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Sample records for molecular virulence determinants

  1. Listeria Pathogenesis and Molecular Virulence Determinants

    PubMed Central

    Vázquez-Boland, José A.; Kuhn, Michael; Berche, Patrick; Chakraborty, Trinad; Domínguez-Bernal, Gustavo; Goebel, Werner; González-Zorn, Bruno; Wehland, Jürgen; Kreft, Jürgen

    2001-01-01

    , rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research. PMID:11432815

  2. Exploiting molecular virulence determinants in Burkholderia to develop vaccine antigens.

    PubMed

    Casey, William Thomas; McClean, Siobhán

    2015-01-01

    The Burkholderia genus is a highly diverse group of species that are distributed throughout a wide range of environments and habitats. Among this group, which is remarkable for its adaptability to a wider range of environmental conditions including disinfectants and organic solvents, are a subgroup that represents some of the most difficult to treat infections. This subgroup includes Burkholderia pseudomallei, the causative agent of melioidosis; B. mallei, the causative agent of glanders and B. cepacia complex (Bcc) which causes opportunistic infections in people with cystic fibrosis and immunocompromised patients. The latter pathogen is itself a group of 18 distinct, but, closely related species. The adaptability of this group allows the expression of a rich selection of molecular virulence determinants to facilitate its survival in the diverse habitats that it colonises. This review will describe a selection of these associated with human infection; comparing them across the three pathogens and highlighting their potential roles as vaccine candidates. Better integration of the knowledge on the pathogenesis and molecular determinants of virulence for these Burkholderia spp may allow the development of more efficacious vaccines. PMID:25850766

  3. Virulence Determination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter reviews the in vitro and in vivo assays that are available for determination of pathogenic potential of Listeria monocytogenes bacteria, highlighting the value of using multiplex PCR for rapid and accurate assessment of listerial virulence....

  4. Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence

    PubMed Central

    Smith, Issar

    2003-01-01

    Tuberculosis (TB), one of the oldest known human diseases. is still is one of the major causes of mortality, since two million people die each year from this malady. TB has many manifestations, affecting bone, the central nervous system, and many other organ systems, but it is primarily a pulmonary disease that is initiated by the deposition of Mycobacterium tuberculosis, contained in aerosol droplets, onto lung alveolar surfaces. From this point, the progression of the disease can have several outcomes, determined largely by the response of the host immune system. The efficacy of this response is affected by intrinsic factors such as the genetics of the immune system as well as extrinsic factors, e.g., insults to the immune system and the nutritional and physiological state of the host. In addition, the pathogen may play a role in disease progression since some M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines and drugs and more specific and rapid diagnostics. Researchers are utilizing information obtained from the complete sequence of the M. tuberculosis genome and from new genetic and physiological methods to identify targets in M. tuberculosis that will aid in the development of these sorely needed antitubercular agents. PMID:12857778

  5. In vitro and in vivo characterization of molecular determinants of virulence in reassortant betanodavirus.

    PubMed

    Souto, Sandra; Mérour, Emilie; Biacchesi, Stéphane; Brémont, Michel; Olveira, José G; Bandín, Isabel

    2015-06-01

    We previously reported that betanodavirus reassortant strains [redspotted grouper nervous necrosis virus/striped jack nervous necrosis virus (SJNNV)] isolated from Senegalese sole (Solea senegalensis) exhibited a modified SJNNV capsid amino acid sequence, with changes at aa 247 and 270. In the current study, we investigated the possible role of both residues as putative virulence determinants. Three recombinant viruses harbouring site-specific mutations in the capsid protein sequence, rSs160.03247 (S247A), rSs160.03270 (S270N) and rSs160.03247+270 (S247A/S270N), were generated using a reverse genetics system. These recombinant viruses were studied in cell culture and in vivo in the natural fish host. The three mutant viruses were shown to be infectious and able to replicate in E-11 cells, reaching final titres similar to the WT virus, although with a somewhat slower kinetics of replication. When the effect of the amino acid substitutions on virus pathogenicity was evaluated in Senegalese sole, typical clinical signs of betanodavirus infection were observed in all groups. However, fish mortality induced by all three mutant viruses was clearly affected. Roughly 40 % of the fish survived in these three groups in contrast with the WT virus which killed 100 % of the fish. These data demonstrated that aa 247 and 270 play a major role in betanodavirus virulence although when both mutated aa 247 and 270 are present, corresponding recombinant virus was not further attenuated. PMID:25626678

  6. Antimicrobial susceptibility, virulence determinant carriage and molecular characteristics of Staphylococcus aureus isolates associated with skin and soft tissue infections.

    PubMed

    Yu, Fangyou; Liu, Yunling; Lv, Jinnan; Qi, Xiuqin; Lu, Chaohui; Ding, Yu; Li, Dan; Liu, Huanle; Wang, Liangxing

    2015-01-01

    A better understanding of the antimicrobial susceptibility, carriage of virulence determinants and molecular characteristics of Staphylococcus aureus isolates associated with skin and soft tissue infections (SSTIs) may provide further insights related to clinical outcomes with these infections. From January 2012 to September 2013, a total of 128 non-duplicate S. aureus isolates were recovered from patients with SSTIs. All 128 S. aureus SSTI isolates carried at least five virulence genes tested. Virulence genes detected among at least 70% of all tested isolates included hld (100%), hla (95.3%), icaA (96.9%), clf (99.2%), sdrC (79.7%), sdrD (70.3%), and sdrE (72.7%). The prevalence of MRSA isolates with 10 virulence genes tested (54.4%, 31/56) was significantly higher than that among MSSA isolates (35.2%, 25/71) (p<0.05). The positive rates of seb, sen, sem, sdrE and pvl among MRSA isolates were significantly higher than among MSSA isolates (p<0.05). ST7 and ST630 accounting for 10.9% were found to be the predominant STs. The most prevalent spa type was t091 (8.6%). MRSA-ST59-SCCmec IV was the most common clone (12.3%) among MRSA isolates whereas among MSSA isolates the dominant clone was MSSA-ST7 (15.5%). Six main clonal complexes (CCs) were found, including CC5 (52.3%), CC7 (11.7%), CC59 (8.6%), CC88 (6.3%), CC398 (4.7%), and CC121 (3.1%). A higher carriage of seb and sec was found among CC59 isolates. In comparison to CC5 and CC7 isolates, those with the highest carriage rates (>80.0%) of sdrC and sdrD, CC59 isolates had lower prevalence of these two virulence genes. All CC59 isolates were susceptible to gentamicin and trimethoprim/sulfamethoxazole, while CC5 and CC7 isolates had resistance rates to these two antimicrobials of 25.4% and 20.9%, and 40.0% and 40.0%, respectively. The resistance rates for tetracycline, clindamycin, and erythromycin among CC5 isolates were lower than among CC7 and CC59 isolates. In conclusion, the molecular typing of S. aureus SSTI

  7. Molecular Determinants of Virulence and Stability of a Reporter-Expressing H5N1 Influenza A Virus

    PubMed Central

    Zhao, Dongming; Fukuyama, Satoshi; Yamada, Shinya; Lopes, Tiago J. S.; Maemura, Tadashi; Katsura, Hiroaki; Ozawa, Makoto; Watanabe, Shinji; Neumann, Gabriele

    2015-01-01

    ABSTRACT We previously reported that an H5N1 virus carrying the Venus reporter gene, which was inserted into the NS gene segment from the A/Puerto Rico/8/1934(H1N1) virus (Venus-H5N1 virus), became more lethal to mice, and the reporter gene was stably maintained after mouse adaptation compared with the wild-type Venus-H5N1 (WT-Venus-H5N1) virus. However, the basis for this difference in virulence and Venus stability was unclear. Here, we investigated the molecular determinants behind this virulence and reporter stability by comparing WT-Venus-H5N1 virus with a mouse-adapted Venus-H5N1 (MA-Venus-H5N1) virus. To determine the genetic basis for these differences, we used reverse genetics to generate a series of reassortants of these two viruses. We found that reassortants with PB2 from MA-Venus-H5N1 (MA-PB2), MA-PA, or MA-NS expressed Venus more stably than did WT-Venus-H5N1 virus. We also found that a single mutation in PB2 (V25A) or in PA (R443K) increased the virulence of the WT-Venus-H5N1 virus in mice and that the presence of both of these mutations substantially enhanced the pathogenicity of the virus. Our results suggest roles for PB2 and PA in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus. IMPORTANCE The ability to visualize influenza viruses has far-reaching benefits in influenza virus research. Previously, we reported that an H5N1 virus bearing the Venus reporter gene became more pathogenic to mice and that its reporter gene was more highly expressed and more stably maintained after mouse adaptation. Here, we investigated the molecular determinants behind this enhanced virulence and reporter stability. We found that mutations in PB2 (V25A) and PA (R443K) play crucial roles in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus and in the virulence of influenza virus in mice. Our findings further our knowledge of the pathogenicity of influenza virus in mammals and will help

  8. [Virulence determinant of Chromobacterium violaceum].

    PubMed

    Miki, Tsuyoshi

    2014-01-01

    Chromobacterium violaceum is a Gram-negative bacterium that infects humans and animals with fatal sepsis. The infection with C. violaceum is rare in case of those who are healthy, but once established, C. violaceum causes sever disease accompanied by abscess formation in the lungs, liver and spleen. Furthermore, C. violaceum is resistant to a broad range of antibiotics, which in some cases renders the antimicrobial therapy for this infection difficult. Thus, the infection with C. violaceum displays high mortality rates unless initial proper antimicrobial therapy. In contrast, the infection mechanism had completely remained unknown. To this end, we have tried to identify virulence factors-associated with C. violaceum infection. Two distinct type III secretion systems (TTSSs) were thought to be one of the most important virulence factors, which are encoded by Chromobacterium pathogenicity island 1/1a and 2 (Cpi-1/-1a and -2) respectively. Our results have shown that Cpi-1/-1a-encoded TTSS, but not Cpi-2, is indispensable for the virulence in a mouse infection model. C. violaceum caused fulminant hepatitis in a Cpi-1/-1a-encoded TTSS-dependent manner. We next have identified 16 novel effectors secreted from Cpi-1/-1a-encoded TTS machinery. From these effectors, we found that CopE (Chromobacterium outer protein E) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases. CopE acts as GEF for Rac1 and Cdc42, leading to induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades cultured human epithelial cells in a CopE-dependent manner. Finally, an inactivation of CopE by disruption of copE gene or amino acid point mutation leading to loss of GEF activity attenuates significantly the mouse virulence of C. violaceum. These results suggest that Cpi-1/-1a-encoded TTSS is a major virulence determinant for C. violaceum infection, and that CopE contributes to the virulence in part of this pathogen. PMID:25447983

  9. Molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease.

    PubMed

    Frieman, Matthew; Yount, Boyd; Agnihothram, Sudhakar; Page, Carly; Donaldson, Eric; Roberts, Anjeanette; Vogel, Leatrice; Woodruff, Becky; Scorpio, Diana; Subbarao, Kanta; Baric, Ralph S

    2012-01-01

    SARS coronavirus (SARS-CoV) causes severe acute respiratory tract disease characterized by diffuse alveolar damage and hyaline membrane formation. This pathology often progresses to acute respiratory distress (such as acute respiratory distress syndrome [ARDS]) and atypical pneumonia in humans, with characteristic age-related mortality rates approaching 50% or more in immunosenescent populations. The molecular basis for the extreme virulence of SARS-CoV remains elusive. Since young and aged (1-year-old) mice do not develop severe clinical disease following infection with wild-type SARS-CoV, a mouse-adapted strain of SARS-CoV (called MA15) was developed and was shown to cause lethal infection in these animals. To understand the genetic contributions to the increased pathogenesis of MA15 in rodents, we used reverse genetics and evaluated the virulence of panels of derivative viruses encoding various combinations of mouse-adapted mutations. We found that mutations in the viral spike (S) glycoprotein and, to a much less rigorous extent, in the nsp9 nonstructural protein, were primarily associated with the acquisition of virulence in young animals. The mutations in S likely increase recognition of the mouse angiotensin-converting enzyme 2 (ACE2) receptor not only in MA15 but also in two additional, independently isolated mouse-adapted SARS-CoVs. In contrast to the findings for young animals, mutations to revert to the wild-type sequence in nsp9 and the S glycoprotein were not sufficient to significantly attenuate the virus compared to other combinations of mouse-adapted mutations in 12-month-old mice. This panel of SARS-CoVs provides novel reagents that we have used to further our understanding of differential, age-related pathogenic mechanisms in mouse models of human disease. PMID:22072787

  10. Molecular Determinants of Severe Acute Respiratory Syndrome Coronavirus Pathogenesis and Virulence in Young and Aged Mouse Models of Human Disease

    PubMed Central

    Yount, Boyd; Agnihothram, Sudhakar; Page, Carly; Donaldson, Eric; Roberts, Anjeanette; Vogel, Leatrice; Woodruff, Becky; Scorpio, Diana; Subbarao, Kanta; Baric, Ralph S.

    2012-01-01

    SARS coronavirus (SARS-CoV) causes severe acute respiratory tract disease characterized by diffuse alveolar damage and hyaline membrane formation. This pathology often progresses to acute respiratory distress (such as acute respiratory distress syndrome [ARDS]) and atypical pneumonia in humans, with characteristic age-related mortality rates approaching 50% or more in immunosenescent populations. The molecular basis for the extreme virulence of SARS-CoV remains elusive. Since young and aged (1-year-old) mice do not develop severe clinical disease following infection with wild-type SARS-CoV, a mouse-adapted strain of SARS-CoV (called MA15) was developed and was shown to cause lethal infection in these animals. To understand the genetic contributions to the increased pathogenesis of MA15 in rodents, we used reverse genetics and evaluated the virulence of panels of derivative viruses encoding various combinations of mouse-adapted mutations. We found that mutations in the viral spike (S) glycoprotein and, to a much less rigorous extent, in the nsp9 nonstructural protein, were primarily associated with the acquisition of virulence in young animals. The mutations in S likely increase recognition of the mouse angiotensin-converting enzyme 2 (ACE2) receptor not only in MA15 but also in two additional, independently isolated mouse-adapted SARS-CoVs. In contrast to the findings for young animals, mutations to revert to the wild-type sequence in nsp9 and the S glycoprotein were not sufficient to significantly attenuate the virus compared to other combinations of mouse-adapted mutations in 12-month-old mice. This panel of SARS-CoVs provides novel reagents that we have used to further our understanding of differential, age-related pathogenic mechanisms in mouse models of human disease. PMID:22072787

  11. Comparison of Saccharomyces cerevisiae strains of clinical and nonclinical origin by molecular typing and determination of putative virulence traits

    PubMed Central

    Klingberg, Trine Danø; Lesnik, Urska; Arneborg, Nils; Raspor, Peter; Jespersen, Lene

    2008-01-01

    Saccharomyces cerevisiae strains of clinical and nonclinical origin were compared by pulse field gel electrophoresis. Complete separation between strains of clinical origin and food strains by their chromosome length polymorphism was not obtained even though there was a tendency for the clinical and food strains to cluster separately. All the investigated strains, except for one food strain, were able to grow at temperatures ≥37 °C but not at 42 °C. Great strain variations were observed in pseudohyphal growth and invasiveness, but the characters were not linked to strains of clinical origin. The adhesion capacities of the yeast strains to a human intestinal epithelial cell line (Caco-2) in response to different nutritional availabilities were determined, as were the effects of the strains on the transepithelial electrical resistance (TER) across polarized monolayers of Caco-2 cells. The yeast strains displayed very low adhesion capacities to Caco-2 cells (0.6–6.2%), and no significant difference was observed between the strains of clinical and nonclinical origin. Both S. cerevisiae strains of clinical and non-clinical origin increased the TER of polarized monolayers of Caco-2 cells. Based on the results obtained in this study, no specific virulence factor was found that clearly separated the strains of clinical origin from the strains of nonclinical origin. On the contrary, all investigated strains of S. cerevisiae were found to strengthen the epithelial barrier function. PMID:18355272

  12. Viral determinants of simian immunodeficiency virus (SIV) virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac.

    PubMed Central

    Marthas, M L; Ramos, R A; Lohman, B L; Van Rompay, K K; Unger, R E; Miller, C J; Banapour, B; Pedersen, N C; Luciw, P A

    1993-01-01

    To identify viral determinants of simian immunodeficiency virus (SIV) virulence, two pairs of reciprocal recombinants constructed from a pathogenic (SIVmac239) and a nonpathogenic (SIVmac1A11) molecular clone of SIV were tested in rhesus macaques. A large 6.2-kb fragment containing gag, pol, env, and the regulatory genes from each of the cloned (parental) viruses was exchanged to produce one pair of recombinant viruses (designated SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11gag-env/239 to indicate the genetic origins of the 5'/internal/3' regions, respectively, of the virus). A smaller 1.4-kb fragment containing the external env domain of each of the parental viruses was exchanged to create the second pair (SIVmac1A11/239env/1A11 and SIVmac239/1A11env/239) of recombinant viruses. Each of the two parental and four recombinant viruses was inoculated intravenously into four rhesus macaques, and all 24 animals were viremic by 4 weeks postinoculation (p.i.). Virus could not be isolated from peripheral blood mononuclear cells (PBMC) of any animals infected with SIVmac1A11 after 6 weeks p.i. but was consistently isolated from all macaques inoculated with SIVmac239 for 92 weeks p.i. Virus isolation was variable from animals infected with recombinant viruses; SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11env/239 were isolated most frequently. Animals inoculated with SIVmac239 had 10 to 100 times more virus-infected PBMC than those infected with recombinant viruses. Three animals infected with SIVmac239 died with simian AIDS (SAIDS) during the 2-year observation period after inoculation, and the fourth SIVmac239-infected animal had clinical signs of SAIDS. Two animals infected with recombinant viruses died with SAIDS; one was infected with SIVmac239/1A11gag-env/239, and the other was infected with SIVmac1A11/239gag-env/1A11. The remaining 18 macaques remained healthy by 2 years p.i., and 13 were aviremic. One year after inoculation, peripheral lymph nodes of some of these

  13. Multiple Genome Segments Determine Virulence of Bluetongue Virus Serotype 8

    PubMed Central

    Janowicz, Anna; Caporale, Marco; Shaw, Andrew; Gulletta, Salvatore; Di Gialleonardo, Luigina; Ratinier, Maxime

    2015-01-01

    ABSTRACT Bluetongue virus (BTV) causes bluetongue, a major hemorrhagic disease of ruminants. In order to investigate the molecular determinants of BTV virulence, we used a BTV8 strain minimally passaged in tissue culture (termed BTV8L in this study) and a derivative strain passaged extensively in tissue culture (BTV8H) in in vitro and in vivo studies. BTV8L was pathogenic in both IFNAR−/− mice and in sheep, while BTV8H was attenuated in both species. To identify genetic changes which led to BTV8H attenuation, we generated 34 reassortants between BTV8L and BTV8H. We found that partial attenuation of BTV8L in IFNAR−/− mice was achieved by simply replacing genomic segment 2 (Seg2, encoding VP2) or Seg10 (encoding NS3) with the BTV8H homologous segments. Fully attenuated viruses required at least two genome segments from BTV8H, including Seg2 with either Seg1 (encoding VP1), Seg6 (encoding VP6 and NS4), or Seg10 (encoding NS3). Conversely, full reversion of virulence of BTV8H required at least five genomic segments of BTV8L. We also demonstrated that BTV8H acquired an increased affinity for glycosaminoglycan receptors during passaging in cell culture due to mutations in its VP2 protein. Replication of BTV8H was relatively poor in interferon (IFN)-competent primary ovine endothelial cells compared to replication of BTV8L, and this phenotype was determined by several viral genomic segments, including Seg4 and Seg9. This study demonstrated that multiple viral proteins contribute to BTV8 virulence. VP2 and NS3 are primary determinants of BTV pathogenesis, but VP1, VP5, VP4, VP6, and VP7 also contribute to virulence. IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants, and it is listed as a notifiable disease by the World Organization for Animal Health (OIE). The clinical outcome of BTV infection varies considerably and depends on environmental and host- and virus-specific factors. Over the years, BTV serotypes/strains with various degrees of

  14. Virulence and Fitness Determinants of Uropathogenic Escherichia coli

    PubMed Central

    Subashchandrabose, Sargurunathan; Mobley, Harry L T.

    2015-01-01

    Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is a major global public health concern. Increasing antibiotic resistance found in clinical UPEC isolates underscores the immediate need for development of novel therapeutics against this pathogen. Better understanding of the fitness and virulence mechanisms that are integral to the pathogenesis of UTI will facilitate identification of novel strategies to prevent and treat infection with UPEC. Working towards that goal, the global UPEC research community has made great strides at unraveling various virulence and fitness genes. Here, we summarize major findings on virulence and fitness determinants that enable UPEC to successfully survive and colonize the urinary tract of mammalian hosts. Major sections of this chapter are devoted to the role of iron acquisition systems, metabolic pathways, fimbriae, flagella, toxins, biofilm formation, capsule, and strain-specific genes in the initiation and progression of UTIs. Transcriptomes of UPEC during experimental UTI in a murine model and naturally occurring UTI in women are compared to elucidate virulence mechanisms specifically involved in human UTI. Capitalizing on the advances in molecular pathogenesis research by translating these findings will help develop better clinical strategies for prevention and management of UTIs. PMID:26350328

  15. Virulence determinants of pandemic influenza viruses

    PubMed Central

    Tscherne, Donna M.; García-Sastre, Adolfo

    2011-01-01

    Influenza A viruses cause recurrent, seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. The ability of influenza A viruses to adapt to various hosts and undergo reassortment events ensures constant generation of new strains with unpredictable degrees of pathogenicity, transmissibility, and pandemic potential. Currently, the combination of factors that drives the emergence of pandemic influenza is unclear, making it impossible to foresee the details of a future outbreak. Identification and characterization of influenza A virus virulence determinants may provide insight into genotypic signatures of pathogenicity as well as a more thorough understanding of the factors that give rise to pandemics. PMID:21206092

  16. Molecular characteristics and evolutionary analysis of a very virulent infectious bursal disease virus.

    PubMed

    Li, Zan; Qi, XiaoLe; Ren, XianGang; Cui, Lei; Wang, XiaoMei; Zhu, Ping

    2015-08-01

    Infectious bursal disease virus (IBDV) poses a significant threat to the poultry industry. Viral protein 2 (VP2), the major structural protein of IBDV, has been subjected to frequent mutations that have imparted tremendous genetic diversity to the virus. To determine how amino acid mutations may affect the virulence of IBDV, we built a structural model of VP2 of a very virulent strain of IBDV identified in China, vvIBDV Gx, and performed a molecular dynamics simulation of the interaction between virulence sites. The study showed that the amino acid substitutions that distinguish vvIBDV from attenuated IBDV (H253Q and T284A) favor a hydrophobic and flexible conformation of β-barrel loops in VP2, which could promote interactions between the virus and potential IBDV-specific receptors. Population sequence analysis revealed that the IBDV strains prevalent in East Asia show a significant signal of positive selection at virulence sites 253 and 284. In addition, a signal of co-evolution between sites 253 and 284 was identified. These results suggest that changes in the virulence of IBDV may result from both the interaction and the co-evolution of multiple amino acid substitutions at virulence sites. PMID:26245145

  17. Enhanced IRES activity by the 3’UTR element determines the virulence of FMDV isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A reverse genetics approach was used to identify viral genetic determinants of the differential virulence displayed by two field foot-and-mouth disease virus (FMDV) strains (A/Arg/00 and A/Arg/01) isolated in Argentina during the 2000-2001 epidemics. A molecular clone of A/Arg/01 strain and viral ch...

  18. Virulence determinants associated with the Asian community-associated methicillin-resistant Staphylococcus aureus lineage ST59

    PubMed Central

    Li, Min; Dai, Yingxin; Zhu, Yuanjun; Fu, Chih-Lung; Tan, Vee Y.; Wang, Yanan; Wang, Xing; Hong, Xufen; Liu, Qian; Li, Tianming; Qin, Juanxiu; Ma, Xiaowei; Fang, Jingyuan; Otto, Michael

    2016-01-01

    Understanding virulence is vital for the development of novel therapeutics to target infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), which cause an ongoing epidemic in the United States and are on a global rise. However, what defines virulence particularly of global CA-MRSA lineages is poorly understood. Threatening a vast population, the predominant Asian CA-MRSA lineage ST59 is of major epidemiological importance. However, there have been no molecular analyses using defined virulence gene deletion mutants in that lineage as of yet. Here, we compared virulence in skin, lung, and blood infection models of ST59 CA-MRSA isolates with geographically matched hospital-associated MRSA isolates. We selected a representative ST59 CA-MRSA isolate based on toxin expression and virulence characteristics, and produced isogenic gene deletion mutants of important CA-MRSA virulence determinants (α-toxin, PSM α, Agr) in that isolate for in-vitro and in-vivo analyses. Our results demonstrate strongly enhanced virulence of ST59 CA-MRSA over hospital-associated lineages, supporting the notion that enhanced virulence is characteristic for CA-MRSA. Furthermore, they show strong and significant contribution of Agr, α-toxin, and PSMα to pathogenesis of ST59 CA-MRSA skin, lung, and blood infection, emphasizing the value of drug development efforts targeted toward those virulence determinants. PMID:27296890

  19. 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

  20. Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil

    PubMed Central

    Medeiros, A.W.; Pereira, R.I.; Oliveira, D.V.; Martins, P.D.; d’Azevedo, P.A.; Van der Sand, S.; Frazzon, J.; Frazzon, A.P.G

    2014-01-01

    The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student’s t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains. PMID:24948952

  1. Molecular characterisation of virulence graded field isolates of myxoma virus

    PubMed Central

    2010-01-01

    Background Myxoma virus (MV) has been endemic in Europe since shortly after its deliberate release in France in 1952. While the emergence of more resistant hosts and more transmissible and attenuated virus is well documented, there have been relatively few studies focused on the sequence changes incurred by the virus as it has adapted to its new host. In order to identify regions of variability within the MV genome to be used for phylogenetic studies and to try to investigate causes of MV strain attenuation we have molecularly characterised nine strains of MV isolated in Spain between the years 1992 and 1995 from wide ranging geographic locations and which had been previously graded for virulence by experimental infection of rabbits. Results The findings reported here show the analysis of 16 genomic regions accounting for approximately 10% of the viral genomes. Of the 20 genes analysed 5 (M034L, M069L, M071L, M130R and M135R) were identical in all strains and 1 (M122R) contained only a single point mutation in an individual strain. Four genes (M002L/R, M009L, M036L and M017L) showed insertions or deletions that led to disruption of the ORFs. Conclusions The findings presented here provide valuable tools for strain differentiation and phylogenetic studies of MV isolates and some clues as to the reasons for virus attenuation in the field. PMID:20187925

  2. Molecular characterization of the virulent infectious hematopoietic necrosis virus (IHNV) strain 220-90

    PubMed Central

    2010-01-01

    Background Infectious hematopoietic necrosis virus (IHNV) is the type species of the genus Novirhabdovirus, within the family Rhabdoviridae, infecting several species of wild and hatchery reared salmonids. Similar to other rhabdoviruses, IHNV has a linear single-stranded, negative-sense RNA genome of approximately 11,000 nucleotides. The IHNV genome encodes six genes; the nucleocapsid, phosphoprotein, matrix protein, glycoprotein, non-virion protein and polymerase protein genes, respectively. This study describes molecular characterization of the virulent IHNV strain 220-90, belonging to the M genogroup, and its phylogenetic relationships with available sequences of IHNV isolates worldwide. Results The complete genomic sequence of IHNV strain 220-90 was determined from the DNA of six overlapping clones obtained by RT-PCR amplification of genomic RNA. The complete genome sequence of 220-90 comprises 11,133 nucleotides (GenBank GQ413939) with the gene order of 3'-N-P-M-G-NV-L-5'. These genes are separated by conserved gene junctions, with di-nucleotide gene spacers. An additional uracil nucleotide was found at the end of the 5'-trailer region, which was not reported before in other IHNV strains. The first 15 of the 16 nucleotides at the 3'- and 5'-termini of the genome are complementary, and the first 4 nucleotides at 3'-ends of the IHNV are identical to other novirhadoviruses. Sequence homology and phylogenetic analysis of the glycoprotein genes show that 220-90 strain is 97% identical to most of the IHNV strains. Comparison of the virulent 220-90 genomic sequences with less virulent WRAC isolate shows more than 300 nucleotides changes in the genome, which doesn't allow one to speculate putative residues involved in the virulence of IHNV. Conclusion We have molecularly characterized one of the well studied IHNV isolates, 220-90 of genogroup M, which is virulent for rainbow trout, and compared phylogenetic relationship with North American and other strains

  3. Anatomical site-specific contributions of pneumococcal virulence determinants

    PubMed Central

    Shenoy, Anukul T.; Orihuela, Carlos J.

    2016-01-01

    Streptococcus pneumoniae is an opportunistic pathogen globally associated with significant morbidity and mortality. It is capable of causing a wide range of diseases including sinusitis, conjunctivitis, otitis media, pneumonia, bacteraemia, sepsis, and meningitis. While its capsular polysaccharide is indispensible for invasive disease, and opsonising antibodies against the capsule are the basis for the current vaccines, a long history of biomedical research indicates that other components of this Gram-positive bacterium are also critical for virulence. Herein we review the contribution of pneumococcal virulence determinants to survival and persistence in the context of distinct anatomical sites. We discuss how these determinants allow the pneumococcus to evade mucociliary clearance during colonisation, establish lower respiratory tract infection, resist complement deposition and opsonophagocytosis in the bloodstream, and invade secondary tissues such as the central nervous system leading to meningitis. We do so in a manner that highlights both the critical role of the capsular polysaccharide and the accompanying and necessary protein determinants. Understanding the complex interplay between host and pathogen is necessary to find new ways to prevent pneumococcal infection. This review is an attempt to do so with consideration for the latest research findings.

  4. Genetic Analysis and Molecular Identification of Virulence in Xanthomonas oryzae pv. oryzae Isolates

    PubMed Central

    Onasanya, Amos; Onasanya, R. O.; Ojo, Abiodun A.; Adewale, B. O.

    2013-01-01

    Bacterial leaf blight (BLB) of rice is a very destructive disease worldwide and is caused by Xanthomonas oryzae pv. oryzae (Xoo). The aim of the present study was to examine if the Xoo virulence pathotypes obtained using phenotypic pathotyping could be confirmed using molecular approach. After screening of 60 Operon primers with genomic DNA of two Xoo isolates (virulent pathotype, Vr, and mildly virulent pathotype, MVr), 12 Operon primers that gave reproducible and useful genetic information were selected and used to analyze 50 Xoo isolates from 7 West African countries. Genetic analysis revealed two major Xoo virulence genotypes (Mta and Mtb) with Mta having two subgroups (Mta1 and Mta2). Mta1 (Vr1) subgroup genotype has occurrence in six countries and Mta2 (Vr2) in three countries while Mtb genotype characterized mildly virulence (MVr) Xoo isolates present in five countries. The study revealed possible linkage and correlation between phenotypic pathotyping and molecular typing of Xoo virulence. Xoo virulence genotypes were known to exist within country and there was evidence of Xoo pathogen migration between countries. Durable resistance rice cultivars would need to overcome both Mta and Mtb Xoo virulence genotypes in order to survive after their deployment into different rice ecologies in West Africa. PMID:27335673

  5. Deciphering the Molecular Variations of Pine Wood Nematode Bursaphelenchus xylophilus with Different Virulence.

    PubMed

    Ding, Xiaolei; Ye, Jianren; Lin, Sixi; Wu, Xiaoqin; Li, Dewei; Nian, Bo

    2016-01-01

    Bursaphelenchus xylophilus is the causative agent of pine wilt disease which has caused huge economic losses in many countries. It has been reported that two forms of pine wood nematodes existed in its native region, i.e., with strong virulence and weak virulence. However, little is known about the molecular differences between the two forms. To better understand their molecular variations, transcriptome and genome sequences of three strongly virulent and one weakly virulent strains were analyzed. We found 238 transcripts and 84 exons which showed notable changes between the two virulent forms. Functional analyses of both differentially expressed transcripts and exons indicated that different virulence strains showed dissimilar nematode growth, reproduction, and oxidoreductase activities. In addition, we also detected a small number of exon-skipping events in B. xylophilus. Meanwhile, 117 SNPs were identified as potential genetic markers in distinguishing the two forms. Four of them were further proved to have undergone allele specific expressions and possibly interrupted the target site of evolutionary conserved B. xylophilus miR-47. These particular SNPs were experimentally verified by including eight additional strains to ensure the validity of our sequencing results. These results could help researchers to better diagnose nematode species with different virulence and facilitate the control of pine wilt disease. PMID:27224277

  6. Deciphering the Molecular Variations of Pine Wood Nematode Bursaphelenchus xylophilus with Different Virulence

    PubMed Central

    Ding, Xiaolei; Ye, Jianren; Lin, Sixi; Wu, Xiaoqin; Li, Dewei; Nian, Bo

    2016-01-01

    Bursaphelenchus xylophilus is the causative agent of pine wilt disease which has caused huge economic losses in many countries. It has been reported that two forms of pine wood nematodes existed in its native region, i.e., with strong virulence and weak virulence. However, little is known about the molecular differences between the two forms. To better understand their molecular variations, transcriptome and genome sequences of three strongly virulent and one weakly virulent strains were analyzed. We found 238 transcripts and 84 exons which showed notable changes between the two virulent forms. Functional analyses of both differentially expressed transcripts and exons indicated that different virulence strains showed dissimilar nematode growth, reproduction, and oxidoreductase activities. In addition, we also detected a small number of exon-skipping events in B. xylophilus. Meanwhile, 117 SNPs were identified as potential genetic markers in distinguishing the two forms. Four of them were further proved to have undergone allele specific expressions and possibly interrupted the target site of evolutionary conserved B. xylophilus miR-47. These particular SNPs were experimentally verified by including eight additional strains to ensure the validity of our sequencing results. These results could help researchers to better diagnose nematode species with different virulence and facilitate the control of pine wilt disease. PMID:27224277

  7. The Salmonella typhimurium mar locus: molecular and genetic analyses and assessment of its role in virulence.

    PubMed Central

    Sulavik, M C; Dazer, M; Miller, P F

    1997-01-01

    The marRAB operon is a regulatory locus that controls multiple drug resistance in Escherichia coli. marA encodes a positive regulator of the antibiotic resistance response, acting by altering the expression of unlinked genes. marR encodes a repressor of marRAB transcription and controls the production of MarA in response to environmental signals. A molecular and genetic study of the homologous operon in Salmonella typhimurium was undertaken, and the role of marA in virulence in a murine model was assessed. Expression of E. coli marA (marAEC) present on a multicopy plasmid in S. typhimurium resulted in a multiple antibiotic resistance (Mar) phenotype, suggesting that a similar regulon exists in this organism. A genomic plasmid library containing S. typhimurium chromosomal sequences was introduced into an E. coli strain that was deleted for the mar locus and contained a single-copy marR'-'lacZ translational fusion. Plasmid clones that contained both S. typhimurium marR (marRSt) and marA (marASt) genes were identified as those that were capable of repressing expression of the fusion and which resulted in a Mar phenotype. The predicted amino acid sequences of MarRSt, MarASt, and MarBSt were 91, 86, and 42% identical, respectively, to the same genes from E. coli, while the operator/promoter region of the operon was 86% identical to the same 98-nucleotide-upstream region in E. coli. The marRAB transcriptional start sites for both organisms were determined by primer extension, and a marRABSt transcript of approximately 1.1 kb was identified by Northern blot analysis. Its accumulation was shown to be inducible by sodium salicylate. Open reading frames flanking the marRAB operon were also conserved. An S. typhimurium marA disruption strain was constructed by an allelic exchange method and compared to the wild-type strain for virulence in a murine BALB/c infection model. No effect on virulence was noted. The endogenous S. typhimurium plasmid that is associated with virulence

  8. Flagellar Biosynthesis Exerts Temporal Regulation of Secretion of Specific Campylobacter jejuni Colonization and Virulence Determinants

    PubMed Central

    Barrero-Tobon, Angelica M.; Hendrixson, David R.

    2014-01-01

    Summary The Campylobacter jejuni flagellum exports both proteins that form the flagellar organelle for swimming motility and colonization and virulence factors that promote commensal colonization of the avian intestinal tract or invasion of human intestinal cells, respectively. We explored how the C. jejuni flagellum is a versatile secretory organelle by examining molecular determinants that allow colonization and virulence factors to exploit the flagellum for their own secretion. Flagellar biogenesis was observed to exert temporal control of secretion of these proteins, indicating that a bolus of secretion of colonization and virulence factors occurs during hook biogenesis with filament polymerization itself reducing secretion of these factors. Furthermore, we found that intramolecular and intermolecular requirements for flagellar-dependent secretion of these proteins were most reminiscent to those for flagellin secretion. Importantly, we discovered that secretion of one colonization and virluence factor, CiaI, was not required for invasion of human colonic cells, which counters previous hypotheses for how this protein functions during invasion. Instead, secretion of CiaI was essential for C. jejuni to facilitate commensal colonization of the natural avian host. Our work provides insight into the versatility of the bacterial flagellum as a secretory machine that can export proteins promoting diverse biological processes. PMID:25041103

  9. Virulence determinants of uropathogenic Escherichia coli and Proteus mirabilis.

    PubMed

    Mobley, H L; Island, M D; Massad, G

    1994-11-01

    The urinary tract is among the most common sites of bacterial infection and E. coli is by far the most common infecting agent. In patients with urinary catheters in place or structural abnormalities of the urinary tract, Proteus mirabilis is also a frequent isolate. To study virulence of these bacterial species, we have isolated the genes that encode putative virulence factors, constructed specific mutations within these genes, introduced the mutation back into the wild type strain by allelic exchange, and analyzed these mutants for virulence in appropriate in vitro and in vivo models. Specific virulence markers have been identified for strains that cause urinary tract infection. For E. coli, these include P fimbriae, S fimbriae, hemolysin, aerobactin, serum resistance, and a small group of O-serotypes. Redundant virulence factors must be present in these organisms as mutation of the most clearly identified epidemiological marker, P fimbriae, does not result in attenuation of a virulent strain. For P. mirabilis, urease appears to contribute most significantly to virulence. Fimbriae play a significant but more subtle role in colonization. Hemolysin, although potently cytotoxic to renal cells in vitro, does not appear to contribute significantly to the pathogenesis of ascending urinary tract infection. We can conclude that the pathogenesis of urinary tract infection and acute pyelonephritis caused by uropathogenic E. coli and P. mirabilis are multifactorial, as mutation of single genes rarely causes significant attenuation of virulence. PMID:7869662

  10. FDA Escherichia coli Identification (FDA-ECID) Microarray: a Pangenome Molecular Toolbox for Serotyping, Virulence Profiling, Molecular Epidemiology, and Phylogeny

    PubMed Central

    Patel, Isha R.; Gangiredla, Jayanthi; Lacher, David W.; Mammel, Mark K.; Jackson, Scott A.; Lampel, Keith A.

    2016-01-01

    ABSTRACT Most Escherichia coli strains are nonpathogenic. However, for clinical diagnosis and food safety analysis, current identification methods for pathogenic E. coli either are time-consuming and/or provide limited information. Here, we utilized a custom DNA microarray with informative genetic features extracted from 368 sequence sets for rapid and high-throughput pathogen identification. The FDA Escherichia coli Identification (FDA-ECID) platform contains three sets of molecularly informative features that together stratify strain identification and relatedness. First, 53 known flagellin alleles, 103 alleles of wzx and wzy, and 5 alleles of wzm provide molecular serotyping utility. Second, 41,932 probe sets representing the pan-genome of E. coli provide strain-level gene content information. Third, approximately 125,000 single nucleotide polymorphisms (SNPs) of available whole-genome sequences (WGS) were distilled to 9,984 SNPs capable of recapitulating the E. coli phylogeny. We analyzed 103 diverse E. coli strains with available WGS data, including those associated with past foodborne illnesses, to determine robustness and accuracy. The array was able to accurately identify the molecular O and H serotypes, potentially correcting serological failures and providing better resolution for H-nontypeable/nonmotile phenotypes. In addition, molecular risk assessment was possible with key virulence marker identifications. Epidemiologically, each strain had a unique comparative genomic fingerprint that was extended to an additional 507 food and clinical isolates. Finally, a 99.7% phylogenetic concordance was established between microarray analysis and WGS using SNP-level data for advanced genome typing. Our study demonstrates FDA-ECID as a powerful tool for epidemiology and molecular risk assessment with the capacity to profile the global landscape and diversity of E. coli. IMPORTANCE This study describes a robust, state-of-the-art platform developed from available

  11. Virulence and Molecular Polymorphism in International Collections of the Wheat Leaf Rust Fungus Puccinia triticina.

    PubMed

    Kolmer, J A; Liu, J Q

    2000-04-01

    ABSTRACT Collections of Puccinia triticina, the wheat leaf rust fungus, were obtained from Great Britain, Slovakia, Israel, Germany, Australia, Italy, Spain, Hungary, South Africa, Uruguay, New Zealand, Brazil, Pakistan, Nepal, and eastern and western Canada. All single-uredinial isolates derived from the collections were tested for virulence polymorphism on 22 Thatcher wheat lines that are near-isogenic for leaf rust resistance genes. Based on virulence phenotype, selected isolates were also tested for randomly amplified polymorphic DNA (RAPD) using 11 primers. The national collections were placed into 11 groups based on previously established epidemiological zones. Among the 131 single-uredinial isolates, 105 virulence phenotypes and 82 RAPD phenotypes were described. In a modified analysis of variance, 26% of the virulence variation was due to differences in isolates between groups, with the remainder attributable to differences within groups. Of the RAPD variation, 36% was due to differences in isolates between groups. Clustering based on the average virulence distance (simple distance coefficient) within and between groups resulted in eight groups that differed significantly. Collections from Australia-New Zealand, Spain, Italy, and Britain did not differ significantly for virulence. Clustering of RAPD marker differences (1 - Dice coefficient) distinguished nine groups that differed significantly. Collections from Spain and Italy did not differ significantly for RAPD variation, neither did collections from western Canada and South America. Groups of isolates distinguished by avirulent/virulent infection types to wheat lines with resistance genes Lr1, Lr2a, Lr2c, and Lr3 also differed significantly for RAPD distance, showing a general relationship between virulence and RAPD phenotype. The results indicated that on a worldwide level collections of P. triticina differ for virulence and molecular backgrounds. PMID:18944595

  12. Lineage-specific Virulence Determinants of Haemophilus influenzae Biogroup aegyptius

    PubMed Central

    Strouts, Fiona R.; Power, Peter; Croucher, Nicholas J.; Corton, Nicola; van Tonder, Andries; Quail, Michael A.; Langford, Paul R.; Hudson, Michael J.; Parkhill, Julian; Bentley, Stephen D.

    2012-01-01

    An emergent clone of Haemophilus influenzae biogroup aegyptius (Hae) is responsible for outbreaks of Brazilian purpuric fever (BPF). First recorded in Brazil in 1984, the so-called BPF clone of Hae caused a fulminant disease that started with conjunctivitis but developed into septicemic shock; mortality rates were as high as 70%. To identify virulence determinants, we conducted a pan-genomic analysis. Sequencing of the genomes of the BPF clone strain F3031 and a noninvasive conjunctivitis strain, F3047, and comparison of these sequences with 5 other complete H. influenzae genomes showed that >77% of the F3031 genome is shared among all H. influenzae strains. Delineation of the Hae accessory genome enabled characterization of 163 predicted protein-coding genes; identified differences in established autotransporter adhesins; and revealed a suite of novel adhesins unique to Hae, including novel trimeric autotransporter adhesins and 4 new fimbrial operons. These novel adhesins might play a critical role in host–pathogen interactions. PMID:22377449

  13. Rescue of Very Virulent and Mosaic Infectious Bursal Disease Virus from Cloned cDNA: VP2 Is Not the Sole Determinant of the Very Virulent Phenotype

    PubMed Central

    Boot, Hein J.; ter Huurne, A. Agnes H. M.; Hoekman, Arjan J. W.; Peeters, Ben P. H.; Gielkens, Arno L. J.

    2000-01-01

    Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young

  14. Virulence Phenotypes and Molecular Genotypes of Puccinia triticina Isolates from Italy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty-four isolates of Puccinia triticina from Italy were characterized for virulence to seedlings of 22 common wheat cv. Thatcher isolines each with a different leaf rust resistance gene, and for molecular genotypes at 15 simple sequence repeat (SSR) loci. The isolates were compared with a set of ...

  15. A Nonessential African Swine Fever Virus Gene UK Is a Significant Virulence Determinant in Domestic Swine

    PubMed Central

    Zsak, L.; Caler, E.; Lu, Z.; Kutish, G. F.; Neilan, J. G.; Rock, D. L.

