, Olga; Naumann, Michael

2017-01-01

Gastric cancer is a leading cause of cancer death worldwide. Diet, obesity, smoking and chronic infections, especially with Helicobacter pylori, contribute to stomach cancer development. H. pylori possesses a variety of virulence factors including encoded factors from the cytotoxin-associated gene pathogenicity island (cagPAI) or vacuolating cytotoxin A (VacA). Most of the cagPAI-encoded products form a type 4 secretion system (T4SS), a pilus-like macromolecular transporter, which translocates CagA into the cytoplasm of the host cell. Only H. pylori strains carrying the cagPAI induce the transcription factor NF-κB, but CagA and VacA are dispensable for direct NF-κB activation. NF-κB-driven gene products include cytokines/chemokines, growth factors, anti-apoptotic factors, angiogenesis regulators and metalloproteinases. Many of the genes transcribed by NF-κB promote gastric carcinogenesis. Since it has been shown that chemotherapy-caused cellular stress could elicit activation of the survival factor NF-κB, which leads to acquisition of chemoresistance, the NF-κB system is recommended for therapeutic targeting. Research is motivated for further search of predisposing conditions, diagnostic markers and efficient drugs to improve significantly the overall survival of patients. In this review, we provide an overview about mechanisms and consequences of NF-κB activation in gastric mucosa in order to understand the role of NF-κB in gastric carcinogenesis. PMID:28350359

A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease

PubMed Central

Bacci, Giovanni; Fiscarelli, Ersilia; Taccetti, Giovanni; Dolce, Daniela; Paganin, Patrizia; Morelli, Patrizia; Tuccio, Vanessa; De Alessandri, Alessandra; Lucidi, Vincenzina

2017-01-01

In recent years, next-generation sequencing (NGS) was employed to decipher the structure and composition of the microbiota of the airways in cystic fibrosis (CF) patients. However, little is still known about the overall gene functions harbored by the resident microbial populations and which specific genes are associated with various stages of CF lung disease. In the present study, we aimed to identify the microbial gene repertoire of CF microbiota in twelve patients with severe and normal/mild lung disease by performing sputum shotgun metagenome sequencing. The abundance of metabolic pathways encoded by microbes inhabiting CF airways was reconstructed from the metagenome. We identified a set of metabolic pathways differently distributed in patients with different pulmonary function; namely, pathways related to bacterial chemotaxis and flagellar assembly, as well as genes encoding efflux-mediated antibiotic resistance mechanisms and virulence-related genes. The results indicated that the microbiome of CF patients with low pulmonary function is enriched in virulence-related genes and in genes encoding efflux-mediated antibiotic resistance mechanisms. Overall, the microbiome of severely affected adults with CF seems to encode different mechanisms for the facilitation of microbial colonization and persistence in the lung, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in patients with severe lung disease. PMID:28758937

Nucleic acid immunization protects dogs against challenge with virulent canine parvovirus.

PubMed

Jiang, W; Baker, H J; Swango, L J; Schorr, J; Self, M J; Smith, B F

1998-04-01

Nucleic acid vaccines (NAVs) use expression vectors encoding one or more antigen genes to transfect host cells inducing both humoral and cellular immunity against the expressed antigen. NAV offers major advantages over conventional vaccines for the protection of humans and animals. This study shows that a plasmid DNA (pGT36VP1) encoding the full length VP1 region of canine parvovirus (CPV) induces immunity that protects dogs against challenge with virulent virus. Five dogs without anti-CPV antibodies were injected at 9 months of age with increasing doses of pGT36VP1 or saline. NAV vaccinated dogs showed an increase of serum IgG titer starting 1 week post-injection which peaked at week 2 and remained detectable for at least 14 weeks. A second dose of NAV resulted in an anamnestic response within 1 week. IgG titers peaked at week 3 and 4 after the second injection. All pGT36VP1 vaccinated dogs were protected against infection after virulent CPV challenge regardless of dose and the unvaccinated control dog was fully susceptible. This study demonstrated for the first time that a NAV can protect dogs against an infectious disease.

Feline Immunodeficiency Virus Evolutionarily Acquires Two Proteins, Vif and Protease, Capable of Antagonizing Feline APOBEC3.

PubMed

Yoshikawa, Rokusuke; Takeuchi, Junko S; Yamada, Eri; Nakano, Yusuke; Misawa, Naoko; Kimura, Yuichi; Ren, Fengrong; Miyazawa, Takayuki; Koyanagi, Yoshio; Sato, Kei

2017-06-01

The interplay between viral and host proteins has been well studied to elucidate virus-host interactions and their relevance to virulence. Mammalian genes encode apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins, which act as intrinsic restriction factors against lentiviruses. To overcome APOBEC3-mediated antiviral actions, lentiviruses have evolutionarily acquired an accessory protein, viral infectivity factor (Vif), and Vif degrades host APOBEC3 proteins via a ubiquitin/proteasome-dependent pathway. Although the Vif-APOBEC3 interaction and its evolutionary significance, particularly those of primate lentiviruses (including HIV) and primates (including humans), have been well investigated, those of nonprimate lentiviruses and nonprimates are poorly understood. Moreover, the factors that determine lentiviral pathogenicity remain unclear. Here, we focus on feline immunodeficiency virus (FIV), a pathogenic lentivirus in domestic cats, and the interaction between FIV Vif and feline APOBEC3 in terms of viral virulence and evolution. We reveal the significantly reduced diversity of FIV subtype B compared to that of other subtypes, which may associate with the low pathogenicity of this subtype. We also demonstrate that FIV subtype B Vif is less active with regard to feline APOBEC3 degradation. More intriguingly, we further reveal that FIV protease cleaves feline APOBEC3 in released virions. Taken together, our findings provide evidence that a lentivirus encodes two types of anti-APOBEC3 factors, Vif and viral protease. IMPORTANCE During the history of mammalian evolution, mammals coevolved with retroviruses, including lentiviruses. All pathogenic lentiviruses, excluding equine infectious anemia virus, have acquired the vif gene via evolution to combat APOBEC3 proteins, which are intrinsic restriction factors against exogenous lentiviruses. Here we demonstrate that FIV, a pathogenic lentivirus in domestic cats, antagonizes feline APOBEC3 proteins by both Vif and a viral protease. Furthermore, the Vif proteins of an FIV subtype (subtype B) have attenuated their anti-APOBEC3 activity through evolution. Our findings can be a clue to elucidate the complicated evolutionary processes by which lentiviruses adapt to mammals. Copyright © 2017 Yoshikawa et al.

Feline Immunodeficiency Virus Evolutionarily Acquires Two Proteins, Vif and Protease, Capable of Antagonizing Feline APOBEC3

PubMed Central

Yoshikawa, Rokusuke; Takeuchi, Junko S.; Yamada, Eri; Nakano, Yusuke; Misawa, Naoko; Kimura, Yuichi; Ren, Fengrong; Miyazawa, Takayuki; Koyanagi, Yoshio

2017-01-01

ABSTRACT The interplay between viral and host proteins has been well studied to elucidate virus-host interactions and their relevance to virulence. Mammalian genes encode apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins, which act as intrinsic restriction factors against lentiviruses. To overcome APOBEC3-mediated antiviral actions, lentiviruses have evolutionarily acquired an accessory protein, viral infectivity factor (Vif), and Vif degrades host APOBEC3 proteins via a ubiquitin/proteasome-dependent pathway. Although the Vif-APOBEC3 interaction and its evolutionary significance, particularly those of primate lentiviruses (including HIV) and primates (including humans), have been well investigated, those of nonprimate lentiviruses and nonprimates are poorly understood. Moreover, the factors that determine lentiviral pathogenicity remain unclear. Here, we focus on feline immunodeficiency virus (FIV), a pathogenic lentivirus in domestic cats, and the interaction between FIV Vif and feline APOBEC3 in terms of viral virulence and evolution. We reveal the significantly reduced diversity of FIV subtype B compared to that of other subtypes, which may associate with the low pathogenicity of this subtype. We also demonstrate that FIV subtype B Vif is less active with regard to feline APOBEC3 degradation. More intriguingly, we further reveal that FIV protease cleaves feline APOBEC3 in released virions. Taken together, our findings provide evidence that a lentivirus encodes two types of anti-APOBEC3 factors, Vif and viral protease. IMPORTANCE During the history of mammalian evolution, mammals coevolved with retroviruses, including lentiviruses. All pathogenic lentiviruses, excluding equine infectious anemia virus, have acquired the vif gene via evolution to combat APOBEC3 proteins, which are intrinsic restriction factors against exogenous lentiviruses. Here we demonstrate that FIV, a pathogenic lentivirus in domestic cats, antagonizes feline APOBEC3 proteins by both Vif and a viral protease. Furthermore, the Vif proteins of an FIV subtype (subtype B) have attenuated their anti-APOBEC3 activity through evolution. Our findings can be a clue to elucidate the complicated evolutionary processes by which lentiviruses adapt to mammals. PMID:28331087

Role of the 85-Kilobase Plasmid and Plasmid-Encoded Virulence-Associated Protein A in Intracellular Survival and Virulence of Rhodococcus equi

PubMed Central

Giguère, Steeve; Hondalus, Mary K.; Yager, Julie A.; Darrah, Patricia; Mosser, David M.; Prescott, John F.

1999-01-01

Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype. PMID:10377138

Transcriptome Reprogramming by Plasmid-Encoded Transcriptional Regulators Is Required for Host Niche Adaption of a Macrophage Pathogen

PubMed Central

Coulson, Garry B.; Miranda-CasoLuengo, Aleksandra A.; Miranda-CasoLuengo, Raúl; Wang, Xiaoguang; Oliver, Jenna; Willingham-Lane, Jennifer M.

2015-01-01

Rhodococcus equi is a facultative intracellular pathogen of macrophages, relying on the presence of a conjugative virulence plasmid harboring a 21-kb pathogenicity island (PAI) for growth in host macrophages. The PAI encodes a family of 6 virulence-associated proteins (Vaps) in addition to 20 other proteins. The contribution of these to virulence has remained unclear. We show that the presence of only 3 virulence plasmid genes (of 73 in total) is required and sufficient for intracellular growth. These include a single vap family member, vapA, and two PAI-located transcriptional regulators, virR and virS. Both transcriptional regulators are essential for wild-type-level expression of vapA, yet vapA expression alone is not sufficient to allow intracellular growth. A whole-genome microarray analysis revealed that VirR and VirS substantially integrate themselves into the chromosomal regulatory network, significantly altering the transcription of 18% of all chromosomal genes. This pathoadaptation involved significant enrichment of select gene ontologies, in particular, enrichment of genes involved in transport processes, energy production, and cellular metabolism, suggesting a major change in cell physiology allowing the bacterium to grow in the hostile environment of the host cell. The results suggest that following the acquisition of the virulence plasmid by an avirulent ancestor of R. equi, coevolution between the plasmid and the chromosome took place, allowing VirR and VirS to regulate the transcription of chromosomal genes in a process that ultimately promoted intracellular growth. Our findings suggest a mechanism for cooption of existing chromosomal traits during the evolution of a pathogenic bacterium from an avirulent saprophyte. PMID:26015480

Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete Mucor circinelloides.

PubMed

Lee, Soo Chan; Li, Alicia; Calo, Silvia; Heitman, Joseph

2013-01-01

Many pathogenic fungi are dimorphic and switch between yeast and filamentous states. This switch alters host-microbe interactions and is critical for pathogenicity. However, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high CO₂ conditions. We found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits exclusively multi-budded yeast growth. We also found that M. circinelloides encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Mutations in the latch region of CnbR and in the FKBP12-FK506 binding domain of CnaA result in hyphal growth of Mucor in the presence of FK506. Disruption of the cnbR gene encoding the sole calcineurin B subunit necessary for calcineurin activity yielded mutants locked in permanent yeast phase growth. These findings reveal that the calcineurin pathway plays key roles in the dimorphic transition from yeast to hyphae. The cnbR yeast-locked mutants are less virulent than the wild-type strain in a heterologous host system, providing evidence that hyphae or the yeast-hyphal transition are linked to virulence. Protein kinase A activity (PKA) is elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, suggesting a novel connection between PKA and calcineurin. cnaA mutants lacking the CnaA catalytic subunit are hypersensitive to calcineurin inhibitors, display a hyphal polarity defect, and produce a mixture of yeast and hyphae in aerobic culture. The cnaA mutants also produce spores that are larger than wild-type, and spore size is correlated with virulence potential. Our results demonstrate that the calcineurin pathway orchestrates the yeast-hyphal and spore size dimorphic transitions that contribute to virulence of this common zygomycete fungal pathogen.

Genomic insights of Pannonibacter phragmitetus strain 31801 isolated from a patient with a liver abscess.

PubMed

Zhou, Yajun; Jiang, Tao; Hu, Shaohua; Wang, Mingxi; Ming, Desong; Chen, Shicheng

2017-12-01

Pannonibacter phragmitetus is a bioremediation reagent for the detoxification of heavy metals and polycyclic aromatic compounds (PAHs) while it rarely infects healthy populations. However, infection by the opportunistic pathogen P. phragmitetus complicates diagnosis and treatments, and poses a serious threat to immunocompromised patients owing to its multidrug resistance. Unfortunately, genome features, antimicrobial resistance, and virulence potentials in P. phragmitetus have not been reported before. A predominant colony (31801) was isolated from a liver abscess patient, indicating that it accounted for the infection. To investigate its infection mechanism(s) in depth, we sequenced this bacterial genome and tested its antimicrobial resistance. Average nucleotide identity (ANI) analysis assigned the bacterium to the species P. phragmitetus (ANI, >95%). Comparative genomics analyses among Pannonibacter spp. representing the different living niches were used to describe the Pannonibacter pan-genomes and to examine virulence factors, prophages, CRISPR arrays, and genomic islands. Pannonibacter phragmitetus 31801 consisted of one chromosome and one plasmid, while the plasmid was absent in other Pannonibacter isolates. Pannonibacter phragmitetus 31801 may have a great infection potential because a lot of genes encoding toxins, flagellum formation, iron uptake, and virulence factor secretion systems in its genome. Moreover, the genome has 24 genomic islands and 2 prophages. A combination of antimicrobial susceptibility tests and the detailed antibiotic resistance gene analysis provide useful information about the drug resistance mechanisms and therefore can be used to guide the treatment strategy for the bacterial infection. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

[Genetic diversity of extraintestinal Escherichia coli strains producers of beta-lactamases TEM, SHV and CTX-M associated with healthcare].