    1998-01-01

    Sequence analysis of the right variable genomic region of the pathogenic African swine fever virus (ASFV) isolate E70 revealed a novel gene, UK, that is immediately upstream from the previously described ASFV virulence-associated gene NL-S (L. Zsak, Z. Lu, G. F. Kutish, J. G. Neilan, and D. L. Rock, J. Virol. 70:8865–8871, 1996). UK, transcriptionally oriented toward the right end of the genome, predicts a protein of 96 amino acids with a molecular mass of 10.7 kDa. Searches of genetic databases did not find significant similarity between UK and other known genes. Sequence analysis of the UK genes from several pathogenic ASFVs from Europe, the Caribbean, and Africa demonstrated that this gene was highly conserved among diverse pathogenic isolates, including those from both tick and pig sources. Polyclonal antibodies raised against the UK protein specifically precipitated a 15-kDa protein from ASFV-infected macrophage cell cultures as early as 2 h postinfection. A recombinant UK gene deletion mutant, ΔUK, and its revertant, UK-R, were constructed from the E70 isolate to study gene function. Although deletion of UK did not affect the growth characteristics of the virus in macrophage cell cultures, ΔUK exhibited reduced virulence in infected pigs. While mortality among parental E70- or UK-R-infected animals was 100%, all ΔUK-infected pigs survived infection. Fever responses were comparable in E70-, UK-R-, and ΔUK-infected groups; however, ΔUK-infected animals exhibited significant, 100- to 1,000-fold, reductions in viremia titers. These data indicate that the highly conserved UK gene of ASFV, while being nonessential for growth in macrophages in vitro, is an important viral virulence determinant for domestic pigs. PMID:9444996

  16. A nonessential African swine fever virus gene UK is a significant virulence determinant in domestic swine.

    PubMed

    Zsak, L; Caler, E; Lu, Z; Kutish, G F; Neilan, J G; Rock, D L

    1998-02-01

    Sequence analysis of the right variable genomic region of the pathogenic African swine fever virus (ASFV) isolate E70 revealed a novel gene, UK, that is immediately upstream from the previously described ASFV virulence-associated gene NL-S (L. Zsak, Z. Lu, G. F. Kutish, J. G. Neilan, and D. L. Rock, J. Virol. 70:8865-8871, 1996). UK, transcriptionally oriented toward the right end of the genome, predicts a protein of 96 amino acids with a molecular mass of 10.7 kDa. Searches of genetic databases did not find significant similarity between UK and other known genes. Sequence analysis of the UK genes from several pathogenic ASFVs from Europe, the Caribbean, and Africa demonstrated that this gene was highly conserved among diverse pathogenic isolates, including those from both tick and pig sources. Polyclonal antibodies raised against the UK protein specifically precipitated a 15-kDa protein from ASFV-infected macrophage cell cultures as early as 2 h postinfection. A recombinant UK gene deletion mutant, deltaUK, and its revertant, UK-R, were constructed from the E70 isolate to study gene function. Although deletion of UK did not affect the growth characteristics of the virus in macrophage cell cultures, deltaUK exhibited reduced virulence in infected pigs. While mortality among parental E70- or UK-R-infected animals was 100%, all deltaUK-infected pigs survived infection. Fever responses were comparable in E70-, UK-R-, and deltaUK-infected groups; however, deltaUK-infected animals exhibited significant, 100- to 1,000-fold, reductions in viremia titers. These data indicate that the highly conserved UK gene of ASFV, while being nonessential for growth in macrophages in vitro, is an important viral virulence determinant for domestic pigs. PMID:9444996

  17. PSM-Mec-A Virulence Determinant that Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in Staphylococci.

    PubMed

    Qin, Li; McCausland, Joshua W; Cheung, Gordon Y C; Otto, Michael

    2016-01-01

    PSM-mec is a secreted virulence factor that belongs to the phenol-soluble modulin (PSM) family of amphipathic, alpha-helical peptide toxins produced by Staphylococcus species. All known PSMs are core genome-encoded with the exception of PSM-mec, whose gene is found in specific sub-types of SCCmec methicillin resistance mobile genetic elements present in methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci. In addition to the cytolytic translational product, PSM-mec, the psm-mec locus encodes a regulatory RNA. In S. aureus, the psm-mec locus influences cytolytic capacity, methicillin resistance, biofilm formation, cell spreading, and the expression of other virulence factors, such as other PSMs, which results in a significant impact on immune evasion and disease. However, these effects are highly strain-dependent, which is possibly due to differences in PSM-mec peptide vs. psm-mec RNA-controlled effects. Here, we summarize the functional properties of PSM-mec and the psm-mec RNA molecule and their roles in staphylococcal pathogenesis and physiology. PMID:27597849

  18. PSM-Mec—A Virulence Determinant that Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in Staphylococci

    PubMed Central

    Qin, Li; McCausland, Joshua W.; Cheung, Gordon Y. C.; Otto, Michael

    2016-01-01

    PSM-mec is a secreted virulence factor that belongs to the phenol-soluble modulin (PSM) family of amphipathic, alpha-helical peptide toxins produced by Staphylococcus species. All known PSMs are core genome-encoded with the exception of PSM-mec, whose gene is found in specific sub-types of SCCmec methicillin resistance mobile genetic elements present in methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci. In addition to the cytolytic translational product, PSM-mec, the psm-mec locus encodes a regulatory RNA. In S. aureus, the psm-mec locus influences cytolytic capacity, methicillin resistance, biofilm formation, cell spreading, and the expression of other virulence factors, such as other PSMs, which results in a significant impact on immune evasion and disease. However, these effects are highly strain-dependent, which is possibly due to differences in PSM-mec peptide vs. psm-mec RNA-controlled effects. Here, we summarize the functional properties of PSM-mec and the psm-mec RNA molecule and their roles in staphylococcal pathogenesis and physiology. PMID:27597849

  19. Directed Screen of Francisella novicida Virulence Determinants Using Drosophila melanogaster▿ †

    PubMed Central

    Åhlund, Monika K.; Rydén, Patrik; Sjöstedt, Anders; Stöven, Svenja

    2010-01-01

    Francisella tularensis is a highly virulent, facultative intracellular human pathogen whose virulence mechanisms are not well understood. Occasional outbreaks of tularemia and the potential use of F. tularensis as a bioterrorist agent warrant better knowledge about the pathogenicity of this bacterium. Thus far, genome-wide in vivo screens for virulence factors have been performed in mice, all however restricted by the necessity to apply competition-based, negative-selection assays. We wanted to individually evaluate putative virulence determinants suggested by such assays and performed directed screening of 249 F. novicida transposon insertion mutants by using survival of infected fruit flies as a measure of bacterial virulence. Some 20% of the genes tested were required for normal virulence in flies; most of these had not previously been investigated in detail in vitro or in vivo. We further characterized their involvement in bacterial proliferation and pathogenicity in flies and in mouse macrophages. Hierarchical cluster analysis of mutant phenotypes indicated a functional linkage between clustered genes. One cluster grouped all but four genes of the Francisella pathogenicity island and other loci required for intracellular survival. We also identified genes involved in adaptation to oxidative stress and genes which might induce host energy wasting. Several genes related to type IV pilus formation demonstrated hypervirulent mutant phenotypes. Collectively, the data demonstrate that the bacteria in part use similar virulence mechanisms in mammals as in Drosophila melanogaster but that a considerable proportion of the virulence factors active in mammals are dispensable for pathogenicity in the insect model. PMID:20479082

  20. A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence

    PubMed Central

    Balasubramanian, Deepak; Schneper, Lisa; Kumari, Hansi; Mathee, Kalai

    2013-01-01

    Pseudomonas aeruginosa is a metabolically versatile bacterium that is found in a wide range of biotic and abiotic habitats. It is a major human opportunistic pathogen causing numerous acute and chronic infections. The critical traits contributing to the pathogenic potential of P. aeruginosa are the production of a myriad of virulence factors, formation of biofilms and antibiotic resistance. Expression of these traits is under stringent regulation, and it responds to largely unidentified environmental signals. This review is focused on providing a global picture of virulence gene regulation in P. aeruginosa. In addition to key regulatory pathways that control the transition from acute to chronic infection phenotypes, some regulators have been identified that modulate multiple virulence mechanisms. Despite of a propensity for chaotic behaviour, no chaotic motifs were readily observed in the P. aeruginosa virulence regulatory network. Having a ‘birds-eye’ view of the regulatory cascades provides the forum opportunities to pose questions, formulate hypotheses and evaluate theories in elucidating P. aeruginosa pathogenesis. Understanding the mechanisms involved in making P. aeruginosa a successful pathogen is essential in helping devise control strategies. PMID:23143271

  1. Molecular Basis of Virulence in Staphylococcus aureus Mastitis

    PubMed Central

    Le Maréchal, Caroline; Seyffert, Nubia; Jardin, Julien; Hernandez, David; Jan, Gwenaël; Rault, Lucie; Azevedo, Vasco; François, Patrice; Schrenzel, Jacques; van de Guchte, Maarten; Even, Sergine; Berkova, Nadia; Thiéry, Richard; Fitzgerald, J. Ross

    2011-01-01

    Background S. aureus is one of the main pathogens involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable, ranging from subclinical to gangrenous mastitis. This work represents an in-depth characterization of S. aureus mastitis isolates to identify bacterial factors involved in severity of mastitis infection. Methodology/Principal Findings We employed genomic, transcriptomic and proteomic approaches to comprehensively compare two clonally related S. aureus strains that reproducibly induce severe (strain O11) and milder (strain O46) mastitis in ewes. Variation in the content of mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production was observed. In particular, O11 produced relatively high levels of exoproteins, including toxins and proteases known to be important in virulence. A characteristic we observed in other S. aureus strains isolated from clinical mastitis cases. Conclusions/Significance Our data are consistent with a dose-dependant role of some staphylococcal factors in the hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins and iron acquisition pathways constitute good targets for further research to define the underlying mechanisms of mastitis severity. PMID:22096559

  2. Detection of Bacterial Virulence Genes by Subtractive Hybridization: Identification of Capsular Polysaccharide of Burkholderia pseudomallei as a Major Virulence Determinant

    PubMed Central

    Reckseidler, Shauna L.; DeShazer, David; Sokol, Pamela A.; Woods, Donald E.

    2001-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic. Burkholderia thailandensis is a nonpathogenic environmental organism closely related to B. pseudomallei. Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in B. pseudomallei. Screening of the subtraction library revealed A-T-rich DNA sequences unique to B. pseudomallei, suggesting they may have been acquired by horizontal transfer. One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase from Pseudomonas aeruginosa. This gene was insertionally inactivated in wild-type B. pseudomallei to create SR1015. It was determined by enzyme-linked immunosorbent assay and immunoelectron microscopy that the inactivated gene was involved in the production of a major surface polysaccharide. The 50% lethal dose (LD50) for wild-type B. pseudomallei is <10 CFU; the LD50 for SR1015 was determined to be 3.5 × 105 CFU, similar to that of B. thailandensis (6.8 × 105 CFU). DNA sequencing of the region flanking the glycosyltransferase gene revealed open reading frames similar to capsular polysaccharide genes in Haemophilus influenzae, Escherichia coli, and Neisseria meningitidis. In addition, DNA from Burkholderia mallei and Burkholderia stabilis hybridized to a glycosyltransferase fragment probe, and a capsular structure was identified on the surface of B. stabilis via immunoelectron microscopy. Thus, the combination of PCR-based subtractive hybridization, insertional inactivation, and animal virulence studies has facilitated the identification of an important virulence determinant in B. pseudomallei. PMID:11119486

  3. In vivo correlates of molecularly inferred virulence among extraintestinal pathogenic Escherichia coli (ExPEC) in the wax moth Galleria mellonella model system

    PubMed Central

    Williamson, Deborah A; Mills, Grant; Johnson, James R; Porter, Stephen; Wiles, Siouxsie

    2014-01-01

    In contrast to commensal Escherichia coli, extraintestinal pathogenic E. coli (ExPEC) strains possess an array of virulence-associated genes. We sought to establish the feasibility of using the invertebrate Galleria mellonella (greater wax moth) for assessing ExPEC virulence and to investigate the correlation between genotypic determinants of virulence and in vivo pathogenicity. We observed a correlation between the number of virulence genes and larval survival, such that ExPEC isolates with higher virulence scores killed larvae significantly faster than isolates with lower virulence scores. By correlating genotypic and phenotypic virulence, we provide preliminary validation of this model for future studies investigating ExPEC virulence. PMID:24518442

  4. Virulence determinants in the PB2 gene of a mouse-adapted H9N2 virus.

    PubMed

    Liu, Qingtao; Huang, Junqing; Chen, Yuxin; Chen, Hongzhi; Li, Qunhui; He, Liang; Hao, Xiaoli; Liu, Jingjing; Gu, Min; Hu, Jiao; Wang, Xiaoquan; Hu, Shunlin; Liu, Xiaowen; Liu, Xiufan

    2015-01-01

    The molecular bases of adaptation and pathogenicity of H9N2 influenza virus in mammals are largely unknown. Here, we show that a mouse-adapted PB2 gene with a phenylalanine-to-leucine mutation (F404L) mainly contributes to enhanced polymerase activity, replication, and pathogenicity of H9N2 in mice and also increases the virulence of the H5N1 and 2009 pandemic H1N1 influenza viruses. Therefore, we defined a novel pathogenic determinant, providing further insights into the pathogenesis of influenza viruses in mammals. PMID:25339773

  5. Novel swine virulence determinant in the left variable region of the African swine fever virus genome.

    PubMed

    Neilan, J G; Zsak, L; Lu, Z; Kutish, G F; Afonso, C L; Rock, D L

    2002-04-01

    Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70DeltaNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70DeltaNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalDeltaNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalDeltaNL genome was capable of restoring full virulence to E70DeltaNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70DeltaNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalDeltaNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70DeltaNL. Comparative nucleotide sequence analysis of the left variable region of the E70DeltaNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70DeltaNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalDeltaNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence

  6. Novel Swine Virulence Determinant in the Left Variable Region of the African Swine Fever Virus Genome

    PubMed Central

    Neilan, J. G.; Zsak, L.; Lu, Z.; Kutish, G. F.; Afonso, C. L.; Rock, D. L.

    2002-01-01

    Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70ΔNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70ΔNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalΔNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalΔNL genome was capable of restoring full virulence to E70ΔNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70ΔNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalΔNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70ΔNL. Comparative nucleotide sequence analysis of the left variable region of the E70ΔNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70ΔNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalΔNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine

  7. Phenotypic differences of Cryptococcus molecular types and their implications for virulence in a Drosophila model of infection.

    PubMed

    Thompson, George R; Albert, Nathaniel; Hodge, Greg; Wilson, Machelle D; Sykes, Jane E; Bays, Derek J; Firacative, Carolina; Meyer, Wieland; Kontoyiannis, Dimitrios P

    2014-07-01

    Compared to Cryptococcus neoformans, little is known about the virulence of the molecular types in Cryptococcus gattii. We compared in vitro virulence factor production and survival data using a Drosophila model of infection to further characterize the phenotypic features of different cryptococcal molecular types. Forty-nine different isolates were inoculated into wild-type flies and followed for survival. In vitro, isolates were assessed for growth at 30 and 37°C, melanin production, capsule size, resistance to H(2)O(2), and antifungal susceptibility. A mediator model was used to assess molecular type and virulence characteristics as predictors of survival in the fly model. VGIII was the most virulent molecular type in flies (P < 0.001). At 30°C, VGIII isolates grew most rapidly; at 37°C, VNI isolates grew best. C. gattii capsules were larger than those of C. neoformans (P < 0.001). Mediator model analysis found a strong correlation of Drosophila survival with molecular type and with growth at 30°C. We found molecular-type-specific differences in C. gattii in growth at different temperatures, melanin production, capsule size, ability to resist hydrogen peroxide, and antifungal susceptibility, while growth at 30°C and the VGIII molecular type were strongly associated with virulence in a Drosophila model of infection. PMID:24799631

  8. Phenotypic Differences of Cryptococcus Molecular Types and Their Implications for Virulence in a Drosophila Model of Infection

    PubMed Central

    Albert, Nathaniel; Hodge, Greg; Wilson, Machelle D.; Sykes, Jane E.; Bays, Derek J.; Firacative, Carolina; Meyer, Wieland

    2014-01-01

    Compared to Cryptococcus neoformans, little is known about the virulence of the molecular types in Cryptococcus gattii. We compared in vitro virulence factor production and survival data using a Drosophila model of infection to further characterize the phenotypic features of different cryptococcal molecular types. Forty-nine different isolates were inoculated into wild-type flies and followed for survival. In vitro, isolates were assessed for growth at 30 and 37°C, melanin production, capsule size, resistance to H2O2, and antifungal susceptibility. A mediator model was used to assess molecular type and virulence characteristics as predictors of survival in the fly model. VGIII was the most virulent molecular type in flies (P < 0.001). At 30°C, VGIII isolates grew most rapidly; at 37°C, VNI isolates grew best. C. gattii capsules were larger than those of C. neoformans (P < 0.001). Mediator model analysis found a strong correlation of Drosophila survival with molecular type and with growth at 30°C. We found molecular-type-specific differences in C. gattii in growth at different temperatures, melanin production, capsule size, ability to resist hydrogen peroxide, and antifungal susceptibility, while growth at 30°C and the VGIII molecular type were strongly associated with virulence in a Drosophila model of infection. PMID:24799631

  9. Environmental regulation of expression of virulence determinants in Bordetella pertussis.

    PubMed Central

    Melton, A R; Weiss, A A

    1989-01-01

    The trans-activator vir is required for expression of all virulence-associated genes in Bordetella pertussis. The nature of the global regulation of these factors by vir and environmental signals was examined by Northern blot analysis and with beta-galactosidase transcriptional fusions in five vir-regulated genes. Northern blots suggested that vir regulates at the level of transcription since Vir- organisms did not exhibit detectable mRNA from vir-regulated loci. Environmental signals such as high levels of salts, nicotinic acid, and 6-chloronicotinic acid or growth at low temperatures were examined. Of all of the cations and anions examined, only SO4 ions eliminated transcription of vir-regulated genes and reduced transcription of vir itself, suggesting that global regulation is obtained by modifying expression of the essential component, vir. Organisms grown on 6-chloronicotinic acid or quinaldic acid did not have detectable transcription from vir-regulated loci. Modulation by nicotinic acid, on the other hand, was strain dependent, acting at the level of transcription in strain 18-323 but not in Tohama I derivatives. Growth at lower temperatures reduced, but did not eliminate, transcription from vir-regulated loci. At 28 degrees C the ratio of pertussis toxin mRNA to recA mRNA (a non-vir-regulated factor) was equivalent to that at 37 degrees C, suggesting that transcription at low temperatures is reduced in a proportional manner and need not involve vir. Images PMID:2478524

  10. Innate immune responses of porcine macrophage cell line (Cdelts2+) to virus-associated virulence determinants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The goal of this study was to define the changes in the expression of immune genes in response to virus-associated virulence determinants. We stimulated a monocyte-derived porcine macrophage cell line (Cdelta2+) for 3 and 24h with Imiquimod, Poly IC and Poly IC with Lyovec. Cell lysates were process...

  11. VIRULENCE RELATIONSHIPS OF AEROMONAS SPECIES AS DETERMINED BY EXPOSURES TO IMMUNOCOMPROMISED MICE

    EPA Science Inventory

    Our laboratory is currently determining the virulence of opportunistic pathogens reported in treated drinking water and drinking water sources. Aeromonas hydrophila is currently on the EPA's Contaminant Candidate List (CCL) and is an example of those types of bacteria that conta...

  12. Molecular characterization and LD(50) identify virulent isolates of Staphylococcus epidermidis from adult sepsis.

    PubMed

    Herndona, B L; Bialkowska-Hobrzanska, H; Dall, L

    2000-02-01

    Staphylococcus epidermidis plays an important role in infections of patients with implanted prosthetic devices. The exact clinical significance of recovered S. epidermidis from clinical specimens is difficult to assess, as they are inhabitants of the normal skin. In this study, 11 adults with clinical sepsis and blood cultures that grew only S. epidermidis were the host population. Bacterial virulence in vivo was determined by using the mouse LD(50) assay where the intravenous lethality was determined for each patient isolate. Bacterial dose (CFU x 10(9)) that produced lethality in 50% of the animals at 12 h was the value used for comparison. Restriction fragment length polymorphism (RFLP) analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE) was used for identification of individual strains and their clonal organization. Confirmation of species assignment was done by RFLP analysis of 16S + 23S rRNA gene regions (ribotyping). Plasmid profile analysis was also conducted. Four of 11 blood isolates from adults with S. epidermidis sepsis had indistinguishable or closely related DNA patterns and were considered clone A. The same clone was previously seen to account for the majority of sepsis in a neonatal intensive care unit. There were significant differences in virulence characteristics of the S. epidermidis isolates. Clone A isolates produced lethality by LD(50) in mice at a dose averaging 2.35; clone B isolate at a dose of 2.54, and the remaining isolates, representing six distinct clones, were lethal to mice at significantly larger doses (3.51-5.17, average 4.16). These data suggest that individual clones of S. epidermidis isolated from septic adults have detectable differences in virulence as defined by an animal bioassay, and the more virulent clone is widespread. PMID:10705046

  13. Molecular Typing and Virulence Characteristic of Methicillin-Resistant Staphylococcus Aureus Isolates from Pediatric Patients in Bucaramanga, Colombia

    PubMed Central

    Machuca, Mayra Alejandra; Sosa, Luis Miguel; González, Clara Isabel

    2013-01-01

    Background Staphylococcus aureus is among the most common global nosocomial pathogens. The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide that causes nosocomial and community infections. The goals of this study were to establish the clonal complexes (CC) of the isolates of MRSA obtained from pediatric patients in a university hospital in Colombia and to investigate its molecular characteristics based on the virulence genes and the genes of staphylococcal toxins and adhesins. Methods A total of 53 MRSA isolates from pediatric patients with local or systemic infections were collected. The MRSA isolates were typed based on the SCCmec, MLST, spa and agr genes. The molecular characterization included the detection of Panton-Valentine Leukocidin, superantigenic and exfoliative toxins, and adhesin genes. The correlation between the molecular types identified and the profile of virulence factors was determined for all isolates. Results Four CC were identified, including CC8, CC5, CC80 and CC78. The ST8-MRSA-IVc-agrI was the predominant clone among the isolates, followed by the ST5-MRSA-I-agrII and ST5-MRSA-IVc-agrII clones. Twelve spa types were identified, of which t10796 and t10799 were new repeat sequences. The isolates were carriers of toxin genes, and hlg (100%), sek (92%) and pvl (88%) were the most frequent. Ten toxin gene profiles were observed, and the most frequent were seq-sek-hlg (22.6%), sek-hlg (22.6%), seb-seq-sek-hlg (18.9%) and seb-sek-hlg (15.1%). The adhesion genes were present in most of the MRSA isolates, including the following: clf-A (89%), clf-B (87%), fnb-A (83%) and ica (83%). The majority of the strains carried SCCmec-IVc and were identified as causing nosocomial infection. No significant association between a molecular type and the virulence factors was found. Conclusion Four major MRSA clone complexes were identified among the isolates. ST8-MRSA-IVc-agrI pvl+ (USA300-LV) was the

  14. Surface antigens of the syphilis spirochete and their potential as virulence determinants.

    PubMed Central

    Blanco, D. R.; Miller, J. N.; Lovett, M. A.

    1997-01-01

    A unique physical feature of Treponema pallidum, the venereally transmitted agent of human syphilis, is that its outer membrane contains 100-fold less membrane-spanning protein than the outer membranes of typical gram-negative bacteria, a property that has been related to the chronicity of syphilitic infection. These membrane-spanning T. pallidum rare outer membrane proteins, termed TROMPs, represent potential surface-exposed virulence determinants and targets of host immunity. Only recently has the outer membrane of T. pallidum been isolated and its constituent proteins identified. Five proteins of molecular mass 17-, 28-, 31-, 45-, and 65-kDa were outer membrane associated. The 17- and 45-kDa proteins, which are also present in greater amounts with the T. pallidum inner membrane protoplasmic cylinder complex, had been previously characterized lipoproteins and are, therefore, not membrane-spanning but rather membrane-anchored by their lipid moiety. In contrast, the 28-, 31-, and 65-kDa proteins are exclusively associated with the outer membrane. Both the purified native and an Escherichia coli recombinant outer membrane form of the 31-kDa protein, designated Tromp1, exhibit porin activity, thereby confirming the membrane-spanning outer membrane topology of Tromp1. The 28-kDa protein, designated Tromp2, has sequence characteristics in common with membrane-spanning outer membrane proteins and has also been recombinantly expressed in E. coli, where it targets exclusively to the E. coli outer membrane. The 65-kDa protein, designated Tromp3, is present in the least amount relative to Tromps1 and 2. Tromps 1, 2, and 3 were antigenic when tested with serum from infection and immune syphilitic rabbits and humans. These newly identified TROMPs provide a molecular foundation for the future study of syphilis pathogenesis and immunity. PMID:9126440

  15. Low-shear modelled microgravity alters expression of virulence determinants of Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Rosado, Helena; Doyle, Marie; Hinds, Jason; Taylor, Peter W.

    2010-02-01

    Microbiological monitoring of air and surfaces within the ISS indicate that bacteria of the genus Staphylococcus are found with high frequency. Staphylococcus aureus, an opportunistic pathogen with the capacity to cause severe debilitating infection, constitutes a significant proportion of these isolates. Experiments conducted during short-term flight suggest that growth in microgravity leads to increases in bacterial antibiotic resistance and to cell wall changes. Growth under low-shear modelled microgravity (LSMMG) indicated that a reduced gravitational field acts as an environmental signal for expression of enhanced bacterial virulence in gram-negative pathogens. We therefore examined the effect of simulated microgravity on parameters of antibiotic susceptibility and virulence in methicillin-susceptible S. aureus isolates RF1, RF6 and RF11; these strains were grown in a high aspect ratio vessel under LSMMG and compared with cells grown under normal gravity (NG). There were no significant differences in antibiotic susceptibility of staphylococci grown under LSMMG compared to NG. LSMMG-induced reductions in synthesis of the pigment staphyloxanthin and the major virulence determinant α-toxin were noted. Significant changes in global gene expression were identified by DNA microarray analysis; with isolate RF6, the expression of hla and genes of the regulatory system saeR/saeS were reduced approximately two-fold. These data provide strong evidence that growth of S. aureus under modelled microgravity leads to a reduction in expression of virulence determinants.

  16. Molecular Characterization of Virulence Genes in Vancomycin-Resistant and Vancomycin-Sensitive Enterococci

    PubMed Central

    Biswas, Priyanka Paul; Dey, Sangeeta; Sen, Aninda; Adhikari, Luna

    2016-01-01

    Background: The aim of this study was to find out the correlation between presence of virulence (gelatinase [gel E], enterococcal surface protein [esp], cytolysin A [cyl A], hyaluronidase [hyl], and aggregation substance [asa1]) and vancomycin-resistant genes (van A and van B) in enterococci, with their phenotypic expression. Materials and Methods: A total of 500 isolates (250 each clinical and fecal) were processed. Enterococci were isolated from various clinical samples and from fecal specimens of colonized patients. Various virulence determinants namely asa1, esp, hyl, gel E, and cyl were detected by phenotypic methods. Minimum inhibitory concentration (MIC) of vancomycin was determined by agar dilution method. Multiplex polymerase chain reaction (PCR) was used to detect the presence of virulence and van genes. Results: Out of all the samples processed, 12.0% (60/500) isolates carried van A or van B genes as confirmed by MIC test and PCR methods. Genes responsible for virulence were detected by multiplex PCR and at least one of the five was detected in all the clinical vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE). gel E, esp, and hyl genes were found to be significantly higher in clinical VRE. Of the fecal isolates, presence of gel E, esp, and asa1 was significantly higher in VRE as compared to VSE. The presence of hyl gene in the clinical VRE was found to be statistically significant (P = 0.043) as against the fecal VRE. Correlation between the presence of virulence genes and their expression as detected by phenotypic tests showed that while biofilm production was seen in 61.1% (22/36) of clinical VRE, the corresponding genes, i.e., asa1 and esp were detected in 30.5% (11/36) and 27.8% (10/36) of strains only. Conclusion: Enterococcus faecium isolates were found to carry esp gene, a phenomenon that has been described previously only for Enterococcus faecalis, but we were unable to correlate the presence of esp with their

  17. Sequence determination of a mildly virulent strain (CU-2) of Gallid herpesvirus type 2 using 454 pyrosequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete DNA sequence of the mildly virulent Gallid herpesvirus type 2 strain CU-2 was determined and consists of 176,922 bp with an overall gene organization typical of class E alphaherpesviruses. Phylogenetically, this strain partitions in its own branch between the virulent strains RB-1B, Md1...

  18. Differential Expression and Roles of Staphylococcus aureus Virulence Determinants during Colonization and Disease

    PubMed Central

    Jenkins, Amy; Diep, Binh An; Mai, Thuy T.; Vo, Nhung H.; Warrener, Paul; Suzich, Joann; Stover, C. Kendall

    2015-01-01

    ABSTRACT Staphylococcus aureus is a Gram-positive, commensal bacterium known to asymptomatically colonize the human skin, nares, and gastrointestinal tract. Colonized individuals are at increased risk for developing S. aureus infections, which range from mild skin and soft tissue infections to more severe diseases, such as endocarditis, bacteremia, sepsis, and osteomyelitis. Different virulence factors are required for S. aureus to infect different body sites. In this study, virulence gene expression was analyzed in two S. aureus isolates during nasal colonization, bacteremia and in the heart during sepsis. These models were chosen to represent the stepwise progression of S. aureus from an asymptomatic colonizer to an invasive pathogen. Expression of 23 putative S. aureus virulence determinants, representing protein and carbohydrate adhesins, secreted toxins, and proteins involved in metal cation acquisition and immune evasion were analyzed. Consistent upregulation of sdrC, fnbA, fhuD, sstD, and hla was observed in the shift between colonization and invasive pathogen, suggesting a prominent role for these genes in staphylococcal pathogenesis. Finally, gene expression data were correlated to the roles of the genes in pathogenesis by using knockout mutants in the animal models. These results provide insights into how S. aureus modifies virulence gene expression between commensal and invasive pathogens. PMID:25691592

  19. Temperature control of molecular circuit switch responsible for virulent phenotype expression in uropathogenic Escherichia coli

    NASA Astrophysics Data System (ADS)

    Samoilov, Michael

    2010-03-01

    The behavior and fate of biological organisms are to a large extent dictated by their environment, which can be often viewed as a collection of features and constraints governed by physics laws. Since biological systems comprise networks of molecular interactions, one such key physical property is temperature, whose variations directly affect the rates of biochemical reactions involved. For instance, temperature is known to control many gene regulatory circuits responsible for pathogenicity in bacteria. One such example is type 1 fimbriae (T1F) -- the foremost virulence factor in uropathogenic E. coli (UPEC), which accounts for 80-90% of all community-acquired urinary tract infections (UTIs). The expression of T1F is randomly `phase variable', i.e. individual cells switch between virulent/fimbriate and avirulent/afimbriate phenotypes, with rates regulated by temperature. Our computational investigation of this process, which is based on FimB/FimE recombinase-mediated inversion of fimS DNA element, offers new insights into its discrete-stochastic kinetics. In particular, it elucidates the logic of T1F control optimization to the host temperature and contributes further understanding toward the development of novel therapeutic approaches to UPEC-caused UTIs.

  20. Low-molecular-weight metabolites secreted by Paenibacillus larvae as potential virulence factors of American foulbrood.

    PubMed

    Schild, Hedwig-Annabell; Fuchs, Sebastian W; Bode, Helge B; Grünewald, Bernd

    2014-04-01

    The spore-forming bacterium Paenibacillus larvae causes a severe and highly infective bee disease, American foulbrood (AFB). Despite the large economic losses induced by AFB, the virulence factors produced by P. larvae are as yet unknown. To identify such virulence factors, we experimentally infected young, susceptible larvae of the honeybee, Apis mellifera carnica, with different P. larvae isolates. Honeybee larvae were reared in vitro in 24-well plates in the laboratory after isolation from the brood comb. We identified genotype-specific differences in the etiopathology of AFB between the tested isolates of P. larvae, which were revealed by differences in the median lethal times. Furthermore, we confirmed that extracts of P. larvae cultures contain low-molecular-weight compounds, which are toxic to honeybee larvae. Our data indicate that P. larvae secretes metabolites into the medium with a potent honeybee toxic activity pointing to a novel pathogenic factor(s) of P. larvae. Genome mining of P. larvae subsp. larvae BRL-230010 led to the identification of several biosynthesis gene clusters putatively involved in natural product biosynthesis, highlighting the potential of P. larvae to produce such compounds. PMID:24509920

  1. Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

    PubMed Central

    Green, Jeffrey; Rolfe, Matthew D; Smith, Laura J

    2014-01-01

    Molecular oxygen (O2) and nitric oxide (NO) are diatomic gases that play major roles in infection. The host innate immune system generates reactive oxygen species and NO as bacteriocidal agents and both require O2 for their production. Furthermore, the ability to adapt to changes in O2 availability is crucial for many bacterial pathogens, as many niches within a host are hypoxic. Pathogenic bacteria have evolved transcriptional regulatory systems that perceive these gases and respond by reprogramming gene expression. Direct sensors possess iron-containing co-factors (iron–sulfur clusters, mononuclear iron, heme) or reactive cysteine thiols that react with O2 and/or NO. Indirect sensors perceive the physiological effects of O2 starvation. Thus, O2 and NO act as environmental cues that trigger the coordinated expression of virulence genes and metabolic adaptations necessary for survival within a host. Here, the mechanisms of signal perception by key O2- and NO-responsive bacterial transcription factors and the effects on virulence gene expression are reviewed, followed by consideration of these aspects of gene regulation in two major pathogens, Staphylococcus aureus and Mycobacterium tuberculosis. PMID:25603427

  2. Comparative virulence and molecular diversity of stripe rust (Puccinia striiformis f. sp. tritici) collections from Pakistan and United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Information on virulence and molecular diversity of Puccinia striiformis f. sp. tritici (Pst) is a pre-requisite for mitigating the substantial yield losses caused by the stripe rust pathogen in Pakistan, the United States and other countries of the world. This study was undertaken to analyze both v...

  3. The Capsule Is a Virulence Determinant in the Pathogenesis of Pasteurella multocida M1404 (B:2)

    PubMed Central

    Boyce, John D.; Adler, Ben

    2000-01-01

    Capsules from a range of pathogenic bacteria are key virulence determinants, and the capsule has been implicated in virulence in Pasteurella multocida. We have previously identified and determined the nucleotide sequence of the P. multocida M1404 (B:2) capsule biosynthetic locus (J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121–134, 2000). The cap locus consists of 15 genes, which can be grouped into three functional regions. Regions 1 and 3 contain genes proposed to encode proteins involved in capsule export, and region 2 contains genes proposed to encode proteins involved in polysaccharide biosynthesis. In order to construct a mutant impaired in capsule export, the final gene of region 1, cexA, was disrupted by insertion of a tetracycline resistance cassette by allelic replacement. The genotype of the tet(M) ΩcexA mutant was confirmed by Southern hybridization and PCR. The acapsular phenotype was confirmed by immunofluorescence, and the strain could be complemented and returned to capsule production by the presence of a cloned uninterrupted copy of cexA. Wild-type, mutant, and complemented strains were tested for virulence by intraperitoneal challenge of mice; the presence of the capsule was shown to be a crucial virulence determinant. Following intraperitoneal challenge of mice, the acapsular bacteria were removed efficiently from the blood, spleen, and liver, while wild-type bacteria multiplied rapidly. Acapsular bacteria were readily taken up by murine peritoneal macrophages, but wild-type bacteria were significantly resistant to phagocytosis. Both wild-type and acapsular bacteria were resistant to complement in bovine and murine serum. PMID:10816499

  4. Quantitative Trait Locus Based Virulence Determinant Mapping of the HSV-1 Genome in Murine Ocular Infection: Genes Involved in Viral Regulatory and Innate Immune Networks Contribute to Virulence

    PubMed Central

    Larsen, Inna; Craven, Mark; Brandt, Curtis R.

    2016-01-01

    Herpes simplex virus type 1 causes mucocutaneous lesions, and is the leading cause of infectious blindness in the United States. Animal studies have shown that the severity of HSV-1 ocular disease is influenced by three main factors; innate immunity, host immune response and viral strain. We previously showed that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) resulted in recombinants that exhibit a range of disease phenotypes from severe to avirulent, suggesting epistatic interactions were involved. The goal of this study was to develop a quantitative trait locus (QTL) analysis of HSV-1 ocular virulence determinants and to identify virulence associated SNPs. Blepharitis and stromal keratitis quantitative scores were characterized for 40 OD4:CJ994 recombinants. Viral titers in the eye were also measured. Virulence quantitative trait locus mapping (vQTLmap) was performed using the Lasso, Random Forest, and Ridge regression methods to identify significant phenotypically meaningful regions for each ocular disease parameter. The most predictive Ridge regression model identified several phenotypically meaningful SNPs for blepharitis and stromal keratitis. Notably, phenotypically meaningful nonsynonymous variations were detected in the UL24, UL29 (ICP8), UL41 (VHS), UL53 (gK), UL54 (ICP27), UL56, ICP4, US1 (ICP22), US3 and gG genes. Network analysis revealed that many of these variations were in HSV-1 regulatory networks and viral genes that affect innate immunity. Several genes previously implicated in virulence were identified, validating this approach, while other genes were novel. Several novel polymorphisms were also identified in these genes. This approach provides a framework that will be useful for identifying virulence genes in other pathogenic viruses, as well as epistatic effects that affect HSV-1 ocular virulence. PMID:26962864

  5. Quantitative Trait Locus Based Virulence Determinant Mapping of the HSV-1 Genome in Murine Ocular Infection: Genes Involved in Viral Regulatory and Innate Immune Networks Contribute to Virulence.

    PubMed

    Kolb, Aaron W; Lee, Kyubin; Larsen, Inna; Craven, Mark; Brandt, Curtis R

    2016-03-01

    Herpes simplex virus type 1 causes mucocutaneous lesions, and is the leading cause of infectious blindness in the United States. Animal studies have shown that the severity of HSV-1 ocular disease is influenced by three main factors; innate immunity, host immune response and viral strain. We previously showed that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) resulted in recombinants that exhibit a range of disease phenotypes from severe to avirulent, suggesting epistatic interactions were involved. The goal of this study was to develop a quantitative trait locus (QTL) analysis of HSV-1 ocular virulence determinants and to identify virulence associated SNPs. Blepharitis and stromal keratitis quantitative scores were characterized for 40 OD4:CJ994 recombinants. Viral titers in the eye were also measured. Virulence quantitative trait locus mapping (vQTLmap) was performed using the Lasso, Random Forest, and Ridge regression methods to identify significant phenotypically meaningful regions for each ocular disease parameter. The most predictive Ridge regression model identified several phenotypically meaningful SNPs for blepharitis and stromal keratitis. Notably, phenotypically meaningful nonsynonymous variations were detected in the UL24, UL29 (ICP8), UL41 (VHS), UL53 (gK), UL54 (ICP27), UL56, ICP4, US1 (ICP22), US3 and gG genes. Network analysis revealed that many of these variations were in HSV-1 regulatory networks and viral genes that affect innate immunity. Several genes previously implicated in virulence were identified, validating this approach, while other genes were novel. Several novel polymorphisms were also identified in these genes. This approach provides a framework that will be useful for identifying virulence genes in other pathogenic viruses, as well as epistatic effects that affect HSV-1 ocular virulence. PMID:26962864

  6. Burkholderia cepacia Complex: Emerging Multihost Pathogens Equipped with a Wide Range of Virulence Factors and Determinants

    PubMed Central

    Sousa, Sílvia A.; Ramos, Christian G.; Leitão, Jorge H.

    2011-01-01

    The Burkholderia cepacia complex (Bcc) comprises at least 17 closely-related species of the β-proteobacteria subdivision, widely distributed in natural and man-made inhabitats. Bcc bacteria are endowed with an extraordinary metabolic diversity and emerged in the 1980s as life-threatening and difficult-to-treat pathogens among patients suffering from cystic fibrosis. More recently, these bacteria became recognized as a threat to hospitalized patients suffering from other diseases, in particular oncological patients. In the present paper, we review these and other traits of Bcc bacteria, as well as some of the strategies used to identify and validate the virulence factors and determinants used by these bacteria. The identification and characterization of these virulence factors is expected to lead to the design of novel therapeutic strategies to fight the infections caused by these emergent multidrug resistant human pathogens. PMID:20811541

  7. The lipopolysaccharide O side chain of Vibrio vulnificus serogroup E is a virulence determinant for eels.

    PubMed Central

    Amaro, C; Fouz, B; Biosca, E G; Marco-Noales, E; Collado, R

    1997-01-01

    Vibrio vulnificus is a gram-negative bacterium capable of producing septicemic infections in eels and immunocompromised humans. Two biotypes are classically recognized, with the virulence for eels being specific to strains belonging to biotype 2, which constitutes a homogeneous lipopolysaccharide (LPS)-based O serogroup (which we have designated serogroup E). In the present study we demonstrated that the O side chain of this LPS determines the selective virulence of biotype 2 for eels: (i) biotype 1 strains (which do not belong to serogroup E) are destroyed by the bactericidal action of nonimmune eel serum (NIS) through activation of the alternative pathway of complement, (ii) biotype 2 strains (of serogroup E) are resistant to NIS, and (iii) rough mutants of biotype 2 lacking the O polysaccharide side chain are sensitive to NIS and avirulent for eels. PMID:9169795

  8. Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages

    PubMed Central

    Wehrly, Tara D.; Chong, Audrey; Virtaneva, Kimmo; Sturdevant, Dan E.; Child, Robert; Edwards, Jessica A.; Brouwer, Dedeke; Nair, Vinod; Fischer, Elizabeth R.; Wicke, Luke; Curda, Alissa J.; Kupko, John J.; Martens, Craig; Crane, Deborah D.; Bosio, Catharine M.; Porcella, Stephen F.; Celli, Jean

    2009-01-01

    Summary The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis subsp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile. PMID:19388904

  9. Antimicrobial Resistance, Virulence Profile, and Molecular Characterization of Listeria monocytogenes Isolated from Ready-to-eat Food in China, 2013-2014.

    PubMed

    Yan, Shao Fei; Wang, Wei; Bai, Li; Hu, Yu Jie; Dong, Yin Ping; Xu, Jin; Li, Feng Qin

    2016-06-01

    We aimed to investigate the potential pathogenic profile and antibiotic resistance of Listeria monocytogenes isolated from ready-to-eat food in China. Antimicrobial resistance was determined by broth microdilution following the Clinical and Laboratory Standards Institute protocol. Molecular serotyping, virulence, and resistance genes were identified using PCR. Multi-locus sequence typing was performed on resistant strains. A total of 11.53% (113/980) isolates were resistant, from which 82.3% (93/113) harbored all the virulence genes tested. The resistant strains were subtyped into 18 sequence types (STs), from which ST2, ST5, ST8, and ST9 were involved in listeriosis. This study indicated that several L. monocytogenes isolates from ready-to-eat foods in China have pathogenic potential and are resistant to antibiotics, including antibiotics used as medicines by humans for listeriosis treatment. PMID:27470106

  10. Molecular analysis of Helicobacter pylori virulent-associated genes in hepatobiliary patients

    PubMed Central

    Boonyanugomol, Wongwarut; Chomvarin, Chariya; Sripa, Banchob; Chau-in, Siri; Pugkhem, Ake; Namwat, Wises; Wongboot, Warawan; Khampoosa, Bandit

    2012-01-01

    Objectives The Helicobacter pylori virulence-associated genes in hepatobiliary patients, including vacA, iceA, babA2, cagA and cagE, have not been reported. The aim of this study was to investigate these genes and the association of those and the clinical outcomes in hepatobiliary diseases. Methods Eighty H. pylori-PCR-positive cases were obtained from hepatobiliary patients, representing both cholangiocarcinoma (CCA) (n = 58) and cholelithiasis (n = 22). The diversity of virulence genes was examined by polymerase chain reaction and DNA sequencing. Phylogenetic analysis of cagA was determined using the maximum parsimony method. Results The vacAs1a + c/m1, iceA1 and babA2 genes were the most predominant genotypes in both CCA and cholelithiasis patients. The cagA and cagE genes were found significantly more frequently in patients with CCA than those with cholelithiasis (P < 0.05). The cagA positive samples were the Western-type cagA and showed that almost all of the detected sequences in Thai hepatobiliary and Thai gastric cancer patients were classified in the same cluster but separated from the cluster of Japan and other countries. Conclusions The cagA and cagE genes may be associated in the pathogenesis of hepatobiliary diseases, especially of CCA. Besides the bacterial variation, other host factors may be involved in the pathogenesis of hepatobiliary cancer. PMID:23043664

  11. Virulence and plasmidic resistance determinants of Escherichia coli isolated from municipal and hospital wastewater treatment plants.

    PubMed

    Calhau, Vera; Mendes, Catarina; Pena, Angelina; Mendonça, Nuno; Da Silva, Gabriela Jorge

    2015-06-01

    Escherichia coli is simultaneously an indicator of water contamination and a human pathogen. This study aimed to characterize the virulence and resistance of E. coli from municipal and hospital wastewater treatment plants (WWTPs) in central Portugal. From a total of 193 isolates showing reduced susceptibility to cefotaxime and/or nalidixic acid, 20 E. coli with genetically distinct fingerprint profiles were selected and characterized. Resistance to antimicrobials was determined using the disc diffusion method. Extended spectrum β-lactamase and plasmid-mediated quinolone resistance genes, phylogroups, pathogenicity islands (PAIs) and virulence genes were screened by polymerase chain reaction (PCR). CTX-M producers were typed by multilocus sequence typing. Resistance to beta-lactams was associated with the presence of bla(TEM), bla(SHV), bla(CTX-M-15) and bla(CTX-M-32). Plasmid-mediated quinolone resistance was associated with qnrA, qnrS and aac(6')-Ib-cr. Aminoglycoside resistance and multidrug-resistant phenotypes were also detected. PAI IV(536), PAI II(CFT073), PAI II(536) and PAI I(CFT073), and uropathogenic genes iutA, papAH and sfa/foc were detected. With regard to the clinical ST131 clone, it carried bla(CTX-M-15), blaTEM-type, qnrS and aac(6')-lb-cr; IncF and IncP plasmids, and virulence factors PAI IV(536), PAI I(CFT073), PAI II(CFT073), iutA, sfa/foc and papAH were identified in the effluent of a hospital plant. WWTPs contribute to the dissemination of virulent and resistant bacteria in water ecosystems, constituting an environmental and public health risk. PMID:26042965

  12. Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story.

    PubMed

    Stathopoulos, C; Hendrixson, D R; Thanassi, D G; Hultgren, S J; St Geme, J W; Curtiss, R

    2000-07-01

    Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence. PMID:10967286

  13. Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

    PubMed

    Tamura, Tomokazu; Ruggli, Nicolas; Nagashima, Naofumi; Okamatsu, Masatoshi; Igarashi, Manabu; Mine, Junki; Hofmann, Martin A; Liniger, Matthias; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-09-01

    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs. PMID:26018962

  14. Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence.

    PubMed

    Silvestre, Bruna T; Silveira, Júlia A G; Meneses, Rodrigo M; Facury-Filho, Elias J; Carvalho, Antônio U; Ribeiro, Múcio F B

    2016-02-01

    Bovine anaplasmosis is a disease caused by the intraerythrocytic rickettsia species Anaplasma marginale and results in great economic losses in tropical and subtropical regions. Vertical transmission is an important phenomenon that contributes to the persistence of different strains of the agent within the same herd. The identification of new strains and genetic characterization studies are essential to understanding their epidemiology and virulence and for vaccine development. The aim of this study was to perform molecular and phylogenetic characterizations of a new vertically transmitted strain from A. marginale and to evaluate its virulence by experimental inoculation of rickettsia-free calves. Thirty newborn Holstein calves were subjected to molecular tests for the detection of A. marginale, Babesia bovis and Babesia bigemina. Calves positive for A. marginale (n=3) were splenectomized and monitored for the clinical manifestations of anaplasmosis. Blood samples from one of the calves that presented rickettsemia of 42.8% and spontaneous recovery of clinical parameters were used for molecular and phylogenetic characterization (msp1a gene), and inoculum production was used for the evaluation of virulence. This strain was identified as UFMG3. Three tandem repeat forms (13 and MGI19) were identified from the analysis of the msp1a gene, in which the form MGI19 appeared twice. Analysis of these repeats revealed the presence of the sequences QASTSS and SSASGQQQESS and of aspartic acid (D) at position 20 of both repeats. Phylogenetic analysis showed a close relationship among the UFMG3, MGI19 and UFMG2 strains. For virulence evaluation, six Holstein calves were inoculated intravenously with 2×10(7)A. marginale UFMG3-infected erythrocytes. The calves showed maximum rickettsemia of 5.1%, a moderate decrease in packed cell volume and spontaneous recovery of clinical parameters without the need for treatment. The results of experimental inoculation suggest that the strain A

  15. Molecular determinants of alphavirus neuropathogenesis in mice.

    PubMed

    Atkins, Gregory J; Sheahan, Brian J

    2016-06-01

    Alphaviruses are enveloped viruses with a positive-stranded RNA genome, of the family Togaviridae. In mammals and birds they are mosquito-transmitted and are of veterinary and medical importance. They cause primarily two types of disease: encephalitis and polyarthritis. Here we review attempts to understand the molecular basis of encephalitis and virulence for the central nervous system (CNS) in mouse models. Sindbis virus (SINV) was the first virus to be studied in this way. Other viruses analysed are Semliki Forest virus (SFV), Venezuelan equine encephalitis virus, Eastern equine encephalitis virus and Western equine encephalitis virus. Neurovirulence was found to be associated with damage to neurons in the CNS. It mapped mainly to the E2 region of the genome, and to the nsP3 gene. Also, avirulent natural isolates of both SINV and SFV have been found to have more rapid cleavage of nonstructural proteins due to mutations in the nsP1-nsP2 cleavage site. Immune-mediated demyelination for avirulent SFV has been shown to be associated with infection of oligodendrocytes. For Chikungunya virus, an emerging alphavirus that uncommonly causes encephalitis, analysis of the molecular basis of CNS pathogenicity is beginning. Experiments on SINV and SFV have indicated that virulence may be related to the resistance of virulent virus to interferon action. Although the E2 protein may be involved in tropism for neurons and passage across the blood-brain barrier, the role of the nsP3 protein during infection of neurons is unknown. More information in these areas may help to further explain the neurovirulence of alphaviruses. PMID:27028153

  16. Molecular characterization and virulence of Beauveria spp. from the pine processionary moth, Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae).

    PubMed

    Sevim, Ali; Demir, Ismail; Demirbağ, Zihni

    2010-10-01

    The pine processionary moth Thaumetopoea pityocampa (Den. & Schiff.) is one of the most harmful pests to pine species in Mediterranean countries including Turkey. Caterpillars of T. pityocampa are not only significantly harmful to forest trees but also responsible for various allergic reactions in humans and animals. In this study, in order to find a more effective and safe biological control agent against T. pityocampa, we investigated fungal pathogens of T. pityocampa in the Black Sea Region of Turkey and tested their pathogenicity on it. Five different fungi were isolated and identified based on their morphological and molecular characteristics including ITS and partial sequence of EF1-[alpha]. Based on these characteristics, four isolates were identified as Beauveria bassiana cf. Clade C (Rehner and Buckley in Mycologia 97:84-98, 2005) and one isolate was identified as Beauveria bassiana. Among these isolates, B. bassiana KTU-24, B. bassiana cf. Clade C KTU-66 and KTU-67 showed the highest virulence with 100% mortality within 10 days after application. B. bassiana isolate KTU-24 produced the highest mycosis value with %100. Consequently, B. bassiana KTU-24 seems to be good candidate for further investigation as a possible biological control agent against this pest. PMID:20499189

  17. Antibiotic Resistance, Virulence Gene, and Molecular Profiles of Shiga Toxin-Producing Escherichia coli Isolates from Diverse Sources in Calcutta, India

    PubMed Central

    Khan, Asis; Das, S. C.; Ramamurthy, T.; Sikdar, A.; Khanam, J.; Yamasaki, S.; Takeda, Y.; Nair, G. Balakrish

    2002-01-01

    Antibiotic resistance, virulence gene, and molecular profiles of Shiga toxin-producing Escherichia coli (STEC) non-O157 strains isolated from human stool samples, cow stool samples, and beef samples over a period of 2 years in Calcutta, India, were determined. Resistance to one or more antibiotics was observed in 49.2% of the STEC strains, with some of the strains exhibiting multidrug resistance. The dominant combinations of virulence genes present in the strains studied were stx1 and stx2 (44.5% of strains) and stx1, stx2, and hlyA (enterohemorrhagic E. coli hemolysin gene) (19% of strains). Only 6.4% of the STEC strains harbored eae. The diversity of STEC strains from various sources was assessed by random amplification of polymorphic DNA (RAPD). STEC strains that gave identical or nearly similar DNA fingerprints in RAPD-PCR and had similar virulence genotypes were further characterized by pulsed-field gel electrophoresis (PFGE). Identical RAPD and PFGE profiles were observed in four sets of strains, with each set comprising two strains. There was no match in the RAPD and PFGE profiles between strains of STEC isolated from cows and those isolated from humans. It appears that the clones present in bovine sources are not transmitted to humans in the Calcutta setting although these strains showed evolutionary relatedness. Maybe for this reason, STEC has still not become a major problem in India. PMID:12037056

  18. A structural comparison of Listeria monocytogenes protein chaperones PrsA1 and PrsA2 reveals molecular features required for virulence.

    PubMed

    Cahoon, Laty A; Freitag, Nancy E; Prehna, Gerd

    2016-07-01

    Listeria monocytogenes is a Gram-positive environmental bacterium that lives within soil but transitions into a pathogen upon contact with a mammalian host. The transition of L. monocytogenes from soil dweller to cytosolic pathogen is dependent upon secreted virulence factors that mediate cell invasion and intracellular growth. PrsA1 and PrsA2 are secreted bacterial lipoprotein chaperones that contribute to the folding of proteins translocated across the bacterial membrane; PrsA2 is required for L. monocytogenes virulence, whereas the function of PrsA1 remains to be determined. We have solved an X-ray crystal structure of PrsA1 and have used this model to guide comparison structure-based mutagenesis studies with PrsA2. Targeted mutagenesis of PrsA2 demonstrates that oligomerization of PrsA2 as well as molecular features of the foldase domain are required for protein secretion and virulence, whereas a functional role was uncovered for PrsA1 in bacterial resistance to alcohol. Interestingly, PrsA2 membrane localization is not required for all PrsA2-dependent activities, suggesting that the lipoprotein retains function when released from the bacterial cell. PrsA chaperones are thus multifaceted proteins with distinct domains adapted to accommodate the functional needs of a diverse array of secreted substrates. PMID:27007641

  19. Novel Model To Study Virulence Determinants of Escherichia coli K1▿

    PubMed Central

    Khan, Naveed Ahmed; Goldsworthy, Graham John

    2007-01-01

    It is shown here for the first time that locusts can be used as a model to study Escherichia coli K1 pathogenesis. E. coli K-12 strain HB101 has very low pathogenicity to locusts and does not invade the locust brain, whereas the injection of 2 × 106 E. coli K1 strain RS218 (O18:K1:H7) kills almost 100% of locusts within 72 h and invades the brain within 24 h of injection. Both mortality and invasion of the brain in locusts after injection of E. coli K1 require at least two of the known virulence determinants shown for mammals. Thus, deletion mutants that lack outer membrane protein A or cytotoxic necrotizing factor 1 have reduced abilities to kill locusts and to invade the locust brain compared to the parent E. coli K1. Interestingly, deletion mutants lacking FimH or the NeuDB gene cluster are still able to cause high mortality. It is argued that the likely existence of additional virulence determinants can be investigated in vivo by using this insect system. PMID:17875634

  20. Identification of virulence determinants in uropathogenic Proteus mirabilis using signature-tagged mutagenesis.

    PubMed

    Himpsl, Stephanie D; Lockatell, C Virginia; Hebel, J Richard; Johnson, David E; Mobley, Harry L T

    2008-09-01

    The Gram-negative bacterium Proteus mirabilis causes urinary tract infections (UTIs) in individuals with long-term indwelling catheters or those with functional or structural abnormalities of the urinary tract. Known virulence factors include urease, haemolysin, fimbriae, flagella, DsbA, a phosphate transporter and genes involved in cell-wall synthesis and metabolism, many of which have been identified using the technique of signature-tagged mutagenesis (STM). To identify additional virulence determinants and to increase the theoretical coverage of the genome, this study generated and assessed 1880 P. mirabilis strain HI4320 mutants using this method. Mutants with disruptions in genes vital for colonization of the CBA mouse model of ascending UTI were identified after performing primary and secondary in vivo screens in approximately 315 CBA mice, primary and secondary in vitro screens in both Luria broth and minimal A medium to eliminate mutants with minor growth deficiencies, and co-challenge competition experiments in approximately 500 CBA mice. After completion of in vivo screening, a total of 217 transposon mutants were attenuated in the CBA mouse model of ascending UTI. Following in vitro screening, this number was reduced to 196 transposon mutants with a probable role in virulence. Co-challenge competition experiments confirmed significant attenuation for 37 of the 93 transposon mutants tested, being outcompeted by wild-type HI4320. Following sequence analysis of the 37 mutants, transposon insertions were identified in genes including the peptidyl-prolyl isomerases surA and ppiA, glycosyltransferase cpsF, biopolymer transport protein exbD, transcriptional regulator nhaR, one putative fimbrial protein, flagellar M-ring protein fliF and hook protein flgE, and multiple metabolic genes. PMID:18719175

  1. RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus.

    PubMed

    Gaupp, Rosmarie; Wirf, Jessica; Wonnenberg, B; Biegel, Tanja; Eisenbeis, J; Graham, J; Herrmann, M; Lee, C Y; Beisswenger, C; Wolz, C; Tschernig, T; Bischoff, M; Somerville, G A

    2016-07-01

    In Staphylococcus aureus, metabolism is intimately linked with virulence determinant biosynthesis, and several metabolite-responsive regulators have been reported to mediate this linkage. S. aureus possesses at least three members of the RpiR family of transcriptional regulators. Of the three RpiR homologs, RpiRc is a potential regulator of the pentose phosphate pathway, which also regulates RNAIII levels. RNAIII is the regulatory RNA of the agr quorum-sensing system that controls virulence determinant synthesis. The effect of RpiRc on RNAIII likely involves other regulators, as the regulators that bind the RNAIII promoter have been intensely studied. To determine which regulators might bridge the gap between RpiRc and RNAIII, sarA, sigB, mgrA, and acnA mutations were introduced into an rpiRc mutant background, and the effects on RNAIII were determined. Additionally, phenotypic and genotypic differences were examined in the single and double mutant strains, and the virulence of select strains was examined using two different murine infection models. The data suggest that RpiRc affects RNAIII transcription and the synthesis of virulence determinants in concert with σ(B), SarA, and the bacterial metabolic status to negatively affect virulence. PMID:27113358

  2. Yersinia enterocolitica: Mode of Transmission, Molecular Insights of Virulence, and Pathogenesis of Infection

    PubMed Central

    Sabina, Yeasmin; Rahman, Atiqur; Ray, Ramesh Chandra; Montet, Didier

    2011-01-01

    Although Yersinia enterocolitica is usually transmitted through contaminated food and untreated water, occasional transmission such as human-to-human, animal-to-human and blood transfusion associated transmission have also identified in human disease. Of the six Y. enterocolitica biotypes, the virulence of the pathogenic biotypes, namely, 1B and 2–5 is attributed to the presence of a highly conserved 70-kb virulence plasmid, termed pYV/pCD and certain chromosomal genes. Some biotype 1A strains, despite lacking virulence plasmid (pYV) and traditional chromosomal virulence genes, are isolated frequently from humans with gastrointestinal diseases similar to that produced by isolates belonging known pathogenic biotypes. Y. enterocolitica pathogenic biotypes have evolved two major properties: the ability to penetrate the intestinal wall, which is thought to be controlled by plasmid genes, and the production of heat-stable enterotoxin, which is controlled by chromosomal genes. PMID:22567333

  3. Morphological and Molecular Characterization of Pomegranate Fruit Rot Pathogen, Chaetomella raphigera, and its Virulence Factors.

    PubMed

    Gajbhiye, Milind; Sathe, Shivaji; Shinde, Vikas; Kapadnis, Balu

    2016-03-01

    A new fungal pathogen was isolated from rotten pomegranates collected from the orchards of different parts of Maharashtra. The pathogen was morphologically identified as Chaetomella raphigera followed by sequencing of ITS and D1/D2 hypervariable region of LSU (28S) of rRNA gene. The pathogen produced pectinase, cellulase, xylanase and protease in liquid medium at a concentration of 71, 13.8, 54.3 and 7 U/ml respectively. Enzyme activity was also determined during pathogenesis in the tissues artificially infected by C. raphigera. Xylanase activity was maximum (25.1 U/g) followed by pectinase (19.2 U/g) and cellulase (1.5 U/g), whereas, protease activity was unnoticed. There was significant correlation (P < 0.05) between disease rating scale and pectinase, xylanase and cellulase activity in infected tissues. This indicates the simultaneous production of hydrolytic enzymes that aids in necrosis of fruit tissues. The elevated levels of these enzymes in infected tissues as compared with control suggest their possible role in pathogenesis. Thus, pectinase, cellulase and xylanase produced by C. raphigera acts as major virulence factors in the development of fruit rot in pomegranates. This is a first report of fungal fruit rot caused by C. raphigera in pomegranate. PMID:26843702

  4. Microsecond Molecular Dynamics Simulations of Influenza Neuraminidase Suggest a Mechanism for the Increased Virulence of Stalk-Deletion Mutants

    PubMed Central

    2016-01-01

    Deletions in the stalk of the influenza neuraminidase (NA) surface protein are associated with increased virulence, but the mechanisms responsible for this enhanced virulence are unclear. Here we use microsecond molecular dynamics simulations to explore the effect of stalk deletion on enzymatic activity, contrasting NA proteins from the A/swine/Shandong/N1/2009 strain both with and without a stalk deletion. By modeling and simulating neuraminidase apo glycoproteins embedded in complex-mixture lipid bilayers, we show that the geometry and dynamics of the neuraminidase enzymatic pocket may differ depending on stalk length, with possible repercussions on the binding of the endogenous sialylated-oligosaccharide receptors. We also use these simulations to predict previously unrecognized druggable “hotspots” on the neuraminidase surface that may prove useful for future efforts aimed at structure-based drug design. PMID:27141956

  5. Microsecond Molecular Dynamics Simulations of Influenza Neuraminidase Suggest a Mechanism for the Increased Virulence of Stalk-Deletion Mutants.

    PubMed

    Durrant, Jacob D; Bush, Robin M; Amaro, Rommie E

    2016-08-25

    Deletions in the stalk of the influenza neuraminidase (NA) surface protein are associated with increased virulence, but the mechanisms responsible for this enhanced virulence are unclear. Here we use microsecond molecular dynamics simulations to explore the effect of stalk deletion on enzymatic activity, contrasting NA proteins from the A/swine/Shandong/N1/2009 strain both with and without a stalk deletion. By modeling and simulating neuraminidase apo glycoproteins embedded in complex-mixture lipid bilayers, we show that the geometry and dynamics of the neuraminidase enzymatic pocket may differ depending on stalk length, with possible repercussions on the binding of the endogenous sialylated-oligosaccharide receptors. We also use these simulations to predict previously unrecognized druggable "hotspots" on the neuraminidase surface that may prove useful for future efforts aimed at structure-based drug design. PMID:27141956

  6. The usefulness of molecular techniques to assess the presence of Aeromonas spp. harboring virulence markers in foods.

    PubMed

    Bin Kingombe, César I; Huys, Geert; Howald, Denise; Luthi, Elisabeth; Swings, Jean; Jemmi, Thomas

    2004-07-15

    A total of 78 raw and 123 processed and ready-to-eat retail food samples were used to assess the presence of motile Aeromonas spp. harboring virulence genes (cytotoxic enterotoxin and hemolysin genes) using a recently described PCR method in comparison with the conventional cultivation method based on the use of Ampicillin-Dextrin Agar (ADA) medium. With the ADA-based method, 65/201 (32.3%) samples showed presumptive Aeromonas spp. colonies whereas the PCR method revealed the presence of Aeromonas spp. harboring the targeted virulence genes in 51/201 (25.4%) of the tested samples. The rate of contaminated samples and the presence of pathogenic Aeromonas were significantly lower with both methods for processed than in case of raw samples. A polyphasic identification approach including biochemical and molecular techniques was applied to a selection of 34 PCR-positive presumptive Aeromonas isolates. Following fatty acid methyl ester (FAME) analysis and amplified fragment length polymorphism (AFLP) fingerprinting, a total of 33 isolates (97%) could be identified to the DNA hybridization group (HG) level. The majority of these isolates belonged to the species Aeromonas hydrophila HG3 (50%) and Aeromonas veronii biovar sobria (HG8/10) (38%). Molecular characterization of PCR amplicons obtained from these strains by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) fingerprinting and PCR-Amplicon Sequence Analysis (PCR-ASA) allowed classification of all strains in a known PCR-RFLP and PCR-ASA type. In conclusion, the current findings demonstrate that the combined use of PCR-based virulence marker detection, PCR-RFLP and PCR-ASA offers a rapid, sensitive, and specific system to assess the presence and prevalence of Aeromonas spp. harboring virulence markers in food samples. PMID:15193799

  7. How do Lyme Borrelia Organisms Cause Disease? The Quest for Virulence Determinants#

    PubMed Central

    Norris, Steven J

    2012-01-01

    Lyme disease Borrelia are invasive, nontoxigenic, persistent pathogens, and little is known about their mechanisms of pathogenesis. In our laboratory, a signature-tagged mutagenesis (STM) library of over 4,000 Borrelia burgdorferi transposon mutants has been constructed and is being screened for infectivity in mice. In this manner, a global view of the virulence determinants (factors required for full infectivity) is being developed. Additionally, the mechanisms of immune evasion involving the VMP-like system (vls) are under analysis, and cryo-electron microscopy is providing a detailed view of the three-dimensional structure of B. burgdorferi. These approaches will contribute to the improved understanding of how Lyme disease Borrelia cause disease. PMID:23091573

  8. The enhancin gene: One of the genetic determinants of population variation in baculoviral virulence.

    PubMed

    Martemyanov, V V; Kabilov, M R; Tupikin, A E; Baturina, O A; Belousova, I A; Podgwaite, J D; Ilynykh, A V; Vlassov, V V

    2015-01-01

    It was established that the virulence of the North American baculovirus strain LdMNPV-45 is almost two orders of magnitude higher than the virulence of the Asian strain LdMNPV-27 and does not depend on the test host population (gypsy moth). The Asian strain carries deletions in bro-p and vef-1 genes (82 and 91%, respectively). In accordance with the published data, the product of the latter can greatly increase the virulence of the virus. This result indicates that the population polymorphism of the virulence of baculoviruses can be explained by the vef-1 gene deletion. PMID:26728722

  9. Virulence determinants in enteroaggregative Escherichia coli from North India and their interaction in in vitro organ culture system.

    PubMed

    Gupta, Deepika; Sharma, Monica; Sarkar, Subendu; Thapa, B R; Chakraborti, Anuradha

    2016-09-01

    Enteroaggregative Escherichia coli (EAEC) is an important diarrhoeal pathogen causing diseases in multiple epidemiological and clinical settings. In developing countries like India, diarrhoeal diseases are one of the major killers among paediatric population and oddly, few studies are available from Indian paediatric population on the variability of EAEC virulence genes. In this study, we examined the distribution of plasmid and chromosomal-encoded virulence determinants in EAEC isolates, and analysed cytokines response generated against EAEC with specific aggregative adherence fimbriae (AAF) type in duodenal biopsies using in vitro organ culture (IVOC) mimicking in vivo conditions. Different virulence marker combinations among strains were reflected as a function of specific adhesins signifying EAEC heterogeneity. fis gene emerged as an important genetic marker apart from aggA and aap Further, EAEC infection in IVOC showed upregulation of IL-8, IL-1β, IL-6, TNF-α and TLR-5 expression. EAEC with AAFII induced significant TLR-5 and IL-8 response, conceivably owing to more pathogenicity markers. This study sheds light on the pattern of EAEC pathotypes prevalent in North Indian paediatric population and highlights the presence of unique virulence combinations in pathogenic strains. Thus, evident diversity in EAEC virulence and multifaceted bacteria-host crosstalk can provide useful insights for the strategic management of diarrhoeal diseases in India, where diarrhoeal outbreaks are more frequent. PMID:27493010

  10. 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. PMID:26980093

  11. Roadmap to new virulence determinants in Pseudomonas syringae: Insights from Comparative Genomics and Genome Organization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Systematic comparison of the current repertoire of virulence and host-association genes for three Pseudomonas syringae strains with complete genome sequences P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a, is prompted by recent advances in virul...

  12. Antibiotic Resistant and Virulence Determinants of Staphylococcus haemolyticus C10A as Revealed by Whole Genome Sequencing

    PubMed Central

    Chan, Kok-Gan; Ng, Kim Tien; Chong, Teik Min; Pang, Yong Kek; Kamarulzaman, Adeeba; Yin, Wai-Fong; Tee, Kok Keng

    2015-01-01

    Staphylococcus haemolyticus is one of the pathogens that harbor a high level of antibiotic resistance. Here, we highlighted the potential determinants for multidrug resistance and virulence from the draft genome of Staphylococcus haemolyticus strain C10A, isolated from a patient with chronic obstructive pulmonary disease exacerbation. PMID:26157506

  13. Virulence and molecular variation of Flavobacterium columnare affecting rainbow trout in ID, USA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Columnaris disease is an emerging problem in the rainbow trout (Oncorhychus mykiss) aquaculture industry of Idaho. The epidemiology of this pathogen in the area, and for rainbow trout, is all isolates taken from disease outbreaks are genomovar I and similar based on basic typing protocols. Virulence...

  14. Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence

    PubMed Central

    Ratinier, Maxime; Shaw, Andrew E.; Barry, Gerald; Gu, Quan; Di Gialleonardo, Luigina; Janowicz, Anna; Varela, Mariana; Randall, Richard E.; Caporale, Marco

    2016-01-01

    ABSTRACT Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-β promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence. IMPORTANCE Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein

  15. The catabolite control protein E (CcpE) affects virulence determinant production and pathogenesis of Staphylococcus aureus.

    PubMed

    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

    2014-10-24

    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

  16. 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

  17. Colony types and virulence traits of Legionella feeleii determined by exopolysaccharide materials.

    PubMed

    Wang, Changle; Saito, Mitsumasa; Ogawa, Midori; Yoshida, Shin-Ichi

    2016-05-01

    Legionella feeleii is a Gram-negative pathogenic bacterium that causes Pontiac fever and pneumonia in humans. When L. feeleii serogroup 1 (ATCC 35072) was cultured on BCYE agar plates, two types of colonies were observed and exhibited differences in color, opacity and morphology. Since the two colony types are white rugose and brown translucent, they were termed as white rugose L. feeleii (WRLf) and brown translucent L. feeleii (BTLf), respectively. They exhibited different growth capacities in BYE broth in vitro, and it was found that WRLf could transform to BTLf. Under the electron microscope, it was observed that WRLf secreted materials which could be stained with ruthenium red, which was absent in BTLf. When U937 macrophages and HeLa cells were infected with the bacteria, WRLf manifested stronger internalization ability than BTLf. Intracellular growth in murine macrophages and Acanthamoeba cells was affected by the level of initial phagocytosis. WRLf was more resistant to human serum bactericidal action than BTLf. After being inoculated to guinea pigs, both organisms caused fever in the animals. These results suggest that ruthenium red-stained materials secreted in the surroundings may play a crucial role in determining L. feeleii colony morphology and virulence traits. PMID:27190244

  18. Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity.

    PubMed Central

    Tan, C; Prescott, J F; Patterson, M C; Nicholson, V M

    1995-01-01

    Virulent strains of Rhodococcus equi produce plasmid-mediated 15- and 17-kDa proteins, which are thermoregulated and apparently surface-expressed. We demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that R. equi produce three antigenically-related virulence-associated proteins, a diffuse 18-22-kDa, a 17.5-kDa and a 15-kDa protein. Phase partitioning of whole cells of R. equi strain 103 with Triton X-114 (TX-114) and labelling with [3H]-labelled palmitic acid showed that the two higher molecular weight proteins are hydrophobic and lipid modified. The 15-kDa protein did not partition into TX-114 and was not lipid modified. Cloning and expression of a fragment of the R. equi virulence plasmid in Escherichia coli showed that the three proteins were expressed from a single gene. Sequence analysis of this gene (designated vapA) revealed a 570-bp open reading frame encoding a polypeptide of 189 amino acids with a calculated molecular mass of 19,175 Da. The mature, nonlipid modified protein had a calculated mass of 16,246 Da. The 17.5- and 18-22-kDa forms of the protein are therefore due to lipid modification. No significant sequence homology of the vapA gene with other reported nucleotide sequences were found. Opsonization of virulent R. equi with an IgG1 mouse monoclonal antibody (MAb103) to the VapA protein significantly enhanced uptake in the murine macrophage cell line IC-21. Intraperitoneal injection of mice with Mab103 enhanced initial clearance from the liver of mice challenged intravenously with R. equi. Immunization of mice with the lipid-modified VapA purified by SDS-PAGE fractionation or with acetone precipitated VapA protein following TX-114 extraction resulted in significantly enhanced clearance from the liver and spleen following intravenous challenge. The VapA protein of R. equi appears therefore to be a protective immunogen. Images Fig. 1. Fig. 4. PMID:7704843

  19. Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity.

    PubMed

    Tan, C; Prescott, J F; Patterson, M C; Nicholson, V M

    1995-01-01

    Virulent strains of Rhodococcus equi produce plasmid-mediated 15- and 17-kDa proteins, which are thermoregulated and apparently surface-expressed. We demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that R. equi produce three antigenically-related virulence-associated proteins, a diffuse 18-22-kDa, a 17.5-kDa and a 15-kDa protein. Phase partitioning of whole cells of R. equi strain 103 with Triton X-114 (TX-114) and labelling with [3H]-labelled palmitic acid showed that the two higher molecular weight proteins are hydrophobic and lipid modified. The 15-kDa protein did not partition into TX-114 and was not lipid modified. Cloning and expression of a fragment of the R. equi virulence plasmid in Escherichia coli showed that the three proteins were expressed from a single gene. Sequence analysis of this gene (designated vapA) revealed a 570-bp open reading frame encoding a polypeptide of 189 amino acids with a calculated molecular mass of 19,175 Da. The mature, nonlipid modified protein had a calculated mass of 16,246 Da. The 17.5- and 18-22-kDa forms of the protein are therefore due to lipid modification. No significant sequence homology of the vapA gene with other reported nucleotide sequences were found. Opsonization of virulent R. equi with an IgG1 mouse monoclonal antibody (MAb103) to the VapA protein significantly enhanced uptake in the murine macrophage cell line IC-21. Intraperitoneal injection of mice with Mab103 enhanced initial clearance from the liver of mice challenged intravenously with R. equi. Immunization of mice with the lipid-modified VapA purified by SDS-PAGE fractionation or with acetone precipitated VapA protein following TX-114 extraction resulted in significantly enhanced clearance from the liver and spleen following intravenous challenge. The VapA protein of R. equi appears therefore to be a protective immunogen. PMID:7704843

  20. Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex, gilthead sea bream and European sea bass.

    PubMed

    Pujalte, M J; Sitjà-Bobadilla, A; Macián, M C; Belloch, C; Alvarez-Pellitero, P; Pérez-Sánchez, J; Uruburu, F; Garay, E

    2003-06-01

    Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass. PMID:12866856

  1. Molecular characterization and virulence gene profiling of pathogenic Streptococcus agalactiae populations from tilapia (Oreochromis sp.) farms in Thailand.

    PubMed

    Kayansamruaj, Pattanapon; Pirarat, Nopadon; Katagiri, Takayuki; Hirono, Ikuo; Rodkhum, Channarong

    2014-05-19

    Streptococcus spp. were recovered from diseased tilapia in Thailand during 2009-2010 (n = 33), and were also continually collected from environmental samples (sediment and water) from tilapia farms for 9 months in 2011 (n = 25). The relative percent recovery of streptococci from environmental samples was 13-67%. All streptococcal isolates were identified as S. agalactiae (group B streptococci [GBS]) by a species-specific polymerase chain reaction. In molecular characterization assays, 4 genotypic categories comprised of 1) molecular serotypes, 2) the infB allele, 3) virulence gene profiling patterns (cylE, hylB, scpB, lmb, cspA, dltA, fbsA, fbsB, bibA, gap, and pili backbone-encoded genes), and 4) randomly amplified polymorphic DNA (RAPD) fingerprinting patterns, were used to describe the genotypic diversity of the GBS isolates. There was only 1 isolate identified as molecular serotype III, while the others were serotype Ia. Most GBS serotype Ia isolates had an identical infB allele and virulence gene profiling patterns, but a large diversity was established by RAPD analysis with diversity tending to be geographically dependent. Experimental infection of Nile tilapia (Oreochromis niloticus) revealed that the GBS serotype III isolate was nonpathogenic in the fish, while all 5 serotype Ia isolates (3 fish and 2 environmental isolates) were pathogenic, with a median lethal dose of 6.25-7.56 log10 colony-forming units. In conclusion, GBS isolates from tilapia farms in Thailand showed a large genetic diversity, which was associated with the geographical origins of the bacteria. PMID:24842288

  2. Revised Phylogeny and Novel Horizontally Acquired Virulence Determinants of the Model Soft Rot Phytopathogen Pectobacterium wasabiae SCC3193

    PubMed Central

    Koskinen, Patrik; Nokso-Koivisto, Jussi; Pasanen, Miia; Broberg, Martin; Plyusnin, Ilja; Törönen, Petri; Holm, Liisa; Pirhonen, Minna; Palva, E. Tapio

    2012-01-01

    Soft rot disease is economically one of the most devastating bacterial diseases affecting plants worldwide. In this study, we present novel insights into the phylogeny and virulence of the soft rot model Pectobacterium sp. SCC3193, which was isolated from a diseased potato stem in Finland in the early 1980s. Genomic approaches, including proteome and genome comparisons of all sequenced soft rot bacteria, revealed that SCC3193, previously included in the species Pectobacterium carotovorum, can now be more accurately classified as Pectobacterium wasabiae. Together with the recently revised phylogeny of a few P. carotovorum strains and an increasing number of studies on P. wasabiae, our work indicates that P. wasabiae has been unnoticed but present in potato fields worldwide. A combination of genomic approaches and in planta experiments identified features that separate SCC3193 and other P. wasabiae strains from the rest of soft rot bacteria, such as the absence of a type III secretion system that contributes to virulence of other soft rot species. Experimentally established virulence determinants include the putative transcriptional regulator SirB, two partially redundant type VI secretion systems and two horizontally acquired clusters (Vic1 and Vic2), which contain predicted virulence genes. Genome comparison also revealed other interesting traits that may be related to life in planta or other specific environmental conditions. These traits include a predicted benzoic acid/salicylic acid carboxyl methyltransferase of eukaryotic origin. The novelties found in this work indicate that soft rot bacteria have a reservoir of unknown traits that may be utilized in the poorly understood latent stage in planta. The genomic approaches and the comparison of the model strain SCC3193 to other sequenced Pectobacterium strains, including the type strain of P. wasabiae, provides a solid basis for further investigation of the virulence, distribution and phylogeny of soft rot

  3. Revised phylogeny and novel horizontally acquired virulence determinants of the model soft rot phytopathogen Pectobacterium wasabiae SCC3193.

    PubMed

    Nykyri, Johanna; Niemi, Outi; Koskinen, Patrik; Nokso-Koivisto, Jussi; Pasanen, Miia; Broberg, Martin; Plyusnin, Ilja; Törönen, Petri; Holm, Liisa; Pirhonen, Minna; Palva, E Tapio

    2012-01-01

    Soft rot disease is economically one of the most devastating bacterial diseases affecting plants worldwide. In this study, we present novel insights into the phylogeny and virulence of the soft rot model Pectobacterium sp. SCC3193, which was isolated from a diseased potato stem in Finland in the early 1980s. Genomic approaches, including proteome and genome comparisons of all sequenced soft rot bacteria, revealed that SCC3193, previously included in the species Pectobacterium carotovorum, can now be more accurately classified as Pectobacterium wasabiae. Together with the recently revised phylogeny of a few P. carotovorum strains and an increasing number of studies on P. wasabiae, our work indicates that P. wasabiae has been unnoticed but present in potato fields worldwide. A combination of genomic approaches and in planta experiments identified features that separate SCC3193 and other P. wasabiae strains from the rest of soft rot bacteria, such as the absence of a type III secretion system that contributes to virulence of other soft rot species. Experimentally established virulence determinants include the putative transcriptional regulator SirB, two partially redundant type VI secretion systems and two horizontally acquired clusters (Vic1 and Vic2), which contain predicted virulence genes. Genome comparison also revealed other interesting traits that may be related to life in planta or other specific environmental conditions. These traits include a predicted benzoic acid/salicylic acid carboxyl methyltransferase of eukaryotic origin. The novelties found in this work indicate that soft rot bacteria have a reservoir of unknown traits that may be utilized in the poorly understood latent stage in planta. The genomic approaches and the comparison of the model strain SCC3193 to other sequenced Pectobacterium strains, including the type strain of P. wasabiae, provides a solid basis for further investigation of the virulence, distribution and phylogeny of soft rot

  4. Exploration of Modulated Genetic Circuits Governing Virulence Determinants in Staphylococcus aureus.

    PubMed

    Arya, Rekha; Princy, S Adline

    2016-03-01

    The expression of virulence genes in the human pathogen Staphylococcus aureus is strongly influenced by the multiple global regulators. The signal transduction cascade of these global regulators is accountable for recognizing and integrating the environmental cues to regulate the virulence regulon. While the production of virulent factors by individual global regulators are comparatively straightforward to define, auto-regulation of these global regulators and their impact on other regulators is more complex process. There are several reports on the production of virulent factors that are precisely regulated by switching processes of multiple global regulators including some prominent accessory regulators such as agr, sae and sar which allows S. aureus to coordinate the gene expression, and thus, provide organism an ability to act collectively. This review implicates the mechanisms involved in the global regulation of various virulence factors along with a comprehensive discussion on the differences between these signal transduction systems, their auto-induction and, coordination of classical and some comparatively new bacterial signal transduction systems. PMID:26843693

  5. Molecular characterization of virulent Leptospira interrogans serogroup icterohaemorrhagiae isolated from Cavia aperea.

    PubMed

    Monte, Leonardo G; Jorge, Sérgio; Xavier, Marina A; Leal, Fernanda M A; Amaral, Marta G; Seixas, Fabiana K; Dellagostin, Odir A; Hartleben, Cláudia P

    2013-05-01

    Leptospirosis is a worldwide zoonotic infection caused by pathogenic Leptospira. Synanthropic rodents are recognized carriers of leptospires; however, the role of wild rodents in the epidemiology of the disease is still incipient. In this work, we describe Leptospira strain isolated from Cavia aperea (Brazilian guinea pig). The isolated strain was characterized by partial rpoB gene sequencing, variable-number tandem-repeats and histopathological analysis. The strain was identified as Leptospira interrogans, serogroup Icterohaemorrhagiae and caused clinical signs of leptospirosis in the hamster model, attesting to its virulence. In conclusion, these findings could be useful for elucidating the epidemiological role of C. aperea in leptospirosis. PMID:23435256

  6. Molecular weight determinations of biosolubilized coals

    SciTech Connect

    Linehan, J.C.; Clauss, S.; Bean, R.; Campbell, J.

    1991-05-01

    We have compared several different methods for determining the molecular weight of biosolubilized coals: Aqueous gel permeation Chromatography (GPC), organic GPC, preparative GPC, dynamic laser light scattering (LLS), static LLS, static LLS, mass spectrometry, vapor phase osmometry (VPO) and ultrafiltration. We have found that careful consideration must be given to the molecular weight result obtained from each method. The average molecular weight and the molecular weight distribution were found to be dependent upon many factors, including the technique used; molecular weight standards, pH, and the percentage of sample analyzed. Weight average molecular weights, M{sub w}, obtained for biosolubilized leonardite range from 800,000 daltons for neutral pH aqueous GPC based on polyethylene glycol molecular weight standards to 570 daltons for pH 11.5 buffered aqueous GPC based on a fulvic acid standard. It is clear that the state of association of the biocoal analyte, as well as the interactions of sample with the separation matrix, can have large influence of the observed result, and these must be understood before reliable GPC measurements can be made. Furthermore, a uniform set of molecular weight standards for biodegraded coals is needed. 10 refs., 1 tab.

  7. The Neuraminidase Stalk Deletion Serves as Major Virulence Determinant of H5N1 Highly Pathogenic Avian Influenza Viruses in Chicken.

    PubMed

    Stech, Olga; Veits, Jutta; Abdelwhab, El-Sayed M; Wessels, Ute; Mettenleiter, Thomas C; Stech, Jürgen

    2015-01-01

    Highly pathogenic avian influenza viruses (HPAIV) cause devastating losses in gallinaceous poultry world-wide and raised concerns of a novel pandemic. HPAIV develop from low-pathogenic precursors by acquisition of a polybasic HA cleavage site (HACS), the prime virulence determinant. Beside that HACS, other adaptive changes accumulate in those precursors prior to transformation into an HPAIV. Here, we aimed to unravel such virulence determinants in addition to the HA gene. Stepwise reduction of HPAIV genes revealed that the HPAIV HA and NA form a minimum set of virulence determinants, sufficient for a lethal phenotype in chicken. Abolishing the NA stalk deletion considerably reduced lethality and prevented transmission. Conversely, the analogous stalk deletion reconstructed in the NA of an LPAIV reassortant carrying only the HPAIV HA resulted in 100% lethality both after primary and contact infection. Remarkably, the unmodified LPAIV NA with its long stalk, when exclusively introduced into the H5N1 HPAIV, still enabled high virulence and efficient transmission. Therefore, irrespective of an NA stalk deletion, minor virulence determinants in addition to the essential polybasic HACS contribute to high virulence, whereas the NA stalk deletion alone may serve as major virulence determinant. PMID:26306544

  8. The Neuraminidase Stalk Deletion Serves as Major Virulence Determinant of H5N1 Highly Pathogenic Avian Influenza Viruses in Chicken

    PubMed Central

    Stech, Olga; Veits, Jutta; Abdelwhab, El-Sayed M.; Wessels, Ute; Mettenleiter, Thomas C.; Stech, Jürgen

    2015-01-01

    Highly pathogenic avian influenza viruses (HPAIV) cause devastating losses in gallinaceous poultry world-wide and raised concerns of a novel pandemic. HPAIV develop from low-pathogenic precursors by acquisition of a polybasic HA cleavage site (HACS), the prime virulence determinant. Beside that HACS, other adaptive changes accumulate in those precursors prior to transformation into an HPAIV. Here, we aimed to unravel such virulence determinants in addition to the HA gene. Stepwise reduction of HPAIV genes revealed that the HPAIV HA and NA form a minimum set of virulence determinants, sufficient for a lethal phenotype in chicken. Abolishing the NA stalk deletion considerably reduced lethality and prevented transmission. Conversely, the analogous stalk deletion reconstructed in the NA of an LPAIV reassortant carrying only the HPAIV HA resulted in 100% lethality both after primary and contact infection. Remarkably, the unmodified LPAIV NA with its long stalk, when exclusively introduced into the H5N1 HPAIV, still enabled high virulence and efficient transmission. Therefore, irrespective of an NA stalk deletion, minor virulence determinants in addition to the essential polybasic HACS contribute to high virulence, whereas the NA stalk deletion alone may serve as major virulence determinant. PMID:26306544

  9. Human Hemorrhagic Fever Causing Arenaviruses: Molecular Mechanisms Contributing to Virus Virulence and Disease Pathogenesis

    PubMed Central

    Shao, Junjie; Liang, Yuying; Ly, Hinh

    2015-01-01

    Arenaviruses include multiple human pathogens ranging from the low-risk lymphocytic choriomeningitis virus (LCMV) to highly virulent hemorrhagic fever (HF) causing viruses such as Lassa (LASV), Junin (JUNV), Machupo (MACV), Lujo (LUJV), Sabia (SABV), Guanarito (GTOV), and Chapare (CHPV), for which there are limited preventative and therapeutic measures. Why some arenaviruses can cause virulent human infections while others cannot, even though they are isolated from the same rodent hosts, is an enigma. Recent studies have revealed several potential pathogenic mechanisms of arenaviruses, including factors that increase viral replication capacity and suppress host innate immunity, which leads to high viremia and generalized immune suppression as the hallmarks of severe and lethal arenaviral HF diseases. This review summarizes current knowledge of the roles of each of the four viral proteins and some known cellular factors in the pathogenesis of arenaviral HF as well as of some human primary cell-culture and animal models that lend themselves to studying arenavirus-induced HF disease pathogenesis. Knowledge gained from these studies can be applied towards the development of novel therapeutics and vaccines against these deadly human pathogens. PMID:26011826

  10. Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus

    PubMed Central

    Dinamarco, Taísa Magnani; Almeida, Ricardo S.; Alves de Castro, Patrícia; Brown, Neil Andrew; dos Reis, Thaila Fernanda; Zambelli Ramalho, Leandra Naira; Savoldi, Marcela; Goldman, Maria Helena S.

    2012-01-01

    Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The ΔsebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA::GFP (SebA::green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the ΔsebA mutant. The A. fumigatus ΔsebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the ΔsebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses. PMID:22345349

  11. Molecular characterization of the putative transcription factor SebA involved in virulence in Aspergillus fumigatus.

    PubMed

    Dinamarco, Taísa Magnani; Almeida, Ricardo S; de Castro, Patrícia Alves; Brown, Neil Andrew; dos Reis, Thaila Fernanda; Ramalho, Leandra Naira Zambelli; Savoldi, Marcela; Goldman, Maria Helena S; Goldman, Gustavo Henrique

    2012-04-01

    Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The ΔsebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA::GFP (SebA::green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the ΔsebA mutant. The A. fumigatus ΔsebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the ΔsebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses. PMID:22345349

  12. Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase (lpd), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

    PubMed Central

    Hudson, P.; Gorton, T. S.; Papazisi, L.; Cecchini, K.; Frasca, S.; Geary, S. J.

    2006-01-01

    To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7. PMID:16428737

  13. Serogroup, virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand.

    PubMed

    Mala, Wanida; Alam, Munirul; Angkititrakul, Sunpetch; Wongwajana, Suwin; Lulitanond, Viraphong; Huttayananont, Sriwanna; Kaewkes, Wanlop; Faksri, Kiatichai; Chomvarin, Chariya

    2016-04-01

    Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n=74) and cockles (Anadara granosa) (n=74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p<0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p<0.001) and trh (10.8% versus 1.4%) (p<0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources. PMID:26773828

  14. Regulation of Virulence in Staphylococcus aureus: Molecular Mechanisms and Remaining Puzzles.

    PubMed

    Wang, Boyuan; Muir, Tom W

    2016-02-18

    The agr locus encodes a quorum-sensing (QS) circuit required for the virulence of a spectrum of Gram-positive pathogens and is, therefore, regarded as an important target for the development of chemotherapeutics. In recent years, many of the biochemical events in the Staphylococcus aureus agr circuit have been reconstituted and subject to quantitative analysis in vitro. This work, in conjunction with structural studies on several key players in the signaling circuit, has furnished mechanistic insights into the regulation and evolution of the agr QS system. Here, we review this progress and discuss the remaining open questions in the area. We also highlight advances in the discovery of small-molecule agr modulators and how the newly available biochemical and structural information might be leveraged for the design of next-generation therapeutics targeting the agr system. PMID:26971873

  15. Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence

    PubMed Central

    Piacenza, L.; Zago, M.P.; Peluffo, G.; Alvarez, M.N.; Basombrio, M.A.; Radi, R.

    2013-01-01

    Virulence of Trypanosoma cruzi depends on a variety of genetic and biochemical factors. It has been proposed that components of the parasites' antioxidant system may play a key part in this process by pre-adapting the pathogen to the oxidative environment encountered during host cell invasion. Using several isolates (10 strains) belonging to the two major phylogenetic lineages (T. cruzi-I and T. cruzi-II), we investigated whether there was an association between virulence (ranging from highly aggressive to attenuated isolates at the parasitemia and histopathological level) and the antioxidant enzyme content. Antibodies raised against trypanothione synthetase (TcTS), ascorbate peroxidase (TcAPX), mitochondrial and cytosolic tryparedoxin peroxidases (TcMPX and TcCPX) and trypanothione reductase (TcTR) were used to evaluate the antioxidant enzyme levels in epimastigote and metacyclic trypomastigote forms in the T. cruzi strains. Levels of TcCPX, TcMPX and TcTS were shown to increase during differentiation from the non-infective epimastigote to the infective metacyclic trypomastigote stage in all parasite strains examined. Peroxiredoxins were found to be present at higher levels in the metacyclic infective forms of the virulent isolates compared with the attenuated strains. Additionally, an increased resistance of epimastigotes from virulent T. cruzi populations to hydrogen peroxide and peroxynitrite challenge was observed. In mouse infection models, a direct correlation was found between protein levels of TcCPX, TcMPX and TcTS, and the parasitemia elicited by the different isolates studied (Pearson's coefficient: 0.617, 0.771, 0.499; respectively, P < 0.01). No correlation with parasitemia was found for TcAPX and TcTR proteins in any of the strains analyzed. Our data support that enzymes of the parasite antioxidant armamentarium at the onset of infection represent new virulence factors involved in the establishment of disease. PMID:19505468

  16. Structure and Interactions of a Dimeric Variant of sHIP, a Novel Virulence Determinant of Streptococcus pyogenes

    PubMed Central

    Diehl, Carl; Wisniewska, Magdalena; Frick, Inga-Maria; Streicher, Werner; Björck, Lars; Malmström, Johan; Wikström, Mats

    2016-01-01

    Streptococcus pyogenes is one of the most significant bacterial pathogens in the human population mostly causing superficial and uncomplicated infections (pharyngitis and impetigo) but also invasive and life-threatening disease. We have previously identified a virulence determinant, protein sHIP, which is secreted at higher levels by an invasive compared to a non-invasive strain of S. pyogenes. The present work presents a further characterization of the structural and functional properties of this bacterial protein. Biophysical and structural studies have shown that protein sHIP forms stable tetramers both in the crystal and in solution. The tetramers are composed of four helix-loop-helix motifs with the loop regions connecting the helices displaying a high degree of flexibility. Owing to interactions at the tetramer interface, the observed tetramer can be described as a dimer of dimers. We identified three residues at the tetramer interface (Leu84, Leu88, Tyr95), which due to largely non-polar side-chains, could be important determinants for protein oligomerization. Based on these observations, we produced a sHIP variant in which these residues were mutated to alanines. Biophysical experiments clearly indicated that the sHIP mutant appear only as dimers in solution confirming the importance of the interfacial residues for protein oligomerisation. Furthermore, we could show that the sHIP mutant interacts with intact histidine-rich glycoprotein (HRG) and the histidine-rich repeats in HRG, and inhibits their antibacterial activity to the same or even higher extent as compared to the wild type protein sHIP. We determined the crystal structure of the sHIP mutant, which, as a result of the high quality of the data, allowed us to improve the existing structural model of the protein. Finally, by employing NMR spectroscopy in solution, we generated a model for the complex between the sHIP mutant and an HRG-derived heparin-binding peptide, providing further molecular

  17. Structure and Interactions of a Dimeric Variant of sHIP, a Novel Virulence Determinant of Streptococcus pyogenes.

    PubMed

    Diehl, Carl; Wisniewska, Magdalena; Frick, Inga-Maria; Streicher, Werner; Björck, Lars; Malmström, Johan; Wikström, Mats

    2016-01-01

    Streptococcus pyogenes is one of the most significant bacterial pathogens in the human population mostly causing superficial and uncomplicated infections (pharyngitis and impetigo) but also invasive and life-threatening disease. We have previously identified a virulence determinant, protein sHIP, which is secreted at higher levels by an invasive compared to a non-invasive strain of S. pyogenes. The present work presents a further characterization of the structural and functional properties of this bacterial protein. Biophysical and structural studies have shown that protein sHIP forms stable tetramers both in the crystal and in solution. The tetramers are composed of four helix-loop-helix motifs with the loop regions connecting the helices displaying a high degree of flexibility. Owing to interactions at the tetramer interface, the observed tetramer can be described as a dimer of dimers. We identified three residues at the tetramer interface (Leu84, Leu88, Tyr95), which due to largely non-polar side-chains, could be important determinants for protein oligomerization. Based on these observations, we produced a sHIP variant in which these residues were mutated to alanines. Biophysical experiments clearly indicated that the sHIP mutant appear only as dimers in solution confirming the importance of the interfacial residues for protein oligomerisation. Furthermore, we could show that the sHIP mutant interacts with intact histidine-rich glycoprotein (HRG) and the histidine-rich repeats in HRG, and inhibits their antibacterial activity to the same or even higher extent as compared to the wild type protein sHIP. We determined the crystal structure of the sHIP mutant, which, as a result of the high quality of the data, allowed us to improve the existing structural model of the protein. Finally, by employing NMR spectroscopy in solution, we generated a model for the complex between the sHIP mutant and an HRG-derived heparin-binding peptide, providing further molecular

  18. Determining the Overpotential for a Molecular Electrocatalyst

    SciTech Connect

    Appel, Aaron M.; Helm, Monte L.