PubMed

Varela, Yasmin; Millán, Beatriz; Araque, María

2017-06-01

There are few reports from Venezuela describing the genetic basis that sustains the pathogenic potential and phylogenetics of Escherichia coli extraintestinal strains isolated in health care units. To establish the genetic diversity of extraintestinal E. coli strains producers of betalactamases TEM, SHV and CTX-M associated with healthcare. We studied a collection of 12 strains of extraintestinal E. coli with diminished sensitivity to broad-spectrum cephalosporins. Antimicrobial susceptibility was determined by minimum inhibitory concentration. We determined the phylogenetic groups, virulence factors and genes encoding antimicrobial resistance using PCR, and clonal characterization by repetitive element palindromic-PCR rep-PCR. All strains showed resistance to cephalosporins and joint resistance to quinolones and aminoglycosides. The phylogenetic distribution showed that the A and B1 groups were the most frequent, followed by D and B2. We found all the virulence factors analyzed in the B2 group, and fimH gene was the most frequent among them. We found blaCTX-M in all strains,with a higher prevalence of blaCTX-M-8; two of these strains showed coproduction of blaCTX-M-9 and were genetically identified as blaCTXM-65 and blaCTX-M-147 by sequencing. The strains under study showed genetic diversity, hosting a variety of virulence genes, as well as antimicrobial resistance with no particular phylogroup prevalence. This is the first report of blaCTX-M alleles in Venezuela and in the world associated to non-genetically related strains isolated in health care units, a situation that deserves attention, as well as the rationalization of antimicrobials use.

Role and regulation of the orphan AphA protein of quorum sensing in pathogenic Vibrios.

PubMed

Lu, Renfei; Osei-Adjei, George; Huang, Xinxiang; Zhang, Yiquan

2018-03-01

Quorum sensing (QS), a cell-to-cell communication process, is widely distributed in the bacterial kingdom. Bacteria use QS to control gene expression in response to cell density by detecting the signal molecules called autoinducers. AphA protein is the master QS regulator of vibrios operating at low cell density. It regulates the expression of a variety of genes, especially those encoding virulence factors, flagella/motility and biofilm formation. The role and regulation of AphA in vibrios, especially in human pathogenic vibrios, are summarized in this review. Clarification of the roles of AphA will help us to understand the pathogenesis of vibrios.

The role of complex carbohydrate catabolism in the pathogenesis of invasive streptococci

PubMed Central

Shelburne, Samuel A.; Davenport, Michael T.; Keith, David B.; Musser, James M.

2009-01-01

Historically, the study of bacterial catabolism of complex carbohydrates has contributed to understanding basic bacterial physiology. Recently, however, genome-wide screens of streptococcal pathogenesis have identified genes encoding proteins involved in complex carbohydrate catabolism as participating in pathogen infectivity. Subsequent studies have focused on specific mechanisms by which carbohydrate utilization proteins might contribute to the ability of streptococci to colonize and infect the host. Moreover, transcriptome and biochemical analyses have uncovered novel regulatory pathways by which streptococci link environmental carbohydrate availability to virulence factor production. Herein we review new insights into the role of complex carbohydrates in streptococcal host-pathogen interaction. PMID:18508271

Effect of sypQ gene on poly-N-acetylglucosamine biosynthesis in Vibrio parahaemolyticus and its role in infection process.

PubMed

Ye, Libin; Zheng, Xiaolin; Zheng, Hongjian

2014-04-01

The syp locus includes four genes encoding putative regulators, six genes encoding glycosyltransferases, two encoding export proteins, and six other genes encoding unidentified functional proteins associated with biofilm formation and symbiotic colonization. However, the individual functions of the respective genes remain unclear. Amino acid alignment indicates that sypQ is presumably involved in biosynthesizing poly-N-acetylglucosamine (PNAG), which is proposed to be a critical virulence factor in pathogen infection and is regarded as a target for protective immunity against a variety of Gram-negative/positive pathogens. However, no evidence showing that Vibrio parahaemolyticus also produces PNAG has been reported. Herein, the V. parahaemolyticus is confirmed to possess potential for producing PNAG for the first time. Our results indicated that gene sypQ is associated with PNAG biosynthesis and PNAG is involved in pathogen colonization. We propose that the function of pgaC in Escherichia coli could be taken over by sypQ from V. parahaemolyticus. We also tested whether PNAG can be used as a target against V. parahaemolyticus when it infects Pseudosciaena crocea. Our results showed that PNAG isolated from V. parahaemolyticus is an effective agent for decreasing V. parahaemolyticus invasion, implying that PNAG could be used to develop an effective vaccine against V. parahaemolyticus infection.

Virulence Factors Detection in Aspergillus Isolates from Clinical and Environmental Samples

PubMed Central

Raksha; Urhekar, A.D.

2017-01-01

Introduction Pathogenesis of aspergillosis is dependent on various factors of the host (immune status) and virulence factors of the pathogen which could play a significant role in the pathogenesis of invasive aspergillosis. Aim To study the virulence factors of Aspergillus species isolated from patient samples and environmental samples. Materials and Methods This prospective and experimental study was carried out at Department of Microbiology, MGM Medical College and Hospital, Mumbai, Maharashtra, India, from July 2014 to June 2015. For detection of virulence factors of Aspergillus species, total 750 samples were included in this study (350 from patients and 400 samples from environment). Patient samples and hospital environment samples were subjected to standard methods for screening of Biofilm, Lipase, α–amylase, proteinase, haemolysin, phospholipase and pectinase. Statistical analysis was done using Chi-square test and SPSS (Version 17.0). Results American Type Culture Collection (ATCC) control of Aspergillus oryzae, Aspergillus niger and Aspergillus brasiliensis showed production of all virulence factors. In patient samples maximum virulence factor was produced i.e., α-amylase activity (89.74%) followed by proteinase activity (87.17%), biofilm production was (82.05%) haemolysin activity (79.48%), lipase activity (66.66%), pectinase activity and phospholipase activity (61.53%). In environment samples maximum virulence factor was produced i.e., proteinase activity (41.02%) followed by biofilm production was (38.46%), α-amylase activity (35.89%), haemolysin activity (33.33%), lipase activity (28.20%), phospholipase (25.64%) and pectinase activity (23.07%). The differences in patient and environment virulence factors were statistically significant (p-value <0.05). Conclusion Overall the presence of virulence factors was found more in Aspergillus species isolated from patient samples then environmental samples. This could be due to invasiveness nature of Aspergilli. Aspergillus niger was common isolates from both patient and environmental samples. Our study highlights the possible transmission of Aspergilli from environment to patient. Detection of virulence factors of Aspergillus species help to differentiate between pathogenic and non-pathogenic Aspergilli. Presence of virulence factors confirmed pathogenicity of the isolates. It also helps the physicians to treat the patient when appropriate treatment is needed. PMID:28892890

Comparison of vaccine efficacy for different antigen delivery systems for infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L.) in a cohabitation challenge model.

PubMed

Munang'andu, Hetron M; Fredriksen, Børge N; Mutoloki, Stephen; Brudeseth, Bjørn; Kuo, Tsun-Yung; Marjara, Inderjit S; Dalmo, Roy A; Evensen, Øystein

2012-06-08

Two strains of IPNV made by reverse genetics on the Norwegian Sp strain NVI-015 (GenBank AY379740) backbone encoding the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs were used to prepare inactivated whole virus (IWV), nanoparticle vaccines with whole virus, Escherichia coli subunit encoding truncated VP2-TA and VP2-PT, VP2-TA and VP2-PT fusion antigens with putative translocating domains of Pseudomonas aeruginosa exotoxin, and plasmid DNA encoding segment A of the TA strain. Post challenge survival percentages (PCSP) showed that IWV vaccines conferred highest protection (PCSP=42-53) while nanoparticle, sub-unit recombinant and DNA vaccines fell short of the IWV vaccines in Atlantic salmon (Salmo salar L.) postsmolts challenged with the highly virulent Sp strain NVI-015 (TA strain) of IPNV after 560 degree days post vaccination. Antibody levels induced by these vaccines did not show antigenic differences between the virulent and avirulent motifs for vaccines made with the same antigen dose and delivery system after 8 weeks post vaccination. Our findings show that fish vaccinated with less potent vaccines comprising of nanoparticle, DNA and recombinant vaccines got infected much earlier and yielded to higher infection rates than fish vaccinated with IWV vaccines that were highly potent. Ability of the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs to limit establishment of infection showed equal protection for vaccines made of the same antigen dose and delivery systems. Prevention of tissue damage linked to viral infection was eminent in the more potent vaccines than the less protective ones. Hence, there still remains the challenge of developing highly efficacious vaccines with the ability to eliminate the post challenge carrier state in IPNV vaccinology. Copyright © 2012 Elsevier Ltd. All rights reserved.

Gene and Protein Expression in Response to Different Growth Temperatures and Oxygen Availability in Burkholderia thailandensis

PubMed Central

Peano, Clelia; Chiaramonte, Fabrizio; Motta, Sara; Pietrelli, Alessandro; Jaillon, Sebastien; Rossi, Elio; Consolandi, Clarissa; Champion, Olivia L.; Michell, Stephen L.; Freddi, Luca; Falciola, Luigi; Basilico, Fabrizio; Garlanda, Cecilia; Mauri, Pierluigi; De Bellis, Gianluca; Landini, Paolo

2014-01-01

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors. PMID:24671187

The Dominant-Negative Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR Increases the Efficacy of Rift Valley Fever Virus MP-12 Vaccine

PubMed Central

Lihoradova, Olga; Kalveram, Birte; Indran, Sabarish V.; Lokugamage, Nandadeva; Juelich, Terry L.; Hill, Terence E.; Tseng, Chien-Te K.; Gong, Bin; Fukushi, Shuetsu; Morikawa, Shigeru; Freiberg, Alexander N.

2012-01-01

Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans. PMID:22573861

Legionella pneumophila strain associated with the first evidence of person-to-person transmission of Legionnaires’ disease: a unique mosaic genetic backbone

PubMed Central

Borges, Vítor; Nunes, Alexandra; Sampaio, Daniel A.; Vieira, Luís; Machado, Jorge; Simões, Maria J.; Gonçalves, Paulo; Gomes, João P.

2016-01-01

A first strong evidence of person-to-person transmission of Legionnaires’ Disease (LD) was recently reported. Here, we characterize the genetic backbone of this case-related Legionella pneumophila strain (“PtVFX/2014”), which also caused a large outbreak of LD. PtVFX/2014 is phylogenetically divergent from the most worldwide studied outbreak-associated L. pneumophila subspecies pneumophila serogroup 1 strains. In fact, this strain is also from serogroup 1, but belongs to the L. pneumophila subspecies fraseri. Its genomic mosaic backbone reveals eight horizontally transferred regions encompassing genes, for instance, involved in lipopolysaccharide biosynthesis or encoding virulence-associated Dot/Icm type IVB secretion system (T4BSS) substrates. PtVFX/2014 also inherited a rare ~65 kb pathogenicity island carrying virulence factors and detoxifying enzymes believed to contribute to the emergence of best-fitted strains in water reservoirs and in human macrophages, as well as a inter-species transferred (from L. oakridgensis) ~37.5 kb genomic island (harboring a lvh/lvr T4ASS cluster) that had never been found intact within L. pneumophila species. PtVFX/2014 encodes another lvh/lvr cluster near to CRISPR-associated genes, which may boost L. pneumophila transition from an environmental bacterium to a human pathogen. Overall, this unique genomic make-up may impact PtVFX/2014 ability to adapt to diverse environments, and, ultimately, to be transmitted and cause human disease. PMID:27196677

Genome sequence of Shigella flexneri strain SP1, a diarrheal isolate that encodes an extended-spectrum β-lactamase (ESBL).

PubMed

Shen, Ping; Fan, Jianzhong; Guo, Lihua; Li, Jiahua; Li, Ang; Zhang, Jing; Ying, Chaoqun; Ji, Jinru; Xu, Hao; Zheng, Beiwen; Xiao, Yonghong

2017-05-12

Shigellosis is the most common cause of gastrointestinal infections in developing countries. In China, the species most frequently responsible for shigellosis is Shigella flexneri. S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on biochemical and serological properties. Moreover, increasing numbers of ESBL-producing Shigella strains have been isolated from clinical samples. Despite this, only a few cases of ESBL-producing Shigella have been described in China. Therefore, a better understanding of ESBL-producing Shigella from a genomic standpoint is required. In this study, a S. flexneri type 1a isolate SP1 harboring bla CTX-M-14 , which was recovered from the patient with diarrhea, was subjected to whole genome sequencing. The draft genome assembly of S. flexneri strain SP1 consisted of 4,592,345 bp with a G+C content of 50.46%. RAST analysis revealed the genome contained 4798 coding sequences (CDSs) and 100 RNA-encoding genes. We detected one incomplete prophage and six candidate CRISPR loci in the genome. In vitro antimicrobial susceptibility testing demonstrated that strain SP1 is resistant to ampicillin, amoxicillin/clavulanic acid, cefazolin, ceftriaxone and trimethoprim. In silico analysis detected genes mediating resistance to aminoglycosides, β-lactams, phenicol, tetracycline, sulphonamides, and trimethoprim. The bla CTX-M-14 gene was located on an IncFII2 plasmid. A series of virulence factors were identified in the genome. In this study, we report the whole genome sequence of a bla CTX-M-14 -encoding S. flexneri strain SP1. Dozens of resistance determinants were detected in the genome and may be responsible for the multidrug-resistance of this strain, although further confirmation studies are warranted. Numerous virulence factors identified in the strain suggest that isolate SP1 is potential pathogenic. The availability of the genome sequence and comparative analysis with other S. flexneri strains provides the basis to further address the evolution of drug resistance mechanisms and pathogenicity in S. flexneri.

The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein.

PubMed

Bleiziffer, Isabelle; Eikmeier, Julian; Pohlentz, Gottfried; McAulay, Kathryn; Xia, Guoqing; Hussain, Muzaffar; Peschel, Andreas; Foster, Simon; Peters, Georg; Heilmann, Christine

2017-01-01

Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff's staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl residues can stimulate biofilm formation. Thus, sugar modifications may represent promising new targets for novel therapeutic or prophylactic measures against life-threatening S. aureus infections.

Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from different origins and sources.