    2014-02-07

    “The additional potential (beyond the thermodynamic requirement) needed to drive a reaction at a certain rate is called the overpotential.”1 Over the last decade there has been considerable interest in the design and testing of molecular electrocatalysis for the interconversion of renewable energy and chemical fuels.2-5 One of the primary motivations for such research is the replacement of expensive and rare precious metal catalysts, such as platinum, with cheaper, more abundant metals.2,6-8 To become competitive with current electrocatalytic energy conversion technologies, new catalysts must be robust, fast, and energy-efficient. This last feature, the energy-efficiency, is dependent upon the overpotential. For molecular catalysts, the determination and reporting of overpotentials can be complicated by the frequent dependence on assumptions, especially when working in nonaqueous solvents. As overpotentials become lower, the meaningful comparison of molecular catalysts will require improved accuracy and precision. The intended purpose of this viewpoint is to provide a clear and concise description of overpotential and recommendation for its determination in molecular electrocatalysis. This material is based upon work supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences.

  19. Phenotypic Assays to Determine Virulence Factors of Uropathogenic Escherichia coli (UPEC) Isolates and their Correlation with Antibiotic Resistance Pattern

    PubMed Central

    Tabasi, Mohsen; Asadi Karam, Mohammad Reza; Habibi, Mehri; Yekaninejad, Mir Saeed; Bouzari, Saeid

    2015-01-01

    Objectives Urinary tract infection caused by uropathogenic Escherichia coli (UPEC) strains is one of the most important infections in the world. UPEC encode widespread virulence factors closely related with pathogenesis of the bacteria. The purpose of this study was to evaluate the presence of different phenotypic virulence markers in UPEC isolates and determine their correlation with antibiotic resistance pattern. Methods UPEC isolates from patients with different clinical symptoms of UTI were collected and screened for biofilm and hemolysin production, mannose resistant, and mannose sensitive hemagglutination (MRHA and MSHA, respectively). In addition, antimicrobial resistance pattern and ESBL-producing isolates were recorded. Results Of the 156 UPEC isolates, biofilm and hemolysin formation was seen in 133 (85.3%) and 53 (34%) isolates, respectively. Moreover, 98 (62.8%) and 58 (37.2%) isolates showed the presence of Types 1 fimbriae (MSHA) and P fimbriae (MRHA), respectively. Our results also showed a relationship between biofilm formation in UPEC isolated from acute cystitis patients and recurrent UTI cases. Occurrence of UTI was dramatically correlated with the patients' profiles. We observed that the difference in antimicrobial susceptibilities of the biofilm and nonbiofilm former isolates was statistically significant. The UPEC isolates showed the highest resistance to ampicillin, tetracycline, amoxicillin, and cotrimoxazole. Moreover, 26.9% of isolates were ESBL producers. Conclusion This study indicated that there is a relationship between the phenotypic virulence traits of the UPEC isolates, patients' profiles, and antibiotic resistance. Detection of the phenotypic virulence factors could help to improve understanding of pathogenesis of UPEC isolates and better medical intervention. PMID:26473094

  20. Pathway analysis of Candida albicans survival and virulence determinants in a murine infection model.

    PubMed

    Becker, Jeffrey M; Kauffman, Sarah J; Hauser, Melinda; Huang, Liyin; Lin, Molly; Sillaots, Susan; Jiang, Bo; Xu, Deming; Roemer, Terry

    2010-12-21

    One potentially rich source of possible targets for antifungal therapy are those Candida albicans genes deemed essential for growth under the standard culture (i.e., in vitro) conditions; however, these genes are largely unexplored as drug targets because essential genes are not experimentally amenable to conventional gene deletion and virulence studies. Using tetracycline-regulatable promoter-based conditional mutants, we investigated a murine model of candidiasis in which repressing essential genes in the host was achieved. By adding doxycycline to the drinking water starting 3 days prior to (dox - 3D) or 2 days post (dox + 2D) infection, the phenotypic consequences of temporal gene inactivation were assessed by monitoring animal survival and fungal burden in prophylaxis and acute infection settings. Of 177 selected conditional shut-off strains tested, the virulence of 102 was blocked under both repressing conditions, suggesting that the corresponding genes are essential for growth and survival in a murine host across early and established infection periods. Among these genes were those previously identified as antifungal drug targets (i.e., FKS1, ERG1, and ERG11), verifying that this methodology can be used to validate potential new targets. We also identify genes either conditionally essential or dispensable for in vitro growth but required for survival and virulence, including those in late stage ergosterol synthesis, or early steps in fatty acid or riboflavin biosynthesis. This study evaluates the role of essential genes with respect to pathogen virulence in a large-scale, systems biology context, and provides a general method for gene target validation and for uncovering unexpected antimicrobial targets. PMID:21135205

  1. Molecular determinants for a cardiovascular collapse in anthrax

    PubMed Central

    Brojatsch, Jurgen; Casadevall, Arturo; Goldman, David L.

    2015-01-01

    Bacillus anthracis releases two bipartite proteins, lethal toxin and edema factor, that contribute significantly to the progression of anthrax-associated shock. As blocking the anthrax toxins prevents disease, the toxins are considered the main virulence factors of the bacterium. The anthrax bacterium and the anthrax toxins trigger multiorgan failure associated with enhanced vascular permeability, hemorrhage and cardiac dysfunction in animal challenge models. A recent study using mice that either lacked the anthrax toxin receptor in specific cells and corresponding mice expressing the receptor in specific cell types demonstrated that cardiovascular cells are critical for disease mediated by anthrax lethal toxin. These studies are consistent with involvement of the cardiovascular system, and with an increase of cardiac failure markers observed in human anthrax and in animal models using B. anthracis and anthrax toxins. This review discusses the current state of knowledge regarding the pathophysiology of anthrax and tries to provide a mechanistic model and molecular determinants for the circulatory shock in anthrax. PMID:24389148

  2. Molecular determinants for a cardiovascular collapse in anthrax.

    PubMed

    Brojatsch, Jurgen; Casadevall, Arturo; Goldman, David L

    2014-01-01

    Bacillus anthracis releases two bipartite proteins, lethal toxin and edema factor, that contribute significantly to the progression of anthrax-associated shock. As blocking the anthrax toxins prevents disease, the toxins are considered the main virulence factors of the bacterium. The anthrax bacterium and the anthrax toxins trigger multi-organ failure associated with enhanced vascular permeability, hemorrhage and cardiac dysfunction in animal challenge models. A recent study using mice that either lacked the anthrax toxin receptor in specific cells and corresponding mice expressing the receptor in specific cell types demonstrated that cardiovascular cells are critical for disease mediated by anthrax lethal toxin. These studies are consistent with involvement of the cardiovascular system, and with an increase of cardiac failure markers observed in human anthrax and in animal models using B. anthracis and anthrax toxins. This review discusses the current state of knowledge regarding the pathophysiology of anthrax and tries to provide a mechanistic model and molecular determinants for the circulatory shock in anthrax. PMID:24389148

  3. Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation.

    PubMed

    Hillmann, Falk; Novohradská, Silvia; Mattern, Derek J; Forberger, Tilmann; Heinekamp, Thorsten; Westermann, Martin; Winckler, Thomas; Brakhage, Axel A

    2015-08-01

    Filamentous fungi represent classical examples for environmentally acquired human pathogens whose major virulence mechanisms are likely to have emerged long before the appearance of innate immune systems. In natural habitats, amoeba predation could impose a major selection pressure towards the acquisition of virulence attributes. To test this hypothesis, we exploited the amoeba Dictyostelium discoideum to study its interaction with Aspergillus fumigatus, two abundant soil inhabitants for which we found co-occurrence in various sites. Fungal conidia were efficiently taken up by D. discoideum, but ingestion was higher when conidia were devoid of the green fungal spore pigment dihydroxynaphtalene melanin, in line with earlier results obtained for immune cells. Conidia were able to survive phagocytic processing, and intracellular germination was initiated only after several hours of co-incubation which eventually led to a lethal disruption of the host cell. Besides phagocytic interactions, both amoeba and fungus secreted cross inhibitory factors which suppressed fungal growth or induced amoeba aggregation with subsequent cell lysis, respectively. On the fungal side, we identified gliotoxin as the major fungal factor killing Dictyostelium, supporting the idea that major virulence attributes, such as escape from phagocytosis and the secretion of mycotoxins are beneficial to escape from environmental predators. PMID:25684622

  4. Molecular and virulence characteristics of multi-drug resistant Salmonella Enteritidis strains isolated from poultry.

    PubMed

    Hur, Jin; Kim, Ji Hee; Park, Jong Ho; Lee, Young-Ju; Lee, John Hwa

    2011-09-01

    Forty-six Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strains were isolated from chicken meat, faeces, and eggshells collected from hatcheries throughout Korea. The strains were examined for the presence of antimicrobial resistance and virulence genes. All 46 isolates were resistant to at least one of 21 antibiotics used in this study, 30 (65.2%) were resistant to three or more antimicrobials, and a single remarkable isolate was resistant to 15 antimicrobials. The isolates were primarily resistant to penicillins, sulfisoxazole, streptomycin, tetracycline and quinolones. The high rate of resistance in S. Enteritidis strains, sometimes to multiple drugs, may complicate future options for treating human infections. Nineteen of the 21 penicillin resistant isolates carried the bla(TEM) gene, while one strain, resistant both to penicillins and ceftriaxone, carried the bla(CTX-M) gene. Thirty-seven of the 45 sulfisoxazole resistant isolates carried sul2, and 23/24 streptomycin resistant isolates carried both strA and strB. All 10 tetracycline resistant isolates carried the tet(A) gene. Most isolates harboured both SPI-1 and SPI-2-associated genes, and the spv operon, which are known to be associated with human infections. The presence of these genes suggests that these strains could give rise to public health problems if dispersed in the general human population. PMID:20822940

  5. Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for....food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Yersinia pestis, Y. pseudotuberculosis, and Y, enterocolitica, phenotypic expression of several virulence plasmid (pYV: 70-kb)-associated genetic determinants may include low calcium response (Lcr, pin point colony, size = 0.36 mm), colony morphology (size = 1.13 mm), crystal violet (CV) binding...

  6. Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for...food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Yersinia pestis, Y. pseudotuberculosis, and Y, enterocolitica, phenotypic expression of virulence plasmid (pYV: 70-kb)-associated genetic determinants may include low calcium response (Lcr, pin point colony, size = 0.36 mm), colony morphology (size = 1.13 mm), crystal violet (CV) binding (dark-v...

  7. Molecular Mechanisms of Sex Determination in Reptiles

    PubMed Central

    Rhen, T.; Schroeder, A.

    2010-01-01

    Charles Darwin first provided a lucid explanation of how gender differences evolve nearly 140 years ago. Yet, a disconnect remains between his theory of sexual selection and the mechanisms that underlie the development of males and females. In particular, comparisons between representatives of different phyla (i.e., flies and mice) reveal distinct genetic mechanisms for sexual differentiation. Such differences are hard to comprehend unless we study organisms that bridge the phylogenetic gap. Analysis of variation within monophyletic groups (i.e., amniotes) is just as important if we hope to elucidate the evolution of mechanisms underlying sexual differentiation. Here we review the molecular, cellular, morphological, and physiological changes associated with sex determination in reptiles. Most research on the molecular biology of sex determination in reptiles describes expression patterns for orthologs of mammalian sex-determining genes. Many of these genes have evolutionarily conserved expression profiles (i.e., DMRT1 and SOX9 are expressed at a higher level in developing testes vs. developing ovaries in all species), which suggests functional conservation. However, expression profiling alone does not test gene function and will not identify novel sex-determining genes or gene interactions. For that reason, we provide a prospectus on various techniques that promise to reveal new sex-determining genes and regulatory interactions among these genes. We offer specific examples of novel candidate genes and a new signaling pathway in support of these techniques. PMID:20145384

  8. Chromosomal islands of Streptococcus pyogenes and related streptococci: molecular switches for survival and virulence

    PubMed Central

    Nguyen, Scott V.; McShan, William M.

    2014-01-01

    Streptococcus pyogenes is a significant pathogen of humans, annually causing over 700,000,000 infections and 500,000 deaths. Virulence in S. pyogenes is closely linked to mobile genetic elements like phages and chromosomal islands (CI). S. pyogenes phage-like chromosomal islands (SpyCI) confer a complex mutator phenotype on their host. SpyCI integrate into the 5′ end of DNA mismatch repair (MMR) gene mutL, which also disrupts downstream operon genes lmrP, ruvA, and tag. During early logarithmic growth, SpyCI excise from the bacterial chromosome and replicate as episomes, relieving the mutator phenotype. As growth slows and the cells enter stationary phase, SpyCI reintegrate into the chromosome, again silencing the MMR operon. This system creates a unique growth-dependent and reversible mutator phenotype. Additional CI using the identical attachment site in mutL have been identified in related species, including Streptococcus dysgalactiae subsp. equisimilis, Streptococcus anginosus, Streptococcus intermedius, Streptococcus parauberis, and Streptococcus canis. These CI have small genomes, which range from 13 to 20 kB, conserved integrase and DNA replication genes, and no identifiable genes encoding capsid proteins. SpyCI may employ a helper phage for packaging and dissemination in a fashion similar to the Staphylococcus aureus pathogenicity islands (SaPI). Outside of the core replication and integration genes, SpyCI and related CI show considerable diversity with the presence of many indels that may contribute to the host cell phenotype or fitness. SpyCI are a subset of a larger family of streptococcal CI who potentially regulate the expression of other host genes. The biological and phylogenetic analysis of streptococcal chromosomal islands provides important clues as to how these chromosomal islands help S. pyogenes and other streptococcal species persist in human populations in spite of antibiotic therapy and immune challenges. PMID:25161960

  9. Chromosomal islands of Streptococcus pyogenes and related streptococci: molecular switches for survival and virulence.

    PubMed

    Nguyen, Scott V; McShan, William M

    2014-01-01

    Streptococcus pyogenes is a significant pathogen of humans, annually causing over 700,000,000 infections and 500,000 deaths. Virulence in S. pyogenes is closely linked to mobile genetic elements like phages and chromosomal islands (CI). S. pyogenes phage-like chromosomal islands (SpyCI) confer a complex mutator phenotype on their host. SpyCI integrate into the 5' end of DNA mismatch repair (MMR) gene mutL, which also disrupts downstream operon genes lmrP, ruvA, and tag. During early logarithmic growth, SpyCI excise from the bacterial chromosome and replicate as episomes, relieving the mutator phenotype. As growth slows and the cells enter stationary phase, SpyCI reintegrate into the chromosome, again silencing the MMR operon. This system creates a unique growth-dependent and reversible mutator phenotype. Additional CI using the identical attachment site in mutL have been identified in related species, including Streptococcus dysgalactiae subsp. equisimilis, Streptococcus anginosus, Streptococcus intermedius, Streptococcus parauberis, and Streptococcus canis. These CI have small genomes, which range from 13 to 20 kB, conserved integrase and DNA replication genes, and no identifiable genes encoding capsid proteins. SpyCI may employ a helper phage for packaging and dissemination in a fashion similar to the Staphylococcus aureus pathogenicity islands (SaPI). Outside of the core replication and integration genes, SpyCI and related CI show considerable diversity with the presence of many indels that may contribute to the host cell phenotype or fitness. SpyCI are a subset of a larger family of streptococcal CI who potentially regulate the expression of other host genes. The biological and phylogenetic analysis of streptococcal chromosomal islands provides important clues as to how these chromosomal islands help S. pyogenes and other streptococcal species persist in human populations in spite of antibiotic therapy and immune challenges. PMID:25161960

  10. Modulation of type I IFN induction by a virulence determinant within the alphavirus nsP1 protein.

    PubMed

    Cruz, Catherine C; Suthar, Mehul S; Montgomery, Stephanie A; Shabman, Reed; Simmons, Jason; Johnston, Robert E; Morrison, Thomas E; Heise, Mark T

    2010-03-30

    Alphaviruses are mosquito-borne viruses that cause serious human and animal diseases. Previous studies demonstrated that a determinant within the nsP1/nsP2 cleavage domain of the virulent Sindbis AR86 virus played a key role in regulating adult mouse virulence without adversely affecting viral replication. Additional characterization of this determinant demonstrated that a virus with the attenuating mutation induced more type I IFN production both in vivo and in vitro. Interestingly, this phenotype was not specific to the Sindbis AR86 virus, as a similar mutation in a distantly related alphavirus, Ross River Virus (RRV), also led to enhanced IFN induction. This effect was independent of virus-induced host shutoff, since IRF-3 phosphorylation, which occurs independently of de novo host transcription/translation, was induced more robustly in cells infected with the mutant viruses. Altogether, these results demonstrate that critical determinants within the nsP1/nsP2 cleavage domain play an important role in regulating alphavirus-induced IFN responses. PMID:20097400

  11. The molecular circuitry governing retinal determination.

    PubMed

    Kumar, Justin P

    2009-04-01

    The developing eye of the fruit fly, Drosophila melanogaster, has become a premier model system for studying the genetic and molecular mechanisms that govern tissue determination. Over the last fifteen years a regulatory circuit consisting of the members of the Pax, Six, Eya and Dach gene families has been identified and shown to govern the specification of a wide range of tissues including the retina of both insects and mammals. These genes are not organized in a simple developmental pathway or cascade in which there is a unidirectional flow of information. Rather, there are multiple feedback loops built into the system rendering its appearance and functionality more in line with the workings of a network. In this review I will attempt to describe the genetic, molecular and biochemical interactions that govern the specification of the Drosophila compound eye. In particular, the primary focus will be on the interactions that have been experimentally verified at the molecular and biochemical levels. During the course of this description I will also attempt to place each discovery in its own historical context. While a number of signaling pathways play significant roles in early eye development this review will focus on the network of nuclear factors that promote retinal determination. PMID:19013263

  12. The Molecular Circuitry Governing Retinal Determination

    PubMed Central

    Kumar, Justin P.

    2009-01-01

    The developing eye of the fruit fly, Drosophila melanogaster, has become a premier model system for studying the genetic and molecular mechanisms that govern tissue determination. Over the last fifteen years a regulatory circuit consisting of the members of the Pax, Six, Eya and Dach gene families has been identified and shown to govern the specification of a wide range of tissues including the retina of both insects and mammals. These genes are not organized in a simple developmental pathway or cascade in which there is a unidirectional flow of information. Rather, there are multiple feedback loops built into the system rendering its appearance and functionality more in line with the workings of a network. In this review I will attempt to describe the genetic, molecular and biochemical interactions that govern the specification of the Drosophila compound eye. In particular, the primary focus will be on the interactions that have been experimentally verified at the molecular and biochemical levels. During the course of this description I will also attempt to place each discovery in its own historical context. While a number of signaling pathways play significant roles in early eye development this review will focus on the network of nuclear factors that promote retinal determination. PMID:19013263

  13. Characterization of the low-molecular-mass proteins of virulent Treponema pallidum.

    PubMed Central

    Stamm, L V; Parrish, E A

    1994-01-01

    We previously demonstrated that Treponema pallidum cells incubated in vitro in the presence of heat-inactivated normal rabbit serum (HINRS) synthesize, in very small quantities, several pathogen-specific, low-molecular-mass proteins that appear to be localized extracellularly. In this study, we have taken advantage of our ability to metabolically radiolabel T. pallidum cells to high specific activity to further characterize these antigens. We found that the low-molecular-mass proteins are not related to the 15- and 17-kDa detergent-phase proteins (J. D. Radolf, N. R. Chamberlain, A. Clausell, and M. V. Norgard, Infect. Immun. 56:490-498, 1988). The low-molecular-mass proteins did not incorporate 3H-labeled fatty acids and were not precipitated by rabbit immunoglobulin G (IgG) antibodies directed against glutathione S-transferase fusions to the nonlipidated 15- and 17-kDa proteins. We prepared polyclonal antisera to the low-molecular-mass proteins by immunizing two rabbits with the concentrated supernatant of T. pallidum cells. IgG antibodies present in the sera of both rabbits precipitated a 21.5-kDa protein from solubilized extracts of T. pallidum supernatant and cells. IgG antibodies in the serum of the second rabbit precipitated an additional 15.5-kDa low-molecular-mass protein only from solubilized extracts of supernatant. While investigating the effect of eliminating HINRS from the extraction medium, we observed that the low-molecular-mass proteins remained associated with treponemal cells that were incubated in the absence of HINRS. These proteins could be eluted from the cells by the addition of HINRS or rabbit serum albumin, suggesting that they are located on or near the treponemal cell surface. The 15.5- and 21.5-kDa low-molecular-mass proteins were not washed off treponemal cells with buffer containing 1 M KCl. Experiments employing selective solubilization of the T. pallidum outer membrane with 0.1% Triton X-114 and proteinase K accessibility indicated

  14. Physiological and molecular determinants of embryo implantation

    PubMed Central

    Zhang, Shuang; Lin, Haiyan; Kong, Shuangbo; Wang, Shumin; Wang, Hongmei; Wang, Haibin; Armant, D. Randall

    2014-01-01

    Embryo implantation involves the intimate interaction between an implantation-competent blastocyst and a receptive uterus, which occurs in a limited time period known as the window of implantation. Emerging evidence shows that defects originating during embryo implantation induce ripple effects with adverse consequences on later gestation events, highlighting the significance of this event for pregnancy success. Although a multitude of cellular events and molecular pathways involved in embryo-uterine crosstalk during implantation have been identified through gene expression studies and genetically engineered mouse models, a comprehensive understanding of the nature of embryo implantation is still missing. This review focuses on recent progress with particular attention to physiological and molecular determinants of blastocyst activation, uterine receptivity, blastocyst attachment and uterine decidualization. A better understanding of underlying mechanisms governing embryo implantation should generate new strategies to rectify implantation failure and improve pregnancy rates in women. PMID:23290997

  15. Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

    PubMed

    Petersen, Lauren M; Tisa, Louis S

    2014-11-01

    A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. PMID:25182493

  16. Molecular Characterization of Protease Activity in Serratia sp. Strain SCBI and Its Importance in Cytotoxicity and Virulence

    PubMed Central

    Petersen, Lauren M.

    2014-01-01

    A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. PMID:25182493

  17. Virulence Attributes and Host Response Assays for Determining Pathogenic Potential of Pseudomonas Strains Used in Biotechnology

    PubMed Central

    Tayabali, Azam F.; Coleman, Gordon; Nguyen, Kathy C.

    2015-01-01

    Pseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications. PMID:26619347

  18. Detection of antibiotic resistance, virulence gene determinants and biofilm formation in Aeromonas species isolated from cattle.

    PubMed

    Igbinosa, Isoken H; Igbinosa, Etinosa O; Okoh, Anthony I

    2015-11-01

    This study aimed to assess the antibiogram of Aeromonas strains recovered from cattle faeces and the potential pathogenic status of the isolates. The antibiogram of the Aeromonas isolates demonstrated total resistance to clindamycin oxacillin, trimethoprim, novobiocin and ticarcillin. However, Aeromonas strains were sensitive to cefotaxime, oxytetracycline and tobramycin. The Aeromonas strains from Lovedale and Fort Cox farms were found to possess some virulence genes. The percentage distribution was aer 71.4%, ast 35.7%, fla 60.7%, lip 35.7% and hlyA 25% for Lovedale farm and aer 63.1%, alt 10.5%, ast 55.2%, fla 78.9%, lip 21% and hlyA 35.9% for Fort Cox farm. Class 1 integron was present in 27% of Aeromonas isolates; the bla TEM gene was present in 34.8%, while the blaP1 class A β-lactamase gene was detected in 12.1% of the isolates. Approximately 86% of the isolates formed a biofilm on microtitre plates. The presence of multiple antibiotic resistance and virulence genes in Aeromonas isolates from cattle faeces reveals the pathogenic and infectious importance of these isolates and is of great significance to public health. The possession of a biofilm-forming capability by such isolates may lead to difficulty during the management of infection related to Aeromonas species. PMID:26143545

  19. Molecular Characterization of Gβ-Like Protein CpcB Involved in Antifungal Drug Susceptibility and Virulence in A. fumigatus.

    PubMed

    Cai, Zhendong; Chai, Yanfei; Zhang, Caiyun; Feng, Ruoyun; Sang, Hong; Lu, Ling

    2016-01-01

    Aspergillus fumigatus is an airborne human fungal pathogen that can survive in a wide range of environmental condition. G protein complex transduces external signals from a variety of stimuli outside a cell to its interior effectors in all eukaryotes. Gβ-like CpcB (cross pathway control B) belongs to a WD40 repeat protein family with the conserved G-H and W-D residues. Previous studies have demonstrated that Gβ-like proteins cooperate with related signal transduction proteins to function during many important developmental processes in A. fumigatus. However, the molecular characteristics of Gβ-like CpcB have not yet been identified. In this study, we demonstrated that the G-H residues in WD repeat 1, 2, 3, and the W-D residue in WD repeat 2 of CpcB are required not only to control normal hyphal growth and conidiation but also to affect antifungal drug susceptibility. The enhanced drug resistance might be due to reduced intracellular drug accumulation and altered ergosterol component. Moreover, we find that the first G-H residue of CpcB plays an important role in the virulence of A. fumigatus. To our knowledge, this is the first report for finding the importance of the conserved G-H and W-D residues for a Gβ-like protein in understanding of G protein functions. PMID:26903985

  20. Molecular Characterization of Gβ-Like Protein CpcB Involved in Antifungal Drug Susceptibility and Virulence in A. fumigatus

    PubMed Central

    Cai, Zhendong; Chai, Yanfei; Zhang, Caiyun; Feng, Ruoyun; Sang, Hong; Lu, Ling

    2016-01-01

    Aspergillus fumigatus is an airborne human fungal pathogen that can survive in a wide range of environmental condition. G protein complex transduces external signals from a variety of stimuli outside a cell to its interior effectors in all eukaryotes. Gβ-like CpcB (cross pathway control B) belongs to a WD40 repeat protein family with the conserved G–H and W–D residues. Previous studies have demonstrated that Gβ-like proteins cooperate with related signal transduction proteins to function during many important developmental processes in A. fumigatus. However, the molecular characteristics of Gβ-like CpcB have not yet been identified. In this study, we demonstrated that the G–H residues in WD repeat 1, 2, 3, and the W–D residue in WD repeat 2 of CpcB are required not only to control normal hyphal growth and conidiation but also to affect antifungal drug susceptibility. The enhanced drug resistance might be due to reduced intracellular drug accumulation and altered ergosterol component. Moreover, we find that the first G–H residue of CpcB plays an important role in the virulence of A. fumigatus. To our knowledge, this is the first report for finding the importance of the conserved G–H and W–D residues for a Gβ-like protein in understanding of G protein functions. PMID:26903985

  1. The Defective Prophage Pool of Escherichia coli O157: Prophage–Prophage Interactions Potentiate Horizontal Transfer of Virulence Determinants

    PubMed Central

    Asadulghani, Md; Ogura, Yoshitoshi; Ooka, Tadasuke; Itoh, Takehiko; Sawaguchi, Akira; Iguchi, Atsushi; Nakayama, Keisuke; Hayashi, Tetsuya

    2009-01-01

    Bacteriophages are major genetic factors promoting horizontal gene transfer (HGT) between bacteria. Their roles in dynamic bacterial genome evolution have been increasingly highlighted by the fact that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes. Enterohemorrhagic Escherichia coli O157 is the most striking case. A sequenced strain (O157 Sakai) possesses 18 prophages (Sp1–Sp18) that encode numerous genes related to O157 virulence, including those for two potent cytotoxins, Shiga toxins (Stx) 1 and 2. However, most of these prophages appeared to contain multiple genetic defects. To understand whether these defective prophages have the potential to act as mobile genetic elements to spread virulence determinants, we looked closely at the Sp1–Sp18 sequences, defined the genetic defects of each Sp, and then systematically analyzed all Sps for their biological activities. We show that many of the defective prophages, including the Stx1 phage, are inducible and released from O157 cells as particulate DNA. In fact, some prophages can even be transferred to other E. coli strains. We also show that new Stx1 phages are generated by recombination between the Stx1 and Stx2 phage genomes. The results indicate that these defective prophages are not simply genetic remnants generated in the course of O157 evolution, but rather genetic elements with a high potential for disseminating virulence-related genes and other genetic traits to other bacteria. We speculate that recombination and various other types of inter-prophage interactions in the O157 prophage pool potentiate such activities. Our data provide new insights into the potential activities of the defective prophages embedded in bacterial genomes and lead to the formulation of a novel concept of inter-prophage interactions in defective prophage communities. PMID:19412337

  2. VIRULENCE AND MOLECULAR CHARACTERIZATION OF A WORLD-WIDE COLLECTION OF PUCCINIA TRITICINA FROM DURUM WHEAT COMPARED TO U.S. BREAD WHEAT RACES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leaf rust caused by Puccinia triticina, has recently become more prevalent on durum wheat in Europe, North and South America. We determined the virulence specificities of P. triticina isolates from durum wheat from Argentina, Chile, Ethiopia, France, Mexico, Spain and the USA, and 10 representative...

  3. VIRULENCE AND MOLECULAR CHARACTERIZATION OF A WORLD-WIDE COLLECTION OF PUCCINIA TRITICINA FROM DURUM WHEAT COMPARED TO US BREAD WHEAT RACES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leaf rust caused by Puccinia triticina, has recently become more prevalent on durum wheat in Europe, North and South America. We determined the virulence specificities of P. triticina isolates from durum wheat from Argentina, Chile, Ethiopia, France, Mexico, Spain and the USA, and 10 representative ...

  4. Molecular pathogenesis of Helicobacter pylori infection: the role of bacterial virulence factors.

    PubMed

    Molnar, Bela; Galamb, Orsolya; Sipos, Ferenc; Leiszter, Katalin; Tulassay, Zsolt

    2010-01-01

    Helicobacter pylori is one of the most common pathogens affecting humankind, infecting approximately 50% of the world's population. Of those infected, many will develop asymptomatic gastritis, but 10% develop gastric or duodenal ulcers. The clinical outcome of the infection may involve a combination of bacterial factors, host factors and environmental factors. In the process of development of gastritis, ulceration and cancer, several cellular and molecular steps follow each other. Infection, acid survival, adhesion, cytotoxicity, epithelial cell turnover changes, inflammation, regeneration or pathological alteration towards erosions, ulceration, and cancer can be observed on the cellular level. Bacterial factors like urease, AmiE, AmiF, hydrogenase and arginase are needed for survival in the acidic gastric environment. The bacterial flagellae are essential to move the bacteria towards the epithelial surface. Adhesive factors like BabA, SabA and ureaseA are necessary for adhesion against MHC-II complexes and Le antigens. The bacteria VacA and CagA are cytotoxic factors. The Cag type IV secretion system delivers these proteins inside the epithelial cells. After disruption of epithelial cell junctions, the bacteria can pass through the gastric wall facing direct immune response from neutrophils, lymphocytes, mast cells and dendritic cells. This review describes and summarizes our present molecular biological information and knowledge about the Helicobacter infective component, cell functions and processes. The possible role of host counter responses and interactions with gastric epithelia and immune cells are also detailed. PMID:21088410

  5. NPS6, Encoding a Nonribosomal Peptide Synthetase Involved in Siderophore-Mediated Iron Metabolism, Is a Conserved Virulence Determinant of Plant Pathogenic Ascomycetes[W

    PubMed Central

    Oide, Shinichi; Moeder, Wolfgang; Krasnoff, Stuart; Gibson, Donna; Haas, Hubertus; Yoshioka, Keiko; Turgeon, B. Gillian

    2006-01-01

    NPS6, encoding a nonribosomal peptide synthetase, is a virulence determinant in the maize (Zea mays) pathogen Cochliobolus heterostrophus and is involved in tolerance to H2O2. Deletion of NPS6 orthologs in the rice (Oryza sativa) pathogen, Cochliobolus miyabeanus, the wheat (Triticum aestivum) pathogen, Fusarium graminearum, and the Arabidopsis thaliana pathogen, Alternaria brassicicola, resulted in reduced virulence and hypersensitivity to H2O2. Introduction of the NPS6 ortholog from the saprobe Neurospora crassa to the Δnps6 strain of C. heterostrophus restored wild-type virulence to maize and tolerance to H2O2, demonstrating functional conservation in filamentous ascomycete phytopathogens and saprobes. Increased sensitivity to iron depletion was identified as a conserved phenotype of Δnps6 strains. Exogenous application of iron enhanced the virulence of Δnps6 strains of C. heterostrophus, C. miyabeanus, F. graminearum, and A. brassicicola to each host. NPS6 is responsible for the biosynthesis of extracellular siderophores by C. heterostrophus, F. graminearum, and A. brassicicola. Application of the extracellular siderophore of A. brassicicola restored wild-type virulence of the ΔAbnps6 strain to Arabidopsis. It is proposed that the role of extracellular siderophores in fungal virulence to plants is to supply an essential nutrient, iron, to their producers in planta and not to act as phytotoxins, depriving their hosts of iron. PMID:17056706

  6. 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

  7. The contribution of group A streptococcal virulence determinants to the pathogenesis of sepsis.

    PubMed

    Reglinski, Mark; Sriskandan, Shiranee

    2014-01-01

    Streptococcus pyogenes (group A streptococcus, GAS) is responsible for a wide range of pathologies ranging from mild pharyngitis and impetigo to severe invasive soft tissue infections. Despite the continuing susceptibility of the bacterium to β-lactam antibiotics there has been an unexplained resurgence in the prevalence of invasive GAS infection over the past 30 years. Of particular importance was the emergence of a GAS-associated sepsis syndrome that is analogous to the systemic toxicosis associated with TSST-1 producing strains of Staphylococcus aureus. Despite being recognized for over 20 years, the etiology of GAS associated sepsis and the streptococcal toxic shock syndrome remains poorly understood. Here we review the virulence factors that contribute to the etiology of GAS associated sepsis with a particular focus on coagulation system interactions and the role of the superantigens in the development of streptococcal toxic shock syndrome. PMID:24157731

  8. The contribution of group A streptococcal virulence determinants to the pathogenesis of sepsis

    PubMed Central

    Reglinski, Mark; Sriskandan, Shiranee

    2014-01-01

    Streptococcus pyogenes (group A streptococcus, GAS) is responsible for a wide range of pathologies ranging from mild pharyngitis and impetigo to severe invasive soft tissue infections. Despite the continuing susceptibility of the bacterium to β-lactam antibiotics there has been an unexplained resurgence in the prevalence of invasive GAS infection over the past 30 years. Of particular importance was the emergence of a GAS-associated sepsis syndrome that is analogous to the systemic toxicosis associated with TSST-1 producing strains of Staphylococcus aureus. Despite being recognized for over 20 years, the etiology of GAS associated sepsis and the streptococcal toxic shock syndrome remains poorly understood. Here we review the virulence factors that contribute to the etiology of GAS associated sepsis with a particular focus on coagulation system interactions and the role of the superantigens in the development of streptococcal toxic shock syndrome. PMID:24157731

  9. The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei.

    PubMed

    Burtnick, Mary N; Brett, Paul J; Harding, Sarah V; Ngugi, Sarah A; Ribot, Wilson J; Chantratita, Narisara; Scorpio, Angelo; Milne, Timothy S; Dean, Rachel E; Fritz, David L; Peacock, Sharon J; Prior, Joanne L; Atkins, Timothy P; Deshazer, David

    2011-04-01

    The Burkholderia pseudomallei K96243 genome encodes six type VI secretion systems (T6SSs), but little is known about the role of these systems in the biology of B. pseudomallei. In this study, we purified recombinant Hcp proteins from each T6SS and tested them as vaccine candidates in the BALB/c mouse model of melioidosis. Recombinant Hcp2 protected 80% of mice against a lethal challenge with K96243, while recombinant Hcp1, Hcp3, and Hcp6 protected 50% of mice against challenge. Hcp6 was the only Hcp constitutively produced by B. pseudomallei in vitro; however, it was not exported to the extracellular milieu. Hcp1, on the other hand, was produced and exported in vitro when the VirAG two-component regulatory system was overexpressed in trans. We also constructed six hcp deletion mutants (Δhcp1 through Δhcp6) and tested them for virulence in the Syrian hamster model of infection. The 50% lethal doses (LD(50)s) for the Δhcp2 through Δhcp6 mutants were indistinguishable from K96243 (<10 bacteria), but the LD(50) for the Δhcp1 mutant was >10(3) bacteria. The hcp1 deletion mutant also exhibited a growth defect in RAW 264.7 macrophages and was unable to form multinucleated giant cells in this cell line. Unlike K96243, the Δhcp1 mutant was only weakly cytotoxic to RAW 264.7 macrophages 18 h after infection. The results suggest that the cluster 1 T6SS is essential for virulence and plays an important role in the intracellular lifestyle of B. pseudomallei. PMID:21300775

  10. Russian populations of Puccinia triticina in distant regions are not differentiated for virulence and molecular genotype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to determine whether genetically distinct groups of Puccinia triticina are present in four regions of Russia. Collections of P. triticina were obtained from the Central, North Caucasus, Volga, and West Siberia regions of Russia from 2006 to 2010. Ninety-nine single ur...

  11. Biochemical, Structural and Molecular Dynamics Analyses of the Potential Virulence Factor RipA from Yersinia pestis

    PubMed Central

    Torres, Rodrigo; Swift, Robert V.; Chim, Nicholas; Wheatley, Nicole; Lan, Benson; Atwood, Brian R.; Pujol, Céline; Sankaran, Banu; Bliska, James B.; Amaro, Rommie E.; Goulding, Celia W.

    2011-01-01

    Human diseases are attributed in part to the ability of pathogens to evade the eukaryotic immune systems. A subset of these pathogens has developed mechanisms to survive in human macrophages. Yersinia pestis, the causative agent of the bubonic plague, is a predominately extracellular pathogen with the ability to survive and replicate intracellularly. A previous study has shown that a novel rip (required for intracellular proliferation) operon (ripA, ripB and ripC) is essential for replication and survival of Y. pestis in postactivated macrophages, by playing a role in lowering macrophage-produced nitric oxide (NO) levels. A bioinformatics analysis indicates that the rip operon is conserved among a distally related subset of macrophage-residing pathogens, including Burkholderia and Salmonella species, and suggests that this previously uncharacterized pathway is also required for intracellular survival of these pathogens. The focus of this study is ripA, which encodes for a protein highly homologous to 4-hydroxybutyrate-CoA transferase; however, biochemical analysis suggests that RipA functions as a butyryl-CoA transferase. The 1.9 Å X-ray crystal structure reveals that RipA belongs to the class of Family I CoA transferases and exhibits a unique tetrameric state. Molecular dynamics simulations are consistent with RipA tetramer formation and suggest a possible gating mechanism for CoA binding mediated by Val227. Together, our structural characterization and molecular dynamic simulations offer insights into acyl-CoA specificity within the active site binding pocket, and support biochemical results that RipA is a butyryl-CoA transferase. We hypothesize that the end product of the rip operon is butyrate, a known anti-inflammatory, which has been shown to lower NO levels in macrophages. Thus, the results of this molecular study of Y. pestis RipA provide a structural platform for rational inhibitor design, which may lead to a greater understanding of the role of RipA in

  12. The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis.

    PubMed Central

    Patti, J M; Bremell, T; Krajewska-Pietrasik, D; Abdelnour, A; Tarkowski, A; Rydén, C; Höök, M

    1994-01-01

    The importance of a collagen-binding adhesin in the pathogenesis of septic arthritis has been examined by comparing the virulence of two sets of Staphylococcus aureus mutants in an animal model. Collagen adhesin-negative mutant PH100 was constructed by replacing the chromosomal collagen adhesin gene (cna) in a clinical strain, Phillips, with an inactivated copy of the gene. Collagen adhesin-positive mutant S. aureus CYL574 was generated by introducing the cna gene into CYL316, a strain that normally lacks the cna gene. Biochemical, immunological, and functional analyses of the generated mutants and their respective parent strains showed that binding of 125I-labeled collagen, expression of an immunoreactive collagen adhesin, and bacterial adherence to cartilage were directly correlated with the presence of a functional cna gene. Greater than 70% of the mice injected with the Cna+ strains developed clinical signs of arthritis, whereas less than 27% of the animals injected with Cna- strains showed symptoms of disease. Furthermore, mice injected with the Cna+ strain Phillips had remarkably elevated levels of immunoglobulin G1 and interleukin-6 compared with mice injected with the Cna- mutant PH100. Taken together, these results demonstrate that collagen adhesin plays an important role in the pathogenesis of septic arthritis induced by S. aureus. Images PMID:8262622

  13. Determination of Helicobacter pylori virulence by analysis of the cag pathogenicity island isolated from Iranian population

    PubMed Central

    Baghaei, Kaveh; Shokrzadeh, Leila; Jafari, Fereshteh; Dabiri, Hossein; Yamaoka, Yoshio; Bolfion, Mehdi; Zojaji, Homayon; Aslani, Mehdi; Zali, Mohammad Reza

    2009-01-01

    Background The cag pathogenicity island (PAI), which can divide into two parts: cagI and cagII, is the most well-known virulence factor of Helicobacter pylori. Aims We investigated the association between genetic variations within the cag PAI (cagA and cagE in the cagI and cagT in the cagII) and clinical outcomes in Iranian population. Subjects A total of 231 patients including 182 patients with gastritis, 41 with peptic ulcer and 8 with gastric cancer. Methods The presences of the cagA, cagE and cagT genes were measured by polymerase chain reaction and the results were compared with clinical outcomes and gastric histology. Results The cagA, cagE and cagT genes were found in 154 (66.7%), 90 (39.0%) and 70 (30.3%) of clinical isolates. At least 144 (62.3%) strains possessed partially deleted cag PAI (e.g., 69 [29.9%] strains were cagA-positive, but cagE and cagT-negative). Conclusion The simple gene as well as the combination of the genes in the cag PAI appeared not to be useful markers to predict H. pylori-related diseases in Iranian population. The genomic sequences of the cag PAI in Iranian strains might be considerably different from those in other geographic locations. PMID:19261552

  14. Recognition of Enteropathogenic Escherichia coli Virulence Determinants by Human Colostrum and Serum Antibodies

    PubMed Central

    Parissi-Crivelli, Aurora; Parissi-Crivelli, Joaquín M.; Girón, Jorge A.

    2000-01-01

    Human colostra and sera collected from Mexican mothers and their children at birth and 6 months thereafter were studied for the presence of antibodies against the bundle-forming pilus and several chromosomal virulence gene products (intimin and secreted proteins EspA and EspB) of enteropathogenic Escherichia coli (EPEC). Among 21 colostrum samples studied, 76, 71.5, 57, and 47% of them contained immunoglobulin A (IgA) antibodies against EspA, intimin, EspB, and BfpA, respectively. Interestingly, there was a difference in IgG response to EPEC antigens between the sera from neonates and sera from the same children 6 months later. While the number of neonates reacting to Esps and intimin diminished when they reached 6 months of age, those reacting with BfpA increased from 9 to 71%. Intimin from an enterohemorrhagic E. coli strain was also recognized by most of the samples reacting with EPEC intimin. These data suggest that Bfp and Esps elicit an antibody response during the early days of life of neonates and support the value of breast-feeding in areas of the world where bacterial diarrheal infections are endemic. PMID:10878066

  15. Molecular method for determining sex of walruses

    USGS Publications Warehouse

    Fischbach, A.S.; Jay, C.V.; Jackson, J.V.; Andersen, L.W.; Sage, G.K.; Talbot, S.L.

    2008-01-01

    We evaluated the ability of a set of published trans-species molecular sexing primers and a set of walrus-specific primers, which we developed, to accurately identify sex of 235 Pacific walruses (Odobenus rosmarus divergens). The trans-species primers were developed for mammals and targeted the X- and Y-gametologs of the zinc finger protein genes (ZFX, ZFY). We extended this method by using these primers to obtain sequence from Pacific and Atlantic walrus (0. r. rosmarus) ZFX and ZFY genes to develop new walrus-specific primers, which yield polymerase chain reaction products of distinct lengths (327 and 288 base pairs from the X- and Y-chromosome, respectively), allowing them to be used for sex determination. Both methods yielded a determination of sex in all but 1-2% of samples with an accuracy of 99.6-100%. Our walrus-specific primers offer the advantage of small fragment size and facile application to automated electrophoresis and visualization.

  16. Molecular characterization of mammalian-adapted Korean-type avian H9N2 virus and evaluation of its virulence in mice.

    PubMed

    Park, Kuk Jin; Song, Min-Suk; Kim, Eun-Ha; Kwon, Hyeok-Il; Baek, Yun Hee; Choi, Eun-Hye; Park, Su-Jin; Kim, Se Mi; Kim, Young-Il; Choi, Won-Suk; Yoo, Dae-Won; Kim, Chul-Joong; Choi, Young Ki

    2015-08-01

    Avian influenza A virus (AIV) is commonly isolated from domestic poultry and wild migratory birds, and the H9N2 subtype is the most prevalent and the major cause of severe disease in poultry in Korea. In addition to the veterinary concerns regarding the H9N2 subtype, it is also considered to be the next potential human pandemic strain due to its rapid evolution and interspecies transmission. In this study, we utilize serial lung-to-lung passage of a low pathogenic avian influenza virus (LPAI) H9N2 (A/Ck/Korea/163/04, WT163) (Y439-lineage) in mice to increase pathogenicity and investigate the potential virulence marker. Mouse-adapted H9N2 virus obtained high virulence (100% mortality) in mice after 98 serial passages. Sequence results show that the mouse adaptation (ma163) possesses several mutations within seven gene segments (PB2, PA, HA, NP, NA, M, and NS) relative to the wild-type strain. The HA gene showed the most mutations (at least 11) with one resulting in the loss of an N-glycosylation site (at amino acid 166). Moreover, reverse genetic studies established that an E627K substitution in PB2 and the loss of the N-glycosylation site in the HA protein (aa166) are critical virulence markers in the mouse-adapted H9N2 virus. Thus, these results add to the increasing body of mutational analysis data defining the function of the viral polymerase and HA genes and their roles in mammalian host adaptation. To our knowledge, this is first report of the generation of a mammalian-adapted Korea H9N2 virus (Y493-lineages). Therefore, this study offers valuable insights into the molecular evolution of the LPAI Korean H9N2 in a new host and adds to the current knowledge of the molecular markers associated with increased virulence. PMID:26224460

  17. Molecular determinants archetypical to the phylum Nematoda

    PubMed Central

    2009-01-01

    Background Nematoda diverged from other animals between 600–1,200 million years ago and has become one of the most diverse animal phyla on earth. Most nematodes are free-living animals, but many are parasites of plants and animals including humans, posing major ecological and economical challenges around the world. Results We investigated phylum-specific molecular characteristics in Nematoda by exploring over 214,000 polypeptides from 32 nematode species including 27 parasites. Over 50,000 nematode protein families were identified based on primary sequence, including ~10% with members from at least three different species. Nearly 1,600 of the multi-species families did not share homology to Pfam domains, including a total of 758 restricted to Nematoda. Majority of the 462 families that were conserved among both free-living and parasitic species contained members from multiple nematode clades, yet ~90% of the 296 parasite-specific families originated only from a single clade. Features of these protein families were revealed through extrapolation of essential functions from observed RNAi phenotypes in C. elegans, bioinformatics-based functional annotations, identification of distant homology based on protein folds, and prediction of expression at accessible nematode surfaces. In addition, we identified a group of nematode-restricted sequence features in energy-generating electron transfer complexes as potential targets for new chemicals with minimal or no toxicity to the host. Conclusion This study identified and characterized the molecular determinants that help in defining the phylum Nematoda, and therefore improved our understanding of nematode protein evolution and provided novel insights for the development of next generation parasite control strategies. PMID:19296854

  18. Antifungal Susceptibility in Serum and Virulence Determinants of Candida Bloodstream Isolates from Hong Kong

    PubMed Central

    Seneviratne, Chaminda J.; Rajan, Suhasini; Wong, Sarah S. W.; Tsang, Dominic N. C.; Lai, Christopher K. C.; Samaranayake, Lakshman P.; Jin, Lijian

    2016-01-01

    Candida bloodstream infections (CBI) are one of the most common nosocomial infections globally, and they account for a high mortality rate. The increasing global prevalence of drug-resistant Candida strains has also been posing a challenge to clinicians. In this study, we comprehensively evaluated the biofilm formation and production of hemolysin and proteinase of 63 CBI isolates derived from a hospital setting in Hong Kong as well as their antifungal susceptibility both in the presence and in the absence of human serum, using standard methodology. Candida albicans was the predominant species among the 63 CBI isolates collected, and non-albicans Candida species accounted for approximately one third of the isolates (36.5%). Of them, Candida tropicalis was the most common non-albicans Candida species. A high proportion (31.7%) of the CBI isolates (40% of C. albicans isolates, 10% of C. tropicalis isolates, 11% of C. parapsilosis isolates, and 100% of C. glabrata isolates) were found to be resistant to fluconazole. One of the isolates (C. tropicalis) was resistant to amphotericin B. A rising prevalence of drug-resistance CBI isolates in Hong Kong was observed with reference to a previous study. Notably, all non-albicans Candida species, showed increased hemolytic activity relative to C. albicans, whilst C. albicans, C. tropicalis, and C. parapsilosis exhibited proteinase activities. Majority of the isolates were capable of forming mature biofilms. Interestingly, the presence of serum distorted the yeast sensitivity to fluconazole, but not amphotericin B. Taken together, our findings demonstrate that CBI isolates of Candida have the potential to express to varying extent their virulence attributes (e.g., biofilm formation, hemolysin production, and proteinase activity) and these, together with perturbations in their antifungal sensitivity in the presence of serum, may contribute to treatment complication in candidemia. The effect of serum on antifungal activity

  19. Antifungal Susceptibility in Serum and Virulence Determinants of Candida Bloodstream Isolates from Hong Kong.

    PubMed

    Seneviratne, Chaminda J; Rajan, Suhasini; Wong, Sarah S W; Tsang, Dominic N C; Lai, Christopher K C; Samaranayake, Lakshman P; Jin, Lijian

    2016-01-01

    Candida bloodstream infections (CBI) are one of the most common nosocomial infections globally, and they account for a high mortality rate. The increasing global prevalence of drug-resistant Candida strains has also been posing a challenge to clinicians. In this study, we comprehensively evaluated the biofilm formation and production of hemolysin and proteinase of 63 CBI isolates derived from a hospital setting in Hong Kong as well as their antifungal susceptibility both in the presence and in the absence of human serum, using standard methodology. Candida albicans was the predominant species among the 63 CBI isolates collected, and non-albicans Candida species accounted for approximately one third of the isolates (36.5%). Of them, Candida tropicalis was the most common non-albicans Candida species. A high proportion (31.7%) of the CBI isolates (40% of C. albicans isolates, 10% of C. tropicalis isolates, 11% of C. parapsilosis isolates, and 100% of C. glabrata isolates) were found to be resistant to fluconazole. One of the isolates (C. tropicalis) was resistant to amphotericin B. A rising prevalence of drug-resistance CBI isolates in Hong Kong was observed with reference to a previous study. Notably, all non-albicans Candida species, showed increased hemolytic activity relative to C. albicans, whilst C. albicans, C. tropicalis, and C. parapsilosis exhibited proteinase activities. Majority of the isolates were capable of forming mature biofilms. Interestingly, the presence of serum distorted the yeast sensitivity to fluconazole, but not amphotericin B. Taken together, our findings demonstrate that CBI isolates of Candida have the potential to express to varying extent their virulence attributes (e.g., biofilm formation, hemolysin production, and proteinase activity) and these, together with perturbations in their antifungal sensitivity in the presence of serum, may contribute to treatment complication in candidemia. The effect of serum on antifungal activity

  20. Molecular Characterization, Morphological Characteristics, Virulence, and Geographic Distribution of Rhizoctonia spp. in Washington State.

    PubMed

    Jaaffar, Ahmad Kamil Mohd; Paulitz, Timothy C; Schroeder, Kurtis L; Thomashow, Linda S; Weller, David M

    2016-05-01

    Rhizoctonia root rot and bare patch, caused by Rhizoctonia solani anastomosis group (AG)-8 and R. oryzae, are chronic and important yield-limiting diseases of wheat and barley in the Inland Pacific Northwest (PNW) of the United States. Major gaps remain in our understanding of the epidemiology of these diseases, in part because multiple Rhizoctonia AGs and species can be isolated from the same cereal roots from the field, contributing to the challenge of identifying the causal agents correctly. In this study, a collection totaling 498 isolates of Rhizoctonia was assembled from surveys conducted from 2000 to 2009, 2010, and 2011 over a wide range of cereal production fields throughout Washington State in the PNW. To determine the identity of the isolates, PCR with AG- or species-specific primers and/or DNA sequence analysis of the internal transcribed spacers was performed. R. solani AG-2-1, AG-8, AG-10, AG-3, AG-4, and AG-11 comprised 157 (32%), 70 (14%), 21 (4%), 20 (4%), 1 (0.2%), and 1 (0.2%), respectively, of the total isolates. AG-I-like binucleate Rhizoctonia sp. comprised 44 (9%) of the total; and 53 (11%), 80 (16%), and 51 (10%) were identified as R. oryzae genotypes I, II, and III, respectively. Isolates of AG-2-1, the dominant Rhizoctonia, occurred in all six agronomic zones defined by annual precipitation and temperature within the region sampled. Isolates of AG-8 also were cosmopolitan in their distribution but the frequency of isolation varied among years, and they were most abundant in zones of low and moderate precipitation. R. oryzae was cosmopolitan, and collectively the three genotypes comprised 37% of the isolates. Only isolates of R. solani AG-8 and R. oryzae genotypes II and III (but not genotype I) caused symptoms typically associated with Rhizoctonia root rot and bare patch of wheat. Isolates of AG-2-1 caused only mild root rot and AG-I-like binucleate isolates and members of groups AG-3, AG-4, and AG-11 showed only slight or no discoloration

  1. Molecular Analysis of Virulence Profiles and Shiga Toxin Genes in Food-Borne Shiga Toxin-Producing Escherichia coli▿

    PubMed Central

    Slanec, T.; Fruth, A.; Creuzburg, K.; Schmidt, H.

    2009-01-01

    In this study, 75 Shiga toxin (Stx)-producing Escherichia coli (STEC) strains originating from foods (n = 73) and drinking water (n = 2) were analyzed for their stx genotype, as well as for further chromosome-, phage-, and plasmid-encoded virulence factors. A broad spectrum of stx genes was detected. Fifty-three strains (70.7%) contained stx2 or stx2 variants, including stx2d, mucus-activatable stx2d, stx2e, and stx2g. Seven strains (9.3%) harbored stx1 or stx1c, and 15 strains (20.0%) carried both stx2 and/or stx2 variants and stx1 or stx1c. Beside stx, the most abundant accessory virulence markers in STEC food isolates were iha (57.3%), ehxA (40.0%), espP (28.0%), and subAB (25.3%). Only four strains were eae positive; three of these belonged to the serogroups O26, O103, and O157 and contained a typical enterohemorrhagic E. coli virulence spectrum. The results of this study show that a number of STEC strains that occur in foods appear to be pathogenic for humans, based on their virulence profiles. Analysis of stx subtypes and detection of additional virulence factors in eae-negative strains may help to better assess the risk of such strains for causing human infection. PMID:19684176

  2. Prevalence of Virulence Determinants and Antimicrobial Resistance among Commensal Escherichia coli Derived from Dairy and Beef Cattle

    PubMed Central

    Bok, Ewa; Mazurek, Justyna; Stosik, Michał; Wojciech, Magdalena; Baldy-Chudzik, Katarzyna

    2015-01-01

    Cattle is a reservoir of potentially pathogenic E. coli, bacteria that can represent a significant threat to public health, hence it is crucial to monitor the prevalence of the genetic determinants of virulence and antimicrobial resistance among the E. coli population. The aim of this study was the analysis of the phylogenetic structure, distribution of virulence factors (VFs) and prevalence of antimicrobial resistance among E. coli isolated from two groups of healthy cattle: 50 cows housed in the conventional barn (147 isolates) and 42 cows living on the ecological pasture (118 isolates). The phylogenetic analysis, identification of VFs and antimicrobial resistance genes were based on either multiplex or simplex PCR. The antimicrobial susceptibilities of E. coli were examined using the broth microdilution method. Two statistical approaches were used to analyse the results obtained for two groups of cattle. The relations between the dependent (VFs profiles, antibiotics) and the independent variables were described using the two models. The mixed logit model was used to characterise the prevalence of the analysed factors in the sets of isolates. The univariate logistic regression model was used to characterise the prevalence of these factors in particular animals. Given each model, the odds ratio (OR) and the 95% confidence interval for the population were estimated. The phylogroup B1 was predominant among isolates from beef cattle, while the phylogroups A, B1 and D occurred with equal frequency among isolates from dairy cattle. The frequency of VFs-positive isolates was significantly higher among isolates from beef cattle. E. coli from dairy cattle revealed significantly higher resistance to antibiotics. Some of the tested resistance genes were present among isolates from dairy cattle. Our study showed that the habitat and diet may affect the genetic diversity of commensal E. coli in the cattle. The results suggest that the ecological pasture habitat is related to

  3. The Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection against Oxidative Stress but Is Not a Determinant of Virulence

    PubMed Central

    Taylor, Martin C.; Lewis, Michael D.; Francisco, Amanda Fortes; Wilkinson, Shane R.; Kelly, John M.

    2015-01-01

    Background The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy. Methodology/Principal Findings To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo. Conclusions/Significance TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design. PMID:25875298

  4. Prevalence of virulence determinants and antimicrobial resistance among commensal Escherichia coli derived from dairy and beef cattle.

    PubMed

    Bok, Ewa; Mazurek, Justyna; Stosik, Michał; Wojciech, Magdalena; Baldy-Chudzik, Katarzyna

    2015-01-01

    Cattle is a reservoir of potentially pathogenic E. coli, bacteria that can represent a significant threat to public health, hence it is crucial to monitor the prevalence of the genetic determinants of virulence and antimicrobial resistance among the E. coli population. The aim of this study was the analysis of the phylogenetic structure, distribution of virulence factors (VFs) and prevalence of antimicrobial resistance among E. coli isolated from two groups of healthy cattle: 50 cows housed in the conventional barn (147 isolates) and 42 cows living on the ecological pasture (118 isolates). The phylogenetic analysis, identification of VFs and antimicrobial resistance genes were based on either multiplex or simplex PCR. The antimicrobial susceptibilities of E. coli were examined using the broth microdilution method. Two statistical approaches were used to analyse the results obtained for two groups of cattle. The relations between the dependent (VFs profiles, antibiotics) and the independent variables were described using the two models. The mixed logit model was used to characterise the prevalence of the analysed factors in the sets of isolates. The univariate logistic regression model was used to characterise the prevalence of these factors in particular animals. Given each model, the odds ratio (OR) and the 95% confidence interval for the population were estimated. The phylogroup B1 was predominant among isolates from beef cattle, while the phylogroups A, B1 and D occurred with equal frequency among isolates from dairy cattle. The frequency of VFs-positive isolates was significantly higher among isolates from beef cattle. E. coli from dairy cattle revealed significantly higher resistance to antibiotics. Some of the tested resistance genes were present among isolates from dairy cattle. Our study showed that the habitat and diet may affect the genetic diversity of commensal E. coli in the cattle. The results suggest that the ecological pasture habitat is related to

  5. Antibiotic resistance, virulence determinants and production of biogenic amines among enterococci from ovine, feline, canine, porcine and human milk

    PubMed Central

    2013-01-01

    Background Recent studies have shown that mammalian milk represents a continuous supply of commensal bacteria, including enterococci. The objectives of this study were to evaluate the presence of enterococci in milk of different species and to screen them for several genetic and phenotypic traits of clinical significance among enterococci. Results Samples were obtained from, at least, nine porcine, canine, ovine, feline and human healthy hosts. Enterococci could be isolated, at a concentration of 1.00 × 102 -1.16 × 103 CFU/ml, from all the porcine samples and, also from 85, 50, 25 and 25% of the human, canine, feline and ovine ones, respectively. They were identified as Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus durans. Among the 120 initial enterococcal isolates, 36 were selected on the basis of their different PFGE profiles and further characterized. MLST analysis revealed a wide diversity of STs among the E. faecalis and E. faecium strains, including some frequently associated to hospital infections and novel STs. All the E. faecalis strains possessed some of the potential virulence determinants (cad, ccf, cob, cpd, efaAfs, agg2, gelE, cylA, espfs) assayed while the E. faecium ones only harboured the efaAfm gene. All the tested strains were susceptible to tigecycline, linezolid and vancomycin, and produced tyramine. Their susceptibility to the rest of the antimicrobials and their ability to produce other biogenic amines varied depending on the strain. Enterococci strains isolated from porcine samples showed the widest spectrum of antibiotic resistance. Conclusions Enterococci isolated from milk of different mammals showed a great genetic diversity. The wide distribution of virulence genes and/or antibiotic resistance among the E. faecalis and E. faecium isolates indicates that they can constitute a reservoir of such traits and a risk to animal and human health. PMID:24325647

  6. Fob1 and Fob2 Proteins Are Virulence Determinants of Rhizopus oryzae via Facilitating Iron Uptake from Ferrioxamine

    PubMed Central

    Liu, Mingfu; Lin, Lin; Gebremariam, Teclegiorgis; Luo, Guanpingsheng; Skory, Christopher D.; French, Samuel W.; Chou, Tsui-Fen; Edwards, John E.; Ibrahim, Ashraf S.

    2015-01-01

    Dialysis patients with chronic renal failure receiving deferoxamine for treating iron overload are uniquely predisposed for mucormycosis, which is most often caused by Rhizopus oryzae. Although the deferoxamine siderophore is not secreted by Mucorales, previous studies established that Rhizopus species utilize iron from ferrioxamine (iron-rich form of deferoxamine). Here we determined that the CBS domain proteins of Fob1 and Fob2 act as receptors on the cell surface of R. oryzae during iron uptake from ferrioxamine. Fob1 and Fob2 cell surface expression was induced in the presence of ferrioxamine and bound radiolabeled ferrioxamine. A R. oryzae strain with targeted reduced Fob1/Fob2 expression was impaired for iron uptake, germinating, and growing on medium with ferrioxamine as the sole source of iron. This strain also exhibited reduced virulence in a deferoxamine-treated, but not the diabetic ketoacidotic (DKA), mouse model of mucormycosis. The mechanism by which R. oryzae obtains iron from ferrioxamine involves the reductase/permease uptake system since the growth on ferrioxamine supplemented medium is associated with elevated reductase activity and the use of the ferrous chelator bathophenanthroline disulfonate abrogates iron uptake and growth on medium supplemented with ferrioxamine as a sole source of iron. Finally, R. oryzae mutants with reduced copies of the high affinity iron permease (FTR1) or with decreased FTR1 expression had an impaired iron uptake from ferrioxamine in vitro and reduced virulence in the deferoxamine-treated mouse model of mucormycosis. These two receptors appear to be conserved in Mucorales, and can be the subject of future novel therapy to maintain the use of deferoxamine for treating iron-overload. PMID:25974051

  7. Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to severe diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in mammal...

  8. NPS6, Encoding a Non-Ribosomal Peptide Synthetase Involved in Siderophore-Mediated Iron Metabolism, is a Conserved Virulence Determinant of Plant Pathogenic Ascomycetes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NPS6, encoding a non-ribosomal peptide synthetase, is a virulence determinant in the corn pathogen Cochliobolus heterostrophus and is also involved in resistance to oxidative stress, generated by hydrogen peroxide. Deletion of NPS6 orthologs in the rice pathogen, Cochliobolus miyabeanus, the cereal...

  9. Dynamic fracture toughness determined using molecular dynamics

    SciTech Connect

    Swadener, J. G.; Baskes, M. I.; Nastasi, Michael Anthony,

    2004-01-01

    Molecular dynamics (MD) simulations of fracture in crystalline silicon are conducted in order to determine the dynamic fracture toughness. The MD simulations show how the potential energy released during fracture is partitioned into surface energy, energy stored in defects and kinetic energy. First, the MD fracture simulations are shown to produce brittle fracture and be in reasonable agreement with experimental results. Then dynamic hcture toughness is calculated as the sum of the surface energy and the energy stored as defects directly from the MD models. Models oriented to produce fracture on either (111) or (101) planes are used. For the (101) fracture orientation, equilibrium crack speeds of greater than 80% of the Rayleigh wave speed are obtained. Crack speeds initially show a steep increase with increasing energy release rate followed by a much more gradual increase. No plateau in crack speed is observed for static energy release rates up to 20 J/m{sup 2}. At the point where the change in crack speed behavior occur, the dynamic fracture toughness (J{sub d}) is still within 10% of two times the surface energy (2{gamma}{sub 0}) and changing very slowly. From these MD simulations, it appears that the change in crack speed behavior is due to a change in the kinetic energy generation during dynamic fracture. In addition, MD simulations of facture in silicon with defects were conducted. The addition of defects increases the inelastic dissipation and the energy stored in defects.

  10. Molecular Screening of Virulence Genes in Extraintestinal Pathogenic Escherichia coli Isolated from Human Blood Culture in Brazil

    PubMed Central

    Koga, Vanessa L.; Cyoia, Paula S.; Neves, Meiriele S.; Vidotto, Marilda C.; Nakazato, Gerson; Kobayashi, Renata K. T.

    2014-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. In the present study, 20 E. coli isolates from human hemocultures were characterized to identify genetic features associated with virulence (pathogenicity islands markers, phylogenetic group, virulence genes, plasmid profiles, and conjugative plasmids) and these results were compared with commensal isolates. The most prevalent pathogenicity island, in strains from hemoculture, were PAI IV536, described by many researchers as a stable island in enterobacteria. Among virulence genes, iutA gene was found more frequently and this gene enconding the aerobactin siderophore receptor. According to the phylogenetic classification, group B2 was the most commonly found. Additionally, through plasmid analysis, 14 isolates showed plasmids and 3 of these were shown to be conjugative. Although in stool samples of healthy people the presence of commensal strains is common, human intestinal tract may serve as a reservoir for ExPEC. PMID:24822211

  11. Evaluation of Brucella abortus S19 vaccine strains by bacteriological tests, molecular analysis of ery loci and virulence in BALB/c mice.

    PubMed

    Mukherjee, Falguni; Jain, Jainendra; Grilló, Maria Jesús; Blasco, José María; Nair, Mrinalini

    2005-09-01

    Two Brucella abortus S19 commercial vaccine strains used for vaccination against brucellosis in India and three S19 strains available as international reference were examined by microbiological assays and molecular analysis of the ery loci involved in erythritol metabolism, and tested for residual virulence in BALB/c mice. According to the sensitivity to penicillin and i-erythritol, the five strains tested had the phenotypic characteristics of strain S19. However, on culture medium containing i-erythritol, all strains developed spontaneous i-erythritol resistant colonies at mutation rates ranging from 1.42x10(-2) to 1.33x10(-6). The S19 characteristic 702 bp deletion in the erythrulose 1-phosphate dehydrogenase gene of the ery locus was present only in the three reference strains but not in the two commercial vaccines. Both commercial strains and one of the reference strains showed reduced virulence in BALB/c mice. The presence or absence in S19 strains of the 702 bp deletion in the ery locus had no correlation with either the rates of spontaneous mutation to erythritol resistance or the residual virulence in mice. PMID:16081301

  12. Comparative analysis of virulence determinants, antibiotic susceptibility patterns and serogrouping of atypical enteropathogenic Escherichia coli versus typical enteropathogenic E. coli in India.

    PubMed

    Malvi, Supriya; Appannanavar, Suma; Mohan, Balvinder; Kaur, Harsimran; Gautam, Neha; Bharti, Bhavneet; Kumar, Yashwant; Taneja, Neelam

    2015-10-01

    The epidemiology of enteropathogenic Escherichia coli (EPEC) and the significance of isolation of atypical EPEC (aEPEC) in childhood diarrhoea have not been well studied in an Indian context. A comparative study was undertaken to investigate virulence determinants, antibiotic susceptibility patterns and serogrouping of typical EPEC (tEPEC) versus aEPEC causing diarrhoea in children. A total of 400 prospective and 500 retrospective E. coli isolates were included. PCR was performed for eae, bfpA, efa, nleB, nleE, cdt, ehxA and paa genes. The Clinical and Laboratory Standards Institute's disc diffusion test was used to determine the antimicrobial susceptibility. Phenotypic screening of extended spectrum β-lactamases (ESBLs), AmpC and Klebsiella pneumoniae carbapenemase (KPC) production, and molecular detection of bla(NDM-1), bla(VIM), bla(CTX-M-15), bla(IMP) and bla(KPC) were performed. aEPEC (57.6 %) were more common as compared with tEPEC (42.3 %). The occurrence of virulence genes was observed to be three times higher in aEPEC as compared with tEPEC, efa1 (14.7 % of aEPEC, 4 % of tEPEC) being the most common. Most of the isolates did not belong to the classical EPEC O-serogroups. The highest resistance was observed against amoxicillin (93.22 %) followed by quinolones (83 %), cephalosporins (37.28 %), cotrimoxazole (35.59 %) and carbapenems (30.5 %). Overall equal numbers of aEPEC (41.17 %) and tEPEC (40 %) were observed to be multidrug-resistant. Fifteen EPEC strains demonstrated presence of ESBLs, five produced AmpC and four each produced metallo-β-lactamases and KPC-type carbapenemases; eight, seven and one isolate(s) each were positive for bla(VIM), bla(CTX-M-15) and bla(NDM-1), respectively. Here, to the best of our knowledge, we report for the first time on carbapenem resistance and the presence of bla(NDM-1) and bla(CTX-M-15) in EPEC isolates from India. PMID:26233663

  13. Molecular characterization of a novobiocin-resistant Aeromonas hydrophila catfish vaccine strain compared to its virulent parent strain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A total of 10 and 13 missense mutations were found in the deduced gyrB and rpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were sign...

  14. Molecular characterization of a novobiocin-resistant Aeromonas hydrophila catfish vaccine strain compared to its virulent parent strain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A total of 10 and 13 missense mutations were found in the deduced GyrB and RpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were sign...

  15. 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. PMID:26654792

  16. The Determination of Molecular Structure from Rotational Spectra

    DOE R&D Accomplishments Database

    Laurie, V. W.; Herschbach, D. R.

    1962-07-01

    An analysis is presented concerning the average molecular configuration variations and their effects on molecular structure determinations. It is noted that the isotopic dependence of the zero-point is often primarily governed by the isotopic variation of the average molecular configuration. (J.R.D.)

  17. Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis, and Concurrently Affect the Pathogen's Growth

    PubMed Central

    Kumar, Krishan; Tharad, Megha; Ganapathy, Swetha; Ram, Geeta; Narayan, Azeet; Khan, Jameel Ahmad; Pratap, Rana; Ghosh, Anamika; Samuchiwal, Sachin Kumar; Kumar, Sushil; Bhalla, Kuhulika; Gupta, Deepti; Natarajan, Krishnamurthy; Singh, Yogendra; Ranganathan, Anand

    2009-01-01

    Background The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung. Methodology/Principal Findings During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage. Conclusions/Significance While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide's binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine

  18. A novel flagellar sheath protein, FcpA, determines filament coiling, translational motility and virulence for the Leptospira spirochete.

    PubMed

    Wunder, Elsio A; Figueira, Cláudio P; Benaroudj, Nadia; Hu, Bo; Tong, Brian A; Trajtenberg, Felipe; Liu, Jun; Reis, Mitermayer G; Charon, Nyles W; Buschiazzo, Alejandro; Picardeau, Mathieu; Ko, Albert I

    2016-08-01

    Leptospira are unique among bacteria based on their helical cell morphology with hook-shaped ends and the presence of periplasmic flagella (PF) with pronounced spontaneous supercoiling. The factors that provoke such supercoiling, as well as the role that PF coiling plays in generating the characteristic hook-end cell morphology and motility, have not been elucidated. We have now identified an abundant protein from the pathogen L. interrogans, exposed on the PF surface, and named it Flagellar-coiling protein A (FcpA). The gene encoding FcpA is highly conserved among Leptospira and was not found in other bacteria. fcpA(-) mutants, obtained from clinical isolates or by allelic exchange, had relatively straight, smaller-diameter PF, and were not able to produce translational motility. These mutants lost their ability to cause disease in the standard hamster model of leptospirosis. Complementation of fcpA restored the wild-type morphology, motility and virulence phenotypes. In summary, we identified a novel Leptospira 36-kDa protein, the main component of the spirochete's PF sheath, and a key determinant of the flagella's coiled structure. FcpA is essential for bacterial translational motility and to enable the spirochete to penetrate the host, traverse tissue barriers, disseminate to cause systemic infection and reach target organs. PMID:27113476

  19. Vru (Sub0144) controls expression of proven and putative virulence determinants and alters the ability of Streptococcus uberis to cause disease in dairy cattle.

    PubMed

    Egan, Sharon A; Ward, Philip N; Watson, Michael; Field, Terence R; Leigh, James A

    2012-06-01

    The regulation and control of gene expression in response to differing environmental stimuli is crucial for successful pathogen adaptation and persistence. The regulatory gene vru of Streptococcus uberis encodes a stand-alone response regulator with similarity to the Mga of group A Streptococcus. Mga controls expression of a number of important virulence determinants. Experimental intramammary challenge of dairy cattle with a mutant of S. uberis carrying an inactivating lesion in vru showed reduced ability to colonize the mammary gland and an inability to induce clinical signs of mastitis compared with the wild-type strain. Analysis of transcriptional differences of gene expression in the mutant, determined by microarray analysis, identified a number of coding sequences with altered expression in the absence of Vru. These consisted of known and putative virulence determinants, including Lbp (Sub0145), SclB (Sub1095), PauA (Sub1785) and hasA (Sub1696). PMID:22383474

  20. Evaluation of ultrafiltration for determining molecular weight of fulvic acid

    USGS Publications Warehouse

    Aiken, G.R.

    1984-01-01

    Two commonly used ultrafiltration membranes are evaluated for the determination of molecular weights of humic substances. Polyacrylic acids of Mr 2000 and 5000 and two well-characterized fulvic acids are used as standards. Molecular size characteristics of standards, as determined by small-angle X-ray scattering, are presented. Great care in evaluating molecular weight data obtained by ultrafiltration is needed because of broad nominal cutoffs and membrane-solute interactions.

  1. Cryptosporidium Pathogenicity and Virulence

    PubMed Central

    Bouzid, Maha; Chalmers, Rachel M.; Tyler, Kevin M.

    2013-01-01

    Cryptosporidium is a protozoan parasite of medical and veterinary importance that causes gastroenteritis in a variety of vertebrate hosts. Several studies have reported different degrees of pathogenicity and virulence among Cryptosporidium species and isolates of the same species as well as evidence of variation in host susceptibility to infection. The identification and validation of Cryptosporidium virulence factors have been hindered by the renowned difficulties pertaining to the in vitro culture and genetic manipulation of this parasite. Nevertheless, substantial progress has been made in identifying putative virulence factors for Cryptosporidium. This progress has been accelerated since the publication of the Cryptosporidium parvum and C. hominis genomes, with the characterization of over 25 putative virulence factors identified by using a variety of immunological and molecular techniques and which are proposed to be involved in aspects of host-pathogen interactions from adhesion and locomotion to invasion and proliferation. Progress has also been made in the contribution of host factors that are associated with variations in both the severity and risk of infection. Here we provide a review comprised of the current state of knowledge on Cryptosporidium infectivity, pathogenesis, and transmissibility in light of our contemporary understanding of microbial virulence. PMID:23297262

  2. Parasitoid wasp virulence

    PubMed Central

    Mortimer, Nathan T

    2013-01-01

    In nature, larvae of the fruit fly Drosophila melanogaster are commonly infected by parasitoid wasps. Following infection, flies mount an immune response termed cellular encapsulation in which fly immune cells form a multilayered capsule that covers and kills the wasp egg. Parasitoids have thus evolved virulence factors to suppress cellular encapsulation. To uncover the molecular mechanisms underlying the antiwasp response, we and others have begun identifying and functionally characterizing these virulence factors. Our recent work on the Drosophila parasitoid Ganaspis sp.1 has demonstrated that a virulence factor encoding a SERCA-type calcium pump plays an important role in Ganaspis sp.1 virulence. This venom SERCA antagonizes fly immune cell calcium signaling and thereby prevents the activation of the encapsulation response. In this way, the study of wasp virulence factors has revealed a novel aspect of fly immunity, namely a role for calcium signaling in fly immune cell activation, which is conserved with human immunity, again illustrating the marked conservation between fly and mammalian immune responses. Our findings demonstrate that the cellular encapsulation response can serve as a model of immune cell function and can also provide valuable insight into basic cell biological processes. PMID:24088661

  3. CovRS-Regulated Transcriptome Analysis of a Hypervirulent M23 Strain of Group A Streptococcus pyogenes Provides New Insights into Virulence Determinants

    PubMed Central

    Bao, Yun-Juan; Liang, Zhong; Mayfield, Jeffrey A.; Lee, Shaun W.; Ploplis, Victoria A.

    2015-01-01

    ABSTRACT The two-component control of virulence (Cov) regulator (R)-sensor (S) (CovRS) regulates the virulence of Streptococcus pyogenes (group A Streptococcus [GAS]). Inactivation of CovS during infection switches the pathogenicity of GAS to a more invasive form by regulating transcription of diverse virulence genes via CovR. However, the manner in which CovRS controls virulence through expression of extended gene families has not been fully determined. In the current study, the CovS-regulated gene expression profiles of a hypervirulent emm23 GAS strain (M23ND/CovS negative [M23ND/CovS−]) and a noninvasive isogenic strain (M23ND/CovS+), under different growth conditions, were investigated. RNA sequencing identified altered expression of ∼349 genes (18% of the chromosome). The data demonstrated that M23ND/CovS− achieved hypervirulence by allowing enhanced expression of genes responsible for antiphagocytosis (e.g., hasABC), by abrogating expression of toxin genes (e.g., speB), and by compromising gene products with dispensable functions (e.g., sfb1). Among these genes, several (e.g., parE and parC) were not previously reported to be regulated by CovRS. Furthermore, the study revealed that CovS also modulated the expression of a broad spectrum of metabolic genes that maximized nutrient utilization and energy metabolism during growth and dissemination, where the bacteria encounter large variations in available nutrients, thus restructuring metabolism of GAS for adaption to diverse growth environments. From constructing a genome-scale metabolic model, we identified 16 nonredundant metabolic gene modules that constitute unique nutrient sources. These genes were proposed to be essential for pathogen growth and are likely associated with GAS virulence. The genome-wide prediction of genes associated with virulence identifies new candidate genes that potentially contribute to GAS virulence. IMPORTANCE The CovRS system modulates transcription of ∼18% of the genes in

  4. Preparation, characterization, and immunogenicity of conjugate vaccines directed against Actinobacillus pleuropneumoniae virulence determinants.

    PubMed

    Byrd, W; Kadis, S

    1992-08-01

    Conjugate vaccines were prepared in an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP). Two subunit conjugates were prepared by coupling the A. pleuropneumoniae 4074 serotype 1 capsular polysaccharide (CP) to the hemolysin protein (HP) and the lipopolysaccharide (LPS) to the HP. Adipic acid dihydrazide was used as a spacer to facilitate the conjugation in a carbodiimide-mediated reaction. The CP and the LPS were found to be covalently coupled to the HP in the conjugates as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detergent gel chromatography analyses. Following a booster vaccination, pigs exhibited significantly high (P less than 0.05) immunoglobulin G antibodies against CP, LPS, and HP. The anti-CP and anti-LPS immunoglobulin G antibodies were found to function as opsonins in the phagocytosis of A. pleuropneumoniae by polymorphonuclear leukocytes, whereas antibodies to the HP neutralized the cytotoxic effect of the HP on polymorphonuclear leukocytes. No killing of A. pleuropneumoniae was observed when the effects of the antibodies were tested in the presence of complement. Thus, polysaccharide-protein A. pleuropneumoniae conjugates elicit significant antibody responses against each component of each conjugate, which could be instrumental in protecting swine against SPAP. PMID:1639471

  5. Genome Sequence of Brucella abortus Vaccine Strain S19 Compared to Virulent Strains Yields Candidate Virulence Genes

    PubMed Central

    Crasta, Oswald R.; Folkerts, Otto; Fei, Zhangjun; Mane, Shrinivasrao P.; Evans, Clive; Martino-Catt, Susan; Bricker, Betsy; Yu, GongXin; Du, Lei; Sobral, Bruno W.

    2008-01-01

    The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9–941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism. PMID:18478107

  6. Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes.

    PubMed

    Crasta, Oswald R; Folkerts, Otto; Fei, Zhangjun; Mane, Shrinivasrao P; Evans, Clive; Martino-Catt, Susan; Bricker, Betsy; Yu, GongXin; Du, Lei; Sobral, Bruno W

    2008-01-01

    The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism. PMID:18478107

  7. Identification of a virulence determinant that is conserved in the Jawetz and Heyl biotypes of [Pasteurella] pneumotropica.

    PubMed

    Sasaki, Hiraku; Ishikawa, Hiroki; Terayama, Hayato; Asano, Ryoki; Kawamoto, Eiichi; Ishibashi, Hidetoshi; Boot, Ron

    2016-08-01

    [Pasteurella] pneumotropica is a ubiquitous bacterium frequently isolated from laboratory rodents. Although this bacterium causes various diseases in immunosuppressed animals, little is known about major virulence factors and their roles in pathogenicity. To identify virulence factors, we sequenced the genome of [P.] pneumotropica biotype Heyl strain ATCC 12555, and compared the resulting non-contiguous draft genome sequence with the genome of biotype Jawetz strain ATCC 35149. Among a large number of genes encoding virulence-associated factors in both strains, four genes encoding for YadA-like proteins, which are known virulence factors that function in host cell adherence and invasion in many pathogens. In this study, we assessed YadA distribution and biological activity as an example of one of virulence-associated factor shared, with biotype Jawetz and Heyl. More than half of mouse isolates were found to have at least one of these genes; whereas, the majority of rat isolates did not. Autoagglutination activity, and ability to bind to mouse collagen type IV and mouse fibroblast cells, was significantly higher in YadA-positive than YadA-negative strains. To conclude, we identified a large number of candidate genes predicted to influence [P.] pneumotropica pathogenesis. PMID:27402782

  8. Development of a DNA Microarray for Enterococcal Species, Virulence, and Antibiotic Resistance Gene Determinations among Isolates from Poultry▿

    PubMed Central

    Champagne, J.; Diarra, M. S.; Rempel, H.; Topp, E.; Greer, C. W.; Harel, J.; Masson, L.

    2011-01-01

    A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential. PMID:21335389

  9. Virulence determinants, antimicrobial susceptibility, and molecular profiles of Erysipelothrix rhusiopathiae strains isolated from China

    PubMed Central

    Ding, Yi; Zhu, Dongmei; Zhang, Jianmin; Yang, Longsheng; Wang, Xiangru; Chen, Huanchun; Tan, Chen

    2015-01-01

    The aim of this study was to understand the epidemiology, serotype, antibiotic sensitivity, and clonal structure of Erysipelothrix rhusiopathiae strains in China. Forty-eight strains were collected from seven provinces during the period from 2012 to 2013. Pulse-field electrophoresis identified 32 different patterns which were classified into clonal groups A–D. Most pulsed-field gel electrophoresis (PFGE) patterns were observed in clonal complex A and B, suggesting high diversity of genetic characterization in these two predominant clonal complexes. Antibiotic sensitivity test shows that all the stains were susceptible to ampicillin, erythromycin, and cefotaxime, and resistant to kanamycin, cefazolin, sulfadiazine, and amikacin. Erythromycin and ampicillin are recommended as first-line antibiotics for treatment of E. rhusiopathiae in China. The high variation in PFGE pattern among the main clonal groups shows that the E. rhusiopathiae in China may originate from different lineages and sources instead of from expansion of a single clonal lineage across different regions. PMID:26975059

  10. Molecular determinants of acute kidney injury

    PubMed Central

    Husi, Holger; Human, Christin

    2015-01-01

    Abstract: Background: Acute kidney injury (AKI) is a condition that leads to a rapid deterioration of renal function associated with impairment to maintain electrolyte and acid balance, and, if left untreated, ultimately irreversible kidney damage and renal necrosis. There are a number of causes that can trigger AKI, ranging from underlying conditions as well as trauma and surgery. Specifically, the global rise in surgical procedures led to a substantial increase of AKI incidence rates, which in turn impacts on mortality rates, quality of life and economic costs to the healthcare system. However, no effective therapy for AKI exists. Current approaches, such as pharmacological intervention, help in alleviating symptoms in slowing down the progression, but do not prevent or reverse AKI-induced organ damage. Methods: An in-depth understanding of the molecular machinery involved in and modulated by AKI induction and progression is necessary to specifically pharmacologically target key molecules. A major hurdle to devise a successful strategy is the multifactorial and complex nature of the disorder itself, whereby the activation of a number of seemingly independent molecular pathways in the kidney leads to apoptotic and necrotic events. Results: The renin-angiotensin-aldosterone-system (RAAS) axis appears to be a common element, leading to downstream events such as triggers of immune responses via the NFB pathway. Other pathways intricately linked with AKI-induction and progression are the tumor necrosis factor alpha (TNF α) and transforming growth factor beta (TGF β) signaling cascades, as well as a number of other modulators. Surprisingly, it has been shown that the involvement of the glutamatergic axis, believed to be mainly a component of the neurological system, is also a major contributor. Conclusions: Here we address the current understanding of the molecular pathways evoked in AKI, their interplay, and the potential to pharmacologically intervene in the

  11. Proteomic profiling of Pseudomonas aeruginosa AES-1R, PAO1 and PA14 reveals potential virulence determinants associated with a transmissible cystic fibrosis-associated strain

    PubMed Central

    2012-01-01

    Background Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-person transmissible strains have been identified in CF clinics worldwide. The molecular basis for transmissibility and colonization of the CF lung remains poorly understood. Results A dual proteomics approach consisting of gel-based and gel-free comparisons were undertaken to analyse protein profiles in a transmissible, early (acute) isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1. Over 1700 P. aeruginosa proteins were confidently identified. AES-1R protein profiles revealed elevated abundance of proteins associated with virulence and siderophore biosynthesis and acquisition, antibiotic resistance and lipopolysaccharide and fatty acid biosynthesis. The most abundant protein in AES-1R was confirmed as a previously hypothetical protein with sequence similarity to carbohydrate-binding proteins and database search revealed this gene is only found in the CF-associated strain PA2192. The link with CF infection may suggest that transmissible strains have acquired an ability to rapidly interact with host mucosal glycoproteins. Conclusions Our data suggest that AES-1R expresses higher levels of proteins, such as those involved in antibiotic resistance, iron acquisition and virulence that may provide a competitive advantage during early infection in the CF lung. Identification of novel proteins associated with transmissibility and acute infection may aid in deciphering new strategies for intervention to limit P. aeruginosa infections in CF patients. PMID:22264352

  12. Molecular Determinants in Phagocyte-Bacteria Interactions.

    PubMed

    Kaufmann, Stefan H E; Dorhoi, Anca

    2016-03-15

    Phagocytes are crucial for host defense against bacterial pathogens. As first demonstrated by Metchnikoff, neutrophils and mononuclear phagocytes share the capacity to engulf, kill, and digest microbial invaders. Generally, neutrophils focus on extracellular, and mononuclear phagocytes on intracellular, pathogens. Reciprocally, extracellular pathogens often capitalize on hindering phagocytosis and killing of phagocytes, whereas intracellular bacteria frequently allow their engulfment and then block intracellular killing. As foreseen by Metchnikoff, phagocytes become highly versatile by acquiring diverse phenotypes, but still retaining some plasticity. Further, phagocytes engage in active crosstalk with parenchymal and immune cells to promote adjunctive reactions, including inflammation, tissue healing, and remodeling. This dynamic network allows the host to cope with different types of microbial invaders. Here we present an update of molecular and cellular mechanisms underlying phagocyte functions in antibacterial defense. We focus on four exemplary bacteria ranging from an opportunistic extracellular to a persistent intracellular pathogen. PMID:26982355

  13. Presynaptic Molecular Determinants of Quantal Size

    PubMed Central

    Takamori, Shigeo

    2016-01-01

    The quantal hypothesis for the release of neurotransmitters at the chemical synapse has gained wide acceptance since it was first worked out at the motor endplate in frog skeletal muscle in the 1950’s. Considering the morphological identification of synaptic vesicles (SVs) at the nerve terminals that appeared to be homogeneous in size, the hypothesis proposed that signal transduction at synapses is mediated by the release of neurotransmitters packed in SVs that are individually uniform in size; the amount of transmitter in a synaptic vesicle is called a quantum. Although quantal size—the amplitude of the postsynaptic response elicited by the release of neurotransmitters from a single vesicle—clearly depends on the number and sensitivity of the postsynaptic receptors, accumulating evidence has also indicated that the amount of neurotransmitters stored in SVs can be altered by various presynaptic factors. Here, I provide an overview of the concepts and underlying presynaptic molecular underpinnings that may regulate quantal size. PMID:26903855

  14. Application of multilocus sequence analysis for molecular characterization of enterococci with virulence factors recovered from a tropical recreational beach.

    PubMed

    Ahmad, Asmat; Dada, Ayokunle Christopher; Usup, Gires

    2014-05-01

    Partial gene sequences of phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) were evaluated for species delineation and detection of recombination among enterococci populations recovered from a bathing beach impacted by low tide river flow. At inter-species level, a maximum similarity of 86.5% and 94.8% was observed among the enterococci pheS and rpoA sequence, respectively. A superimposed plot of delimited pairwise similarity values obtained for 266 pair-wise observations revealed that while there was a harmony between species identity obtained from both genes, pheS was more discriminatory than rpoA. The difference was more pronounced for inter-species comparison. A number of putative recombination events between indigenous and non-indigenous strains was detected based on a library of aligned sequences. Virulence genes cyl, esp, gelE and asa were detected in 7, 22, 100 and 63%, respectively among river isolates but at lower proportion of 0, 20, 67 and 42%, respectively among beach water isolates. Random amplified polymorphic DNA profiling presented evidence suggesting low tide river as a source of fecal enterococci entering the recreation beach water. Multilocus sequence typing analysis of a number of Enterococcus faecalis isolates presented four sequence types, ST59, 117, 181 and 474. The presence of genetically diverse fecal enterococci with associated virulence traits and a background of recombination events in surface recreational water could present a potential public health risk. PMID:24974655

  15. MOLECULAR CHARACTERIZATION OF VIRULENCE AND ANTIMICROBIAL SUSCEPTIBILITY PROFILES OF UROPATHOGENIC ESCHERICHIA COLI FROM PATIENTS IN A TERTIARY HOSPITAL, SOUTHERN THAILAND.