PubMed

Miko, Angelika; Delannoy, Sabine; Fach, Patrick; Strockbine, Nancy A; Lindstedt, Björn Arne; Mariani-Kurkdjian, Patricia; Reetz, Jochen; Beutin, Lothar

2013-12-01

Sixty-two Escherichia coli strains carrying the wzxO104-gene from different sources, origins and time periods were analyzed for their serotypes, virulence genes and compared for genomic similarity by pulsed-field gel-electrophoresis (PFGE). The O104 antigen was present in 55 strains and the structurally and genetically related capsular antigen K9 in five strains. The presence of 49 genes associated with enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC) was investigated. Fifty-four strains of serotypes O104:H2 (n=1), O104:H4 (n=37), O104:H7 (n=5) and O104:H21 (n=11) produced Shiga-toxins (Stx). Among STEC O104, a close association between serotype, virulence gene profile and genomic similarity was found. EAEC virulence genes were only present in STEC O104:H4 strains. EHEC-O157 plasmid-encoded genes were only found in STEC O104:H2, O104:H7 and O104:H21 strains. None of the 62 O104 or K9 strains carried an eae-gene involved in the attaching and effacing phenotype. The 38 O104:H4 strains formed a single PFGE-cluster (>83.7% similarity). Thirty-one of these strains were from the European O104:H4 outbreak in 2011. The outbreak strains and older O104:H4 strains from Germany (2001), Georgia and France (2009) clustered together at>86.2% similarity. O104:H4 strains isolated between 2001 and 2009 differed for some plasmid-encoded virulence genes compared to the outbreak strains from 2011. STEC O104:H21 and STEC O104:H7 strains isolated in the U.S. and in Europe showed characteristic differences in their Stx-types, virulence gene and PFGE profiles indicating that these have evolved separately. E. coli K9 strains were not associated with virulence and were heterogeneous for their serotypes and PFGE profiles. Copyright © 2013 Elsevier GmbH. All rights reserved.

Dissecting the machinery that introduces disulfide bonds in Pseudomonas aeruginosa.

PubMed

Arts, Isabelle S; Ball, Geneviève; Leverrier, Pauline; Garvis, Steven; Nicolaes, Valérie; Vertommen, Didier; Ize, Bérengère; Tamu Dufe, Veronica; Messens, Joris; Voulhoux, Romé; Collet, Jean-François

2013-12-10

Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors. The human pathogen Pseudomonas aeruginosa causes life-threatening infections in immunodepressed and cystic fibrosis patients. The emergence of P. aeruginosa strains resistant to all of the available antibacterial agents calls for the urgent development of new antibiotics active against this bacterium. The pathogenic power of P. aeruginosa is mediated by an arsenal of extracellular virulence factors, most of which are stabilized by disulfide bonds. Thus, targeting the machinery that introduces disulfide bonds appears to be a promising strategy to combat P. aeruginosa. Here, we unraveled the oxidative protein folding system of P. aeruginosa in full detail. The system uniquely consists of two membrane proteins that generate disulfide bonds de novo to deliver them to P. aeruginosa DsbA1 (PaDsbA1), a soluble oxidoreductase. PaDsbA1 in turn donates disulfide bonds to secreted proteins, including virulence factors. Disruption of the disulfide bond formation machinery dramatically decreases P. aeruginosa virulence, confirming that disulfide formation systems are valid targets for the design of antimicrobial drugs.

Rift Valley Fever Virus Lacking the NSs and NSm Genes Is Highly Attenuated, Confers Protective Immunity from Virulent Virus Challenge, and Allows for Differential Identification of Infected and Vaccinated Animals▿

PubMed Central

Bird, Brian H.; Albariño, César G.; Hartman, Amy L.; Erickson, Bobbie Rae; Ksiazek, Thomas G.; Nichol, Stuart T.

2008-01-01

Rift Valley fever (RVF) virus is a mosquito-borne human and veterinary pathogen associated with large outbreaks of severe disease throughout Africa and more recently the Arabian peninsula. Infection of livestock can result in sweeping “abortion storms” and high mortality among young animals. Human infection results in self-limiting febrile disease that in ∼1 to 2% of patients progresses to more serious complications including hepatitis, encephalitis, and retinitis or a hemorrhagic syndrome with high fatality. The virus S segment-encoded NSs and the M segment-encoded NSm proteins are important virulence factors. The development of safe, effective vaccines and tools to screen and evaluate antiviral compounds is critical for future control strategies. Here, we report the successful reverse genetics generation of multiple recombinant enhanced green fluorescent protein-tagged RVF viruses containing either the full-length, complete virus genome or precise deletions of the NSs gene alone or the NSs/NSm genes in combination, thus creating attenuating deletions on multiple virus genome segments. These viruses were highly attenuated, with no detectable viremia or clinical illness observed with high challenge dosages (1.0 × 104 PFU) in the rat lethal disease model. A single-dose immunization regimen induced robust anti-RVF virus immunoglobulin G antibodies (titer, ∼1:6,400) by day 26 postvaccination. All vaccinated animals that were subsequently challenged with a high dose of virulent RVF virus survived infection and could be serologically differentiated from naïve, experimentally infected animals by the lack of NSs antibodies. These rationally designed marker RVF vaccine viruses will be useful tools for in vitro screening of therapeutic compounds and will provide a basis for further development of RVF virus marker vaccines for use in endemic regions or following the natural or intentional introduction of the virus into previously unaffected areas. PMID:18199647

Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in mice.

PubMed

Monteiro, Gaby E R; Jansen van Vuren, Petrus; Wichgers Schreur, Paul J; Odendaal, Lieza; Clift, Sarah J; Kortekaas, Jeroen; Paweska, Janusz T

2018-04-02

The NSs protein encoded by the S segment of Rift Valley fever virus (RVFV) is the major virulence factor, counteracting the host innate antiviral defence. It contains five highly conserved cysteine residues at positions 39, 40, 149, 178 and 194, which are thought to stabilize the tertiary and quaternary structure of the protein. Here, we report significant differences between clinical, virological, histopathological and host gene responses in BALB/c mice infected with wild-type RVFV (wtRVFV) or a genetic mutant having a double cysteine-to-serine substitution at residues 39 and 40 of the NSs protein (RVFV-C39S/C40S). Mice infected with the wtRVFV developed a fatal acute disease; characterized by high levels of viral replication, severe hepatocellular necrosis, and massive up-regulation of transcription of genes encoding type I and -II interferons (IFN) as well as pro-apoptotic and pro-inflammatory cytokines. The RVFV-C39S/C40S mutant did not cause clinical disease and its attenuated virulence was consistent with virological, histopathological and host gene expression findings in BALB/c mice. Clinical signs in mice infected with viruses containing cysteine-to-serine substitutions at positions 178 or 194 were similar to those occurring in mice infected with the wtRVFV, while a mutant containing a substitution at position 149 caused mild, non-fatal disease in mice. As mutant RVFV-C39S/C40S showed an attenuated phenotype in mice, the molecular mechanisms behind this attenuation were further investigated. The results show that two mechanisms are responsible for the attenuation; (1) loss of the IFN antagonistic propriety characteristic of the wtRVFV NSs and (2) the inability of the attenuated mutant to degrade Proteine Kinase R (PKR). Copyright © 2018. Published by Elsevier B.V.

Biofilm formation and virulence factor analysis of Staphylococcus aureus isolates collected from ovine mastitis.

PubMed

Azara, E; Longheu, C; Sanna, G; Tola, S

2017-08-01

To perform a phenotypic and genotypic characterization of 258 Staphylococcus aureus isolates from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines. The potential for biofilm production was determined by phenotypic test of Congo Red Agar (CRA) and by PCR for the detection of icaA/D genes. Isolates were also screened by PCR for the presence of enterotoxins (sea, seb, sec, sed and see), toxic shock syndrome toxin (tsst), leukotoxins (lukD-E, lukM and lukPV83), haemolysins (hly-β and hly-γ), autolysin (atlA) genes and encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: clfA, clfB, fnbA, fnbB, bbp, cna, eno, fib, epbs, sdrC, sdrD and SdrE). None of the 258 isolates showed biofilm-forming ability on CRA and harboured icaA/D genes. The most frequent pyrogenic toxin superantigen genes amplified were sec plus tsst-1, which were found strictly in combination with 71·3% of the Staph. aureus isolates tested. None of the isolates harboured the genes encoding sea and see. Of the 258 isolates tested, 159 (61·6%) possessed all lukD-E/lukM/lukPV83 genes, 123 (47·7%) harboured both hly-β/hly-γ genes, whereas almost all (97·3%) were PCR positive for atlA gene. With respect to adhesion determinants, 179 (69·4%) isolates presented simultaneously four genes (fnbA, fib, clfA and clfB) for fibronectin- and fibrinogen-binding proteins. In this search, several putative virulence determinants have been identified in ovine Staph. aureus isolates collected in Sardinia. Some of the putative virulence determinants could be considered as components of a vaccine because of their role in ovine mastitis pathogenesis. © 2017 The Society for Applied Microbiology.

Directed mutagenesis of the Rickettsia prowazekii pld gene encoding phospholipase D.

PubMed

Driskell, Lonnie O; Yu, Xue-jie; Zhang, Lihong; Liu, Yan; Popov, Vsevolod L; Walker, David H; Tucker, Aimee M; Wood, David O

2009-08-01

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

Disruption of the Phospholipase D Gene Attenuates the Virulence of Aspergillus fumigatus

PubMed Central

Li, Xianping; Gao, Meihua; Han, Xuelin; Tao, Sha; Zheng, Dongyu; Cheng, Ying; Yu, Rentao; Han, Gaige; Schmidt, Martina

2012-01-01

Aspergillus fumigatus is the most prevalent airborne fungal pathogen that induces serious infections in immunocompromised patients. Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, knowledge of the impact of phospholipases on A. fumigatus virulence is rather limited. In this study, disruption of the pld gene encoding phospholipase D (PLD), an important member of the phospholipase protein family in A. fumigatus, was confirmed to significantly decrease both intracellular and extracellular PLD activity of A. fumigatus. The pld gene disruption did not alter conidial morphological characteristics, germination, growth, and biofilm formation but significantly suppressed the internalization of A. fumigatus into A549 epithelial cells without affecting conidial adhesion to epithelial cells. Importantly, the suppressed internalization was fully rescued in the presence of 100 μM phosphatidic acid, the PLD product. Indeed, complementation of pld restored the PLD activity and internalization capacity of A. fumigatus. Phagocytosis of A. fumigatus conidia by J774 macrophages was not affected by the absence of the pld gene. Pretreatment of conidia with 1-butanol and a specific PLD inhibitor decreased the internalization of A. fumigatus into A549 epithelial cells but had no effect on phagocytosis by J774 macrophages. Finally, loss of the pld gene attenuated the virulence of A. fumigatus in mice immunosuppressed with hydrocortisone acetate but not with cyclophosphamide. These data suggest that PLD of A. fumigatus regulates its internalization into lung epithelial cells and may represent an important virulence factor for A. fumigatus infection. PMID:22083709

Interaction of phase variation, host and pressure/gas composition: pneumococcal gene expression of PsaA, SpxB, Ply and LytA in simulated middle ear environments.

PubMed

Li-Korotky, Ha-Sheng; Lo, Chia-Yee; Zeng, Fan-Rui; Lo, David; Banks, Juliane M

2009-10-01

Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variants are more efficient in colonizing the nasopharynx while opaque (O) variants exhibit greater virulence during systemic infections. This study was intended to delineate the underlying molecular mechanisms by which the predominant S. pneumoniae variant efficiently infects the middle ear (ME) mucosa. Human ME epithelial cells were preconditioned for 24h under one of the three gas/pressure conditions designed to simulate those for (1) normal ME (NME), (2) ME with Eustachian tube obstruction (ETO) and (3) ME with tympanostomy tube placement (TT), and then were incubated with ∼ 10(7)CFU/ml of either T or O variants of S. pneumoniae (6A) for 3h. Relative expression levels of genes encoding virulence factors, PsaA (surface adhesion), SpxB (pyruvate oxidase), Ply (pneumolysin), and LytA (autolysin) were assessed separately in epithelium-attached and supernatant bacteria 3h post infection using real-time PCR. Basal levels of the virulence molecules in inocula were comparable between two variants. However, relative expression levels of the gene transcripts were significantly induced in epithelium-attached T variants 3h after infection. Comparing with NME and TT conditions, ETO environment produced the largest effect on the differential expression of the virulence genes in the infected ME epithelial cells between T (induced) and O (suppressed) phenotypic pneumococci. T variant is a predominant phenotype responsible for the pathogenesis of pneumococcal OM.

A ToxA-like protein from Cochliobolus heterostrophus induces light-dependent leaf necrosis and acts as a virulence factor with host selectivity on maize.

PubMed

Lu, Shunwen; Gillian Turgeon, B; Edwards, Michael C

2015-08-01

ToxA, the first discovered fungal proteinaceous host-selective toxin (HST), was originally identified in 1989 from the tan spot fungus Pyrenophora tritici-repentis (Ptr). About 25years later, a homolog was identified in the leaf/glume blotch fungus Stagonospora nodorum (Parastagonospora nodorum), also a pathogen of wheat. Here we report the identification and function of a ToxA-like protein from the maize pathogen Cochliobolus heterostrophus (Ch) that possesses necrosis-inducing activity specifically against maize. ChToxA is encoded by a 535-bp open reading frame featuring a ToxA-specific intron with unusual splicing sites (5'-ATAAGT…TAC-3') at conserved positions relative to PtrToxA. The protein shows 64% similarity to PtrToxA and is predicted to adopt a similar three-dimensional structure, although lacking the arginyl-glycyl-aspartic acid (RGD) motif reported to be required for internalization into sensitive wheat mesophyll cells. Reverse-transcriptase PCR revealed that the ChTOXA gene expression is up-regulated in planta, relative to axenic culture. Plant assays indicated that the recombinant ChToxA protein induces light-dependent leaf necrosis in a host-selective manner on maize inbred lines. Gene deletion experiments confirmed that ChtoxA mutants are reduced in virulence on specific ChToxA-sensitive maize lines, relative to virulence caused by wild-type strains. Database searches identified potential ChToxA homologues in other plant-pathogenic ascomycetes. Sequence and phylogenetic analyses revealed that the corresponding ToxA-like proteins include one member recently shown to be associated with formation of penetration hypha. These results provide the first evidence that C. heterostrophus is capable of producing proteinaceous HSTs as virulence factors in addition to well-known secondary metabolite-type toxins produced biosynthetically by polyketide synthase megaenzymes. Further studies on ChToxA may provide new insights into effector evolution in host-pathogen interactions. Published by Elsevier Inc.

The Aspergillus fumigatus conidial melanin production is regulated by the bifunctional bHLH DevR and MADS-box RlmA transcription factors.

PubMed

Valiante, Vito; Baldin, Clara; Hortschansky, Peter; Jain, Radhika; Thywißen, Andreas; Straßburger, Maria; Shelest, Ekaterina; Heinekamp, Thorsten; Brakhage, Axel A

2016-10-01

Melanins play a crucial role in defending organisms against external stressors. In several pathogenic fungi, including the human pathogen Aspergillus fumigatus, melanin production was shown to contribute to virulence. A. fumigatus produces two different types of melanins, i.e., pyomelanin and dihydroxynaphthalene (DHN)-melanin. DHN-melanin forms the gray-green pigment characteristic for conidia, playing an important role in immune evasion of conidia and thus for fungal virulence. The DHN-melanin biosynthesis pathway is encoded by six genes organized in a cluster with the polyketide synthase gene pksP as a core element. Here, cross-species promoter analysis identified specific DNA binding sites in the DHN-melanin biosynthesis genes pksP-arp1 intergenic region that can be recognized by bHLH and MADS-box transcriptional regulators. Independent deletion of two genes coding for the transcription factors DevR (bHLH) and RlmA (MADS-box) interfered with sporulation and reduced the expression of the DHN-melanin gene cluster. In vitro and in vivo experiments proved that these transcription factors cooperatively regulate pksP expression acting both as repressors and activators in a mutually exclusive manner. The dual role executed by each regulator depends on specific DNA motifs recognized in the pksP promoter region. © 2016 John Wiley & Sons Ltd.