    PubMed

    Themphachanal, Monchanok; Kongpheng, Suttiporn; Rattanachuay, Pattamarat; Khianngam, Saowapar; Singkhamanan, Kamonnut; Sukhumungoon, Pharanai

    2015-11-01

    Among uropathogens, uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI) worldwide, but clinical aspects due to this bacterial species is not fully understood in southern Thailand. Two hundred fifty-four UPEC isolates from patients admitted to Maharaj Nakhon Si Thammarat Hospital, southern Thailand were examined for crucial virulence genes, showing that 33.5% contained at least one of the virulence, genes tested. Genes encoding P fimbria, cytotoxic necrotizing factor-1 and α-hemolysin constituted the majority (15.8%) carried by UPEC isolates. Phylogenetic group classification revealed that 57.5% of UPEC belonged to group D. Antimicrobial susceptibility tests showed that 70.5% and 65.1% of the isolates were resistant to ciprofloxacin and norfloxacin, respectively. Moreover, 50.0% of UPEC were capable of producing extended spectrum beta-lactamases. These findings should be of benefit for more appropriate treatment of UTI patients in this region of Thailand. Keywords: uropathogenic Escherichia coli, antibiotics resistance, cnfl, hlyA, pap, Thailand PMID:26867360

  16. Complete genome sequence of hypervirulent and outbreak-associated Acinetobacter baumannii strain LAC-4: epidemiology, resistance genetic determinants and potential virulence factors

    PubMed Central

    Ou, Hong-Yu; Kuang, Shan N.; He, Xinyi; Molgora, Brenda M.; Ewing, Peter J.; Deng, Zixin; Osby, Melanie; Chen, Wangxue; Xu, H. Howard

    2015-01-01

    Acinetobacter baumannii is an important human pathogen due to its multi-drug resistance. In this study, the genome of an ST10 outbreak A. baumannii isolate LAC-4 was completely sequenced to better understand its epidemiology, antibiotic resistance genetic determinants and potential virulence factors. Compared with 20 other complete genomes of A. baumannii, LAC-4 genome harbors at least 12 copies of five distinct insertion sequences. It contains 12 and 14 copies of two novel IS elements, ISAba25 and ISAba26, respectively. Additionally, three novel composite transposons were identified: Tn6250, Tn6251 and Tn6252, two of which contain resistance genes. The antibiotic resistance genetic determinants on the LAC-4 genome correlate well with observed antimicrobial susceptibility patterns. Moreover, twelve genomic islands (GI) were identified in LAC-4 genome. Among them, the 33.4-kb GI12 contains a large number of genes which constitute the K (capsule) locus. LAC-4 harbors several unique putative virulence factor loci. Furthermore, LAC-4 and all 19 other outbreak isolates were found to harbor a heme oxygenase gene (hemO)-containing gene cluster. The sequencing of the first complete genome of an ST10 A. baumannii clinical strain should accelerate our understanding of the epidemiology, mechanisms of resistance and virulence of A. baumannii. PMID:25728466

  17. Molecular Diffusion Coefficients: Experimental Determination and Demonstration.

    ERIC Educational Resources Information Center

    Fate, Gwendolyn; Lynn, David G.

    1990-01-01

    Presented are laboratory methods which allow the demonstration and determination of the diffusion coefficients of compounds ranging in size from water to small proteins. Included are the procedures involving the use of a spectrometer, UV cell, triterated agar, and oxygen diffusion. Results including quantification are described. (CW)

  18. NDV HN gene C-terminal extension is not the determinant of the enteric tropism but influences the virus virulence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many asymptomatic enteric Newcastle disease virus (NDV) strains contain a larger hemagglutinin-neuraminidase (HN) protein (616 amino acids, aa) than that (571 aa) of virulent respirotropic NDV strains. Therefore, it has been suspected that the 45 aa extension at the C-terminus of HN influences the v...

  19. 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

  20. Distribution of Virulence Factors and Molecular Fingerprinting of Aeromonas Species Isolates from Water and Clinical Samples: Suggestive Evidence of Water-to-Human Transmission ▿ †

    PubMed Central

    Khajanchi, Bijay K.; Fadl, Amin A.; Borchardt, Mark A.; Berg, Richard L.; Horneman, Amy J.; Stemper, Mary E.; Joseph, Sam W.; Moyer, Nelson P.; Sha, Jian; Chopra, Ashok K.

    2010-01-01

    A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans. PMID:20154106

  1. Crowding of Molecular Motors Determines Microtubule Depolymerization

    PubMed Central

    Reese, Louis; Melbinger, Anna; Frey, Erwin

    2011-01-01

    The assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT-associated proteins. Here, we investigate how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. Using an individual-based model, we reproduce experimental findings. Moreover, crowding is identified as the key regulatory mechanism of depolymerization dynamics. Our analysis reveals two qualitatively distinct regimes. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise that cancel out the effect of the depolymerization kinetics such that the depolymerization speed is solely determined by the motor density. Because this density changes over the MT length, length-dependent regulation is possible. Remarkably, motor cooperativity affects only the end-residence time of depolymerases and not the depolymerization speed. PMID:22067158

  2. Structural and Molecular Mechanism of CdpR Involved in Quorum-Sensing and Bacterial Virulence in Pseudomonas aeruginosa

    PubMed Central

    Zhu, Miao; Kang, Huaping; Ma, Jinbiao; Wu, Min; Gan, Jianhua; Deng, Xin; Liang, Haihua

    2016-01-01

    Although quorum-sensing (QS) systems are important regulators of virulence gene expression in the opportunistic human pathogen Pseudomonas aeruginosa, their detailed regulatory mechanisms have not been fully characterized. Here, we show that deletion of PA2588 resulted in increased production of pyocyanin and biofilm, as well as enhanced pathogenicity in a mouse model. To gain insights into the function of PA2588, we performed a ChIP-seq assay and identified 28 targets of PA2588, including the intergenic region between PA2588 and pqsH, which encodes the key synthase of Pseudomonas quinolone signal (PQS). Though the C-terminal domain was similar to DNA-binding regions of other AraC family members, structural studies revealed that PA2588 has a novel fold at the N-terminal region (NTR), and its C-terminal HTH (helix-turn-helix) domain is also unique in DNA recognition. We also demonstrated that the adaptor protein ClpS, an essential regulator of ATP-dependent protease ClpAP, directly interacted with PA2588 before delivering CdpR to ClpAP for degradation. We named PA2588 as CdpR (ClpAP-degradation and pathogenicity Regulator). Moreover, deletion of clpP or clpS/clpA promotes bacterial survival in a mouse model of acute pneumonia infection. Taken together, this study uncovered that CdpR is an important QS regulator, which can interact with the ClpAS-P system to regulate the expression of virulence factors and pathogenicity. PMID:27119725

  3. Glucose- but Not Rice-Based Oral Rehydration Therapy Enhances the Production of Virulence Determinants in the Human Pathogen Vibrio cholerae

    PubMed Central

    Kühn, Juliane; Finger, Flavio; Bertuzzo, Enrico; Borgeaud, Sandrine; Gatto, Marino; Rinaldo, Andrea; Blokesch, Melanie

    2014-01-01

    Despite major attempts to prevent cholera transmission, millions of people worldwide still must address this devastating disease. Cholera research has so far mainly focused on the causative agent, the bacterium Vibrio cholerae, or on disease treatment, but rarely were results from both fields interconnected. Indeed, the treatment of this severe diarrheal disease is mostly accomplished by oral rehydration therapy (ORT), whereby water and electrolytes are replenished. Commonly distributed oral rehydration salts also contain glucose. Here, we analyzed the effects of glucose and alternative carbon sources on the production of virulence determinants in the causative agent of cholera, the bacterium Vibrio cholerae during in vitro experimentation. We demonstrate that virulence gene expression and the production of cholera toxin are enhanced in the presence of glucose or similarly transported sugars in a ToxR-, TcpP- and ToxT-dependent manner. The virulence genes were significantly less expressed if alternative non-PTS carbon sources, including rice-based starch, were utilized. Notably, even though glucose-based ORT is commonly used, field studies indicated that rice-based ORT performs better. We therefore used a spatially explicit epidemiological model to demonstrate that the better performing rice-based ORT could have a significant impact on epidemic progression based on the recent outbreak of cholera in Haiti. Our results strongly support a change of carbon source for the treatment of cholera, especially in epidemic settings. PMID:25474211

  4. Multiple determinants in the coding region of Pea seed-borne mosaic virus P3 are involved in virulence against sbm-2 resistance.

    PubMed

    Hjulsager, Charlotte Kristiane; Olsen, Birgit Schlichter; Jensen, Ditte Marie Kjaer; Cordea, Mirela Irina; Krath, Britta N; Johansen, I Elisabeth; Lund, Ole Søgaard

    2006-11-10

    Viral determinants for overcoming Pisum sativum recessive resistance, sbm-2, against the potyvirus Pea seed-borne mosaic virus (PSbMV) were identified in the region encoding the N-terminal part of the P3 protein. Codons conserved between sbm-2 virulent isolates in this region: Q21, K30 and H122 were found to specifically impair sbm-2 virulence when mutated in selected genetic backgrounds. The corresponding amino acids, Gln21 and Lys30, are neighbored by P3 residues strongly conserved among potyviruses and His122 is conserved particularly in potyviral species infecting legumes. The strongest selective inhibition of sbm-2 virulence, however, was observed by elimination of isolate specific length polymorphisms also located in the N-terminal part of the P3 protein. Length variation in N-terminal P3 is common between potyviral species. However, intra-species length polymorphism in this region was found only among PSbMV isolates. Our findings comply with a model for PSbMV pathotypes having evolved by a diversification of the P3 protein likely to extend to the level of function. PMID:16908044

  5. Virulence determinants of Salmonella Gallinarum biovar Pullorum identified by PCR signature-tagged mutagenesis and the spiC mutant as a candidate live attenuated vaccine.

    PubMed

    Geng, Shizhong; Jiao, Xinan; Barrow, Paul; Pan, Zhiming; Chen, Xiang

    2014-01-31

    Salmonella Gallinarum biovar Pullorum (S. Gallinarum biovar Pullorum) is the causative agent of pullorum disease (PD) in chickens which results in considerable economic losses to the poultry industries in developing countries. PCR-Signature Tagged Mutagenesis was used to identify virulence determinants of S. Gallinarum biovar Pullorum and novel attenuated live vaccine candidates for use against this disease. A library of 1800 signature-tagged S. Gallinarum biovar Pullorum mutants was constructed and screened for virulence-associated genes in chickens. The attenuation of 10 mutants was confirmed by in vivo and in vitro competitive index (CI) studies. The transposons were found to be located in SPI-1 (2/10 mutants), SPI-2 (3/10), the virulence plasmid (1/10) and non-SPI genes (4/10). One highly attenuated spiC mutant persisted in spleen and liver for less than 10 days and induced high levels of circulating antibody and protective immunity against oral challenge in young broiler chickens. The spiC mutant is a potential new vaccine candidate for use with chickens against this disease. PMID:24355532

  6. Multidrug-resistant Escherichia coli from canine urinary tract infections tend to have commensal phylotypes, lower prevalence of virulence determinants and ampC-replicons☆

    PubMed Central

    Wagner, Samuel; Gally, David L.; Argyle, Sally A.

    2014-01-01

    Multidrug-resistant Escherichia coli is an emerging clinical challenge in domestic species. Treatment options in many cases are limited. This study characterized MDR E. coli isolates from urinary tract infections in dogs, collected between 2002 and 2011. Isolates were evaluated in terms of β-lactamase production, phylogenetic group, ST type, replicon type and virulence marker profile. Comparisons were made with antibiotic susceptible isolates also collected from dogs with urinary tract infections. AmpC β-lactamase was produced in 67% of the MDR isolates (12/18). Of these, 8 could be specifically attributed to the CMY-2 gene. None of the isolates tested in either group expressed ESBLs. Phylo-group distribution was as expected in the susceptible isolates, with an over representation of the pathogenic B2 phylo-group (67%). In contrast, the phylogenetic background for the MDR group was mixed, with representation of commensal phylo-groups A and B1. The B2 phylo-group represented the smallest proportion (A, B1, B2 or D was 28%, 22%, 11% and 33%, respectively). Virulence marker profiles, evaluated using Identibac® microarray, discriminated between the two groups. Marker sequences for a core panel of virulence determinants were identified in most of the susceptible isolates, but not in most of the MDR isolates. These findings indicate that for MDR isolates, plasmid-mediated AmpC is an important resistance mechanism, and while still capable of causing clinical disease, there is evidence for a shift towards phylogenetic groups of reduced inferred virulence potential. There was no evidence of zoonotic potential in either the susceptible or MDR urinary tract isolates in this study. PMID:24485933

  7. Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains

    PubMed Central

    2009-01-01

    Background Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. Results We found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes (STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population. Conclusion Despite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal

  8. Cryptococcus gattii Virulence Composite: Candidate Genes Revealed by Microarray Analysis of High and Less Virulent Vancouver Island Outbreak Strains

    PubMed Central

    Ngamskulrungroj, Popchai; Price, Jennifer; Sorrell, Tania; Perfect, John R.; Meyer, Wieland

    2011-01-01

    Human and animal cryptococcosis due to an unusual molecular type of Cryptococcus gattii (VGII) emerged recently on Vancouver Island, Canada. Unlike C. neoformans, C. gattii causes disease mainly in immunocompetent hosts, despite producing a similar suite of virulence determinants. To investigate a potential relationship between the regulation of expression of a virulence gene composite and virulence, we took advantage of two subtypes of VGII (a and b), one highly virulent (R265) and one less virulent (R272), that were identified from the Vancouver outbreak. By expression microarray analysis, 202 genes showed at least a 2-fold difference in expression with 108 being up- and 94 being down-regulated in strain R265 compared with strain R272. Specifically, expression levels of genes encoding putative virulence factors (e.g. LAC1, LAC2, CAS3 and MPK1) and genes encoding proteins involved in cell wall assembly, carbohydrate and lipid metabolism were increased in strain R265, whereas genes involved in the regulation of mitosis and ergosterol biosynthesis were suppressed. In vitro phenotypic studies and transcription analysis confirmed the microarray results. Gene disruption of LAC1 and MPK1 revealed defects in melanin synthesis and cell wall integrity, respectively, where CAS3 was not essential for capsule production. Moreover, MPK1 also controls melanin and capsule production and causes a severe attenuation of the virulence in a murine inhalational model. Overall, this study provides the basis for further genetic studies to characterize the differences in the virulence composite of strains with minor evolutionary divergences in gene expression in the primary pathogen C. gattii, that have led to a major invasive fungal infection outbreak. PMID:21249145

  9. Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant.

    PubMed

    Amich, Jorge; Dümig, Michaela; O'Keeffe, Gráinne; Binder, Jasmin; Doyle, Sean; Beilhack, Andreas; Krappmann, Sven

    2016-04-01

    Fungal infections are of major relevance due to the increased numbers of immunocompromised patients, frequently delayed diagnosis, and limited therapeutics. To date, the growth and nutritional requirements of fungi during infection, which are relevant for invasion of the host, are poorly understood. This is particularly true for invasive pulmonary aspergillosis, as so far, sources of (macro)elements that are exploited during infection have been identified to only a limited extent. Here, we have investigated sulfur (S) utilization by the human-pathogenic mold Aspergillus fumigatus during invasive growth. Our data reveal that inorganic S compounds or taurine is unlikely to serve as an S source during invasive pulmonary aspergillosis since a sulfate transporter mutant strain and a sulfite reductase mutant strain are fully virulent. In contrast, the S-containing amino acid cysteine is limiting for fungal growth, as proven by the reduced virulence of a cysteine auxotroph. Moreover, phenotypic characterization of this strain further revealed the robustness of the subordinate glutathione redox system. Interestingly, we demonstrate that methionine synthase is essential for A. fumigatus virulence, defining the biosynthetic route of this proteinogenic amino acid as a potential antifungal target. In conclusion, we provide novel insights into the nutritional requirements ofA. fumigatus during pathogenesis, a prerequisite to understanding and fighting infection. PMID:26787716

  10. Bacillus anthracis-Like Bacteria and Other B. cereus Group Members in a Microbial Community Within the International Space Station: A Challenge for Rapid and Easy Molecular Detection of Virulent B. anthracis

    PubMed Central

    van Tongeren, Sandra P.; Roest, Hendrik I. J.; Degener, John E.; Harmsen, Hermie J. M.

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods. PMID:24945323

  11. DETERMINATION OF MOLECULAR WEIGHT CITRUS PECTIN USING ION CHROMATOGRAPHY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective was to investigate the use of ELS as a mass detector coupled with MALLS for determining the molecular weights of pectins and other polysaccharides under changing buffer concentrations using HPLC. This would permit the direct determination of the charge to size ratio of pectin which is imp...

  12. Virulence Potential of Fusogenic Orthoreoviruses

    PubMed Central

    Cheng, Peter K.C.; Lai, Mary Y.Y.; Leung, Peter C.K.; Wong, Kitty K.Y.; Lee, W.Y.; Lim, Wilina W.L.

    2012-01-01

    Several severe respiratory virus infections that have emerged during the past decade originated in animals, including bats. In Indonesia, exposure to bats has been associated with increased risk of acquiring orthoreovirus infection. Although orthoreovirus infections are mild and self-limiting, we explored their potential for evolution into a more virulent form. We used conventional virus culture, electron microscopy, and molecular sequencing to isolate and identify orthoreoviruses from 3 patients in whom respiratory tract infection developed after travel to Indonesia. Virus characterization by plaque-reduction neutralization testing showed antigenic similarity, but sequencing of the small segment genes suggested virus reassortment, which could lead to increased virulence. Bats as a reservoir might contribute to virus evolution and genetic diversity, giving orthoreoviruses the potential to become more virulent. Evolution of this virus should be closely monitored so that prevention and control measures can be taken should it become more virulent. PMID:22608100

  13. Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis.

    PubMed

    Ye, Yingwang; Li, Hui; Ling, Na; Han, Yongjia; Wu, Qingping; Xu, Xiaoke; Jiao, Rui; Gao, Jina

    2016-01-18

    Cronobacter is a group of important foodborne pathogens associated with neonatal meningitis, septicemia, and necrotizing enterocolitis. Among Cronobacter species, Cronobacter sakazakii is the most common species in terms of isolation frequency. However, the molecular basis involved in virulence differences among C. sakazakii isolates is still unknown. In this study, based on the determination of virulence differences of C. sakazakii G362 (virulent isolate) and L3101 (attenuated isolate) through intraperitoneal injection, histopathologic analysis (small intestine, kidney, and liver) further confirmed virulence differences. Thereafter, the potential virulence factors were determined using two-dimensional electrophoresis (2-DE) coupled with MALDI/TOP/TOF mass spectrometry. Among a total of 36 protein spots showing differential expression (fold change>1.2), we identified 31 different proteins, of which the expression abundance of 22 was increased in G362. These up-regulated proteins in G362 mainly contained DNA starvation/stationary phase protection protein Dps, OmpA, LuxS, ATP-dependent Clp protease ClpC, and ABC transporter substrate-binding proteins, which might be involved in virulence of C. sakazakii. This is the first report to determine the potential virulence factors of C. sakazakii isolates at the proteomic levels. PMID:26546912

  14. Molecular Evolution of Typical Enteropathogenic Escherichia coli: Clonal Analysis by Multilocus Sequence Typing and Virulence Gene Allelic Profiling▿ †

    PubMed Central

    Lacher, David W.; Steinsland, Hans; Blank, T. Eric; Donnenberg, Michael S.; Whittam, Thomas S.

    2007-01-01

    Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Typical EPEC isolates are differentiated from other types of pathogenic E. coli by two distinctive phenotypes, attaching effacement and localized adherence. The genes specifying these phenotypes are found on the locus of enterocyte effacement (LEE) and the EPEC adherence factor (EAF) plasmid. To describe how typical EPEC has evolved, we characterized a diverse collection of strains by multilocus sequence typing (MLST) and performed restriction fragment length polymorphism (RFLP) analysis of three virulence genes (eae, bfpA, and perA) to assess allelic variation. Among 129 strains representing 20 O-serogroups, 21 clonal genotypes were identified using MLST. RFLP analysis resolved nine eae, nine bfpA, and four perA alleles. Each bfpA allele was associated with only one perA allele class, suggesting that recombination has not played a large role in shuffling the bfpA and perA loci between separate EAF plasmids. The distribution of eae alleles among typical EPEC strains is more concordant with the clonal relationships than the distribution of the EAF plasmid types. These results provide further support for the hypothesis that the EPEC pathotype has evolved multiple times within E. coli through separate acquisitions of the LEE island and EAF plasmid. PMID:17098897

  15. Molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli in Tunisia and characterization of their virulence factors and plasmid addiction systems

    PubMed Central

    2013-01-01

    Background Extended-spectrum β-lactamases (ESBLs), particularly CTX-M- type ESBLs, are among the most important resistance determinants spreading worldwide in Enterobacteriaceae. The aim of this study was to characterize a collection of 163 ESBL-producing Escherichia coli collected in Tunisia, their ESBL-encoding plasmids and plasmid associated addiction systems. Results The collection comprised 163 ESBL producers collected from two university hospitals of Sfax between 1989 and 2009. 118 isolates harbored blaCTX-M gene (101 blaCTX-M-15 gene and 17 blaCTX-M-14 gene). 49 isolates carried blaSHV-12 gene, 9 blaSHV-2a gene and only 3 blaTEM-26 gene. 16 isolates produced both CTX-M and SHV-12. The 101 CTX-M-15-producing isolates were significantly associated to phylogroup B2 and exhibiting a high number of virulence factors. 24 (23.7%) of the group B2 isolates belonged to clonal complex ST131. Pulsed-field gel electrophoresis (PFGE) typing revealed a genetic diversity of the isolates. 144 ESBL determinants were transferable mostly by conjugation. The majority of plasmid carrying blaCTX-M-15 genes (72/88) were assigned to various single replicon or multireplicon IncF types and had significantly a higher frequency of addiction systems, notably the VagCD module. Conclusion This study demonstrates that the dissemination of CTX-M-15 producing E. coli in our setting was due to the spread of various IncF-type plasmids harboring multiple addiction systems, into related clones with high frequency of virulence determinants. PMID:23800277

  16. Molecular characterization of Salmonella enterica serotype Enteritidis isolates from humans by antimicrobial resistance, virulence genes, and pulsed-field gel electrophoresis.

    PubMed

    Zou, Ming; Keelara, Shivaramu; Thakur, Siddhartha

    2012-03-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major serovar associated with human salmonellosis. A total of 425 clinical S. Enteritidis isolates of human origin were collected between June 2009 and September 2010 from North Carolina. The isolates were further characterized for antimicrobial susceptibility, antimicrobial resistance coding determinants, virulence genes, and fingerprint profiles to determine whether they were similar or different to the S. Enteritidis strain responsible for the human outbreak due to consumption of contaminated eggs. Ten different antimicrobial resistance phenotypes were observed with the highest frequency of resistance exhibited to ampicillin (n=10; 2.35%). The isolates were predominantly pansusceptible (n=409; 96.23%); however, seven isolates were multidrug resistant (MDR; i.e., resistant to three or more antimicrobials). Extended spectrum β-lactamase (ESBL) coding genes (bla(TEM) and bla(PSE)) were detected in the ampicillin-resistant isolates, whereas a single MDR isolate tested positive for class 1 integron (1 kb). The majority of the isolates (n=422; 99.3%) carried the invA, mgtC, stn, sopB, sopE1, and sefA virulence genes. However, 37 (8.7%) and 46 (10.82%) S. Enteritidis isolates tested negative for the plasmid encoded genes spvC and rck, respectively. Pulsed-field gel electrophoresis (PFGE) typing of 118 S. Enteritidis isolates by restriction enzymes XbaI and BlnI resulted in seven clusters, each with a discriminatory index (DI) of 0.715 and 0.785, respectively. The combination of XbaI-BlnI patterns generated a dendrogram with 14 clusters and a higher DI of 0.914. The PFGE profile of 80 isolates matched 100% with the S. Enteritidis strain that has been cited for the recent outbreak in the United States due to consumption of contaminated eggs. In conclusion, we identified a genotypic similar S. Enteritidis population in our study based on antimicrobial susceptibility, virulence gene, and PFGE fingerprint

  17. Risk factors in enterococci isolated from foods in Morocco: determination of antimicrobial resistance and incidence of virulence traits.

    PubMed

    Valenzuela, Antonio Sánchez; Omar, Nabil Ben; Abriouel, Hikmate; López, Rosario Lucas; Ortega, Elena; Cañamero, Magdalena Martínez; Gálvez, Antonio

    2008-08-01

    A collection of enterococci isolated from meat, dairy and vegetable foods from Morocco including 23 Enterococus faecalis and 15 Enterococcus faecium isolates was studied. All isolates were sensitive to ampicillin, penicillin, and gentamicin. Many E. faecalis isolates were resistant to tetracycline (86.95%), followed by rifampicin (78.26% ciprofloxacin (60.87%), quinupristin/dalfopristin (56.52%), nitrofurantoin (43.47%), levofloxacin (39.13%), erythromycin (21.73%), streptomycin (17.39%), chloramphenicol (8.69%), vancomycin (8.69%), and teicoplanin (4.34%). E. faecium isolates showed a different antibiotic resistance profile: a high percentage were resistant to nitrofurantoin (73.33%), followed by erythromycin (66.60%), ciprofloxacin (66.66%), levofloxacin (60.00%), and rifampicin (26.66%), and only a very low percentage were resistant to tetracycline (6.66%). One isolate was resistant to vancomycin and teicoplanin. The incidence of virulence factors was much higher among E. faecalis isolates, especially for genes encoding for sex pheromones, collagen adhesin, enterococcal endocarditis antigen, and enterococcal surface protein. Isolates with multiple factors (both antibiotic resistance and virulence traits) were also more frequent among E. faecalis isolates, in which one isolate cumulated up to 15 traits. By contrast, several isolates of E. faecium had only very few unwanted traits as compared to only two isolates in E. faecalis. The high abundance of isolates carrying virulence factors and antibiotic resistance traits suggests that the sanitary quality of foods should be improved in order to decrease the incidence of enterococci. PMID:18514994

  18. Rapid and Specific Detection, Molecular Epidemiology, and Experimental Virulence of the O16 Subgroup within Escherichia coli Sequence Type 131

    PubMed Central

    Clermont, Olivier; Johnston, Brian; Clabots, Connie; Tchesnokova, Veronika; Sokurenko, Evgeni; Junka, Adam F.; Maczynska, Beata; Denamur, Erick

    2014-01-01

    Escherichia coli sequence type 131 (ST131), a widely disseminated multidrug-resistant extraintestinal pathogen, typically exhibits serotype O25b:H4. However, certain ST131 isolates exhibit serotype O16:H5 and derive from a phylogenetic clade that is distinct from the classic O25b:H4 ST131 clade. Both clades are assigned to ST131 by the Achtman multilocus sequence typing (MLST) system and a screening PCR assay that targets ST131-specific sequence polymorphisms in the mdh and gyrB genes. However, they are classified as separate STs by the Pasteur Institute MLST system, and an ST131 PCR method that targets the O25b rfb region and an ST131-specific polymorphism in pabB detects only the O25b-associated clade. Here, we describe a novel PCR-based method that allows for rapid and specific detection of the O16-associated ST131 clade. The clade members uniformly contained allele 41 of fimH (type 1 fimbrial adhesin) and a narrow range of alleles of gyrA and parC (fluoroquinolone target genes). The virulence genotypes of the clade members resembled those of classic O25b:H4 ST131 isolates; representative isolates were variably lethal in a mouse subcutaneous sepsis model. Several pulsotypes spanned multiple sources (adults, children, pets, and human fecal samples) and locales. An analysis of recent clinical E. coli collections showed that the O16 ST131 clade is globally distributed, accounts for 1 to 5% of E. coli isolates overall, and, when compared with other ST131 isolates, it is associated with resistance to ampicillin, gentamicin, and trimethoprim-sulfamethoxazole and with susceptibility to fluoroquinolones and extended-spectrum cephalosporins. Attention to this O16-associated ST131 clade, which is facilitated by our novel PCR-based assay, is warranted in future epidemiological studies of ST131 and, conceivably, in clinical applications. PMID:24501035

  19. Genomics-Based Exploration of Virulence Determinants and Host-Specific Adaptations of Pseudomonas syringae Strains Isolated from Grasses

    PubMed Central

    Dudnik, Alexey; Dudler, Robert

    2014-01-01

    The Pseudomonas syringae species complex has recently been named the number one plant pathogen, due to its economic and environmental impacts, as well as for its role in scientific research. The bacterium has been repeatedly reported to cause outbreaks on bean, cucumber, stone fruit, kiwi and olive tree, as well as on other crop and non-crop plants. It also serves as a model organism for research on the Type III secretion system (T3SS) and plant-pathogen interactions. While most of the current work on this pathogen is either carried out on one of three model strains found on dicot plants with completely sequenced genomes or on isolates obtained from recent outbreaks, not much is known about strains isolated from grasses (Poaceae). Here, we use comparative genomics in order to identify putative virulence-associated genes and other Poaceae-specific adaptations in several newly available genome sequences of strains isolated from grass species. All strains possess only a small number of known Type III effectors, therefore pointing to the importance of non-Type III secreted virulence factors. The implications of this finding are discussed. PMID:25437611

  20. The Glycoprotein Precursor Gene of Junin Virus Determines the Virulence of the Romero Strain and the Attenuation of the Candid #1 Strain in a Representative Animal Model of Argentine Hemorrhagic Fever

    PubMed Central

    Seregin, Alexey V.; Yun, Nadezhda E.; Miller, Milagros; Aronson, Judith; Smith, Jennifer K.; Walker, Aida G.; Smith, Jeanon N.; Huang, Cheng; Manning, John T.; de la Torre, Juan C.

    2015-01-01

    ABSTRACT The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), a potentially deadly disease endemic to central regions of Argentina. The live-attenuated Candid #1 (Can) strain of JUNV is currently used to vaccinate the human population at risk. However, the mechanism of attenuation of this strain is still largely unknown. Therefore, the identification and functional characterization of viral genetic determinants dictating JUNV virulence or attenuation would significantly improve the understanding of the mechanisms underlying AHF and facilitate the development of novel, more effective, and safer vaccines. Here, we utilized a reverse genetics approach to generate recombinant JUNV (rJUNV) strains encoding different gene combinations of the pathogenic Romero (Rom) and attenuated Can strains of JUNV. All strains of rJUNV exhibited in vitro growth kinetics similar to those of their parental counterparts. Analysis of virulence of the rJUNV in a guinea pig model of lethal infection that closely reproduces the features of AHF identified the envelope glycoproteins (GPs) as the major determinants of pathogenesis and attenuation of JUNV. Accordingly, rJUNV strains expressing the full-length GPs of Rom and Can exhibited virulent and attenuated phenotypes, respectively, in guinea pigs. Mutation F427I in the transmembrane region of JUNV envelope glycoprotein GP2 has been shown to attenuate the neurovirulence of JUNV in suckling mice. We document that in the guinea pig model of AHF, mutation F427I in GP2 is also highly attenuating but insufficient to prevent virus dissemination and development of mild clinical and pathological symptoms, indicating that complete attenuation of JUNV requires additional mutations present in Can glycoprotein precursor (GPC). IMPORTANCE Development of antiviral strategies against viral hemorrhagic fevers, including AHF, is one of the top priorities within the Implementation Plan of the U.S. Department

  1. Molecular diversity and high virulence of Legionella pneumophila strains isolated from biofilms developed within a warm spring of a thermal spa

    PubMed Central

    2013-01-01

    Background Several cases of legionellosis have been diagnosed in the same French thermal spa in 1986, 1994 and 1997. L. pneumophila serogroup 1 (Lp1) strains have been isolated from several patients, but the source of contamination was not identified despite the presence of different Lp1 in water samples of the three natural springs feeding the spa at this period. Results Our strategy was to investigate L. pneumophila (Lp) strains from natural biofilms developed in a sulphur-rich warm spring of this contaminated site. Biofilm analysis revealed the presence of three Lp serogroups (Lp1, Lp10 and Lp12). Surprisingly, Lp10 and Lp12 were not reported in the previous described studies from water samples. Besides, the new seven Lp1 we isolated exhibit a high molecular diversity and have been differentiated in five classes according to their DNA genome patterns obtained by PFGE and mip sequences. It must be noted that these DNA patterns are original and unknown in databases. Interestingly, the 27 Lp environmental strains we isolated display a higher cytotoxicity and virulence towards the amoeba Acanthamoeba castellanii than those of known Lp1 epidemic strains. Conclusion The characteristics of Legionella pneumophila Lp1 strains isolated from the warm spring are in agreement with their presence in biofilms and their probable long-term persistence in this ecosystem. PMID:23350929

  2. Stress tolerance and virulence of insect-pathogenic fungi are determined by environmental conditions during conidial formation.

    PubMed

    Rangel, Drauzio E N; Braga, Gilberto U L; Fernandes, Éverton K K; Keyser, Chad A; Hallsworth, John E; Roberts, Donald W

    2015-08-01

    The virulence to insects and tolerance to heat and UV-B radiation of conidia of entomopathogenic fungi are greatly influenced by physical, chemical, and nutritional conditions during mycelial growth. This is evidenced, for example, by the stress phenotypes of Metarhizium robertsii produced on various substrates. Conidia from minimal medium (Czapek's medium without sucrose), complex medium, and insect (Lepidoptera and Coleoptera) cadavers had high, moderate, and poor tolerance to UV-B radiation, respectively. Furthermore, conidia from minimal medium germinated faster and had increased heat tolerance and were more virulent to insects than those from complex medium. Low water-activity or alkaline culture conditions also resulted in production of conidia with high tolerance to heat or UV-B radiation. Conidia produced on complex media exhibited lower stress tolerance, whereas those from complex media supplemented with NaCl or KCl (to reduce water activity) were more tolerant to heat and UV-B than those from the unmodified complex medium. Osmotic and nutritive stresses resulted in production of conidia with a robust stress phenotype, but also were associated with low conidial yield. Physical conditions such as growth under illumination, hypoxic conditions, and heat shock before conidial production also induced both higher UV-B and heat tolerance; but conidial production was not decreased. In conclusion, physical and chemical parameters, as well as nutrition source, can induce great variability in conidial tolerance to stress for entomopathogenic fungi. Implications are discussed in relation to the ecology of entomopathogenic fungi in the field, and to their use for biological control. This review will cover recent technologies on improving stress tolerance of entomopathogenic fungi for biological control of insects. PMID:25791499

  3. Virulence of Escherichia coli Clinical Isolates in a Murine Sepsis Model in Relation to Sequence Type ST131 Status, Fluoroquinolone Resistance, and Virulence Genotype

    PubMed Central

    Porter, Stephen B.; Zhanel, George; Kuskowski, Michael A.; Denamur, Erick

    2012-01-01

    Escherichia coli sequence type ST131 (O25b:H4) has emerged over the past decade as a globally disseminated, multidrug-resistant pathogen. Unlike traditional antimicrobial-resistant E. coli, ST131 derives from virulence-associated phylogenetic group B2 and exhibits extraintestinal virulence factors. This, plus preliminary evidence of virulence in experimental animals, has suggested that ST131's epidemic emergence may be due to high virulence potential, compared with other E. coli types. To test this hypothesis, we compared a large number of matched ST131 and non-ST131 E. coli clinical isolates, both fluoroquinolone resistant and susceptible, plus isolates from classic extraintestinal pathogenic E. coli (ExPEC) sequence types (STs) and case report ST131 household transmission isolates, for virulence in a mouse subcutaneous sepsis model. Overall, in mice, the study isolates produced a wide range of lethality and clinical illness. However, neither ST131 status nor fluoroquinolone phenotype correlated with this diversity of illness severity, which occurred within each of the 6 study groups. In contrast, multiple known or suspected ExPEC virulence genes, including pap (P fimbriae), vat (vacuolating toxin), kpsM II (group 2 capsule), ibeA (invasion of brain endothelium), and clbB/N (colibactin synthesis), plus molecularly defined ExPEC status, were significantly associated with virulence. These findings point away from ST131 isolates as having higher virulence potential compared with other E. coli types in causing invasive extraintestinal infections and suggest instead that ST131's epidemiological success may reflect enhanced fitness for upstream steps in pathogenesis or in colonization and transmission. Additionally, the extensive within-ST virulence diversity suggests an opportunity to compare closely related strains to identify the responsible genetic determinants. PMID:22311928

  4. Surface proteins of Bordetella pertussis: comparison of virulent and avirulent strains and effects of phenotypic modulation.

    PubMed Central

    Armstrong, S K; Parker, C D

    1986-01-01

    The surface proteins of several Bordetella strains and their modulated derivatives were examined by surface radioiodination, cell fractionation, and Western blotting. A surface protein with a high Mr, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis and Bordetella parapertussis cells and was absent in avirulent B. pertussis strains. The electrophoretic profiles of lipopolysaccharide and the 40,000-Mr anion-selective porin were not determinants which correlated with phase variation or phenotypic modulation. At least three envelope proteins (91,000, 32,000, and 30,000 molecular weight) were found only in virulent B. pertussis strains and were absent or diminished in the avirulent phase and most phenotypically modulated strains. Two transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Images PMID:2876957

  5. Molecular Determinants of GS-9620-Dependent TLR7 Activation

    PubMed Central

    Rebbapragada, Indrani; Birkus, Gabriel; Perry, Jason; Xing, Weimei; Kwon, HyockJoo; Pflanz, Stefan

    2016-01-01

    GS-9620 is an orally administered agonist of Toll-like receptor (TLR)7 currently being evaluated in clinical studies for the treatment of chronic HBV and HIV patients. GS-9620 has shown antiviral efficacy in preclinical models of chronic hepadnavirus infection in woodchuck as well as chimpanzee. However, the molecular determinants of GS-9620-dependent activation of TLR7 are not well defined. The studies presented here elucidate GS-9620 subcellular distribution and characterize its molecular interactions with human TLR7 using structure-guided mutational analysis. Based on our results we present a molecular model of TLR7 bound to GS-9620. We also determine that several coding SNPs had no effect on GS-9620-dependent TLR7 activation. In addition, our studies provide evidence that TLR7 exists in a ligand-independent oligomeric state and that, TLR7 activation by GS-9620 is likely associated with compound-induced conformational changes. Finally, we demonstrate that activation of NF-κB and Akt pathways in primary plasmacytoid dendritic cells occur as immediate downstream cellular responses to GS-9620 stimulation. The data presented here further our understanding of the molecular parameters governing TLR7 activation by GS-9620, and more generally by nucleos/tide-related ligands. PMID:26784926

  6. Mutations in the bvgA gene of Bordetella pertussis that differentially affect regulation of virulence determinants.

    PubMed Central

    Stibitz, S

    1994-01-01

    By using chemical mutagenesis and genetic mapping, a search was undertaken for previously undescribed genes which may be involved in different regulatory mechanisms governing different virulence factors of Bordetella pertussis. Previous studies have shown that the fha locus encoding filamentous hemagglutinin is regulated directly by the bvgAS two component system, while regulation of ptx encoding pertussis toxin is less direct or occurs by a different mechanism. With a strain containing gene fusions to each of these regulated loci, screening was done for mutations which were defective for ptx expression but maintained normal or nearly normal levels of fha expression. Two mutations which had such a phenotype and were also deficient in adenylate cyclase toxin/hemolysin expression were found and characterized more fully. Both were found to affect residues in the C-terminal portion of the BvgA response regulator protein, a domain which shares sequence similarity with a family of regulatory proteins including FixJ, UhpA, MalT, RcsA, RcsB, and LuxR. The residues affected are within a region which, by extension from studies on the LuxR protein, may be involved in transcriptional activation. Images PMID:8083156

  7. The Classical Lancefield Antigen of Group A Streptococcus is a Virulence Determinant with Implications for Vaccine Design

    PubMed Central

    Kuipers, Kirsten; Henningham, Anna; Aziz, Ramy K.; Kasirer-Friede, Ana; Lin, Leo; Berends, Evelien T.M.; Davies, Mark R.; Dougan, Gordon; Zhang, Fan; Dahesh, Samira; Shaw, Laura; Gin, Jennifer; Cunningham, Madeleine; Merriman, Joseph A.; Hütter, Julia; Lepenies, Bernd; Rooijakkers, Suzan H.M.; Malley, Richard; Walker, Mark J.; Shattil, Sanford J.; Schlievert, Patrick M.; Choudhury, Biswa; Nizet, Victor

    2014-01-01

    SUMMARY Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes,gacA-L. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAcside chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis and its understanding has implications for vaccine development. PMID:24922575

  8. Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence.

    PubMed

    Pérez-Pascual, David; Gómez, Esther; Guijarro, José A

    2015-01-01

    Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD50 experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine. PMID:25582708

  9. The classical lancefield antigen of group a Streptococcus is a virulence determinant with implications for vaccine design.

    PubMed

    van Sorge, Nina M; Cole, Jason N; Kuipers, Kirsten; Henningham, Anna; Aziz, Ramy K; Kasirer-Friede, Ana; Lin, Leo; Berends, Evelien T M; Davies, Mark R; Dougan, Gordon; Zhang, Fan; Dahesh, Samira; Shaw, Laura; Gin, Jennifer; Cunningham, Madeleine; Merriman, Joseph A; Hütter, Julia; Lepenies, Bernd; Rooijakkers, Suzan H M; Malley, Richard; Walker, Mark J; Shattil, Sanford J; Schlievert, Patrick M; Choudhury, Biswa; Nizet, Victor

    2014-06-11

    Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development. PMID:24922575

  10. Morphological, molecular and virulence characterization of three Lencanicillium species infecting Asian citrus psyllids in Huangyan citrus groves.

    PubMed

    Lu, Lianming; Cheng, Baoping; Du, Danchao; Hu, Xiurong; Peng, Aitian; Pu, Zhanxu; Zhang, Xiaoya; Huang, Zhendong; Chen, Guoqing

    2015-02-01

    Citrus greening or Huanglongbing (HLB) is caused by the infection of Candidatus Liberibacter spp. in citrus plants. Since Asian citrus psyllid is the primary vector of this bacterial pathogen, the spread of HLB can be mitigated by suppressing Asian citrus psyllid populations in citrus groves using entomopathogens. To expand the current data on entomopathogens infecting Asian citrus psyllids, we isolated and characterized three different entomopathogens. Strains ZJLSP07, ZJLA08, and ZJLP09 infected the Asian citrus psyllid, Diaphorina citri Kuwayama, in Huangyan citrus groves. Based on molecular and morphological analyses, two were identified as Lecanicillium attenuatum and Lecanicillium psalliotae, and the third was recognized as an unidentified species of the genus, Lecanicillium. The corrected mortalities caused by strains ZJLSP07, ZJLA08 were 100% at 7days post-inoculation, while by ZJLP09 complete mortality occurred at 6days after inoculation, with 1.0×10(8)conidia/ml at 25°C and a relative humidity of 90% in the laboratory. Under the same condition, the corrected mortalities caused by strains ZJLSP07, ZJLA08 and ZJLP09 were 100%, 92.55% and 100%, respectively at 9days post-inoculation in the greenhouse. Our findings also revealed that these fungal strains infected D. citri using hyphae that penetrated deep into the insect tissues. Further, all three strains secreted the enzymes proteinases, chitinases and lipases with a potential to destroy insect tissues. Interestingly, strain ZJLP09 had an earlier invasion time and the highest levels of enzyme activities when compared to the other two strains. These findings have expanded the existing pool of entomopathogenic fungi that infect D. citri and can be potentially used for the management of D. citri populations. PMID:25593036

  11. Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8.

    PubMed

    Zhang, L; Radziejewska-Lebrecht, J; Krajewska-Pietrasik, D; Toivanen, P; Skurnik, M

    1997-01-01

    The Y. enterocolitica O:8(YeO8) O-antigen repeat units consist of five sugar residues: N-acetyl-D-galactosamine (GalNAc), D-galactose (Gal), D-mannose (Man), L-fucose (Fuc), and 6-deoxy-D-gulose (6d-Gul). The nucleotide sequence of the O-antigen gene cluster of the YeO8 strain 8081-c was determined. Altogether, 18 open reading frames (ORFs) were identified and shown to be essential for O-antigen biosynthesis. We previously characterized the 3'-end of the O-antigen gene cluster and identified four genes: two for GDP-Man biosynthesis, one for UDP-Gal biosynthesis, and one for O-antigen polymerase. Based on sequence similarity, Tn5-insertion phenotypes and chemical analysis, the 14 new genes were assigned the following functions: four genes are involved in the biosynthesis of CDP-6d-Gul and two in GDP-Fuc biosynthesis. Five gene products were assigned sugar transferase functions and one gene product was similar to Wzx, the O-antigen flippase. Two genes remained unassigned. By genetic complementation we also showed that YeO8 O-antigen biosynthesis was dependent on N-acetyl-glucosaminyl:undecaprenylphosphate transferase (GlcNAc transferase), the WecA (formerly known as Rfe) protein. Data obtained from chemical-composition analysis suggest that in addition to being GlcNAc transferase, WecA may also function as a GalNAc transferase. Using a restriction-deficient derivative of Y. enterocolitica O:8 strain 8081, a rough mutant, designated 8081-R2, was isolated. 8081-R2 was complemented in trans with a cloned O-antigen gene cluster restoring surface O-antigen expression. The virulence of the wild-type strain and that of the complemented strain were significantly higher (approx. 100-fold) than that of the rough mutant in an orally infected mouse model, showing that YeO8 O-antigen is a virulence factor. PMID:9004221

  12. Molecular characterization and virus neutralization patterns of severe, non-epizootic forms of feline calicivirus infections resembling virulent systemic disease in cats in Switzerland and in Liechtenstein.

    PubMed

    Willi, Barbara; Spiri, Andrea M; Meli, Marina L; Samman, Ayman; Hoffmann, Karolin; Sydler, Titus; Cattori, Valentino; Graf, Felix; Diserens, Kevin A; Padrutt, Isabelle; Nesina, Stefanie; Berger, Alice; Ruetten, Maja; Riond, Barbara; Hosie, Margaret J; Hofmann-Lehmann, Regina

    2016-01-15

    Feline calicivirus (FCV) infections are associated with oral ulceration, chronic stomatitis and a limping syndrome. Epizootic outbreaks of virulent systemic disease (VSD) have been reported in the USA and Europe. Here, the molecular characterization and neutralization patterns of FCV isolates from cases of severe, non-epizootic infection associated with skin ulceration and edema are presented. Samples from eleven symptomatic cats, four in-contact cats and 27 cats with no contact with symptomatic cats were collected and tested for FCV, feline herpesvirus-1 (FHV-1), feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). Phylogenetic analyses based on the capsid (VP1) gene of FCV and virus neutralization with antisera raised against four FCV vaccine strains were performed. Nine kittens and two adult cats in two shelters and two veterinary clinics in four geographically distinct locations in Switzerland and Liechtenstein were affected. The cats showed fever, tongue and skin ulceration, head and paw edema, and occasionally jaundice, generalized edema and dyspnea. All symptomatic cats tested FCV-positive but were negative for FHV-1, FeLV and FIV, with the exception of one FIV-positive kitten. All kittens of one litter and both adult cats died. The disease did not spread to cats in the environment. Cats in the environment displayed phylogenetically distinct, but related, FCV strains. Virus neutralization patterns suggested that some cases might have been potentially prevented by vaccination with the optimal vaccine strain. In conclusion, clinicians should be aware of severe, non-epizootic forms of FCV infections with initial clinical presentations similar to VSD. PMID:26711049

  13. Fungal virulence genes as targets for antifungal chemotherapy.

    PubMed Central

    Perfect, J R

    1996-01-01

    Fungal virulence genes have now met the age of molecular pathogenesis. The definition of virulence genes needs to be broad so that it encompasses the focus on molecular antifungal targets and vaccine epitopes. However, in the broad but simple definition of a virulence gene, there will be many complex genetic and host interactions which investigators will need to carefully define. Nevertheless, with the increasing numbers of serious fungal infections produced by old and newly reported organisms, the paucity of present antifungal drugs, and the likelihood of increasing drug resistance, the need for investigations into understanding fungal virulence at the molecular level has never been more important. PMID:8807043

  14. Block of Death-Receptor Apoptosis Protects Mouse Cytomegalovirus from Macrophages and Is a Determinant of Virulence in Immunodeficient Hosts

    PubMed Central

    Ebermann, Linda; Ruzsics, Zsolt; Guzmán, Carlos A.; van Rooijen, Nico; Casalegno-Garduño, Rosaely; Koszinowski, Ulrich; Čičin-Šain, Luka

    2012-01-01

    The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC−/−), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system. PMID:23271968

  15. Direct experimental determination of spectral densities of molecular complexes

    SciTech Connect

    Pachón, Leonardo A.; Brumer, Paul

    2014-11-07

    Determining the spectral density of a molecular system immersed in a proteomic scaffold and in contact to a solvent is a fundamental challenge in the coarse-grained description of, e.g., electron and energy transfer dynamics. Once the spectral density is characterized, all the time scales are captured and no artificial separation between fast and slow processes need to be invoked. Based on the fluorescence Stokes shift function, we utilize a simple and robust strategy to extract the spectral density of a number of molecular complexes from available experimental data. Specifically, we show that experimental data for dye molecules in several solvents, amino acid proteins in water, and some photochemical systems (e.g., rhodopsin and green fluorescence proteins), are well described by a three-parameter family of sub-Ohmic spectral densities that are characterized by a fast initial Gaussian-like decay followed by a slow algebraic-like decay rate at long times.

  16. Direct experimental determination of spectral densities of molecular complexes

    NASA Astrophysics Data System (ADS)

    Pachón, Leonardo A.; Brumer, Paul

    2014-11-01

    Determining the spectral density of a molecular system immersed in a proteomic scaffold and in contact to a solvent is a fundamental challenge in the coarse-grained description of, e.g., electron and energy transfer dynamics. Once the spectral density is characterized, all the time scales are captured and no artificial separation between fast and slow processes need to be invoked. Based on the fluorescence Stokes shift function, we utilize a simple and robust strategy to extract the spectral density of a number of molecular complexes from available experimental data. Specifically, we show that experimental data for dye molecules in several solvents, amino acid proteins in water, and some photochemical systems (e.g., rhodopsin and green fluorescence proteins), are well described by a three-parameter family of sub-Ohmic spectral densities that are characterized by a fast initial Gaussian-like decay followed by a slow algebraic-like decay rate at long times.

  17. Molecular mechanisms of mutagenesis determined by the recombinant DNA technology

    SciTech Connect

    Lee, W.R.

    1985-01-01

    A study of the alteration of the DNA in the mutant gene can determine mechanisms of mutation by distinguishing between mutations induced by transition, transversion, frameshifts of a single base and deletions involving many base pairs. The association of a specific pattern of response with a mutagen will permit detecting mutants induced by the mutagen with a reduced background by removing mutations induced by other mechanisms from the pool of potential mutants. From analyses of studies that have been conducted, it is quite apparent that there are substantial differences among mutagens in their modes of action. Of 31 x-ray induced mutants, 20 were large deletions while only 3 showed normal Southern blots. Only one mutant produced a sub-unit polypeptide of normal molecular weight and charge in the in vivo test whereas in vitro synthesis produced a second one. In contrast, nine of thirteen EMS induced mutants produced cross-reacting proteins with sub-unit polypeptide molecular weights equivalent to wild type. Two of three ENU induced mutants recently analyzed in our laboratory produced protein with sub-unit polypeptide molecular weight and electrical charge similar to the wild type stock in which the mutants were induced. One ENU induced mutation is a large deletion. 21 refs., 1 fig.

  18. Comparative genomic analysis of Brucella abortus vaccine strain 104M reveals a set of candidate genes associated with its virulence attenuation

    PubMed Central

    Yu, Dong; Hui, Yiming; Zai, Xiaodong; Xu, Junjie; Liang, Long; Wang, Bingxiang; Yue, Junjie; Li, Shanhu

    2015-01-01

    The Brucella abortus strain 104M, a spontaneously attenuated strain, has been used as a vaccine strain in humans against brucellosis for 6 decades in China. Despite many studies, the molecular mechanisms that cause the attenuation are still unclear. Here, we determined the whole-genome sequence of 104M and conducted a comprehensive comparative analysis against the whole genome sequences of the virulent strain, A13334, and other reference strains. This analysis revealed a highly similar genome structure between 104M and A13334. The further comparative genomic analysis between 104M and A13334 revealed a set of genes missing in 104M. Some of these genes were identified to be directly or indirectly associated with virulence. Similarly, a set of mutations in the virulence-related genes was also identified, which may be related to virulence alteration. This study provides a set of candidate genes associated with virulence attenuation in B.abortus vaccine strain 104M. PMID:26039674

  19. Determinations of molecular weight and molecular weight distribution of high polymers by the rheological properties

    NASA Technical Reports Server (NTRS)

    Huang, J. Y.; Hou, T. H.; Tiwari, S. N.

    1989-01-01

    Several methods are reviewed by which the molecular weight (MW) and the molecular weight distribution (MWD) of polymeric material were determined from the rheological properties. A poly(arylene ether) polymer with six different molecular weights was used in this investigation. Experimentally measured MW and MWD were conducted by GPC/LALLS (gel permeation chromatography/low angle laser light scattering), and the rheological properties of the melts were measured by a Rheometric System Four rheometer. It was found that qualitative information of the MW and MWD of these polymers could be derived from the viscoelastic properties, with the methods proposed by Zeichner and Patel, and by Dormier et al., by shifting the master curves of the dynamic storage modulus, G', and the loss modulus, G'', along the frequency axis. Efforts were also made to calculate quantitative profiles of MW and MWD for these polymers from their rheological properties. The technique recently proposed by Wu was evaluated. It was found that satisfactory results could only be obtained for polymers with single modal distribution in the molecular weight.

  20. Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus.

    PubMed Central

    Taubenberger, J K; Reid, A H; Janczewski, T A; Fanning, T G

    2001-01-01

    The Spanish influenza pandemic of 1918-1919 caused acute illness in 25-30% of the world's population and resulted in the death of 40 million people. The complete genomic sequence of the 1918 influenza virus will be deduced using fixed and frozen tissues of 1918 influenza victims. Sequence and phylogenetic analyses of the complete 1918 haemagglutinin (HA) and neuraminidase (NA) genes show them to be the most avian-like of mammalian sequences and support the hypothesis that the pandemic virus contained surface protein-encoding genes derived from an avian influenza strain and that the 1918 virus is very similar to the common ancestor of human and classical swine H1N1 influenza strains. Neither the 1918 HA genes nor the NA genes possessed mutations that are known to increase tissue tropicity, which accounts for the virulence of other influenza strains such as A/WSN/33 or fowl plague viruses. The complete sequence of the nonstructural (NS) gene segment of the 1918 virus was deduced and tested for the hypothesis that the enhanced virulence in 1918 could have been due to type I interferon inhibition by the NS1 protein. The results from these experiments were inconclusive. Sequence analysis of the 1918 pandemic influenza virus is allowing us to test hypotheses as to the origin and virulence of this strain. This information should help to elucidate how pandemic influenza strains emerge and what genetic features contribute to their virulence. PMID:11779381

  1. Determination of molecular weight distributions in native and pretreated wood.

    PubMed

    Leskinen, Timo; Kelley, Stephen S; Argyropoulos, Dimitris S

    2015-03-30

    The analysis of native wood components by size-exclusion chromatography (SEC) is challenging. Isolation, derivatization and solubilization of wood polymers is required prior to the analysis. The present approach allowed the determination of molecular weight distributions of the carbohydrates and of lignin in native and processed woods, without preparative component isolation steps. For the first time a component selective SEC analysis of sawdust preparations was made possible by the combination of two selective derivatization methods, namely; ionic liquid assisted benzoylation of the carbohydrate fraction and acetobromination of the lignin in acetic acid media. These were optimized for wood samples. The developed method was thus used to examine changes in softwood samples after degradative mechanical and/or chemical treatments, such as ball milling, steam explosion, green liquor pulping, and chemical oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The methodology can also be applied to examine changes in molecular weight and lignin-carbohydrate linkages that occur during wood-based biorefinery operations, such as pretreatments, and enzymatic saccharification. PMID:25563943

  2. Species differences and molecular determinant of TRPA1 cold sensitivity

    PubMed Central

    Chen, Jun; Kang, Dawon; Xu, Jing; Lake, Marc; Hogan, James O.; Sun, Chaohong; Walter, Karl; Yao, Betty; Kim, Donghee

    2013-01-01

    TRPA1 is an ion channel and has been proposed as a thermosensor across species. In invertebrate and ancestral vertebrates such as fly, mosquito, frog, lizard and snakes, TRPA1 serves as a heat receptor, a sensory input utilized for heat avoidance or infrared detection. However, in mammals, whether TRPA1 is a receptor for noxious cold is highly controversial, as channel activation by cold was observed by some groups but disputed by others. Here we attribute the discrepancy to species differences. We show that cold activates rat and mouse TRPA1 but not human or rhesus monkey TRPA1. At the molecular level, a single residue within the S5 transmembrane domain (G878 in rodent but V875 in primate) accounts for the observed difference in cold sensitivity. This residue difference also underlies the species-specific effects of menthol. Together, our findings identify the species-specific cold activation of TRPA1 and reveal a molecular determinant of cold-sensitive gating. PMID:24071625

  3. Apparatus and method of determining molecular weight of large molecules

    DOEpatents

    Fuerstenau, Stephen; Benner, W. Henry; Madden, Norman; Searles, William

    1998-01-01

    A mass spectrometer determines the mass of multiply charged high molecular weight molecules. This spectrometer utilizes an ion detector which is capable of simultaneously measuring the charge z and transit time of a single ion as it passes through the detector. From this transit time, the velocity of the single ion may then be derived, thus providing the mass-to-charge ratio m/z for a single ion which has been accelerated through a known potential. Given z and m/z, the mass m of the single ion can then be calculated. Electrospray ions with masses in excess of 1 MDa and charge numbers greater than 425 e.sup.- are readily detected. The on-axis single ion detection configuration enables a duty cycle of nearly 100% and extends the practical application of electrospray mass spectrometry to the analysis of very large molecules with relatively inexpensive instrumentation.

  4. Apparatus and method of determining molecular weight of large molecules

    DOEpatents

    Fuerstenau, S.; Benner, W.H.; Madden, N.M.; Searles, W.

    1998-06-23

    A mass spectrometer determines the mass of multiply charged high molecular weight molecules. This spectrometer utilizes an ion detector which is capable of simultaneously measuring the charge z and transit time of a single ion as it passes through the detector. From this transit time, the velocity of the single ion may then be derived, thus providing the mass-to-charge ratio m/z for a single ion which has been accelerated through a known potential. Given z and m/z, the mass m of the single ion can then be calculated. Electrospray ions with masses in excess of 1 MDa and charge numbers greater than 425 e{sup {minus}} are readily detected. The on-axis single ion detection configuration enables a duty cycle of nearly 100% and extends the practical application of electrospray mass spectrometry to the analysis of very large molecules with relatively inexpensive instrumentation. 14 figs.

  5. [Molecular genetics and determination of time since death - short communication].

    PubMed

    Šaňková, Markéta; Račanská, Michaela

    2016-01-01

    Estimation of time since death, i.e. the post-mortem interval (PMI), is one of the most problematic issues in forensic practice. Accurate determination of the PMI still remains very complicated task even for an experienced forensic pathologist.Physical changes including algor, livor and rigor mortis can be observed already during the first hours after death of an individual. Unfortunately, the estimation of PMI on the basis of these changes is often burdened with a certain degree of inaccuracy, which is caused by the temperature of surrounding environment, constitution of the body, cause of the death, location of the body, drug abuse etc.Accurate PMI estimation requires assessment of such parameters, which change constantly from the moment of death, but independently on ambient factors. According to current research in the field of molecular biology, it appears that a post-mortem degradation of nucleic acids (both DNA and RNA) will correspond to this definition. PMID:27526264

  6. Molecular determinants of mouse neurovirulence and mosquito infection for Western equine encephalitis virus.

    PubMed

    Mossel, Eric C; Ledermann, Jeremy P; Phillips, Aaron T; Borland, Erin M; Powers, Ann M; Olson, Ken E

    2013-01-01

    Western equine encephalitis virus (WEEV) is a naturally occurring recombinant virus derived from ancestral Sindbis and Eastern equine encephalitis viruses. We previously showed that infection by WEEV isolates McMillan (McM) and IMP-181 (IMP) results in high (∼90-100%) and low (0%) mortality, respectively, in outbred CD-1 mice when virus is delivered by either subcutaneous or aerosol routes. However, relatively little is known about specific virulence determinants of WEEV. We previously observed that IMP infected Culex tarsalis mosquitoes at a high rate (app. 80%) following ingestion of an infected bloodmeal but these mosquitoes were infected by McM at a much lower rate (10%). To understand the viral role in these phenotypic differences, we characterized the pathogenic phenotypes of McM/IMP chimeras. Chimeras encoding the E2 of McM on an IMP backbone (or the reciprocal) had the most significant effect on infection phenotypes in mice or mosquitoes. Furthermore, exchanging the arginine, present on IMP E2 glycoprotein at position 214, for the glutamine present at the same position on McM, ablated mouse mortality. Curiously, the reciprocal exchange did not confer mouse virulence to the IMP virus. Mosquito infectivity was also determined and significantly, one of the important loci was the same as the mouse virulence determinant identified above. Replacing either IMP E2 amino acid 181 or 214 with the corresponding McM amino acid lowered mosquito infection rates to McM-like levels. As with the mouse neurovirulence, reciprocal exchange of amino acids did not confer mosquito infectivity. The identification of WEEV E2 amino acid 214 as necessary for both IMP mosquito infectivity and McM mouse virulence indicates that they are mutually exclusive phenotypes and suggests an explanation for the lack of human or equine WEE cases even in the presence of active transmission. PMID:23544138

  7. A single nucleotide polymorphism at the right terminal region of Mexican papita viroid is a virulent determinant factor on tomato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mexican papita viroid (MPVd), a pospiviroid, causes serious disease outbreaks on greenhouse tomato in North America. Two dominant genotypes (MPV-S and MPV-M), sharing 93.8% sequence identity, incited striking different symptom expression (severe and mild) on tomato ‘Rutgers’. To determine genetic ...

  8. Klebsiella pneumoniae Asparagine tDNAs Are Integration Hotspots for Different Genomic Islands Encoding Microcin E492 Production Determinants and Other Putative Virulence Factors Present in Hypervirulent Strains

    PubMed Central

    Marcoleta, Andrés E.; Berríos-Pastén, Camilo; Nuñez, Gonzalo; Monasterio, Octavio; Lagos, Rosalba

    2016-01-01

    Due to the developing of multi-resistant and invasive hypervirulent strains, Klebsiella pneumoniae has become one of the most urgent bacterial pathogen threats in the last years. Genomic comparison of a growing number of sequenced isolates has allowed the identification of putative virulence factors, proposed to be acquirable mainly through horizontal gene transfer. In particular, those related with synthesizing the antibacterial peptide microcin E492 (MccE492) and salmochelin siderophores were found to be highly prevalent among hypervirulent strains. The determinants for the production of both molecules were first reported as part of a 13-kbp segment of K. pneumoniae RYC492 chromosome, and were cloned and characterized in E. coli. However, the genomic context of this segment in K. pneumoniae remained uncharacterized. In this work, we provided experimental and bioinformatics evidence indicating that the MccE492 cluster is part of a highly conserved 23-kbp genomic island (GI) named GIE492, that was integrated in a specific asparagine-tRNA gene (asn-tDNA) and was found in a high proportion of isolates from liver abscesses sampled around the world. This element resulted to be unstable and its excision frequency increased after treating bacteria with mitomycin C and upon the overexpression of the island-encoded integrase. Besides the MccE492 genetic cluster, it invariably included an integrase-coding gene, at least seven protein-coding genes of unknown function, and a putative transfer origin that possibly allows this GI to be mobilized through conjugation. In addition, we analyzed the asn-tDNA loci of all the available K. pneumoniae assembled chromosomes to evaluate them as GI-integration sites. Remarkably, 73% of the strains harbored at least one GI integrated in one of the four asn-tDNA present in this species, confirming them as integration hotspots. Each of these tDNAs was occupied with different frequencies, although they were 100% identical. Also, we identified

  9. Genome-Wide Gene Expression Analysis Identifies the Proto-oncogene Tyrosine-Protein Kinase Src as a Crucial Virulence Determinant of Infectious Laryngotracheitis Virus in Chicken Cells

    PubMed Central

    Li, Hai; Wang, Fengjie; Han, Zongxi; Gao, Qi; Li, Huixin; Shao, Yuhao; Sun, Nana

    2015-01-01

    ABSTRACT Given the side effects of vaccination against infectious laryngotracheitis (ILT), novel strategies for ILT control and therapy are urgently needed. The modulation of host-virus interactions is a promising strategy to combat the virus; however, the interactions between the host and avian ILT herpesvirus (ILTV) are unclear. Using genome-wide transcriptome studies in combination with a bioinformatic analysis, we identified proto-oncogene tyrosine-protein kinase Src (Src) to be an important modulator of ILTV infection. Src controls the virulence of ILTV and is phosphorylated upon ILTV infection. Functional studies revealed that Src prolongs the survival of host cells by increasing the threshold of virus-induced cell death. Therefore, Src is essential for viral replication in vitro and in ovo but is not required for ILTV-induced cell death. Furthermore, our results identify a positive-feedback loop between Src and the tyrosine kinase focal adhesion kinase (FAK), which is necessary for the phosphorylation of either Src or FAK and is required for Src to modulate ILTV infection. To the best of our knowledge, we are the first to identify a key host regulator controlling host-ILTV interactions. We believe that our findings have revealed a new potential therapeutic target for ILT control and therapy. IMPORTANCE Despite the extensive administration of live attenuated vaccines starting from the mid-20th century and the administration of recombinant vaccines in recent years, infectious laryngotracheitis (ILT) outbreaks due to avian ILT herpesvirus (ILTV) occur worldwide annually. Presently, there are no drugs or control strategies that effectively treat ILT. Targeting of host-virus interactions is considered to be a promising strategy for controlling ILTV infections. However, little is known about the mechanisms governing host-ILTV interactions. The results from our study advance our understanding of host-ILTV interactions on a molecular level and provide experimental

  10. Determination of the infectious titer and virulence of an original US porcine epidemic diarrhea virus PC22A strain.

    PubMed

    Liu, Xinsheng; Lin, Chun-Ming; Annamalai, Thavamathi; Gao, Xiang; Lu, Zhongyan; Esseili, Malak A; Jung, Kwonil; El-Tholoth, Mohamed; Saif, Linda J; Wang, Qiuhong

    2015-01-01

    The infectious dose of a virus pool of original US PEDV strain PC22A was determined in 4-day-old, cesarean-derived, colostrum-deprived (CDCD) piglets. The median pig diarrhea dose (PDD50) of the virus pool was determined as 7.35 log10 PDD50/mL, similar to the cell culture infectious titer, 7.75 log10 plaque-forming units (PFU)/mL. 100 PDD50 caused watery diarrhea in all conventional suckling piglets (n = 12) derived from a PEDV-naive sow, whereas 1000 and 10 000 PDD50 did not cause diarrhea in piglets derived from two PEDV-field exposed-recovered sows. This information is important for future PEDV challenge studies and validation of PEDV vaccines. PMID:26408019

  11. [Investigation of pathogenic phenotypes and virulence determinants of food-borne Salmonella enterica strains in Caenorhabditis elegans animal model].

    PubMed

    Aksoy, Deniz; Şen, Ece

    2015-10-01

    Salmonellosis, caused by non-typhoidal Salmonella enterica serovars with the consumption of contaminated food, is one of the leading food-borne disease that makes microbial food safety an important public health issue. This study was performed in order to determine the antibiotic resistance, serotyping, plasmid profiles and pathogenicity potentials of food-borne Salmonella isolates in Caenorhabditis elegans animal model system in Edirne province, located at Thrace region of Turkey. In this study, 32 Salmonella isolates, of which 26 belonged to Infantis, four to Enteritidis, one to Telaviv and one to Kentucky serovars, isolated from chicken carcasses were used. Antibiotic resistance profiles were determined by disc diffusion and broth microdilution methods. A new C.elegans nematode animal model system was used to determine the pathogenicity potential of the isolates. The antibiotic resistance profiles revealed that one (3.1%) isolate was resistant to gentamicin, two (6.2%) to ciprofloxacin, three (9.4%) to ampicillin, 18 (56.3%) to kanamycin, 19 (60.8%) to neomycin, 25 (78.1%) to tetracycline, 25 (78.1%) to trimethoprim, 26 (81.25%) to nalidixic acid, 27 (84.4%) to streptomycin and 32 (100%) to sulfonamide. All of the 32 strains were susceptible to chloramphenicol and ampicillin/sulbactam. High levels of resistance to streptomycin, nalidixic acid, tetracycline, trimethoprim, sulfonamide, kanamycin and neomycin was determined. According to the plasmid analysis, six isolates (18.75%) harboured 1-3 plasmids with sizes between 1.2 and 42.4 kb. In C.elegans nematode animal model system, the time (in days) required to kill 50% (TD50) of nematodes was calculated for each experimental group. TD50 values of the nematode group fed with S.Typhimurium ATCC 14028 that was used as the positive control and another group fed with E.coli OP50 as the negative control were 4.2 ± 0.5 days and 8.0 ± 0.02 days, respectively. TD50 of the groups fed with Salmonella isolates ranged

  12. Virulence in malaria: an evolutionary viewpoint.

    PubMed Central

    Mackinnon, Margaret J; Read, Andrew F

    2004-01-01

    Malaria parasites cause much morbidity and mortality to their human hosts. From our evolutionary perspective, this is because virulence is positively associated with parasite transmission rate. Natural selection therefore drives virulence upwards, but only to the point where the cost to transmission caused by host death begins to outweigh the transmission benefits. In this review, we summarize data from the laboratory rodent malaria model, Plasmodium chabaudi, and field data on the human malaria parasite, P. falciparum, in relation to this virulence trade-off hypothesis. The data from both species show strong positive correlations between asexual multiplication, transmission rate, infection length, morbidity and mortality, and therefore support the underlying assumptions of the hypothesis. Moreover, the P. falciparum data show that expected total lifetime transmission of the parasite is maximized in young children in whom the fitness cost of host mortality balances the fitness benefits of higher transmission rates and slower clearance rates, thus exhibiting the hypothesized virulence trade-off. This evolutionary explanation of virulence appears to accord well with the clinical and molecular explanations of pathogenesis that involve cytoadherence, red cell invasion and immune evasion, although direct evidence of the fitness advantages of these mechanisms is scarce. One implication of this evolutionary view of virulence is that parasite populations are expected to evolve new levels of virulence in response to medical interventions such as vaccines and drugs. PMID:15306410

  13. Metabolism and virulence in Neisseria meningitidis

    PubMed Central

    Schoen, Christoph; Kischkies, Laura; Elias, Johannes; Ampattu, Biju Joseph

    2014-01-01

    A longstanding question in infection biology addresses the genetic basis for invasive behavior in commensal pathogens. A prime example for such a pathogen is Neisseria meningitidis. On the one hand it is a harmless commensal bacterium exquisitely adapted to humans, and on the other hand it sometimes behaves like a ferocious pathogen causing potentially lethal disease such as sepsis and acute bacterial meningitis. Despite the lack of a classical repertoire of virulence genes in N. meningitidis separating commensal from invasive strains, molecular epidemiology suggests that carriage and invasive strains belong to genetically distinct populations. In recent years, it has become increasingly clear that metabolic adaptation enables meningococci to exploit host resources, supporting the concept of nutritional virulence as a crucial determinant of invasive capability. Here, we discuss the contribution of core metabolic pathways in the context of colonization and invasion with special emphasis on results from genome-wide surveys. The metabolism of lactate, the oxidative stress response, and, in particular, glutathione metabolism as well as the denitrification pathway provide examples of how meningococcal metabolism is intimately linked to pathogenesis. We further discuss evidence from genome-wide approaches regarding potential metabolic differences between strains from hyperinvasive and carriage lineages and present new data assessing in vitro growth differences of strains from these two populations. We hypothesize that strains from carriage and hyperinvasive lineages differ in the expression of regulatory genes involved particularly in stress responses and amino acid metabolism under infection conditions. PMID:25191646

  14. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes

    PubMed Central

    Steinberger-Levy, Ida; Shifman, Ohad; Zvi, Anat; Ariel, Naomi; Beth-Din, Adi; Israeli, Ofir; Gur, David; Aftalion, Moshe; Maoz, Sharon; Ber, Raphael

    2016-01-01

    Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test. PMID:27242774

  15. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes.

    PubMed

    Steinberger-Levy, Ida; Shifman, Ohad; Zvi, Anat; Ariel, Naomi; Beth-Din, Adi; Israeli, Ofir; Gur, David; Aftalion, Moshe; Maoz, Sharon; Ber, Raphael

    2016-01-01

    Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test. PMID:27242774

  16. The Immunologically Distinct O Antigens from Francisella tularensis Subspecies tularensis and Francisella novicida Are both Virulence Determinants and Protective Antigens▿

    PubMed Central

    Thomas, Rebecca M.; Titball, Richard W.; Oyston, Petra C. F.; Griffin, Kate; Waters, Emma; Hitchen, Paul G.; Michell, Stephen L.; Grice, I. Darren; Wilson, Jennifer C.; Prior, Joann L.

    2007-01-01

    We have determined the sequence of the gene cluster encoding the O antigen in Francisella novicida and compared it to the previously reported O-antigen cluster in Francisella tularensis subsp. tularensis. Immunization with purified lipopolysaccharide (LPS) from F. tularensis subsp. tularensis or F. novicida protected against challenge with Francisella tularensis subsp. holarctica and F. novicida, respectively. The LPS from F. tularensis subsp. tularensis did not confer protection against challenge with F. novicida, and the LPS from F. novicida did not confer protection against challenge with F. tularensis subsp. holarctica. Allelic replacement mutants of F. tularensis subsp. tularensis or F. novicida which failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen of F. tularensis subsp. tularensis appeared to be important for intracellular survival whereas the O antigen of F. novicida appeared to be critical for serum resistance and less important for intracellular survival. PMID:17074846

  17. Molecular cloning and characterization of cgt, the Brucella abortus cyclic beta-1,2-glucan transporter gene, and its role in virulence.

    PubMed

    Roset, Mara S; Ciocchini, Andrés E; Ugalde, Rodolfo A; Iñón de Iannino, Nora

    2004-04-01

    The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic beta-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic beta-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic beta-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic beta-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor. PMID:15039351

  18. Molecular typing by random amplification of polymorphic DNA (RAPD) and detection of virulence genes of Salmonella enterica subspecies enterica serovar Gallinarum biovar gallinarum.

    PubMed

    Jin, Ji-Dong; Lee, Dong-Seok; Shin, Eun-Kyung; Kim, Sun-Joong; Jung, Rose; Hahn, Tae-Wook

    2006-12-01

    Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum is the causative agent of fowl typhoid in chickens, outbreaks of which have devastated poultry populations in Korea since 1992. In order to identify genetic differences among S. Gallinarum isolates, bacteria were examined using the random amplified polymorphic DNA (RAPD) method. Of 13 arbitrary primers screened initially, the primer designated as universal rice primer-6 (URP-6) was selected for subsequent typing assays because it produced a distinctive and reproducible DNA fingerprint for a S. Gallinarum reference strain. URP-6-based RAPD analysis assigned 30 S. Gallinarum isolates into 6 types, with 26 isolates (86.6%) belonging to 2 major RAPD types. The distribution of virulence genes in S. Gallinarum isolates was examined by Southern hybridization. All tested isolates had the invasion gene, invA, the virulence plasmid gene, spvB, and the S. Enteritidis fimbrial gene, sefC. The distribution of virulence genes among S. Gallinarum isolates did not correlate with any specific RAPD type. PMID:17213701

  19. Proteomic Characterization of Yersinia pestis Virulence

    SciTech Connect

    Chromy, B; Murphy, G; Gonzales, A; Fitch, J P; McCutchen-Maloney, S L

    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 conditions 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.

  20. New molecular imprinted voltammetric sensor for determination of ochratoxin A.

    PubMed

    Yola, Mehmet Lütfi; Gupta, Vinod Kumar; Atar, Necip

    2016-04-01

    In this report, a novel molecular imprinted voltammetric sensor based on silver nanoparticles (AgNPs) involved in a polyoxometalate (H3PW12O40, POM) functionalized reduced graphene oxide (rGO) modified glassy carbon electrode (GCE) was presented for determination of ochrattoxin A (OCH). The developed surfaces were characterized using scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) method. OCH imprinted GCE was prepared via electropolymerization process of 100mM phenol as monomer in the presence of phosphate buffer solution (pH6.0) containing 25 mM OCH. The linearity range and the detection limit of the method were calculated as 5.0 × 10(-11) - 1.5 × 10(-9)M and 1.6 × 10(-11) M, respectively. The voltammetric sensor was applied to grape juice and wine samples with good selectivity and recovery. The stability of the voltammetric sensor was also reported. PMID:26838863

  1. Mechanical Properties of Nanostructured Materials Determined Through Molecular Modeling Techniques

    NASA Technical Reports Server (NTRS)

    Clancy, Thomas C.; Gates, Thomas S.

    2005-01-01

    The potential for gains in material properties over conventional materials has motivated an effort to develop novel nanostructured materials for aerospace applications. These novel materials typically consist of a polymer matrix reinforced with particles on the nanometer length scale. In this study, molecular modeling is used to construct fully atomistic models of a carbon nanotube embedded in an epoxy polymer matrix. Functionalization of the nanotube which consists of the introduction of direct chemical bonding between the polymer matrix and the nanotube, hence providing a load transfer mechanism, is systematically varied. The relative effectiveness of functionalization in a nanostructured material may depend on a variety of factors related to the details of the chemical bonding and the polymer structure at the nanotube-polymer interface. The objective of this modeling is to determine what influence the details of functionalization of the carbon nanotube with the polymer matrix has on the resulting mechanical properties. By considering a range of degree of functionalization, the structure-property relationships of these materials is examined and mechanical properties of these models are calculated using standard techniques.

  2. Molecular determinants of nucleolar translocation of RNA helicase A

    SciTech Connect

    Liu Zhe; Kenworthy, Rachael; Green, Christopher; Tang, Hengli

    2007-10-15

    RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role.

  3. Sensitive determination of citrinin based on molecular imprinted electrochemical sensor

    NASA Astrophysics Data System (ADS)

    Atar, Necip; Yola, Mehmet Lütfi; Eren, Tanju

    2016-01-01

    In this report, a novel molecular imprinted voltammetric sensor based on glassy carbon electrode (GCE) modified with platinum nanoparticles (PtNPs) involved in a polyoxometalate (H3PW12O40, POM) functionalized reduced graphene oxide (rGO) was prepared for the determination of citrinin (CIT). The developed surfaces were characterized by using scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) method. CIT imprinted GCE was prepared via electropolymerization process of 80.0 mM pyrrole as monomer in the presence of phosphate buffer solution (pH 6.0) containing 20.0 mM CIT. The linearity range and the detection limit of the developed method were calculated as 1.0 × 10-12-1.0 × 10-10 M and 2.0 × 10-13 M, respectively. In addition, the voltammetric sensor was applied to rye samples. The stability and selectivity of the voltammetric sensor were also reported.

  4. Molecular determinants of epidermal growth factor binding: a molecular dynamics study.

    PubMed

    Sanders, Jeffrey M; Wampole, Matthew E; Thakur, Mathew L; Wickstrom, Eric

    2013-01-01

    The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF

  5. The effect of milk components and storage conditions on the virulence of Listeria monocytogenes as determined by a Caco-2 cell assay.

    PubMed

    Pricope-Ciolacu, Luminita; Nicolau, Anca Ioana; Wagner, Martin; Rychli, Kathrin

    2013-08-16

    Nearly all cases of human listeriosis have been associated with consumption of contaminated food, therefore the investigation of the virulence of Listeria (L.) monocytogenes after exposure to environmental conditions in food matrices is critical in order to understand and control its impact on public health. As milk and dairy products have been implicated in more than half of the listeriosis outbreaks, we investigated the in vitro virulence of L. monocytogenes incubated in different milk types at various storage conditions. Incubation in pasteurized milk at refrigeration conditions (4°C) revealed a higher invasion and intracellular proliferation of four different L. monocytogenes strains compared to raw milk using human intestinal epithelial Caco-2 cells. Furthermore the period of storage, which increased L. monocytogenes cell numbers, decreased in vitro virulence. However, L. monocytogenes stored for 3weeks at 4°C in milk are still able to invade and proliferate into the host cell. Interestingly abused storage temperatures (25°C and 30°C) for a short time period (2h) revealed an attenuated impact on the in vitro virulence of L. monocytogenes compared to the storage temperature of 4°C. Regarding the major milk compounds, the level of milk fat significantly affected the in vitro virulence of L. monocytogenes. Pre-incubation in milk with high fat content (3.6%) resulted in a lower invasion capability compared to milk with low fat content. In contrast casein and lactose did not influence the invasiveness of L. monocytogenes into the host cell. In conclusion our study shows that the milk environment and different storage conditions influence the in vitro virulence of L. monocytogenes, both of which have to be considered in the risk assessment of contaminated food. PMID:23831732

  6. Screening of virulence-associated genes as a molecular typing method for characterization of Streptococcus suis isolates recovered from wild boars and pigs.

    PubMed

    Sánchez Del Rey, Verónica; Fernández-Garayzábal, José F; Domínguez, Lucas; Gottschalk, Marcelo; Vela, Ana I

    2016-03-01

    Streptococcus suis is an important zoonotic pathogen associated with a wide range of diseases in pigs, but has also been isolated from wild animals such as rabbits and wild boars. In the current study, 126 S. suis isolates recovered from pigs (n = 85) and wild boars (n = 41) were tested by polymerase chain reaction (PCR) for the presence of nine virulence-associated genes. S. suis isolates from wild boars were differentiated by the lower detection rates of the epf, sly, mrp, sao and dltA genes (0%, 2.4%, 2.4%, 4.8% and 21.9%, respectively) compared with the isolates from pigs (56.5%, 75.3%, 56.5%, 88.2.0% and 88.2%, respectively). The differences in the content of these virulence-associated genes were statistically significant (P < 0.05). There was a correlation between the variants saoM and saoL and serotypes 2 and 9, respectively (P < 0.05). Isolates were classified into 31 virulence-associated gene profiles (VPs). Ten VPs were detected among wild boar isolates and 22 VPs among pig isolates, with only two VPs common to wild boars and pigs. The predominant VPs among isolates from wild boars (VP1, VP7) were different from those observed in pig isolates (VP16 and VP26). VP16 was detected exclusively in clinical pig isolates of serotype 9 and VP26 was detected in 71.4% of the serotype 2 clinical pig isolates. Further multilocus sequence typing (MLST) analysis showed a significant correlation association between certain VPs and STs (VP16 and VP17 with ST123 and ST125 and VP26 with ST1). In conclusion, the current study showed that combination of virulence-associated gene profiling and MLST analysis may provide more information of the relatedness of the S. suis strains from different animal species that could be useful for epidemiological purposes. PMID:26831161

  7. Determination of molecular contamination performance for space chamber tests

    NASA Technical Reports Server (NTRS)

    Scialdone, J. J.

    1973-01-01

    The limitations of chamber tests with regard to the molecular contamination of a spacecraft undergoing vacuum test were examined. The molecular flow conditions existing in the chamber and the parameters dictating the degree of contamination were analyzed. Equations and graphs were developed to show the fraction of molecules returning to the spacecraft out of those emitted and to show other chamber flow parameters as a function of chamber and spacecraft surface molecular pumping and geometric configuration. Type and location of instruments required to measure the outgassing, the degree of contamination, and the returning flows are also discussed.

  8. The minimal gene set member msrA, encoding peptide methionine sulfoxide reductase, is a virulence determinant of the plant pathogen Erwinia chrysanthemi.

    PubMed

    Hassouni, M E; Chambost, J P; Expert, D; Van Gijsegem, F; Barras, F

    1999-02-01

    Peptide methionine sulfoxide reductase (MsrA), which repairs oxidized proteins, is present in most living organisms, and the cognate structural gene belongs to the so-called minimum gene set [Mushegian, A. R. & Koonin, E. V., (1996) Proc. Natl. Acad. Sci. USA 93, 10268-10273]. In this work, we report that MsrA is required for full virulence of the plant pathogen Erwinia chrysanthemi. The following differences were observed between the wild-type and a MsrA- mutant: (i) the MsrA- mutant was more sensitive to oxidative stress; (ii) the MsrA- mutant was less motile on solid surface; (iii) the MsrA- mutant exhibited reduced virulence on chicory leaves; and (iv) no systemic invasion was observed when the MsrA- mutant was inoculated into whole Saintpaulia ionantha plants. These results suggest that plants respond to virulent pathogens by producing active oxygen species, and that enzymes repairing oxidative damage allow virulent pathogens to survive the host environment, thereby supporting the theory that active oxygen species play a key role in plant defense. PMID:9927663

  9. The vacuolar-sorting protein Snf7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex.

    PubMed Central

    da C. Godinho, Rodrigo M.; Crestani, Juliana; Kmetzsch, Lívia; de S. Araujo, Glauber; Frases, Susana; Staats, Charley C.; Schrank, Augusto; Vainstein, Marilene H.; Rodrigues, Marcio L.

    2014-01-01

    Fungal pathogenesis requires a number of extracellularly released virulence factors. Recent studies demonstrating that most fungal extracellular molecules lack secretory tags suggest that unconventional secretion mechanisms and fungal virulence are strictly connected. Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans. Snf7 is a key ESCRT operator required for unconventional secretion in Eukaryotes. In this study we generated snf7Δ mutant strains of C. neoformans and its sibling species C. gattii. Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation. This phenotype culminated with loss of virulence in an intranasal model of murine infection in both species. Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic. These results are in agreement with the observation that unconventional secretion is essential for cryptococcal pathogenesis and strongly suggest the occurrence of still obscure mechanisms of exportation of non-protein molecules in Eukaryotes. PMID:25178636

  10. Cranberry-derived proanthocyanidins impair virulence and inhibit quorum sensing of Pseudomonas aeruginosa

    PubMed Central

    Maisuria, Vimal B.; Los Santos, Yossef Lopez-de; Tufenkji, Nathalie; Déziel, Eric

    2016-01-01

    Bacteria have evolved multiple strategies for causing infections that include producing virulence factors, undertaking motility, developing biofilms, and invading host cells. N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) tightly regulates the expression of multiple virulence factors in the opportunistic pathogenic bacterium Pseudomonas aeruginosa. Thus, inhibiting QS could lead to health benefits. In this study, we demonstrate an anti-virulence activity of a cranberry extract rich in proanthocyanidins (cerPAC) against P. aeruginosa in the model host Drosophila melanogaster and show this is mediated by QS interference. cerPAC reduced the production of QS-regulated virulence determinants and protected D. melanogaster from fatal infection by P. aeruginosa PA14. Quantification of AHL production using liquid chromatography-mass spectrometry confirmed that cerPAC effectively reduced the level of AHLs produced by the bacteria. Furthermore, monitoring QS signaling gene expression revealed that AHL synthases LasI/RhlI and QS transcriptional regulators LasR/RhlR genes were inhibited and antagonized, respectively, by cerPAC. Molecular docking studies suggest that cranberry-derived proanthocyanidin binds to QS transcriptional regulators, mainly interacting with their ligand binding sites. These findings provide insights into the underlying mechanisms of action of a cerPAC to restrict the virulence of P. aeruginosa and can have implications in the development of alternative approaches to control infections. PMID:27503003

  11. Cranberry-derived proanthocyanidins impair virulence and inhibit quorum sensing of Pseudomonas aeruginosa.

    PubMed

    Maisuria, Vimal B; Los Santos, Yossef Lopez-de; Tufenkji, Nathalie; Déziel, Eric

    2016-01-01

    Bacteria have evolved multiple strategies for causing infections that include producing virulence factors, undertaking motility, developing biofilms, and invading host cells. N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) tightly regulates the expression of multiple virulence factors in the opportunistic pathogenic bacterium Pseudomonas aeruginosa. Thus, inhibiting QS could lead to health benefits. In this study, we demonstrate an anti-virulence activity of a cranberry extract rich in proanthocyanidins (cerPAC) against P. aeruginosa in the model host Drosophila melanogaster and show this is mediated by QS interference. cerPAC reduced the production of QS-regulated virulence determinants and protected D. melanogaster from fatal infection by P. aeruginosa PA14. Quantification of AHL production using liquid chromatography-mass spectrometry confirmed that cerPAC effectively reduced the level of AHLs produced by the bacteria. Furthermore, monitoring QS signaling gene expression revealed that AHL synthases LasI/RhlI and QS transcriptional regulators LasR/RhlR genes were inhibited and antagonized, respectively, by cerPAC. Molecular docking studies suggest that cranberry-derived proanthocyanidin binds to QS transcriptional regulators, mainly interacting with their ligand binding sites. These findings provide insights into the underlying mechanisms of action of a cerPAC to restrict the virulence of P. aeruginosa and can have implications in the development of alternative approaches to control infections. PMID:27503003

  12. Determination of structure parameters in molecular tunnelling ionisation model

    NASA Astrophysics Data System (ADS)

    Wang, Jun-Ping; Zhao, Song-Feng; Zhang, Cai-Rong; Li, Wei; Zhou, Xiao-Xin

    2014-04-01

    We extracted the accurate structure parameters in a molecular tunnelling ionisation model (the so-called MO-ADK model) for 23 selected linear molecules including some inner orbitals. The molecular wave functions with the correct asymptotic behaviour are obtained by solving the time-independent Schrödinger equation with B-spline functions and molecular potentials numerically constructed using the modified Leeuwen-Baerends (LBα) model. We show that the orientation-dependent ionisation rate reflects the shape of the ionising orbitals in general. The influences of the Stark shifts of the energy levels on the orientation-dependent ionisation rates of the polar molecules are studied. We also examine the angle-dependent ionisation rates (or probabilities) based on the MO-ADK model by comparing with the molecular strong-field approximation calculations and with recent experimental measurements.

  13. Candida Virulence Properties and Adverse Clinical Outcomes in Neonatal Candidiasis

    PubMed Central

    Bliss, Joseph M.; Wong, Angela Y.; Bhak, Grace; Laforce-Nesbitt, Sonia S.; Taylor, Sarah; Tan, Sylvia; Stoll, Barbara J.; Higgins, Rosemary D.; Shankaran, Seetha; Benjamin, Daniel K.

    2012-01-01

    Objective To determine if premature infants with invasive Candida infection caused by strains with increased virulence properties have worse clinical outcomes than those infected with less virulent strains. Study design Clinical isolates were studied from 2 populations; premature infants colonized with Candida (commensal, n=27), and those with invasive candidiasis (n=81). Individual isolates of C. albicans and C. parapsilosis were tested for virulence in each of 3 assays: phenotypic switching, adhesion, and cytotoxicity. Invasive isolates were considered to have enhanced virulence if they measured more than 1 SD above the mean for the commensal isolates in at least 1 assay. Outcomes of patients with invasive isolates with enhanced virulence were compared with those with invasive isolates lacking enhanced virulence characteristics. Results 61% of invasive isolates of C. albicans and 42% of invasive isolates of C. parapsilosis had enhanced virulence. All C. albicans cerebrospinal fluid (CSF) isolates (n=6) and 90% of urine isolates (n=10) had enhanced virulence, compared with 48% of blood isolates (n=40). Infants with more virulent isolates were younger at the time of positive culture and had higher serum creatinine. Conclusions Individual isolates of Candida species vary in their virulence properties. Strains with higher virulence are associated with certain clinical outcomes. PMID:22504098

  14. Comparative molecular analyses of Borrelia burgdorferi sensu stricto strains B31 and N40D10/E9 and determination of their pathogenicity

    PubMed Central

    2012-01-01

    Background Lyme disease in the United States is caused primarily by B. burgdorferi sensu stricto while other species are also prevalent in Europe. Genetic techniques have identified several chromosomal and plasmid-borne regulatory and virulence factors involved in Lyme pathogenesis. B31 and N40 are two widely studied strains of B. burgdorferi, which belong to two different 16 S-23 S rRNA spacer types (RST) and outer surface protein C (OspC) allelic groups. However, the presence of several known virulence factors in N40 has not been investigated. This is the first comprehensive study that compared these two strains both in vitro and using the mouse model of infection. Results Phylogenetic analyses predict B31 to be more infectious. However, our studies here indicate that N40D10/E9 is more infectious than the B31 strain at lower doses of inoculation in the susceptible C3H mice. Based-upon a careful analyses of known adhesins of these strains, it is predicted that the absence of a known fibronectin-glycosaminoglycan binding adhesin, bbk32, in the N40 strain could at least partially be responsible for reduction in its binding to Vero cells in vitro. Nevertheless, this difference does not affect the infectivity of N40D10/E9 strain. The genes encoding known regulatory and virulence factors critical for pathogenesis were detected in both strains. Differences in the protein profiles of these B. burgdorferi strains in vitro suggest that the novel, differentially expressed molecules may affect infectivity of B. burgdorferi. Further exacerbation of these molecular differences in vivo could affect the pathogenesis of spirochete strains. Conclusion Based upon the studies here, it can be predicted that N40D10/E9 disseminated infection at lower doses may be enhanced by its lower binding to epithelial cells at the site of inoculation due to the absence of BBK32. We suggest that complete molecular analyses of virulence factors followed by their evaluation using the mouse infection

  15. Identification of Secreted Exoproteome Fingerprints of Highly-Virulent and Non-Virulent Staphylococcus aureus Strains

    PubMed Central

    Bonar, Emilia; Wojcik, Iwona; Jankowska, Urszula; Kedracka-Krok, Sylwia; Bukowski, Michal; Polakowska, Klaudia; Lis, Marcin W.; Kosecka-Strojek, Maja; Sabat, Artur J.; Dubin, Grzegorz; Friedrich, Alexander W.; Miedzobrodzki, Jacek; Dubin, Adam; Wladyka, Benedykt

    2016-01-01

    Staphylococcus aureus is a commensal inhabitant of skin and mucous membranes in nose vestibule but also an important opportunistic pathogen of humans and livestock. The extracellular proteome as a whole constitutes its major virulence determinant; however, the involvement of particular proteins is still relatively poorly understood. In this study, we compared the extracellular proteomes of poultry-derived S. aureus strains exhibiting a virulent (VIR) and non-virulent (NVIR) phenotype in a chicken embryo experimental infection model with the aim to identify proteomic signatures associated with the particular phenotypes. Despite significant heterogeneity within the analyzed proteomes, we identified alpha-haemolysin and bifunctional autolysin as indicators of virulence, whereas glutamylendopeptidase production was characteristic for non-virulent strains. Staphopain C (StpC) was identified in both the VIR and NVIR proteomes and the latter fact contradicted previous findings suggesting its involvement in virulence. By supplementing NVIR, StpC-negative strains with StpC, and comparing the virulence of parental and supplemented strains, we demonstrated that staphopain C alone does not affect staphylococcal virulence in a chicken embryo model. PMID:27242969

  16. Virulence and antimicrobial resistance determinants of verotoxigenic Escherichia coli (VTEC) and of multidrug-resistant E. coli from foods of animal origin illegally imported to the EU by flight passengers.