Legionella oakridgensis ATCC 33761 genome sequence and phenotypic characterization reveals its replication capacity in amoebae.

PubMed

Brzuszkiewicz, Elzbieta; Schulz, Tino; Rydzewski, Kerstin; Daniel, Rolf; Gillmaier, Nadine; Dittmann, Christine; Holland, Gudrun; Schunder, Eva; Lautner, Monika; Eisenreich, Wolfgang; Lück, Christian; Heuner, Klaus

2013-12-01

Legionella oakridgensis is able to cause Legionnaires' disease, but is less virulent compared to L. pneumophila strains and very rarely associated with human disease. L. oakridgensis is the only species of the family legionellae which is able to grow on media without additional cysteine. In contrast to earlier publications, we found that L. oakridgensis is able to multiply in amoebae. We sequenced the genome of L. oakridgensis type strain OR-10 (ATCC 33761). The genome is smaller than the other yet sequenced Legionella genomes and has a higher G+C-content of 40.9%. L. oakridgensis lacks a flagellum and it also lacks all genes of the flagellar regulon except of the alternative sigma-28 factor FliA and the anti-sigma-28 factor FlgM. Genes encoding structural components of type I, type II, type IV Lvh and type IV Dot/Icm, Sec- and Tat-secretion systems could be identified. Only a limited set of Dot/Icm effector proteins have been recognized within the genome sequence of L. oakridgensis. Like in L. pneumophila strains, various proteins with eukaryotic motifs and eukaryote-like proteins were detected. We could demonstrate that the Dot/Icm system is essential for intracellular replication of L. oakridgensis. Furthermore, we identified new putative virulence factors of Legionella. Copyright © 2013 Elsevier GmbH. All rights reserved.

Virulence factors associated with cytotoxic necrotizing factor type two in bovine diarrheic and septicemic strains of Escherichia coli.

PubMed Central

Oswald, E; de Rycke, J; Lintermans, P; van Muylem, K; Mainil, J; Daube, G; Pohl, P

1991-01-01

Forty-three bovine isolates of Escherichia coli producing a second type of cytotoxic necrotizing factor (CNF2) and three K-12 strains carrying different Vir plasmids coding for CNF2 were tested for the presence of several virulence factors. Most of the strains were serum resistant (79%), produced an aerobactin (70%), and adhered to calf villi (53%); some of them produced a colicin (32%) and a hemolysin (9%). These strains were also tested by a colony hybridization assay with gene probes for six toxins (classical heat-stable [STaP and STb] and heat-labile [LT-I and LT-IIa] enterotoxins and Shiga-like toxins [SLT-I and SLT-II]) and five adhesion factors (K99, K88, 987P, F17, and F41). Only two gene probes, LT-IIa (9%) and F17A (53%), hybridized with the CNF2 strains. However, antibodies raised against F17 fimbriae did not agglutinate the strains hybridizing with the F17A probe. In contrast, all except one of these strains adhered to calf villi. Interestingly, these two properties, F17A positivity and adherence to calf villi, were the only ones expressed by the K-12 strains carrying different Vir plasmids. In conclusion, this study confirmed that CNF2-producing strains are unrelated to previously described toxigenic E. coli strains and also demonstrated that in half of the strains the production of CNF2 was associated with an adhesion factor genetically related to, but different from, F17, which is more than likely encoded by Vir plasmids. PMID:1774259

Filamentous phages of Ralstonia solanacearum: double-edged swords for pathogenic bacteria.

PubMed

Yamada, Takashi

2013-01-01

Some phages from genus Inovirus use host or bacteriophage-encoded site-specific integrases or recombinases establish a prophage state. During integration or excision, a superinfective form can be produced. The three states (free, prophage, and superinfective) of such phages exert different effects on host bacterial phenotypes. In Ralstonia solanacearum, the causative agent of bacterial wilt disease of crops, the bacterial virulence can be positively or negatively affected by filamentous phages, depending on their state. The presence or absence of a repressor gene in the phage genome may be responsible for the host phenotypic differences (virulent or avirulent) caused by phage infection. This strategy of virulence control may be widespread among filamentous phages that infect pathogenic bacteria of plants.

Community-associated and healthcare-associated methicillin-resistant Staphylococcus aureus virulence toward Caenorhabditis elegans compared.

PubMed

Day, Shandra R; Moore, Christopher M; Kundzins, John R; Sifri, Costi D

2012-11-15

Community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) strains have emerged as major human pathogens. CA-MRSA virulence appears to be distinct from healthcare-associated (HA) MRSA with several factors [α-hemolysin (Hla), Panton-Valentine leukocidin (PVL), α-type phenol soluble modulins (PSMα) and SCCmec IV] postulated to enhance virulence or fitness. Using the Caenorhabditis elegans infection model, we compared the virulence of clinical and laboratory isolates of CA-MRSA and HA-MRSA and explored the contribution of CA-MRSA associated virulence factors to nematode killing. All CA-MRSA strains were highly pathogenic to nematodes, while HA-MRSA strains demonstrated variable nematode killing. Nematode killing by isogenic mutants of hla or the loci for PVL, PSMα, PSMβ, PSMδ or SCCmec IV was not different than the parental strains. These results demonstrate that CA-MRSA is highly virulent, shows some strains of HA-MRSA are equally virulent toward nematodes and suggests CA-MRSA virulence in C. elegans is not linked to a single virulence factor.

Community-associated and healthcare-associated methicillin-resistant Staphylococcus aureus virulence toward Caenorhabditis elegans compared

PubMed Central

Day, Shandra R.; Moore, Christopher M.; Kundzins, John R.; Sifri, Costi D.

2012-01-01

Community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) strains have emerged as major human pathogens. CA-MRSA virulence appears to be distinct from healthcare-associated (HA) MRSA with several factors [α-hemolysin (Hla), Panton-Valentine leukocidin (PVL), α-type phenol soluble modulins (PSMα) and SCCmec IV] postulated to enhance virulence or fitness. Using the Caenorhabditis elegans infection model, we compared the virulence of clinical and laboratory isolates of CA-MRSA and HA-MRSA and explored the contribution of CA-MRSA associated virulence factors to nematode killing. All CA-MRSA strains were highly pathogenic to nematodes, while HA-MRSA strains demonstrated variable nematode killing. Nematode killing by isogenic mutants of hla or the loci for PVL, PSMα, PSMβ, PSMδ or SCCmec IV was not different than the parental strains. These results demonstrate that CA-MRSA is highly virulent, shows some strains of HA-MRSA are equally virulent toward nematodes and suggests CA-MRSA virulence in C. elegans is not linked to a single virulence factor. PMID:23076331

Reconstruction of the metabolic network of Pseudomonas aeruginosa to interrogate virulence factor synthesis

NASA Astrophysics Data System (ADS)

Bartell, Jennifer A.; Blazier, Anna S.; Yen, Phillip; Thøgersen, Juliane C.; Jelsbak, Lars; Goldberg, Joanna B.; Papin, Jason A.

2017-03-01

Virulence-linked pathways in opportunistic pathogens are putative therapeutic targets that may be associated with less potential for resistance than targets in growth-essential pathways. However, efficacy of virulence-linked targets may be affected by the contribution of virulence-related genes to metabolism. We evaluate the complex interrelationships between growth and virulence-linked pathways using a genome-scale metabolic network reconstruction of Pseudomonas aeruginosa strain PA14 and an updated, expanded reconstruction of P. aeruginosa strain PAO1. The PA14 reconstruction accounts for the activity of 112 virulence-linked genes and virulence factor synthesis pathways that produce 17 unique compounds. We integrate eight published genome-scale mutant screens to validate gene essentiality predictions in rich media, contextualize intra-screen discrepancies and evaluate virulence-linked gene distribution across essentiality datasets. Computational screening further elucidates interconnectivity between inhibition of virulence factor synthesis and growth. Successful validation of selected gene perturbations using PA14 transposon mutants demonstrates the utility of model-driven screening of therapeutic targets.

Multiplex-PCR-Based Screening and Computational Modeling of Virulence Factors and T-Cell Mediated Immunity in Helicobacter pylori Infections for Accurate Clinical Diagnosis.

PubMed

Oktem-Okullu, Sinem; Tiftikci, Arzu; Saruc, Murat; Cicek, Bahattin; Vardareli, Eser; Tozun, Nurdan; Kocagoz, Tanil; Sezerman, Ugur; Yavuz, Ahmet Sinan; Sayi-Yazgan, Ayca

2015-01-01

The outcome of H. pylori infection is closely related with bacteria's virulence factors and host immune response. The association between T cells and H. pylori infection has been identified, but the effects of the nine major H. pylori specific virulence factors; cagA, vacA, oipA, babA, hpaA, napA, dupA, ureA, ureB on T cell response in H. pylori infected patients have not been fully elucidated. We developed a multiplex- PCR assay to detect nine H. pylori virulence genes with in a three PCR reactions. Also, the expression levels of Th1, Th17 and Treg cell specific cytokines and transcription factors were detected by using qRT-PCR assays. Furthermore, a novel expert derived model is developed to identify set of factors and rules that can distinguish the ulcer patients from gastritis patients. Within all virulence factors that we tested, we identified a correlation between the presence of napA virulence gene and ulcer disease as a first data. Additionally, a positive correlation between the H. pylori dupA virulence factor and IFN-γ, and H. pylori babA virulence factor and IL-17 was detected in gastritis and ulcer patients respectively. By using computer-based models, clinical outcomes of a patients infected with H. pylori can be predicted by screening the patient's H. pylori vacA m1/m2, ureA and cagA status and IFN-γ (Th1), IL-17 (Th17), and FOXP3 (Treg) expression levels. Herein, we report, for the first time, the relationship between H. pylori virulence factors and host immune responses for diagnostic prediction of gastric diseases using computer-based models.

Multiplex-PCR-Based Screening and Computational Modeling of Virulence Factors and T-Cell Mediated Immunity in Helicobacter pylori Infections for Accurate Clinical Diagnosis

PubMed Central

Oktem-Okullu, Sinem; Tiftikci, Arzu; Saruc, Murat; Cicek, Bahattin; Vardareli, Eser; Tozun, Nurdan; Kocagoz, Tanil; Sezerman, Ugur; Yavuz, Ahmet Sinan; Sayi-Yazgan, Ayca

2015-01-01

The outcome of H. pylori infection is closely related with bacteria's virulence factors and host immune response. The association between T cells and H. pylori infection has been identified, but the effects of the nine major H. pylori specific virulence factors; cagA, vacA, oipA, babA, hpaA, napA, dupA, ureA, ureB on T cell response in H. pylori infected patients have not been fully elucidated. We developed a multiplex- PCR assay to detect nine H. pylori virulence genes with in a three PCR reactions. Also, the expression levels of Th1, Th17 and Treg cell specific cytokines and transcription factors were detected by using qRT-PCR assays. Furthermore, a novel expert derived model is developed to identify set of factors and rules that can distinguish the ulcer patients from gastritis patients. Within all virulence factors that we tested, we identified a correlation between the presence of napA virulence gene and ulcer disease as a first data. Additionally, a positive correlation between the H. pylori dupA virulence factor and IFN-γ, and H. pylori babA virulence factor and IL-17 was detected in gastritis and ulcer patients respectively. By using computer-based models, clinical outcomes of a patients infected with H. pylori can be predicted by screening the patient's H. pylori vacA m1/m2, ureA and cagA status and IFN-γ (Th1), IL-17 (Th17), and FOXP3 (Treg) expression levels. Herein, we report, for the first time, the relationship between H. pylori virulence factors and host immune responses for diagnostic prediction of gastric diseases using computer—based models. PMID:26287606

A conserved OmpA-like protein in Legionella pneumophila required for efficient intracellular replication.

PubMed

Goodwin, Ian P; Kumova, Ogan K; Ninio, Shira

2016-08-01

The OmpA-like protein domain has been associated with peptidoglycan-binding proteins, and is often found in virulence factors of bacterial pathogens. The intracellular pathogen Legionella pneumophila encodes for six proteins that contain the OmpA-like domain, among them the highly conserved uncharacterized protein we named CmpA. Here we set out to characterize the CmpA protein and determine its contribution to intracellular survival of L. pneumophila Secondary structure analysis suggests that CmpA is an inner membrane protein with a peptidoglycan-binding domain at the C-teminus. A cmpA mutant was able to replicate normally in broth, but failed to compete with an isogenic wild-type strain in an intracellular growth competition assay. The cmpA mutant also displayed significant intracellular growth defects in both the protozoan host Acanthamoeba castellanii and in primary bone marrow-derived macrophages, where uptake into the cells was also impaired. The cmpA phenotypes were completely restored upon expression of CmpA in trans The data presented here establish CmpA as a novel virulence factor of L. pneumophila that is required for efficient intracellular replication in both mammalian and protozoan hosts. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

[Enterohemorrhagic Escherichia coli as the cause of diarrhea in the Czech Republic, 1965-2013].

PubMed

Marejková, M; Petráš, P

2014-09-01

Enterohemorrhagic Escherichia coli (EHEC) is the cause of diarrhea, bloody diarrhea, and haemolytic uremic syndrome (HUS) worldwide. The role of EHEC in the etiology of HUS in the Czech Republic has recently been described, but the prevalence, characteristics, and epidemiology of EHEC causing diarrhea have not been fully known. Therefore, this study analyzed the serotypes, stx genotypes, and virulence factors in EHEC strains isolated in 1965-2013 from patients with diarrhea or bloody diarrhea and their family contacts. In addition, we characterized diagnostically relevant phenotypes of EHEC strains, their antimicrobial susceptibility, seasonal trends, and distribution by administrative region. Serogrouped E. coli isolates from patients were referred to the National Reference Laboratory (NRL) for E. coli and Shigella for the detection of Stx. Specimens of both human and non-human origin were referred to the NRL for epidemiological investigation. Serotyping was performed by conventional and molecular methods, PCR was applied to stx genotyping and identification of non-stx virulence factors, and standard methods were used for phenotypic analysis and antimicrobial susceptibility testing. The epidemiological link between the human and animal isolates was confirmed using pulsed-field gel electrophoresis (PFGE). Of 50 EHEC strains, 24 were recovered from patients with diarrhea without blood, 19 from patients with bloody diarrhea, six from family contacts, and one from an epidemiologically linked animal. EHEC cases were reported during the whole year, with peaks in May through October, most often in the Central Bohemian and Hradec Králové Regions. EHEC outbreaks occurred in three families: in one of them sheep-to-human transmission of EHEC was detected. The EHEC strains were assigned to five serotypes, with more than half of them being non-sorbitol fermenting (NSF) O157:H7/NM[fliCH7] and a third being strains O26:H11/NM[fliCH11]; serotypes O111:NM[fliCH8], O118:NM[fliCH25], and O104:H4, similarly to sorbitol-fermenting (SF) strains O157:NM[fliCH7], were rare. Of seven stx genotypes identified, all were present in NSF EHEC O157, two in each of EHEC O26 and O111, and one in each of EHEC O118, O104, and SF O157. All but one strain were Stx producers. Genes encoding other virulence factors including toxins (EHEC-hlyA, cdt-V, and espP) and adhesins (eae, efa1, iha, lpf, and sfpA) were detected in all strains and their occurrence was serotype specific. The most common of these genes were eae encoding adhesin intimin and EHEC-hlyA encoding EHEC hemolysin. All EHEC strains but SF O157 harboured terE encoding tellurite resistance. All strains except NSF EHEC O157 and EHEC O118 fermented sorbitol and produced ß-D-glucuronidase. Most (89.8%) EHEC strains were susceptible to all 12 antimicrobials tested. EHEC strains cause diarrhea and bloody diarrhea in the Czech Republic. Nevertheless, only a systematic screening of the stool from patients with diarrhea can make it possible to elucidate their actual role in the etiology of diarrheal diseases (as well as HUS) in the Czech Republic and to consider the data in the European context. EHEC cases are reported to the European Centre for Disease Prevention and Control (ECDC) within the Food and Waterborne Diseases Surveillance Network.

The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.

PubMed

Risatti, G R; Borca, M V; Kutish, G F; Lu, Z; Holinka, L G; French, R A; Tulman, E R; Rock, D L

2005-03-01

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.

Deletion analysis of Streptococcus pneumoniae late competence genes distinguishes virulence determinants that are dependent or independent of competence induction.

PubMed

Zhu, Luchang; Lin, Jingjun; Kuang, Zhizhou; Vidal, Jorge E; Lau, Gee W

2015-07-01

The competence regulon of Streptococcus pneumoniae (pneumococcus) is crucial for genetic transformation. During competence development, the alternative sigma factor ComX is activated, which in turn, initiates transcription of 80 'late' competence genes. Interestingly, only 16 late genes are essential for genetic transformation. We hypothesized that these late genes that are dispensable for competence are beneficial to pneumococcal fitness during infection. These late genes were systematically deleted, and the resulting mutants were examined for their fitness during mouse models of bacteremia and acute pneumonia. Among these, 14 late genes were important for fitness in mice. Significantly, deletion of some late genes attenuated pneumococcal fitness to the same level in both wild-type and ComX-null genetic backgrounds, suggesting that the constitutive baseline expression of these genes was important for bacterial fitness. In contrast, some mutants were attenuated only in the wild-type genetic background but not in the ComX-null background, suggesting that specific expression of these genes during competence state contributed to pneumococcal fitness. Increased virulence during competence state was partially caused by the induction of allolytic enzymes that enhanced pneumolysin release. These results distinguish the role of basal expression versus competence induction in virulence functions encoded by ComX-regulated late competence genes. © 2015 John Wiley & Sons Ltd.

Complete Genome Sequence of the MRSA Isolate HC1335 from ST239 Lineage Displaying a Truncated AgrC Histidine Kinase Receptor

PubMed Central

Botelho, Ana M. N.; Costa, Maiana O. C.; Beltrame, Cristiana O.; Ferreira, Fabienne A.; Lima, Nicholas C. B.; Costa, Bruno S. S.; de Morais, Guilherme L.; Souza, Rangel C.; Almeida, Luiz G. P.; Vasconcelos, Ana T. R.; Nicolás, Marisa F.; Figueiredo, Agnes M. S.

2016-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is still one of the most important hospital pathogen globally. The multiresistant isolates of the ST239-SCCmecIII lineage are spread over large geographic regions, colonizing and infecting hospital patients in virtually all continents. The balance between fitness (adaptability) and virulence potential is likely to represent an important issue in the clonal shift dynamics leading the success of some specific MRSA clones over another. The accessory gene regulator (agr) is the master quorum sensing system of staphylococci playing a role in the global regulation of key virulence factors. Consequently, agr inactivation in S. aureus may represent a significant mechanism of genetic variability in the adaptation of this healthcare-associated pathogen. We report here the complete genome sequence of the methicillin-resistant S. aureus, isolate HC1335, a variant of the ST239 lineage, which presents a natural insertion of an IS256 transposase element in the agrC gene encoding AgrC histidine kinase receptor. PMID:27635055

Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens

PubMed Central

Wright, David P; Ulijasz, Andrew T

2014-01-01

Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential. PMID:25603430

The two-component system GrvRS (EtaRS) regulates ace expression in Enterococcus faecalis OG1RF.

PubMed

Roh, Jung Hyeob; Singh, Kavindra V; La Rosa, Sabina Leanti; Cohen, Ana Luisa V; Murray, Barbara E

2015-01-01

Expression of ace (adhesin to collagen of Enterococcus faecalis), encoding a virulence factor in endocarditis and urinary tract infection models, has been shown to increase under certain conditions, such as in the presence of serum, bile salts, urine, and collagen and at 46 °C. However, the mechanism of ace/Ace regulation under different conditions is still unknown. In this study, we identified a two-component regulatory system GrvRS as the main regulator of ace expression under these stress conditions. Using Northern hybridization and β-galactosidase assays of an ace promoter-lacZ fusion, we found transcription of ace to be virtually absent in a grvR deletion mutant under the conditions that increase ace expression in wild-type OG1RF and in the complemented strain. Moreover, a grvR mutant revealed decreased collagen binding and biofilm formation as well as attenuation in a murine urinary tract infection model. Here we show that GrvR plays a major role in control of ace expression and E. faecalis virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Combinatorial function of velvet and AreA in transcriptional regulation of nitrate utilization and secondary metabolism.

PubMed

López-Berges, Manuel S; Schäfer, Katja; Hera, Concepción; Di Pietro, Antonio

2014-01-01

Velvet is a conserved protein complex that functions as a regulator of fungal development and secondary metabolism. In the soil-inhabiting pathogen Fusarium oxysporum, velvet governs mycotoxin production and virulence on plant and mammalian hosts. Here we report a previously unrecognized role of the velvet complex in regulation of nitrate metabolism. F. oxysporum mutants lacking VeA or LaeA, two key components of the complex, were impaired in growth on the non-preferred nitrogen sources nitrate and nitrite. Both velvet and the general nitrogen response GATA factor AreA were required for transcriptional activation of nitrate (nit1) and nitrite (nii1) reductase genes under de-repressing conditions, as well as for the nitrate-triggered increase in chromatin accessibility at the nit1 locus. AreA also contributed to chromatin accessibility and expression of two velvet-regulated gene clusters, encoding biosynthesis of the mycotoxin beauvericin and of the siderophore ferricrocin. Thus, velvet and AreA coordinately orchestrate primary and secondary metabolism as well as virulence functions in F. oxysporum. Copyright © 2013 Elsevier Inc. All rights reserved.

Cloning, Sequencing, and Role in Virulence of Two Phospholipases (A1 and C) from Mesophilic Aeromonas sp. Serogroup O:34

PubMed Central

Merino, Susana; Aguilar, Alicia; Nogueras, Maria Mercedes; Regue, Miguel; Swift, Simon; Tomás, Juan M.

1999-01-01

Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5α. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process. PMID:10417167

Genome Sequencing of Listeria monocytogenes “Quargel” Listeriosis Outbreak Strains Reveals Two Different Strains with Distinct In Vitro Virulence Potential

PubMed Central

Rychli, Kathrin; Müller, Anneliese; Zaiser, Andreas; Schoder, Dagmar; Allerberger, Franz; Wagner, Martin; Schmitz-Esser, Stephan

2014-01-01

A large listeriosis outbreak occurred in Austria, Germany and the Czech Republic in 2009 and 2010. The outbreak was traced back to a traditional Austrian curd cheese called “Quargel” which was contaminated with two distinct serovar 1/2a Listeria monocytogenes strains (QOC1 and QOC2). In this study we sequenced and analysed the genomes of both outbreak strains in order to investigate the extent of genetic diversity between the two strains belonging to MLST sequence types 398 (QOC2) and 403 (QOC1). Both genomes are highly similar, but also display distinct properties: The QOC1 genome is approximately 74 kbp larger than the QOC2 genome. In addition, the strains harbour 93 (QOC1) and 45 (QOC2) genes encoding strain-specific proteins. A 21 kbp region showing highest similarity to plasmid pLMIV encoding three putative internalins is integrated in the QOC1 genome. In contrast to QOC1, strain QOC2 harbours a vip homologue, which encodes a LPXTG surface protein involved in cell invasion. In accordance, in vitro virulence assays revealed distinct differences in invasion efficiency and intracellular proliferation within different cell types. The higher virulence potential of QOC1 in non-phagocytic cells may be explained by the presence of additional internalins in the pLMIV-like region, whereas the higher invasion capability of QOC2 into phagocytic cells may be due to the presence of a vip homologue. In addition, both strains show differences in stress-related gene content. Strain QOC1 encodes a so-called stress survival islet 1, whereas strain QOC2 harbours a homologue of the uncharacterized LMOf2365_0481 gene. Consistently, QOC1 shows higher resistance to acidic, alkaline and gastric stress. In conclusion, our results show that strain QOC1 and QOC2 are distinct and did not recently evolve from a common ancestor. PMID:24587155

Genome-wide analysis of the regulatory function mediated by the small regulatory psm-mec RNA of methicillin-resistant Staphylococcus aureus.

PubMed

Cheung, Gordon Y C; Villaruz, Amer E; Joo, Hwang-Soo; Duong, Anthony C; Yeh, Anthony J; Nguyen, Thuan H; Sturdevant, Daniel E; Queck, S Y; Otto, M

2014-07-01

Several methicillin resistance (SCCmec) clusters characteristic of hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains harbor the psm-mec locus. In addition to encoding the cytolysin, phenol-soluble modulin (PSM)-mec, this locus has been attributed gene regulatory functions. Here we employed genome-wide transcriptional profiling to define the regulatory function of the psm-mec locus. The immune evasion factor protein A emerged as the primary conserved and strongly regulated target of psm-mec, an effect we show is mediated by the psm-mec RNA. Furthermore, the psm-mec locus exerted regulatory effects that were more moderate in extent. For example, expression of PSM-mec limited expression of mecA, thereby decreasing methicillin resistance. Our study shows that the psm-mec locus has a rare dual regulatory RNA and encoded cytolysin function. Furthermore, our findings reveal a specific mechanism underscoring the recently emerging concept that S. aureus strains balance pronounced virulence and high expression of antibiotic resistance. Published by Elsevier GmbH.

The Clavibacter michiganensis subspecies: molecular investigation of gram-positive bacterial plant pathogens.

PubMed

Eichenlaub, Rudolf; Gartemann, Karl-Heinz

2011-01-01

Clavibacter michiganensis subspecies are actinomycete plant pathogens residing mainly in the xylem vessels that infect economically important host plants. In the Clavibacter subspecies michiganensis and sepedonicus, infecting tomato and potato, respectively, essential factors for disease induction are plasmid encoded and loss of the virulence plasmids converts these biotrophic pathogens into endophytes. The genes responsible for successful colonization of the host plant, including evasion/suppression of plant defense reactions, are chromosomally encoded. Several serine proteases seem to be involved in colonization. They are secreted by Clavibacter, but their targets remain unknown. A type 3 secretion system (T3SS) translocating effectors into the plant cells is absent in these gram-positive pathogens. With the development of the modern 'omics technologies for RNA and proteins based on the known genome sequences, a new phase in the investigation of the mechanisms of plant pathogenicity has begun to allow the genome-wide investigation of the Clavibacter-host interaction. Copyright © 2011 by Annual Reviews. All rights reserved.

Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors.

PubMed

Wallqvist, Anders; Memišević, Vesna; Zavaljevski, Nela; Pieper, Rembert; Rajagopala, Seesandra V; Kwon, Keehwan; Yu, Chenggang; Hoover, Timothy A; Reifman, Jaques

2015-12-29

Francisella tularensis is a select bio-threat agent and one of the most virulent intracellular pathogens known, requiring just a few organisms to establish an infection. Although several virulence factors are known, we lack an understanding of virulence factors that act through host-pathogen protein interactions to promote infection. To address these issues in the highly infectious F. tularensis subsp. tularensis Schu S4 strain, we deployed a combined in silico, in vitro, and in vivo analysis to identify virulence factors and their interactions with host proteins to characterize bacterial infection mechanisms. We initially used comparative genomics and literature to identify and select a set of 49 putative and known virulence factors for analysis. Each protein was then subjected to proteome-scale yeast two-hybrid (Y2H) screens with human and murine cDNA libraries to identify potential host-pathogen protein-protein interactions. Based on the bacterial protein interaction profile with both hosts, we selected seven novel putative virulence factors for mutant construction and animal validation experiments. We were able to create five transposon insertion mutants and used them in an intranasal BALB/c mouse challenge model to establish 50 % lethal dose estimates. Three of these, ΔFTT0482c, ΔFTT1538c, and ΔFTT1597, showed attenuation in lethality and can thus be considered novel F. tularensis virulence factors. The analysis of the accompanying Y2H data identified intracellular protein trafficking between the early endosome to the late endosome as an important component in virulence attenuation for these virulence factors. Furthermore, we also used the Y2H data to investigate host protein binding of two known virulence factors, showing that direct protein binding was a component in the modulation of the inflammatory response via activation of mitogen-activated protein kinases and in the oxidative stress response. Direct interactions with specific host proteins and the ability to influence interactions among host proteins are important components for F. tularensis to avoid host-cell defense mechanisms and successfully establish an infection. Although direct host-pathogen protein-protein binding is only one aspect of Francisella virulence, it is a critical component in directly manipulating and interfering with cellular processes in the host cell.

The PAPI-1 pathogenicity island-encoded small RNA PesA influences Pseudomonas aeruginosa virulence and modulates pyocin S3 production

PubMed Central

Ferrara, Silvia; Falcone, Marilena; Macchi, Raffaella; Bragonzi, Alessandra; Girelli, Daniela; Cariani, Lisa; Cigana, Cristina

2017-01-01

Small non-coding RNAs (sRNAs) are post-transcriptional regulators of gene expression that have been recognized as key contributors to bacterial virulence and pathogenic mechanisms. In this study, we characterized the sRNA PesA of the opportunistic human pathogen Pseudomonas aeruginosa. We show that PesA, which is transcribed within the pathogenicity island PAPI-1 of P. aeruginosa strain PA14, contributes to P. aeruginosa PA14 virulence. In fact, pesA gene deletion resulted in a less pathogenic strain, showing higher survival of cystic fibrosis human bronchial epithelial cells after infection. Moreover, we show that PesA influences positively the expression of pyocin S3 whose genetic locus comprises two structural genes, pyoS3A and pyoS3I, encoding the killing S3A and the immunity S3I proteins, respectively. Interestingly, the deletion of pesA gene results in increased sensitivity to UV irradiation and to the fluoroquinolone antibiotic ciprofloxacin. The degree of UV sensitivity displayed by the PA14 strain lacking PesA is comparable to that of a strain deleted for pyoS3A-I. These results suggest an involvement of pyocin S3 in DNA damage repair and a regulatory role of PesA on this function. PMID:28665976

The FBPase Encoding Gene glpX Is Required for Gluconeogenesis, Bacterial Proliferation and Division In Vivo of Mycobacterium marinum

PubMed Central

Lyu, Liangdong; Wang, Chuan; Li, Yang; Gao, Qian; Yang, Chen

2016-01-01

Lipids have been identified as important carbon sources for Mycobacterium tuberculosis (Mtb) to utilize in vivo. Thus gluconeogenesis bears a key role for Mtb to survive and replicate in host. A rate-limiting enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (FBPase) is encoded by the gene glpX. The functions of glpX were studied in M. marinum, a closely related species to Mtb. The glpX deletion strain (ΔglpX) displayed altered gluconeogenesis, attenuated virulence, and altered bacterial proliferation. Metabolic profiles indicate an accumulation of the FBPase substrate, fructose 1, 6-bisphosphate (FBP) and altered gluconeogenic flux when ΔglpX is cultivated in a gluconeogenic carbon substrate, acetate. In both macrophages and zebrafish, the proliferation of ΔglpX was halted, resulting in dramatically attenuated virulence. Intracellular ΔglpX exhibited an elongated morphology, which was also observed when ΔglpX was grown in a gluconeogenic carbon source. This elongated morphology is also supported by the observation of unseparated multi-nucleoid cell, indicating that a complete mycobacterial division in vivo is correlated with intact gluconeogenesis. Together, our results indicate that glpX has essential functions in gluconeogenesis, and plays an indispensable role in bacterial proliferation in vivo and virulence of M. marinum. PMID:27233038

The FBPase Encoding Gene glpX Is Required for Gluconeogenesis, Bacterial Proliferation and Division In Vivo of Mycobacterium marinum.

PubMed

Tong, Jingfeng; Meng, Lu; Wang, Xinwei; Liu, Lixia; Lyu, Liangdong; Wang, Chuan; Li, Yang; Gao, Qian; Yang, Chen; Niu, Chen

2016-01-01

Lipids have been identified as important carbon sources for Mycobacterium tuberculosis (Mtb) to utilize in vivo. Thus gluconeogenesis bears a key role for Mtb to survive and replicate in host. A rate-limiting enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (FBPase) is encoded by the gene glpX. The functions of glpX were studied in M. marinum, a closely related species to Mtb. The glpX deletion strain (ΔglpX) displayed altered gluconeogenesis, attenuated virulence, and altered bacterial proliferation. Metabolic profiles indicate an accumulation of the FBPase substrate, fructose 1, 6-bisphosphate (FBP) and altered gluconeogenic flux when ΔglpX is cultivated in a gluconeogenic carbon substrate, acetate. In both macrophages and zebrafish, the proliferation of ΔglpX was halted, resulting in dramatically attenuated virulence. Intracellular ΔglpX exhibited an elongated morphology, which was also observed when ΔglpX was grown in a gluconeogenic carbon source. This elongated morphology is also supported by the observation of unseparated multi-nucleoid cell, indicating that a complete mycobacterial division in vivo is correlated with intact gluconeogenesis. Together, our results indicate that glpX has essential functions in gluconeogenesis, and plays an indispensable role in bacterial proliferation in vivo and virulence of M. marinum.

Virulence regulation in Staphylococcus aureus: the need for in vivo analysis of virulence factor regulation.

PubMed

Pragman, Alexa A; Schlievert, Patrick M

2004-10-01

Staphylococcus aureus is a pathogenic microorganism that is responsible for a wide variety of clinical infections. These infections can be relatively mild, but serious, life-threatening infections may result from the expression of staphylococcal virulence factors that are coordinated by virulence regulators. Much work has been done to characterize the actions of staphylococcal virulence regulators in broth culture. Recently, several laboratories showed that transcriptional analyses of virulence regulators in in vivo animal models or in human infection did not correlate with transcriptional analyses accomplished in vitro. In describing the differences between in vitro and in vivo transcription of staphylococcal virulence regulators, we hope to encourage investigators to study virulence regulators using infection models whenever possible.

Listeriolysin S Is a Streptolysin S-Like Virulence Factor That Targets Exclusively Prokaryotic Cells In Vivo.

PubMed

Quereda, Juan J; Nahori, Marie A; Meza-Torres, Jazmín; Sachse, Martin; Titos-Jiménez, Patricia; Gomez-Laguna, Jaime; Dussurget, Olivier; Cossart, Pascale; Pizarro-Cerdá, Javier

2017-04-04

Streptolysin S (SLS)-like virulence factors from clinically relevant Gram-positive pathogens have been proposed to behave as potent cytotoxins, playing key roles in tissue infection. Listeriolysin S (LLS) is an SLS-like hemolysin/bacteriocin present among Listeria monocytogenes strains responsible for human listeriosis outbreaks. As LLS cytotoxic activity has been associated with virulence, we investigated the LLS-specific contribution to host tissue infection. Surprisingly, we first show that LLS causes only weak red blood cell (RBC) hemolysis in vitro and neither confers resistance to phagocytic killing nor favors survival of L. monocytogenes within the blood cells or in the extracellular space (in the plasma). We reveal that LLS does not elicit specific immune responses, is not cytotoxic for eukaryotic cells, and does not impact cell infection by L. monocytogenes Using in vitro cell infection systems and a murine intravenous infection model, we actually demonstrate that LLS expression is undetectable during infection of cells and murine inner organs. Importantly, upon intravenous animal inoculation, L. monocytogenes is found in the gastrointestinal system, and only in this environment LLS expression is detected in vivo Finally, we confirm that LLS production is associated with destruction of target bacteria. Our results demonstrate therefore that LLS does not contribute to L. monocytogenes tissue injury and virulence in inner host organs as previously reported. Moreover, we describe that LlsB, a putative posttranslational modification enzyme encoded in the LLS operon, is necessary for murine inner organ colonization. Overall, we demonstrate that LLS is the first SLS-like virulence factor targeting exclusively prokaryotic cells during in vivo infections. IMPORTANCE The most severe human listeriosis outbreaks are caused by L. monocytogenes strains harboring listeriolysin S (LLS), previously described as a cytotoxin that plays a critical role in host inner tissue infection. Cytotoxic activities have been proposed as a general mode of action for streptolysin S (SLS)-like toxins, including clostridiolysin S and LLS. We now challenge this dogma by demonstrating that LLS does not contribute to virulence in vivo once the intestinal barrier has been crossed. Importantly, we show that intravenous L. monocytogenes inoculation leads to bacterial translocation to the gastrointestinal system, where LLS is specifically expressed, targeting the host gut microbiota. Our study highlights the heterogeneous modes of action of SLS-like toxins, and we demonstrate for the first time a further level of complexity for SLS-like biosynthetic clusters as we reveal that the putative posttranslational modification enzyme LlsB is actually required for inner organ colonization, independently of the LLS activity. Copyright © 2017 Quereda et al.

Insights into virulence factors determining the pathogenicity of Cronobacter sakazakii.

PubMed

Singh, Niharika; Goel, Gunjan; Raghav, Mamta

2015-01-01

Cronobacter sakazakii is an opportunistic pathogen associated with outbreaks of life-threatening necrotizing enterocolitis, meningitis and sepsis in neonates and infants. The pathogen possesses an array of virulence factors which aid in tissue adhesion, invasion and host cell injury. Although the identification and validation of C. sakazakii virulence factors has been hindered by availability of suitable neonatal animal model, various studies has reported outer membrane protein A (ompA) as a potential virulence marker. Various other plasmid associated genes such as filamentous hemagglutinin (fhaBC), Cronobacter plasminogen activator (cpa) and genes responsible for iron acquisition (eitCBAD and iucABD/iutA) have been reported in different strains of C. sakazakii. Besides these proposed virulence factors, several biophysical growth factors such as formation of biofilms and resistance to various environmental stresses also contributes to the pathogenic potential of this pathogen. This review provides an update on virulence determinants associated with the pathogenesis of C. sakazakii. The potential reservoirs of the pathogen, mode of transmission and epidemiology are also discussed.

Insights into virulence factors determining the pathogenicity of Cronobacter sakazakii

PubMed Central

Singh, Niharika; Goel, Gunjan; Raghav, Mamta

2015-01-01

Cronobacter sakazakii is an opportunistic pathogen associated with outbreaks of life-threatening necrotizing enterocolitis, meningitis and sepsis in neonates and infants. The pathogen possesses an array of virulence factors which aid in tissue adhesion, invasion and host cell injury. Although the identification and validation of C. sakazakii virulence factors has been hindered by availability of suitable neonatal animal model, various studies has reported outer membrane protein A (ompA) as a potential virulence marker. Various other plasmid associated genes such as filamentous hemagglutinin (fhaBC), Cronobacter plasminogen activator (cpa) and genes responsible for iron acquisition (eitCBAD and iucABD/iutA) have been reported in different strains of C. sakazakii. Besides these proposed virulence factors, several biophysical growth factors such as formation of biofilms and resistance to various environmental stresses also contributes to the pathogenic potential of this pathogen. This review provides an update on virulence determinants associated with the pathogenesis of C. sakazakii. The potential reservoirs of the pathogen, mode of transmission and epidemiology are also discussed. PMID:25950947

Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

PubMed Central

Caswell, Clayton C.; Elhassanny, Ahmed E. M.; Planchin, Emilie E.; Roux, Christelle M.; Weeks-Gorospe, Jenni N.; Ficht, Thomas A.; Dunman, Paul M.

2013-01-01

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants. PMID:23319565

Identification of a novel gene in ROD9 island of Salmonella Enteritidis involved in the alteration of virulence-associated genes expression.

PubMed

Das, Susmita; Ray, Shilpa; Ryan, Daniel; Sahu, Bikash; Suar, Mrutyunjay

2018-01-01

Salmonella enterica subsp. I serovar Enteritidis (S. Enteritidis), one of the causative agents for non-typhoidal gastrointestinal diseases in humans is an intracellular bacterium and mechanism for its invasion into host cells is critical to cause infection. The virulence of the pathogen is explained by the expression of genes located on its pathogenicity islands, mostly encoded under SPI-1 and SPI-2. However, S. Typhimurium SL1344, despite sharing ∼98% of its genome with S. Enteritidis P125109, lacks few regions of differences (ROD) that are hypothesized to impart virulence potential to S. Enteritidis. In this study, we created different mutants in the ROD9 island of S. Enteritidis, also referred as SPI-19 and identified a novel locus, SEN1005, encoding a hypothetical protein that is involved in its pathogenesis. ΔSEN1005 displayed significantly reduced entry into cultured epithelial cells as well as uptake by macrophages and failed to cause acute colitis in C57BL/6 mice at day 3 post-infection (p.i.). Additionally, the global transcriptome analysis revealed a highly repressed SPI-1 and other down-regulated genes responsible for flagellar assembly, chemotaxis and motility in the mutant which correlated with decreased invasion and abated inflammation as compared to the wild-type. Therefore, our findings revealed that ΔSEN1005 was attenuated in vitro as well as in vivo and we propose this hypothetical protein to play a role in altering the expression of genes involved in Salmonella virulence.

Inhibition effect of tea tree oil on Listeria monocytogenes growth and exotoxin proteins listeriolysin O and p60 secretion.

PubMed

Liu, Z; Meng, R; Zhao, X; Shi, C; Zhang, X; Zhang, Y; Guo, N

2016-12-01

Listeria monocytogenes (L. monocytogenes) is a Gram-positive bacterium that causes infections in humans. In this study, the effects of tea tree oil (TTO) at subinhibitory concentrations on L. monocytogenes growth and two important exotoxin proteins secreted by L. monocytogenes were researched. Treatment with half of minimal inhibitory concentration of TTO demonstrated very little or no reduction in numbers of viable ATCC 19115 cells. Listeriolysin O (LLO) and p60, were investigated. A listeriolysin assay was used to investigate the hemolytic activities of L. monocytogenes exposed to TTO, and the secretion of LLO and p60 was detected by immunoblot analysis. Additionally, real-time RT-PCR was used to analyse the influence of TTO on the transcription of LLO and p60 encoded genes hly and iap respectively. According to our experimental results, we propose that TTO could be used as a promising natural compound against L. monocytogenes and its virulence factors. This is the first report on the influence of subinhibitory concentrations of tea tree oil (TTO) on the secretion of listeriolysin O (LLO) and p60, the critical virulence factors involved in Listeria pathogenesis. The results showed that TTO at 0·25 mg ml -1 reduced the secretion of LLO and p60 to 10 and 34·9% respectively, in addtion, the transcription of hly and iap was reduced to 10 and 4·3% at 0·5 mg ml -1 respectively. We propose that TTO could be used as a promising antimicrobial compound and virulence inhibitor against L. monocytogenes. © 2016 The Society for Applied Microbiology.

Lmo1656 is a secreted virulence factor of Listeria monocytogenes that interacts with the sorting nexin 6-BAR complex.

PubMed

David, Daryl Jason; Pagliuso, Alessandro; Radoshevich, Lilliana; Nahori, Marie-Anne; Cossart, Pascale

2018-06-15

Listeria monocytogenes ( Lm ) is a facultative intracellular bacterial pathogen and the causative agent of listeriosis, a rare but fatal disease. During infection, Lm can traverse several physiological barriers; it can cross the intestine and placenta barrier and, in immunocompromised individuals, the blood-brain barrier. With the recent plethora of sequenced genomes available for Lm , it is clear that the complete repertoire of genes used by Lm to interact with its host remains to be fully explored. Recently, we focused on secreted Lm proteins because they are likely to interact with host cell components. Here, we investigated a putatively secreted protein of Lm , Lmo1656, that is present in most sequenced strains of Lm but absent in the nonpathogenic species Listeria innocua. lmo1656 gene is predicted to encode a small, positively charged protein. We show that Lmo1656 is secreted by Lm Furthermore, deletion of the lmo1656 gene (Δ lmo1656 ) attenuates virulence in mice infected orally but not intravenously, suggesting that Lmo1656 plays a role during oral listeriosis. We identified sorting nexin 6 (SNX6), an endosomal sorting component and BAR domain-containing protein, as a host cell interactor of Lmol656. SNX6 colocalizes with WT Lm during the early steps of infection. This colocalization depends on Lmo1656, and RNAi of SNX6 impairs infection in infected tissue culture cells, suggesting that SNX6 is utilized by Lm during infection. Our results reveal that Lmo1656 is a novel secreted virulence factor of Lm that facilitates recruitment of a specific member of the sorting nexin family in the mammalian host. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Adenylate Cyclase and the Cyclic AMP Receptor Protein Modulate Stress Resistance and Virulence Capacity of Uropathogenic Escherichia coli

PubMed Central

Donovan, Grant T.; Norton, J. Paul; Bower, Jean M.

2013-01-01

In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, the cyaA and crp deletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H2O2) and acid stress. In the mutant strains, H2O2 resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract. PMID:23115037

Streptococcus suis in invasive human infections in Poland: clonality and determinants of virulence and antimicrobial resistance.

PubMed

Bojarska, A; Molska, E; Janas, K; Skoczyńska, A; Stefaniuk, E; Hryniewicz, W; Sadowy, E

2016-06-01

The purpose of this study was to perform an analysis of Streptococcus suis human invasive isolates, collected in Poland by the National Reference Centre for Bacterial Meningitis. Isolates obtained from 21 patients during 2000-2013 were investigated by phenotypic tests, multilocus sequence typing (MLST), analysis of the TR9 locus from the multilocus variable number tandem repeat (VNTR) analysis (MLVA) scheme and pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA. Determinants of virulence and antimicrobial resistance were detected by polymerase chain reaction (PCR) and analysed by sequencing. All isolates represented sequence type 1 (ST1) and were suggested to be serotype 2. PFGE and analysis of the TR9 locus allowed the discrimination of four and 17 types, respectively. Most of the isolates were haemolysis- and DNase-positive, and around half of them formed biofilm. Genes encoding suilysin, extracellular protein factor, fibronectin-binding protein, muramidase-released protein, surface antigen one, enolase, serum opacity factor and pili were ubiquitous in the studied group, while none of the isolates carried sequences characteristic for the 89K pathogenicity island. All isolates were susceptible to penicillin, cefotaxime, imipenem, moxifloxacin, chloramphenicol, rifampicin, gentamicin, linezolid, vancomycin and daptomycin. Five isolates (24 %) were concomitantly non-susceptible to erythromycin, clindamycin and tetracycline, and harboured the tet(O) and erm(B) genes; for one isolate, lsa(E) and lnu(B) were additionally detected. Streptococcus suis isolated in Poland from human invasive infections belongs to a globally distributed clonal complex of this pathogen, enriched in virulence markers. This is the first report of the lsa(E) and lnu(B) resistance genes in S. suis.

Tegumental Phosphodiesterase SmNPP-5 Is a Virulence Factor for Schistosomes ▿

PubMed Central

Bhardwaj, Rita; Krautz-Peterson, Greice; Da'dara, Akram; Tzipori, Saul; Skelly, Patrick J.

2011-01-01

The intravascular trematode Schistosoma mansoni is a causative agent of schistosomiasis, a disease that constitutes a major health problem globally. In this study we cloned and characterized the schistosome tegumental phosphodiesterase SmNPP-5 and evaluated its role in parasite virulence. SmNPP-5 is a 52.5-kDa protein whose gene is rapidly turned on in the intravascular parasitic life stages, following invasion of the definitive host. Highest expression is found in mated adult males. As revealed by immunofluorescence analysis, SmNPP-5 protein is found prominently in the dorsal surface of the tegument of males. Localization by immuno-electron microscopy illustrates a unique pattern of immunogold-labeled SmNPP-5 within the tegument; some immunogold particles are scattered throughout the tissue, but many are clustered in tight arrays. To determine the importance of the protein for the parasites, RNA interference (RNAi) was employed to knock down expression of the SmNPP-5-encoding gene in schistosomula and adult worms. Both quantitative real-time PCR (qRT-PCR) and Western blotting confirmed successful and robust gene suppression. In addition, the suppression and the ectolocalization of this enzyme in live parasites were evident because of a significantly impaired ability of the suppressed parasites to hydrolyze exogenously added phosphodiesterase substrate p-nitrophenyl 5′-dTMP (p-Nph-5′-TMP). The effects of suppressing expression of the SmNPP-5 gene in vivo were tested by injecting parasites into mice. It was found that, unlike controls, parasites whose SmNPP-5 gene was demonstrably suppressed at the time of host infection were greatly impaired in their ability to establish infection. These results demonstrate that SmNPP-5 is a virulence factor for schistosomes. PMID:21825060

Network analysis of S. aureus response to ramoplanin reveals modules for virulence factors and resistance mechanisms and characteristic novel genes.

PubMed

Subramanian, Devika; Natarajan, Jeyakumar

2015-12-10

Staphylococcus aureus is a major human pathogen and ramoplanin is an antimicrobial attributed for effective treatment. The goal of this study was to examine the transcriptomic profiles of ramoplanin sensitive and resistant S. aureus to identify putative modules responsible for virulence and resistance-mechanisms and its characteristic novel genes. The dysregulated genes were used to reconstruct protein functional association networks for virulence-factors and resistance-mechanisms individually. Strong link between metabolic-pathways and development of virulence/resistance is suggested. We identified 15 putative modules of virulence factors. Six hypothetical genes were annotated with novel virulence activity among which SACOL0281 was discovered to be an essential virulence factor EsaD. The roles of MazEF toxin-antitoxin system, SACOL0202/SACOL0201 two-component system and that of amino-sugar and nucleotide-sugar metabolism in virulence are also suggested. In addition, 14 putative modules of resistance mechanisms including modules of ribosomal protein-coding genes and metabolic pathways such as biotin-synthesis, TCA-cycle, riboflavin-biosynthesis, peptidoglycan-biosynthesis etc. are also indicated. Copyright © 2015 Elsevier B.V. All rights reserved.

Mutation of the Erwinia amylovora argD Gene Causes Arginine Auxotrophy, Nonpathogenicity in Apples, and Reduced Virulence in Pears

PubMed Central

Ramos, Laura S.; Lehman, Brian L.; Peter, Kari A.

2014-01-01

Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state. PMID:25172854

Mutation of the Erwinia amylovora argD gene causes arginine auxotrophy, nonpathogenicity in apples, and reduced virulence in pears.

PubMed

Ramos, Laura S; Lehman, Brian L; Peter, Kari A; McNellis, Timothy W

2014-11-01

Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Iron is a signal for Stenotrophomonas maltophilia biofilm formation, oxidative stress response, OMPs expression, and virulence

PubMed Central

García, Carlos A.; Alcaraz, Eliana S.; Franco, Mirta A.; Passerini de Rossi, Beatriz N.

2015-01-01

Stenotrophomonas maltophilia is an emerging nosocomial pathogen. In many bacteria iron availability regulates, through the Fur system, not only iron homeostasis but also virulence. The aim of this work was to assess the role of iron on S. maltophilia biofilm formation, EPS production, oxidative stress response, OMPs regulation, quorum sensing (QS), and virulence. Studies were done on K279a and its isogenic fur mutant F60 cultured in the presence or absence of dipyridyl. This is the first report of spontaneous fur mutants obtained in S. maltophilia. F60 produced higher amounts of biofilms than K279a and CLSM analysis demonstrated improved adherence and biofilm organization. Under iron restricted conditions, K279a produced biofilms with more biomass and enhanced thickness. In addition, F60 produced higher amounts of EPS than K279a but with a similar composition, as revealed by ATR-FTIR spectroscopy. With respect to the oxidative stress response, MnSOD was the only SOD isoenzyme detected in K279a. F60 presented higher SOD activity than the wt strain in planktonic and biofilm cultures, and iron deprivation increased K279a SOD activity. Under iron starvation, SDS-PAGE profile from K279a presented two iron-repressed proteins. Mass spectrometry analysis revealed homology with FepA and another putative TonB-dependent siderophore receptor of K279a. In silico analysis allowed the detection of potential Fur boxes in the respective coding genes. K279a encodes the QS diffusible signal factor (DSF). Under iron restriction K279a produced higher amounts of DSF than under iron rich condition. Finally, F60 was more virulent than K279a in the Galleria mellonella killing assay. These results put in evidence that iron levels regulate, likely through the Fur system, S. maltophilia biofilm formation, oxidative stress response, OMPs expression, DSF production and virulence. PMID:26388863

Elimination of the vitamin B12 uptake or synthesis pathway does not diminish the virulence of Escherichia coli K1 or Salmonella typhimurium in three model systems.

PubMed

Sampson, B A; Gotschlich, E C

1992-09-01

The role of iron in infection is of great importance and is well understood. During infection, both the host and the pathogen go through many complicated changes to regulate iron levels. Iron and vitamin B12 share certain features. For example, Escherichia coli has similar transport systems for both nutrients, and binding proteins for both are located in gastric juice, liver, saliva, granulocytes, and milk. It is because of such parallels between iron and B12 that we have explored the role of B12 in virulence. A btuB::Tn10 insertion which disrupts the gene encoding the vitamin B12 receptor from E. coli K-12 was P1 transduced into a virulent E. coli K1 strain. In both an infant-rat model and a chicken embryo model, no difference in virulence between the wild-type and the mutant strains was found. Strains of Salmonella typhimurium with mutations in the cobalamin synthesis pathway (Cob) and in btuB were used in a mouse model of virulence. Mutation of the Cob locus or of btuB does not decrease virulence. Interestingly, the inability to synthesize vitamin B12 actually increases virulence compared with the wild type in the S. typhimurium model. This effect is independent of the B12 intake of the mice.

Helicobacter pylori virulence factors in development of gastric carcinoma.

PubMed

Wang, Ming-Yi; Liu, Xiao-Fei; Gao, Xiao-Zhong

2015-01-01

Helicobacter pylori plays a vital role in the pathogenesis of gastric carcinoma. However, only a relatively small proportion of individuals infected with H. pylori develop gastric carcinoma. Differences in the incidence of gastric carcinoma among infected individuals can be explained, at least partly, by the different genotypes of H. pylori virulence factors. Thus far, many virulence factors of H. pylori, such as Cag PAI, VacA, OMPs and DupA, have been reported to be involved in the development of gastric cancer. The risk of developing gastric cancer during H. pylori infection is affected by specific host-microbe interactions that are independent of H. pylori virulence factors. In this review, we discuss virulence factors of H. pylori and their role in the development of gastric carcinoma that will provide further understanding of the biological interactions of H. pylori with the host.

Characterization of the pathogenicity island protein PdpA and its role in the virulence of Francisella novicida

PubMed Central

Schmerk, Crystal L.; Duplantis, Barry N.; Wang, Diana; Burke, Robert D.; Chou, Alicia Y.; Elkins, Karen L.; Ludu, Jagjit S.; Nano, Francis E.

2009-01-01

Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice. PMID:19372153

Involvement of NADH Oxidase in Competition and Endocarditis Virulence in Streptococcus sanguinis

PubMed Central

Ge, Xiuchun; Yu, Yang; Zhang, Min; Chen, Lei; Chen, Weihua; Elrami, Fadi; Kong, Fanxiang; Kitten, Todd

2016-01-01

Here, we report for the first time that the Streptococcus sanguinis nox gene encoding NADH oxidase is involved in both competition with Streptococcus mutans and virulence for infective endocarditis. An S. sanguinis nox mutant was found to fail to inhibit the growth of Streptococcus mutans under microaerobic conditions. In the presence of oxygen, the recombinant Nox protein of S. sanguinis could reduce oxygen to water and oxidize NADH to NAD+. The oxidation of NADH to NAD+ was diminished in the nox mutant. The nox mutant exhibited decreased levels of extracellular H2O2; however, the intracellular level of H2O2 in the mutant was increased. Furthermore, the virulence of the nox mutant was attenuated in a rabbit endocarditis model. The nox mutant also was shown to be more sensitive to blood killing, oxidative and acid stresses, and reduced growth in serum. Thus, NADH oxidase contributes to multiple phenotypes related to competitiveness in the oral cavity and systemic virulence. PMID:26930704

Involvement of NADH Oxidase in Competition and Endocarditis Virulence in Streptococcus sanguinis.

PubMed

Ge, Xiuchun; Yu, Yang; Zhang, Min; Chen, Lei; Chen, Weihua; Elrami, Fadi; Kong, Fanxiang; Kitten, Todd; Xu, Ping

2016-05-01

Here, we report for the first time that the Streptococcus sanguinis nox gene encoding NADH oxidase is involved in both competition with Streptococcus mutans and virulence for infective endocarditis. An S. sanguinis nox mutant was found to fail to inhibit the growth of Streptococcus mutans under microaerobic conditions. In the presence of oxygen, the recombinant Nox protein of S. sanguinis could reduce oxygen to water and oxidize NADH to NAD(+) The oxidation of NADH to NAD(+) was diminished in the nox mutant. The nox mutant exhibited decreased levels of extracellular H2O2; however, the intracellular level of H2O2 in the mutant was increased. Furthermore, the virulence of the nox mutant was attenuated in a rabbit endocarditis model. The nox mutant also was shown to be more sensitive to blood killing, oxidative and acid stresses, and reduced growth in serum. Thus, NADH oxidase contributes to multiple phenotypes related to competitiveness in the oral cavity and systemic virulence. Copyright © 2016 Ge et al.

Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

PubMed Central

Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

2016-01-01

Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

AmyR Is a Novel Negative Regulator of Amylovoran Production in Erwinia amylovora

PubMed Central

Wang, Dongping; Korban, Schuyler S.; Pusey, P. Lawrence; Zhao, Youfu

2012-01-01

In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora. PMID:23028751

AmyR is a novel negative regulator of amylovoran production in Erwinia amylovora.

PubMed

Wang, Dongping; Korban, Schuyler S; Pusey, P Lawrence; Zhao, Youfu

2012-01-01

In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora.

Genomic Characterization of Haemophilus parasuis SH0165, a Highly Virulent Strain of Serovar 5 Prevalent in China

PubMed Central

Zhou, Rui; Jin, Qi; Fan, Yang; Bei, Weicheng; Chen, Huanchun

2011-01-01

Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässer's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT), identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis. PMID:21611187

Host-Nonspecific Iron Acquisition Systems and Virulence in the Zoonotic Serovar of Vibrio vulnificus

PubMed Central

Pajuelo, David; Lee, Chung-Te; Roig, Francisco J.; Lemos, Manuel L.; Hor, Lien-I

2014-01-01

The zoonotic serovar of Vibrio vulnificus (known as biotype 2 serovar E) is the etiological agent of human and fish vibriosis. The aim of the present work was to discover the role of the vulnibactin- and hemin-dependent iron acquisition systems in the pathogenicity of this zoonotic serovar under the hypothesis that both are host-nonspecific virulence factors. To this end, we selected three genes for three outer membrane receptors (vuuA, a receptor for ferric vulnibactin, and hupA and hutR, two hemin receptors), obtained single and multiple mutants as well as complemented strains, and tested them in a series of in vitro and in vivo assays, using eels and mice as animal models. The overall results confirm that hupA and vuuA, but not hutR, are host-nonspecific virulence genes and suggest that a third undescribed host-specific plasmid-encoded system could also be used by the zoonotic serovar in fish. hupA and vuuA were expressed in the internal organs of the animals in the first 24 h of infection, suggesting that they may be needed to achieve the population size required to trigger fatal septicemia. vuuA and hupA were sequenced in strains representative of the genetic diversity of this species, and their phylogenies were reconstructed by multilocus sequence analysis of selected housekeeping and virulence genes as a reference. Given the overall results, we suggest that both genes might form part of the core genes essential not only for disease development but also for the survival of this species in its natural reservoir, the aquatic environment. PMID:24478087

A mutation in rcsB, a gene encoding the core component of the Rcs cascade, enhances the virulence of Edwardsiella tarda.

PubMed

Xu, Ying; Xu, Tingting; Wang, Bin; Dong, Xue; Sheng, Aibo; Zhang, Xiao-Hua

2014-04-01

Edwardsiella tarda, a Gram-negative bacterium of the family Enterobacteriaceae, is the causative agent of the systemic disease edwardsiellosis, which is a major problem in aquaculture industry worldwide. Many virulence-related genes in E. tarda have been investigated, but the Rcs phosphorelay, a two-component pathway, which regulates several cell-surface-associated structures related to invasion and survival in host cells, has not yet been thoroughly studied. In the present study, an rcsB in-frame deletion mutant ΔrcsB was constructed through double-crossover allelic exchange. To complement the rcsB mutation, the ΔrcsB (pACYC184K-rcsB) mutant was constructed by transformation of a low-copy plasmid carrying the intact rcsB into the ΔrcsB mutant of E. tarda. Several virulence-associated characters of the mutants and wild-type strain were tested. Compared with wild-type strain EIB202, biofilm formation decreased significantly in ΔrcsB, while ΔrcsB (pACYC184K-rcsB) recovered the phenotype to some extent. In addition, the capacity for autoagglutination, the percentage of adherence and internalization to Epithelioma papulosum cyprini cells and lethality toward zebrafish embryos significantly increased in ΔrcsB. All these phenomena displayed by mutant ΔrcsB showed a certain degree of recovery, though incomplete, in strain ΔrcsB (pACYC184K-rcsB). Present results indicate that rcsB is involved in regulating the gene expression of virulence factors in E. tarda, as shown in other members of Enterobacteriaceae. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

PubMed Central

Quintard, Kévin; Dewitte, Amélie; Reboul, Angéline; Madec, Edwige; Bontemps-Gallo, Sébastien; Dondeyne, Jacqueline; Marceau, Michaël; Simonet, Michel

2015-01-01

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary. PMID:26150539

Streptococcus iniae M-Like Protein Contributes to Virulence in Fish and Is a Target for Live Attenuated Vaccine Development

PubMed Central

Locke, Jeffrey B.; Aziz, Ramy K.; Vicknair, Mike R.; Nizet, Victor; Buchanan, John T.

2008-01-01

Background Streptococcus iniae is a significant pathogen in finfish aquaculture, though knowledge of virulence determinants is lacking. Through pyrosequencing of the S. iniae genome we have identified two gene homologues to classical surface-anchored streptococcal virulence factors: M-like protein (simA) and C5a peptidase (scpI). Methodology/Principal Findings S. iniae possesses a Mga-like locus containing simA and a divergently transcribed putative mga-like regulatory gene, mgx. In contrast to the Mga locus of group A Streptococcus (GAS, S. pyogenes), scpI is located distally in the chromosome. Comparative sequence analysis of the Mgx locus revealed only one significant variant, a strain with an insertion frameshift mutation in simA and a deletion mutation in a region downstream of mgx, generating an ORF which may encode a second putative mga-like gene, mgx2. Allelic exchange mutagenesis of simA and scpI was employed to investigate the potential role of these genes in S. iniae virulence. Our hybrid striped bass (HSB) and zebrafish models of infection revealed that M-like protein contributes significantly to S. iniae pathogenesis whereas C5a peptidase-like protein does not. Further, in vitro cell-based analyses indicate that SiMA, like other M family proteins, contributes to cellular adherence and invasion and provides resistance to phagocytic killing. Attenuation in our virulence models was also observed in the S. iniae isolate possessing a natural simA mutation. Vaccination of HSB with the ΔsimA mutant provided 100% protection against subsequent challenge with a lethal dose of wild-type (WT) S. iniae after 1,400 degree days, and shows promise as a target for live attenuated vaccine development. Conclusions/Significance Analysis of M-like protein and C5a peptidase through allelic replacement revealed that M-like protein plays a significant role in S. iniae virulence, and the Mga-like locus, which may regulate expression of this gene, has an unusual arrangement. The M-like protein mutant created in this research holds promise as live-attenuated vaccine. PMID:18665241

Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus.

PubMed

Gottschalk, Sanne; Gottlieb, Caroline T; Vestergaard, Martin; Hansen, Paul R; Gram, Lone; Ingmer, Hanne; Thomsen, Line E

2015-12-01

The rapid rise in antibiotic-resistant pathogens is causing increased health concerns, and consequently there is an urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs), which have been isolated from a wide range of organisms, represent a very promising class of novel antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl(2) concentrations and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding α-haemolysin. In addition, the S. aureus-encoded natural tolerance mechanisms included peptide cleavage and the addition of positive charge to the cell surface, both of which minimized the antimicrobial activity of FL9. Our results add new information about FL9 and its effect on S. aureus, which may aid in the future development of analogues with improved therapeutic potential.

Identification of Potential Virulence Determinants by Himar1 Transposition of Infectious Borrelia burgdorferi B31▿

PubMed Central

Botkin, Douglas J.; Abbott, April N.; Stewart, Philip E.; Rosa, Patricia A.; Kawabata, Hiroki; Watanabe, Haruo; Norris, Steven J.

2006-01-01

Lyme disease Borrelia organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1 transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferi is feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia. PMID:17015459

Frequency of ace, epa and elrA Genes in Clinical and Environmental Strains of Enterococcus faecalis.

PubMed

Lysakowska, Monika Eliza; Denys, Andrzej; Sienkiewicz, Monika

2012-12-01

Surface proteins play an important role in the pathogenesis of enterococcal infections. Some of them are candidates for a vaccine, e.g., the frequency of endocarditis in rats vaccinated with Ace protein was 75 % as 12 opposed to 100 % in those who weren't. However, there are other components of enterococcal cells, such as Epa antigens or internalin-like proteins, which may be used in the prophylaxis of infections caused by them. However, also other virulence factors and resistance to antibiotics are important during enterococcal infection. Therefore, the relevance of ace, epa, elrA, other virulence genes, as well as resistance to antibiotics was investigated. 161 Enterococcus faecalis strains isolated from teaching hospitals in Lodz, cultured according to standard microbiological methods, were investigated for the presence of genes encoding surface proteins by PCR. Results were analyzed with χ(2) test. The elrA gene was found in all clinical and environmental strains, the ace gene was also widespread among E. faecalis (96.9 %). Both tested epa genes were found in the majority of isolates (83.25 %). There was correlation between the presence of esp and ace genes (p = 0.046) as well as between epa and agg genes (p = 0.0094; χ(2) test). The presence of the genes encoding surface proteins investigated in our study in the great majority of isolates implies that they would appear to be required during E. faecalis infection. Therefore, they could be excellent targets in therapy of enterococcal infections or, as some studies show, candidates for vaccines.

The PB2 Subunit of the Influenza Virus RNA Polymerase Affects Virulence by Interacting with the Mitochondrial Antiviral Signaling Protein and Inhibiting Expression of Beta Interferon▿

PubMed Central

Graef, Katy M.; Vreede, Frank T.; Lau, Yuk-Fai; McCall, Amber W.; Carr, Simon M.; Subbarao, Kanta; Fodor, Ervin

2010-01-01

The PB2 subunit of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. However, the molecular mechanisms involved remain unknown. It was previously shown that the PB2 protein, in addition to its nuclear localization, also accumulates in the mitochondria. Here, we demonstrate that the PB2 protein interacts with the mitochondrial antiviral signaling protein, MAVS (also known as IPS-1, VISA, or Cardif), and inhibits MAVS-mediated beta interferon (IFN-β) expression. In addition, we show that PB2 proteins of influenza viruses differ in their abilities to associate with the mitochondria. In particular, the PB2 proteins of seasonal human influenza viruses localize to the mitochondria while PB2 proteins of avian influenza viruses are nonmitochondrial. This difference in localization is caused by a single amino acid polymorphism in the PB2 mitochondrial targeting signal. In order to address the functional significance of the mitochondrial localization of the PB2 protein in vivo, we have generated two recombinant human influenza viruses encoding either mitochondrial or nonmitochondrial PB2 proteins. We found that the difference in the mitochondrial localization of the PB2 proteins does not affect the growth of these viruses in cell culture. However, the virus encoding the nonmitochondrial PB2 protein induces higher levels of IFN-β and, in an animal model, is attenuated compared to the isogenic virus encoding a mitochondrial PB2. Overall this study implicates the PB2 protein in the regulation of host antiviral innate immune pathways and suggests an important role for the mitochondrial association of the PB2 protein in determining virulence. PMID:20538852

Proteomic Characterization of Yersinia pestis Virulence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chromy, B; Murphy, G; Gonzales, A

2005-01-05

Yersinia pestis, the etiological agent of plague, functions via the Type III secretion mechanism whereby virulence factors are induced upon interactions with a mammalian host. Here, the Y. pestis proteome was studied by two-dimensional differential gel electrophoresis (2-D DIGE) under physiologically relevant growth conditions mimicking the calcium concentrations and temperatures that the pathogen would encounter in the flea vector and upon interaction with the mammalian host. Over 4100 individual protein spots were detected of which hundreds were differentially expressed in the entire comparative experiment. A total of 43 proteins that were differentially expressed between the vector and host growth conditionsmore » were identified by mass spectrometry. Expected differences in expression were observed for several known virulence factors including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as putative virulence factors. Chaperone proteins and signaling molecules hypothesized to be involved in virulence due to their role in Type III secretion were also identified. Other differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants. For example, several sugar metabolism proteins were differentially regulated in response to lower calcium and higher temperature, suggesting these proteins, while not directly connected to virulence, either represent a metabolic switch for survival in the host environment or may facilitate production of virulence factors. Results presented here contribute to a more thorough understanding of the virulence mechanism of Y. pestis through proteomic characterization of the pathogen under induced virulence.« less

Three Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutants with Distinct and Asymmetric Effects on Virulence in Mice Compared with Rabbits

PubMed Central

Perng, Guey-Chuen; Esmaili, Daniel; Slanina, Susan M.; Yukht, Ada; Ghiasi, Homayon; Osorio, Nelson; Mott, Kevin R.; Maguen, Barak; Jin, Ling; Nesburn, Anthony B.; Wechsler, Steven L.

2001-01-01

Herpes simplex virus type 1 latency-associated transcript (LAT)-null mutants have decreased reactivation but normal virulence in rabbits and mice. We report here on dLAT1.5, a mutant with LAT nucleotides 76 to 1667 deleted. Following ocular infection of rabbits, dLAT1.5 reactivated at a lower rate than its wild-type parent McKrae (6.1 versus 11.8%; P = 0.0025 [chi-square test]). Reactivation was restored in the marker-rescued virus dLAT1.5R (12.6%; P = 0.53 versus wild type), confirming the importance of the deleted region in spontaneous reactivation. Compared with wild-type or marker-rescued virus, dLAT1.5 had similar or slightly reduced virulence in rabbits (based on survival following ocular infection). In contrast, in mice, dLAT1.5 had increased virulence (P < 0.0001). Thus, deletion of LAT nucleotides 76 to 1667 increased viral virulence in mice but not in rabbits. In contrast, we also report here that LAT2.9A, a LAT mutant that we previously reported to have increased virulence in rabbits (G. C. Perng, S. M. Slanina, A. Yuhkt, B. S. Drolet, W. J. Keleher, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 73:920–929, 1999), had decreased virulence in mice (P = 0.03). In addition, we also found that dLAT371, a LAT mutant that we previously reported to have wild-type virulence in rabbits (G. C. Perng, S. M. Slanina, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 70:2014–2018, 1996), had decreased virulence in mice (P < 0.05). Thus, these three mutants, each of which encodes a different LAT RNA, have different virulence phenotypes. dLAT1.5 had wild-type virulence in rabbits but increased virulence in mice. In contrast, LAT2.9A had increased virulence in rabbits but decreased virulence in mice, and dLAT371 had wild-type virulence in rabbits but decreased virulence in mice. Taken together, these results suggest that (i) the 5′ end of LAT and/or a gene that overlaps part of this region is involved in viral virulence, (ii) this virulence appears to have species-specific effects, and (iii) regulation of this virulence may be complex. PMID:11533165

Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test.

PubMed

Lobato-Márquez, Damián; Molina-García, Laura; Moreno-Córdoba, Inma; García-Del Portillo, Francisco; Díaz-Orejas, Ramón

2016-01-01

Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50-90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system ( parAB ) and two TA systems ( ccdAB ST and vapBC2 ST ). The TA module ccdAB ST has previously been shown to contribute to pSLT plasmid stability and vapBC2 ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2 ST , in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdAB ST encodes an inactive CcdB ST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdAB ST variant containing a single mutation (R99W) that restores the toxicity of CcdB ST . The "activation" of CcdB ST (R99W) did not increase pSLT stability by ccdAB ST . In contrast, ccdAB ST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdAB ST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdB ST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdA ST antitoxin more than on its toxic activity.

Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins

PubMed Central

Brazier, Andrew J.; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell

2017-01-01

ABSTRACT Plasmodium falciparum, the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi. We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum. Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum-infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum, it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P. falciparum. Our findings suggest that parasite adhesion traits associated with both mild and severe malaria have much earlier origins than previously appreciated and have important implications for virulence evolution in a major human pathogen. PMID:28101534

Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins.

PubMed

Brazier, Andrew J; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Smith, Joseph D

2017-01-01

Plasmodium falciparum , the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi . We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum . Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum -infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum , it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P. falciparum . Our findings suggest that parasite adhesion traits associated with both mild and severe malaria have much earlier origins than previously appreciated and have important implications for virulence evolution in a major human pathogen.

Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

PubMed

Varela, Mariana; Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong; Palmarini, Massimo

2016-06-01

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore the pathogenicity of attenuated mutants resulting from deletions or mutations in the nonstructural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of nonstructural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes. Copyright © 2016 Varela et al.