    PubMed

    Nagy, B; Szmolka, A; Smole Možina, S; Kovač, J; Strauss, A; Schlager, S; Beutlich, J; Appel, B; Lušicky, M; Aprikian, P; Pászti, J; Tóth, I; Kugler, R; Wagner, M

    2015-09-16

    The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx(1) (variants stx1(a) or stx(1c)) or st(x2) (variants stx(2a), stx(2b), stx(2a/d) or stx(2c/d)) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic

  17. Molecular packing determines singlet exciton fission in organic semiconductors.

    PubMed

    Kolata, Kolja; Breuer, Tobias; Witte, Gregor; Chatterjee, Sangam

    2014-07-22

    Carrier multiplication by singlet exciton fission enhances photovoltaic conversion efficiencies in organic solids. This decay of one singlet exciton into two triplet states allows the extraction of up to two electrons per harvested photon and, hence, promises to overcome the Shockley–Queisser limit. However, the microscopic mechanism of singlet exciton fission, especially the relation between molecular packing and electronic response, remains unclear, which therefore hampers the systematic improvement of organic photovoltaic devices. For the model system perfluoropentacene, we experimentally show that singlet exciton fission is greatly enhanced for a slip-stacked molecular arrangement by addressing different crystal axes featuring different packing schemes. This reveals that the fission process strongly depends on the intermolecular coupling: slip-stacking favors delocalization of excitations and allows for efficient exciton fission, while face-to-edge molecular orientations commonly found in the prevailing herringbone molecular stacking patterns even suppress it. Furthermore, we clarify the controversially debated role of excimer states as intermediary rather than competitive or precursory. Our detailed findings serve as a guideline for the design of next-generation molecular materials for application in future organic light-harvesting devices exploiting singlet exciton fission. PMID:24957197

  18. Determination of Molecular Size and Avogadro's Number: A Student Experiment

    ERIC Educational Resources Information Center

    Alexandrakis, George C.

    1978-01-01

    Describes an experiment for estimating molecular size and Avogadro's number. Uses the diffusion length of iodine in air at 100 degrees Celsius as a function of time, and the change in volume of a small quantity of carbon dioxide as it goes from the solid to the gaseous state. (GA)

  19. Molecular Weight Determination by an Improved Temperature-Monitored Vapor-Density Method.

    ERIC Educational Resources Information Center

    Grider, Douglas J.; And Others

    1988-01-01

    Recommends determining molecular weights of liquids by use of a thermocouple. Utilizing a mathematical gas equation, the molecular weight can be determined from the measurement of the vapor temperature upon complete evaporation. Lists benefits as reduced time and cost, and improved safety factors. (ML)

  20. Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids.

    PubMed

    Le Roux, Frédérique; Labreuche, Yannick; Davis, Brigid M; Iqbal, Naeem; Mangenot, Sophie; Goarant, Cyrille; Mazel, Didier; Waldor, Matthew K

    2011-02-01

    Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence-linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non-pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo. PMID:20825454

  1. Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids

    PubMed Central

    Le Roux, Frédérique; Labreuche, Yannick; Davis, Brigid M; Iqbal, Naeem; Mangenot, Sophie; Goarant, Cyrille; Mazel, Didier; Waldor, Matthew K

    2011-01-01

    Summary Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence-linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non-pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo. PMID:20825454

  2. Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.

    PubMed Central

    Day, K P; Karamalis, F; Thompson, J; Barnes, D A; Peterson, C; Brown, H; Brown, G V; Kemp, D J

    1993-01-01

    Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria. Images Fig. 1 Fig. 2 Fig. 3 PMID:8367496

  3. Characterizing the Molecular Basis of Attenuation of Marek's Disease Virus via In Vitro Serial Passage Identifies De Novo Mutations in the Helicase-Primase Subunit Gene UL5 and Other Candidates Associated with Reduced Virulence

    PubMed Central

    Hildebrandt, Evin; Dunn, John R.; Perumbakkam, Sudeep; Niikura, Masahiro

    2014-01-01

    ABSTRACT Marek's disease (MD) is a lymphoproliferative disease of chickens caused by the oncogenic Gallid herpesvirus 2, commonly known as Marek's disease virus (MDV). MD vaccines, the primary control method, are often generated by repeated in vitro serial passage of this highly cell-associated virus to attenuate virulent MDV strains. To understand the genetic basis of attenuation, we used experimental evolution by serially passing three virulent MDV replicates generated from an infectious bacterial artificial chromosome (BAC) clone. All replicates became completely or highly attenuated, indicating that de novo mutation, and not selection among quasispecies existing in a strain, is the primary driving force for the reduction in virulence. Sequence analysis of the attenuated replicates revealed 41 to 95 single-nucleotide variants (SNVs) at 2% or higher frequency in each population and several candidate genes containing high-frequency, nonsynonymous mutations. Five candidate mutations were incorporated into recombinant viruses to determine their in vivo effect. SNVs within UL42 (DNA polymerase auxiliary subunit) and UL46 (tegument) had no measurable influence, while two independent mutations in LORF2 (a gene of unknown function) improved survival time of birds but did not alter disease incidence. A fifth SNV located within UL5 (helicase-primase subunit) greatly reduced in vivo viral replication, increased survival time of birds, and resulted in only 0 to 11% disease incidence. This study shows that multiple genes, often within pathways involving DNA replication and transcriptional regulation, are involved in de novo attenuation of MDV and provides targets for the rational design of future MD vaccines. IMPORTANCE Marek's disease virus (MDV) is a very important pathogen in chickens that costs the worldwide poultry industry $1 billion to $2 billion annually. Marek's disease (MD) vaccines, the primary control method, are often produced by passing virulent strains in cell

  4. Molecular determinants in the biology of liver metastasis.

    PubMed

    Radinsky, R; Ellis, L M

    1996-04-01

    A primary goal of cancer research is an increased understanding of the molecular mechanisms mediating the process of cancer metastasis. Analyses of colon cancer cells (the seeds) and the microenvironment (the soil) has increased our understanding of the biologic mechanisms mediating liver-specific metastasis. Insight into the molecular mechanisms regulating the pathobiology of cancer metastasis as well as a better understanding of the interaction between the metastatic cell and the host environment should produce a foundation for new therapeutic approaches. In this article we summarize experimental observations demonstrating the importance of specific factors that regulate various steps in the metastatic cascade. Furthermore, this article emphasizes the importance of the host organ's microenvironment and its role in liver metastasis formation. The production of metastases depends, in part, on the interaction of particular tumor cells with specific organ environments. Therefore, the successful metastatic cell must be viewed currently as a cell receptive to its environment. The analyses presented herein add important evidence to support the concept that cancer metastasis is not a random process; it is a highly regulated process that can be analyzed on the molecular level. To the clinician, it is readily apparent that by the time metastasis forms, most steps in the metastatic cascade have been completed. Therefore, therapy targeted to downregulate or interrupt the last stages of metastasis, proliferation and angiogenesis, should be the areas of greatest investigation in regards to treating established metastasis, whether they are microscopic or macroscopic. PMID:9019348

  5. Structural features of lipoarabinomannan from Mycobacterium bovis BCG. Determination of molecular mass by laser desorption mass spectrometry.

    PubMed

    Venisse, A; Berjeaud, J M; Chaurand, P; Gilleron, M; Puzo, G

    1993-06-15

    It was recently shown that mycobacterial lipoarabinomannan (LAM) can be classified into two types (Chatterjee, D., Lowell, K., Rivoire B., McNeil M. R., and Brennan, P. J. (1992) J. Biol. Chem. 267, 6234-6239) according to the presence or absence of mannosyl residues (Manp) located at the nonreducing end of the oligoarabinosyl side chains. These two types of LAM were found in a pathogenic Mycobacterium tuberculosis strain and in an avirulent M. tuberculosis strain, respectively, suggesting that LAM with Manp characterizes virulent and "disease-inducing strains." We now report the structure of the LAM from Mycobacterium bovis Bacille Calmette-Guérin (BCG) strain Pasteur, largely used throughout the world as vaccine against tuberculosis. Using an up-to-date analytical approach, we found that the LAM of M. bovis BCG belongs to the class of LAMs capped with Manp. By means of two-dimensional homonuclear and heteronuclear scalar coupling NMR analysis and methylation data, the sugar spin system assignments were partially established, revealing that the LAM contained two types of terminal Manp and 2-O-linked Manp. From the following four-step process: (i) partial hydrolysis of deacylated LAM (dLAM), (ii) oligosaccharide derivatization with aminobenzoic ethyl ester, (iii) HPLC purification, (iv) FAB/MS-MS analysis; it was shown that the dimannosyl unit alpha-D-Manp-(1-->2)-alpha-D-Manp is the major residue capping the termini of the arabinan of the LAM. In this report, LAM molecular mass determination was established using matrix-assisted UV-laser desorption/ionization mass spectrometry which reveals that the LAM molecular mass is around 17.4 kDa. The similarity of the LAM structures between M. bovis BCG and M. tuberculosis H37Rv is discussed in regard to their function in the immunopathology of mycobacterial infection. PMID:8509380

  6. RpoS Is Not Central to the General Stress Response in Borrelia burgdorferi but Does Control Expression of One or More Essential Virulence Determinants

    PubMed Central

    Caimano, Melissa J.; Eggers, Christian H.; Hazlett, Karsten R. O.; Radolf, Justin D.

    2004-01-01

    membrane chambers at a level equivalent to that of the wild type, they were avirulent in mice. Lastly, RT-PCR analysis of the ospE-ospF-elp paralogous lipoprotein families complements earlier findings that many temperature-inducible borrelial loci are controlled in an RpoSBb-independent manner. Together, these data point to fundamental differences between the role(s) of RpoS in B. burgdorferi and that in E. coli. Rather than functioning as a master regulator, RpoSBb appears to serve as a stress-responsive activator of a subset of virulence determinants that, together with the RpoS-independent, differentially expressed regulon, encompass the spirochete's genetic programs required for mammalian host adaptation. PMID:15501774

  7. Virulence Gene Regulation in Escherichia coli.

    PubMed

    Mellies, Jay L; Barron, Alex M S

    2006-01-01

    Escherichia colicauses three types of illnesses in humans: diarrhea, urinary tract infections, and meningitis in newborns. The acquisition of virulence-associated genes and the ability to properly regulate these, often horizontally transferred, loci distinguishes pathogens from the normally harmless commensal E. coli found within the human intestine. This review addresses our current understanding of virulence gene regulation in several important diarrhea-causing pathotypes, including enteropathogenic, enterohemorrhagic,enterotoxigenic, and enteroaggregativeE. coli-EPEC, EHEC, ETEC and EAEC, respectively. The intensely studied regulatory circuitry controlling virulence of uropathogenicE. coli, or UPEC, is also reviewed, as is that of MNEC, a common cause of meningitis in neonates. Specific topics covered include the regulation of initial attachment events necessary for infection, environmental cues affecting virulence gene expression, control of attaching and effacing lesionformation, and control of effector molecule expression and secretion via the type III secretion systems by EPEC and EHEC. How phage control virulence and the expression of the Stx toxins of EHEC, phase variation, quorum sensing, and posttranscriptional regulation of virulence determinants are also addressed. A number of important virulence regulators are described, including the AraC-like molecules PerA of EPEC, CfaR and Rns of ETEC, and AggR of EAEC;the Ler protein of EPEC and EHEC;RfaH of UPEC;and the H-NS molecule that acts to silence gene expression. The regulatory circuitry controlling virulence of these greatly varied E. colipathotypes is complex, but common themes offerinsight into the signals and regulators necessary forE. coli disease progression. PMID:26443571

  8. Determining Geometrically Stable Domains in Molecular Conformation Sets.

    PubMed

    Romanowska, Julia; Nowiński, Krzysztof S; Trylska, Joanna

    2012-08-14

    Detecting significant conformational changes occurring in biomolecules is a challenging task, especially when considering tens to hundreds of thousands of conformations. Conformational variability can be described by dividing a biomolecule into dynamic domains, i.e., by finding compact fragments that move as coherent units. Typical approaches, based on calculating a dynamical cross-correlation matrix, are limited by their inability to reveal correlated rotations and anticorrelated motions. We propose a geometric approach for finding dynamic domains, where we compare traces of atomic movements in a pairwise manner, and search for their best superposition. A quaternion representation of rotation is used to simplify the complex calculations. The algorithm was implemented in a Java graphical program: Geometrically Stable Substructures (GeoStaS). The program processes PDB and DCD binary files with large structural sets for proteins, nucleic acids, and their complexes. We demonstrate its efficiency in analyzing (a) ensembles of structures generated by NMR experiments and (b) conformation sets from biomolecular simulations, such as molecular dynamics. The results provide a clear description of the molecular movements even for large biomolecules. Compared to a standard dynamic cross-correlation matrix, our algorithm detects the correlations in both translational and rotational motions. PMID:26592104

  9. Molecular determinants of platelet delta storage pool deficiencies: an update.

    PubMed

    Masliah-Planchon, Julien; Darnige, Luc; Bellucci, Sylvia

    2013-01-01

    Delta storage pool deficiency (δ-SPD) is a rare heterogeneous group of platelet disorders characterized by a reduction in the number or content of dense granules. δ-SPD causes a mild to moderate bleeding diathesis characterized mainly by mucocutaneous bleeding. Currently, no specific treatment is available and the therapeutic approach is based on prevention of excessive bleeding. However, during the last few years, important insights into the pathophysiology of δ-SPD have been achieved using mouse models and dense granule deficiency-associated congenital diseases, such as Hermansky-Pudlak syndrome and Chediak-Higashi syndrome. It thus appears that δ-SPD represents a genetically heterogeneous group of intracellular vesicle biogenesis and/or trafficking disorders. This review summarizes recent data regarding the molecular mechanisms together with clinical features of the different types of δ-SPD. Although the molecular basis of isolated inherited δ-SPD remains currently unknown, next-generation sequencing strategies should enable researchers to identify the causative genes. Identification of those genes should contribute to our understanding of the pathophysiology, represent useful tools for genetic diagnosis, and eventually lead to new specific therapeutic approaches. PMID:23025459

  10. A Francisella Virulence Factor Catalyzes an Essential Reaction of Biotin Synthesis

    PubMed Central

    Feng, Youjun; Napier, Brooke A.; Manandhar, Miglena; Henke, Sarah K; Weiss, David S.; Cronan, John E.

    2014-01-01

    Summary We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side chain. Expression of bioJ allows growth of an E. coli bioH strain on biotin-free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl-ACP methyl ester carboxyl-esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl-ACP to pimeloyl-ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel sub-clade of the α/β-hydrolase family. Structure-guided mapping combined with site-directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence. PMID:24313380

  11. Structure of the Toxoplasma gondii ROP18 Kinase Domain Reveals a Second Ligand Binding Pocket Required for Acute Virulence*

    PubMed Central

    Lim, Daniel; Gold, Daniel A.; Julien, Lindsay; Rosowski, Emily E.; Niedelman, Wendy; Yaffe, Michael B.; Saeij, Jeroen P. J.

    2013-01-01

    At least a third of the human population is infected with the intracellular parasite Toxoplasma gondii, which contributes significantly to the disease burden in immunocompromised and neutropenic hosts and causes serious congenital complications when vertically transmitted to the fetus. Genetic analyses have identified the Toxoplasma ROP18 Ser/Thr protein kinase as a major factor mediating acute virulence in mice. ROP18 is secreted into the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. However, its precise molecular function and regulation are not fully understood. We have determined the crystal structure of the ROP18 kinase domain, which is inconsistent with a previously proposed autoinhibitory mechanism of regulation. Furthermore, a sucrose molecule bound to our structure identifies an additional ligand-binding pocket outside of the active site cleft. Mutational analysis confirms an important role for this pocket in virulence. PMID:24129568

  12. Molecular players involved in temperature-dependent sex determination and sex differentiation in Teleost fish

    PubMed Central

    2014-01-01

    The molecular mechanisms that underlie sex determination and differentiation are conserved and diversified. In fish species, temperature-dependent sex determination and differentiation seem to be ubiquitous and molecular players involved in these mechanisms may be conserved. Although how the ambient temperature transduces signals to the undifferentiated gonads remains to be elucidated, the genes downstream in the sex differentiation pathway are shared between sex-determining mechanisms. In this paper, we review recent advances on the molecular players that participate in the sex determination and differentiation in fish species, by putting emphasis on temperature-dependent sex determination and differentiation, which include temperature-dependent sex determination and genetic sex determination plus temperature effects. Application of temperature-dependent sex differentiation in farmed fish and the consequences of temperature-induced sex reversal are discussed. PMID:24735220

  13. Molecular determinants of blood–brain barrier permeation

    PubMed Central

    Geldenhuys, Werner J; Mohammad, Afroz S; Adkins, Chris E; Lockman, Paul R

    2015-01-01

    The blood–brain barrier (BBB) is a microvascular unit which selectively regulates the permeability of drugs to the brain. With the rise in CNS drug targets and diseases, there is a need to be able to accurately predict a priori which compounds in a company database should be pursued for favorable properties. In this review, we will explore the different computational tools available today, as well as underpin these to the experimental methods used to determine BBB permeability. These include in vitro models and the in vivo models that yield the dataset we use to generate predictive models. Understanding of how these models were experimentally derived determines our accurate and predicted use for determining a balance between activity and BBB distribution. PMID:26305616

  14. Virulence factors of medically important fungi.

    PubMed Central

    Hogan, L H; Klein, B S; Levitz, S M

    1996-01-01

    Human fungal pathogens have become an increasingly important medical problem with the explosion in the number of immunocompromised patients as a result of cancer, steroid therapy, chemotherapy, and AIDS. Additionally, the globalization of travel and expansion of humankind into previously undisturbed habitats have led to the reemergence of old fungi and new exposure to previously undescribed fungi. Until recently, relatively little was known about virulence factors for the medically important fungi. With the advent of molecular genetics, rapid progress has now been made in understanding the basis of pathogenicity for organisms such as Aspergillus species and Cryptococcus neoformans. The twin technologies of genetic transformation and "knockout" deletion construction allowed for genetic tests of virulence factors in these organisms. Such knowledge will prove invaluable for the rational design of antifungal therapies. Putative virulence factors and attributes are reviewed for Aspergillus species, C. neoformans, the dimorphic fungal pathogens, and others, with a focus upon a molecular genetic approach. Candida species are excluded from coverage, having been the subject of numerous recent reviews. This growing body of knowledge about fungal pathogens and their virulence factors will significantly aid efforts to treat the serious diseases they cause. PMID:8894347

  15. A Longitudinal Study Simultaneously Exploring the Carriage of APEC Virulence Associated Genes and the Molecular Epidemiology of Faecal and Systemic E. coli in Commercial Broiler Chickens

    PubMed Central

    Kemmett, Kirsty; Humphrey, Tom; Rushton, Steven; Close, Andrew; Wigley, Paul; Williams, Nicola J.

    2013-01-01

    Colibacillosis is an economically important syndromic disease of poultry caused by extra-intestinal avian pathogenic Escherichia coli (APEC) but the pathotype remains poorly defined. Combinations of virulence-associated genes (VAGs) have aided APEC identification. The intestinal microbiota is a potential APEC reservoir. Broiler chickens are selectively bred for fast, uniform growth. Here we simultaneously investigate intestinal E. coli VAG carriage in apparently healthy birds and characterise systemic E. coli from diseased broiler chickens from the same flocks. Four flocks were sampled longitudinally from chick placement until slaughter. Phylogrouping, macro-restriction pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed on an isolate subset from one flock to investigate the population structure of faecal and systemic E. coli. Early in production, VAG carriage among chick intestinal E. coli populations was diverse (average Simpson's D value  = 0.73); 24.05% of intestinal E. coli (n = 160) from 1 day old chicks were carrying ≥5 VAGs. Generalised Linear models demonstrated VAG prevalence in potential APEC populations declined with age; 1% of E. coli carrying ≥5 VAGs at slaughter and demonstrated high strain diversity. A variety of VAG profiles and high strain diversity were observed among systemic E. coli. Thirty three new MLST sequence types were identified among 50 isolates and a new sequence type representing 22.2% (ST-2999) of the systemic population was found, differing from the pre-defined pathogenic ST-117 at a single locus. For the first time, this study takes a longitudinal approach to unravelling the APEC paradigm. Our findings, supported by other studies, highlight the difficulty in defining the APEC pathotype. Here we report a high genetic diversity among systemic E. coli between and within diseased broilers, harbouring diverse VAG profiles rather than single and/or highly related pathogenic clones

  16. A Japanese Encephalitis Virus Genotype 5 Molecular Clone Is Highly Neuropathogenic in a Mouse Model: Impact of the Structural Protein Region on Virulence

    PubMed Central

    de Wispelaere, Mélissanne; Frenkiel, Marie-Pascale

    2015-01-01

    ABSTRACT Japanese encephalitis virus (JEV) strains can be separated into 5 genotypes (g1 to g5) based on sequence similarity. JEV g5 strains have been rarely isolated and are poorly characterized. We report here the full characterization of a g5 virus generated using a cDNA-based technology and its comparison with a widely studied g3 strain. We did not observe any major differences between those viruses when their infectious cycles were studied in various cell lines in vitro. Interestingly, the JEV g5 strain was highly pathogenic when inoculated to BALB/c mice, which are known to be largely resistant to JEV g3 infection. The study of chimeric viruses between JEV g3 and g5 showed that there was a poor viral clearance of viruses that express JEV g5 structural proteins in BALB/c mice blood, which correlated with viral invasion of the central nervous system and encephalitis. In addition, using an in vitro model of the blood-brain barrier, we were able to show that JEV g5 does not have an enhanced capacity for entering the central nervous system, compared to JEV g3. Overall, in addition to providing a first characterization of the understudied JEV g5, our work highlights the importance of sustaining an early viremia in the development of JEV encephalitis. IMPORTANCE Genotype 5 viruses are genetically and serologically distinct from other JEV genotypes and can been associated with human encephalitis, which warrants the need for their characterization. In this study, we characterized the in vitro and in vivo properties of a JEV g5 strain and showed that it was more neuropathogenic in a mouse model than a well-characterized JEV g3 strain. The enhanced virulence of JEV g5 was associated with poor viral clearance but not with enhanced crossing of the blood-brain barrier, thus providing new insights into JEV pathogenesis. PMID:25787283

  17. The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

    PubMed Central

    2013-01-01

    Background Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. Results Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. Conclusions We

  18. Molecular Determinants of Magnesium Homeostasis: Insights from Human Disease

    PubMed Central

    Alexander, R. Todd; Hoenderop, Joost G.; Bindels, René J.

    2016-01-01

    The past decade has witnessed multiple advances in our understanding of magnesium (Mg2+) homeostasis. The discovery that mutations in claudin-16/paracellin-1 or claudin-19 are responsible for familial hypomagnesemia with hypercalciuria and nephrocalcinosis provided insight into the molecular mechanisms governing paracellular transport of Mg2+. Our understanding of the transcellular movement of Mg2+ was similarly enhanced by the realization that defects in transient receptor potential melastatin 6 (TRPM6) cause hypomagnesemia with secondary hypocalcemia. This channel regulates the apical entry of Mg2+ into epithelia. In so doing, TRPM6 alters whole-body Mg2+ homeostasis by controlling urinary excretion. Consequently, investigation into the regulation of TRPM6 has increased. Acid-base status, 17β estradiol, and the immunosuppressive agents FK506 and cyclosporine affect plasma Mg2+ levels by altering TRPM6 expression. A mutation in epithelial growth factor is responsible for isolated autosomal recessive hypomagnesemia, and epithelial growth factor activates TRPM6. A defect in the γ-subunit of the Na,K-ATPase causes isolated dominant hypomagnesemia by altering TRPM6 activity through a decrease in the driving force for apical Mg2+ influx. We anticipate that the next decade will provide further detail into the control of the gatekeeper TRPM6 and, therefore, overall whole-body Mg2+ balance. PMID:18562569

  19. Determination of hyaluronan molecular mass distribution in human breast milk.

    PubMed

    Yuan, Han; Amin, Ripal; Ye, Xin; de la Motte, Carol A; Cowman, Mary K

    2015-04-01

    Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M⩽20 kDa, M∼20 to 60 kDa, and M∼60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M⩾110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components. PMID:25579786

  20. Determination of Hyaluronan Molecular Mass Distribution in Human Breast Milk

    PubMed Central

    Yuan, Han; Amin, Ripal; Ye, Xin; De La Motte, Carol A.; Cowman, Mary K.

    2015-01-01

    Hyaluronan (HA) in human milk mediates host responses to microbial infection, via TLR4- and CD44-dependent signaling. Signaling by HA is generally size-specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low M component. Here we report the size distribution of HA in human milk samples from twenty unique donors. A new method for HA analysis, employingion exchange (IEX) chromatography to fractionate HA by size, and specific quantification of each size fraction by competitive Enzyme Linked Sorbent Assay (ELSA), was developed. When separated into four fractions, milk HA with M ≤ 20 kDa, M ≈20-60 kDa, and M ≈ 60-110 kDa comprised an average of 1.5%, 1.4% and 2% of the total HA, respectively. The remaining 95% was HA with M≥110 kDa. Electrophoretic analysis of the higher M HA from thirteen samples showed nearly identical M distributions, with an average M of ∼440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low M HA components. PMID:25579786

  1. Regulatory principles governing Salmonella and Yersinia virulence

    PubMed Central

    Erhardt, Marc; Dersch, Petra

    2015-01-01

    Enteric pathogens such as Salmonella and Yersinia evolved numerous strategies to survive and proliferate in different environmental reservoirs and mammalian hosts. Deciphering common and pathogen-specific principles for how these bacteria adjust and coordinate spatiotemporal expression of virulence determinants, stress adaptation, and metabolic functions is fundamental to understand microbial pathogenesis. In order to manage sudden environmental changes, attacks by the host immune systems and microbial competition, the pathogens employ a plethora of transcriptional and post-transcriptional control elements, including transcription factors, sensory and regulatory RNAs, RNAses, and proteases, to fine-tune and control complex gene regulatory networks. Many of the contributing global regulators and the molecular mechanisms of regulation are frequently conserved between Yersinia and Salmonella. However, the interplay, arrangement, and composition of the control elements vary between these closely related enteric pathogens, which generate phenotypic differences leading to distinct pathogenic properties. In this overview we present common and different regulatory networks used by Salmonella and Yersinia to coordinate the expression of crucial motility, cell adhesion and invasion determinants, immune defense strategies, and metabolic adaptation processes. We highlight evolutionary changes of the gene regulatory circuits that result in different properties of the regulatory elements and how this influences the overall outcome of the infection process. PMID:26441883

  2. Salmonella promotes virulence by repressing cellulose production

    PubMed Central

    Pontes, Mauricio H.; Lee, Eun-Jin; Choi, Jeongjoon; Groisman, Eduardo A.

    2015-01-01

    Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a trade-off between acute virulence and transmission. PMID:25848006

  3. Salmonella promotes virulence by repressing cellulose production.

    PubMed

    Pontes, Mauricio H; Lee, Eun-Jin; Choi, Jeongjoon; Groisman, Eduardo A

    2015-04-21

    Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a trade-off between acute virulence and transmission. PMID:25848006

  4. The Molecular Structure of a Phosphatidylserine Bilayer Determined by Scattering and Molecular Dynamics Simulations

    SciTech Connect

    Pan, Jianjun; Cheng, Xiaolin; Monticelli, Luca; Heberle, Frederick A; Kucerka, Norbert; Tieleman, D. Peter; Katsaras, John

    2014-01-01

    Phosphatidylserine (PS) lipids play essential roles in biological processes, including enzyme activation and apoptosis. We report on the molecular structure and atomic scale interactions of a fluid bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS). A scattering density profile model, aided by molecular dynamics (MD) simulations, was developed to jointly refine different contrast small-angle neutron and X-ray scattering data, which yielded a lipid area of 62.7 A2 at 25 C. MD simulations with POPS lipid area constrained at different values were also performed using all-atom and aliphatic united-atom models. The optimal simulated bilayer was obtained using a model-free comparison approach. Examination of the simulated bilayer, which agrees best with the experimental scattering data, reveals a preferential interaction between Na+ ions and the terminal serine and phosphate moieties. Long-range inter-lipid interactions were identified, primarily between the positively charged ammonium, and the negatively charged carboxylic and phosphate oxygens. The area compressibility modulus KA of the POPS bilayer was derived by quantifying lipid area as a function of surface tension from area-constrained MD simulations. It was found that POPS bilayers possess a much larger KA than that of neutral phosphatidylcholine lipid bilayers. We propose that the unique molecular features of POPS bilayers may play an important role in certain physiological functions.

  5. Molecular distances determined with resonant vibrational energy transfers.

    PubMed

    Chen, Hailong; Wen, Xiewen; Li, Jiebo; Zheng, Junrong

    2014-04-01

    In general, intermolecular distances in condensed phases at the angstrom scale are difficult to measure. We were able to do so by using the vibrational energy transfer method, an ultrafast vibrational analogue of Förster resonance energy transfer. The distances among SCN(-) anions in KSCN crystals and ion clusters of KSCN aqueous solutions were determined with the method. In the crystalline samples, the closest anion distance was determined to be 3.9 ± 0.3 Å, consistent with the XRD result. In the 1.8 and 1 M KSCN aqueous solutions, the anion distances in the ion clusters were determined to be 4.4 ± 0.4 Å. The clustered anion distances in aqueous solutions are very similar to the closest anion distance in the KSCN crystal but significantly shorter than the average anion distance (0.94-1.17 nm) in the aqueous solutions if ion clustering did not occur. The result suggests that ions in the strong electrolyte aqueous solutions can form clusters inside of which they have direct contact with each other. PMID:24641170

  6. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

    PubMed Central

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-01-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6. PMID:27291418

  7. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels.

    PubMed

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-01-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca(2+)-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6. PMID:27291418

  8. Molecular Weight Determinations of Proteins by Californium Plasma Desorption Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Sundqvist, B.; Roepstorff, Peter; Fohlman, J.; Hedin, A.; Hakansson, P.; Kamensky, I.; Lindberg, M.; Salehpour, M.; Sawe, G.

    1984-11-01

    The plasma desorption mass spectrometry method is used to determine the molecular weights of larger molecules than before, to determine the molecular weights of proteins and peptides in mixtures, and to monitor protein modification reactions. Proteins up to molecular weight 25,000 can now be studied with a mass spectrometric technique. Protein-peptide mixtures that could not be resolved with conventional techniques were successfully analyzed by this technique. The precision of the method is good enough to permit one to follow the different steps in the conversion of porcine insulin to human insulin.

  9. Determination of screened Coulomb repulsion energies in organic molecular crystals: A real space approach

    NASA Astrophysics Data System (ADS)

    Cano-Cortés, Laura; Dolfen, Andreas; Merino, Jaime; Koch, Erik

    2010-06-01

    We present a general method for determining screened Coulomb parameters in molecular assemblies, in particular organic molecular crystals. This allows us to calculate the interaction parameters used in a generalized Hubbard model description of correlated organic materials. In such a model only the electrons in levels close to the Fermi level are included explicitly, while the effect of all other electrons is included as a renormalization of the model parameters. For the Coulomb integrals this renormalization is mainly due to screening. For molecular materials we can split the screening into intra- and inter-molecular screening. Here we demonstrate how the inter-molecular screening can be calculated by modeling the molecules by distributed point-polarizabilities and solving the resulting self-consistent electrostatic screening problem in real space. For the example of the quasi one-dimensional molecular metal TTF-TCNQ we demonstrate that the method gives remarkably accurate results.

  10. Molecular Determinants of Cav1.2 Calcium Channel Inactivation

    PubMed Central

    Soldatov, Nikolai M.

    2012-01-01

    Voltage-gated L-type Cav1.2 calcium channels couple membrane depolarization to transient increase in cytoplasmic free Ca2+ concentration that initiates a number of essential cellular functions including cardiac and vascular muscle contraction, gene expression, neuronal plasticity, and exocytosis. Inactivation or spontaneous termination of the calcium current through Cav1.2 is a critical step in regulation of these processes. The pathophysiological significance of this process is manifested in hypertension, heart failure, arrhythmia, and a number of other diseases where acceleration of the calcium current decay should present a benefit function. The central issue of this paper is the inactivation of the Cav1.2 calcium channel mediated by multiple determinants.

  11. Time course Haemophilus parasuis infection reveals pathological differences between virulent and non-virulent strains in the respiratory tract.

    PubMed

    Bello-Orti, Bernardo; Costa-Hurtado, Mar; Martinez-Moliner, Veronica; Segalés, Joaquim; Aragon, Virginia

    2014-06-01

    Haemophilus parasuis is a common inhabitant of the upper respiratory tract of pigs and the etiological agent of Glässer's disease. However, the host-pathogen interaction remains to be well understood. In this work, 33 colostrum-deprived pigs were divided in 4 groups and each group was inoculated intranasally with a different H. parasuis strain (non-virulent strains SW114 and F9, and virulent strains Nagasaki and IT29755). Animals were necropsied at different times in order to determine the location of the bacteria in the respiratory tract of the host during infection. An immunohistochemistry method was developed to detect H. parasuis in nasal turbinates, trachea and lung. Also, the co-localization of H. parasuis with macrophages or neutrophils was examined by double immunohistochemistry and double immunofluorescence. Virulent strains showed a biofilm-like growth in nasal turbinates and trachea and were found easily in lung. Some virulent bacteria were detected in association with macrophages and neutrophils, but also inside pneumocyte-like cells. On the other hand, non-virulent strains were seldom detected in nasal turbinates and trachea, where they showed a microcolony pattern. Non-virulent strains were essentially not detected in lung. In conclusion, this work presents data showing differential localization of H. parasuis bacteria depending on their virulence. Interestingly, the intracellular location of virulent H. parasuis bacteria in non-phagocytic cells in lung could allow the persistence of the bacteria and constitute a virulence mechanism. PMID:24613292

  12. Molecular Determinants of Substrate Specificity in Plant 5-Methylthioadenosine Nucleosidases

    SciTech Connect

    Siu,K.; Lee, J.; Sufrin, J.; Moffatt, B.; McMillan, M.; Cornell, K.; Isom, C.; Howell, L.

    2008-01-01

    5?-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5?-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5?-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Angstroms resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5?-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5?-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pKa of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and

  13. Identification and Manipulation of the Molecular Determinants Influencing Poliovirus Recombination

    PubMed Central

    Runckel, Charles; Westesson, Oscar; Andino, Raul; DeRisi, Joseph L.

    2013-01-01

    The control and prevention of communicable disease is directly impacted by the genetic mutability of the underlying etiological agents. In the case of RNA viruses, genetic recombination may impact public health by facilitating the generation of new viral strains with altered phenotypes and by compromising the genetic stability of live attenuated vaccines. The landscape of homologous recombination within a given RNA viral genome is thought to be influenced by several factors; however, a complete understanding of the genetic determinants of recombination is lacking. Here, we utilize gene synthesis and deep sequencing to create a detailed recombination map of the poliovirus 1 coding region. We identified over 50 thousand breakpoints throughout the genome, and we show the majority of breakpoints to be concentrated in a small number of specific “hotspots,” including those associated with known or predicted RNA secondary structures. Nucleotide base composition was also found to be associated with recombination frequency, suggesting that recombination is modulated across the genome by predictable and alterable motifs. We tested the predictive utility of the nucleotide base composition association by generating an artificial hotspot in the poliovirus genome. Our results imply that modification of these motifs could be extended to whole genome re-designs for the development of recombination-deficient, genetically stable live vaccine strains. PMID:23408891

  14. Unifying model for molecular determinants of the preselection Vβ repertoire.

    PubMed

    Gopalakrishnan, Suhasni; Majumder, Kinjal; Predeus, Alexander; Huang, Yue; Koues, Olivia I; Verma-Gaur, Jiyoti; Loguercio, Salvatore; Su, Andrew I; Feeney, Ann J; Artyomov, Maxim N; Oltz, Eugene M

    2013-08-20

    The primary antigen receptor repertoire is sculpted by the process of V(D)J recombination, which must strike a balance between diversification and favoring gene segments with specialized functions. The precise determinants of how often gene segments are chosen to complete variable region coding exons remain elusive. We quantified Vβ use in the preselection Tcrb repertoire and report relative contributions of 13 distinct features that may shape their recombination efficiencies, including transcription, chromatin environment, spatial proximity to their DβJβ targets, and predicted quality of recombination signal sequences (RSSs). We show that, in contrast to functional Vβ gene segments, all pseudo-Vβ segments are sequestered in transcriptionally silent chromatin, which effectively suppresses wasteful recombination. Importantly, computational analyses provide a unifying model, revealing a minimum set of five parameters that are predictive of Vβ use, dominated by chromatin modifications associated with transcription, but largely independent of precise spatial proximity to DβJβ clusters. This learned model-building strategy may be useful in predicting the relative contributions of epigenetic, spatial, and RSS features in shaping preselection V repertoires at other antigen receptor loci. Ultimately, such models may also predict how designed or naturally occurring alterations of these loci perturb the preselection use of variable gene segments. PMID:23918392

  15. Internal Energy Dependence of Molecular Condensation Coefficients Determined from Molecular Beam Surface Scattering Experiments

    DOE R&D Accomplishments Database

    Sibener, S. J.; Lee, Y. T.

    1978-05-01

    An experiment was performed which confirms the existence of an internal mode dependence of molecular sticking probabilities for collisions of molecules with a cold surface. The scattering of a velocity selected effusive beam of CCl{sub 4} from a 90 K CC1{sub 4} ice surface has been studied at five translational velocities and for two different internal temperatures. At a surface temperature of 90 K (approx. 99% sticking probability) a four fold increase in reflected intensity was observed for the internally excited (560 K) CC1{sub 4} relative to the room temperature (298 K) CC1{sub 4} at a translational velocity of 2.5 X 10{sup 4} cm/sec. For a surface temperature of 90 K all angular distributions were found to peak 15{sup 0} superspecularly independent of incident velocity.

  16. Attenuation of the Virulence of Porphyromonas gingivalis by Using a Specific Synthetic Kgp Protease Inhibitor

    PubMed Central

    Curtis, M. A.; Aduse Opoku, J.; Rangarajan, M.; Gallagher, A.; Sterne, J. A. C.; Reid, C. R.; Evans, H. E. A.; Samuelsson, B.

    2002-01-01

    The Arg- and Lys-gingipains of Porphyromonas gingivalis are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in rgpA and rgpB (encoding Arg-gingipains) and in kgp (encoding Lys-gingipain) and a double mutant with mutations in rgpA and rgpB were prepared by using P. gingivalis W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar Ki was designed and synthesized. Growth of P. gingivalis W50 in the presence of this compound resembled the phenotype of the kgp isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of P. gingivalis and that inhibitors of this enzyme may have therapeutic potential for the control of P. gingivalis infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens. PMID:12438376

  17. GenoType NTM-DR for Identifying Mycobacterium abscessus Subspecies and Determining Molecular Resistance.

    PubMed

    Kehrmann, Jan; Kurt, Nermin; Rueger, Kai; Bange, Franz-Christoph; Buer, Jan

    2016-06-01

    We studied the performance of a new line probe assay for identifying the subspecies and determining the macrolide and aminoglycoside resistance levels of 50 Mycobacterium abscessus isolates. Agreement of GenoType NTM-DR results with sequencing and phenotypic resistance results was 92% for subspecies identification and 98% for determining molecular and phenotypic resistance. PMID:27030487

  18. Determination of the presence of hyaluronic acid in preparations containing amino acids: the molecular weight characterization.

    PubMed

    Bellomaria, A; Nepravishta, R; Mazzanti, U; Marchetti, M; Piccioli, P; Paci, M

    2014-10-15

    Several pharmaceutical preparations contain hyaluronic acid in the presence of a large variety of low molecular weight charged molecules like amino acids. In these mixtures, it is particularly difficult to determine the concentration and the molecular weight of the hyaluronic acid fragments. In fact zwitterionic compounds in high concentration behave by masking the hyaluronic acid due to the electrostatic interactions between amino acids and hyaluronic acid. In such conditions the common colorimetric test of the hyaluronic acid determination appears ineffective and in the (1)H NMR spectra the peaks of the polymer disappear completely. By a simple separation procedure the presence of hyaluronic acid was revealed by the DMAB test and (1)H NMR while its average molecular weight in the final product was determined by DOSY NMR spectroscopy alone. The latter determination is very important due to the healthy effects of some sizes of this polymer's fragments. PMID:25078662

  19. Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression.

    PubMed

    Kriegeskorte, Andre; Block, Desiree; Drescher, Mike; Windmüller, Nadine; Mellmann, Alexander; Baum, Cathrin; Neumann, Claudia; Lorè, Nicola Ivan; Bragonzi, Alessandra; Liebau, Eva; Hertel, Patrick; Seggewiss, Jochen; Becker, Karsten; Proctor, Richard A; Peters, Georg; Kahl, Barbara C

    2014-01-01

    Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed ΔthyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology. Importance: Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus carry mutations in the thymidylate synthase (TS) gene (thyA) responsible for de novo synthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP

  20. Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.

    PubMed

    Liguori, Alessia; Malito, Enrico; Lo Surdo, Paola; Fagnocchi, Luca; Cantini, Francesca; Haag, Andreas F; Brier, Sébastien; Pizza, Mariagrazia; Delany, Isabel; Bottomley, Matthew J

    2016-04-01

    Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in

  1. Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA

    PubMed Central

    Liguori, Alessia; Malito, Enrico; Lo Surdo, Paola; Fagnocchi, Luca; Cantini, Francesca; Haag, Andreas F.; Brier, Sébastien; Pizza, Mariagrazia; Delany, Isabel; Bottomley, Matthew J.

    2016-01-01

    Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in

  2. Transient virulence of emerging pathogens.

    PubMed

    Bolker, Benjamin M; Nanda, Arjun; Shah, Dharmini

    2010-05-01

    Should emerging pathogens be unusually virulent? If so, why? Existing theories of virulence evolution based on a tradeoff between high transmission rates and long infectious periods imply that epidemic growth conditions will select for higher virulence, possibly leading to a transient peak in virulence near the beginning of an epidemic. This transient selection could lead to high virulence in emerging pathogens. Using a simple model of the epidemiological and evolutionary dynamics of emerging pathogens, along with rough estimates of parameters for pathogens such as severe acute respiratory syndrome, West Nile virus and myxomatosis, we estimated the potential magnitude and timing of such transient virulence peaks. Pathogens that are moderately evolvable, highly transmissible, and highly virulent at equilibrium could briefly double their virulence during an epidemic; thus, epidemic-phase selection could contribute significantly to the virulence of emerging pathogens. In order to further assess the potential significance of this mechanism, we bring together data from the literature for the shapes of tradeoff curves for several pathogens (myxomatosis, HIV, and a parasite of Daphnia) and the level of genetic variation for virulence for one (myxomatosis). We discuss the need for better data on tradeoff curves and genetic variance in order to evaluate the plausibility of various scenarios of virulence evolution. PMID:19864267

  3. Fisetin inhibits Listeria monocytogenes virulence by interfering with the oligomerization of listeriolysin O.

    PubMed

    Wang, Jianfeng; Qiu, Jiazhang; Tan, Wei; Zhang, Yu; Wang, Hongshu; Zhou, Xuan; Liu, Shui; Feng, Haihua; Li, Wenhua; Niu, Xiaodi; Deng, Xuming

    2015-05-01

    Listeriolysin O (LLO), an essential virulence determinant of Listeria monocytogenes, is a pore-forming toxin whose primary function is to facilitate cytosolic bacterial replication by breaching the phagosomal membranes, which is critical for the pathogen to evade host immune recognition. The critical role of LLO in the virulence of L. monocytogenes renders it an ideal target for designing novel antivirulence therapeutics. We found that fisetin, a natural flavonoid without antimicrobial activity, is a potent antagonist of LLO-mediated hemolysis. Fisetin effectively inhibits L. monocytogenes infection in both tissue culture and animal infection models. Molecular modeling and mutational analysis revealed that fisetin directly engages loop 2 and loop 3 of LLO, leading to the blockage of cholesterol binding and the reduction of its oligomerization, thus inhibiting its hemolytic activity. Our results establish fisetin as an effective antitoxin agent for LLO, which can be further developed into novel therapeutics against infections caused by L. monocytogenes. PMID:25231018

  4. Direct optical determination of interfacial transport barriers in molecular tunnel junctions.

    PubMed

    Fereiro, Jerry A; McCreery, Richard L; Bergren, Adam Johan

    2013-07-01

    Molecular electronics seeks to build circuitry using organic components with at least one dimension in the nanoscale domain. Progress in the field has been inhibited by the difficulty in determining the energy levels of molecules after being perturbed by interactions with the conducting contacts. We measured the photocurrent spectra for large-area aliphatic and aromatic molecular tunnel junctions with partially transparent copper top contacts. Where no molecular absorption takes place, the photocurrent is dominated by internal photoemission, which exhibits energy thresholds corresponding to interfacial transport barriers, enabling their direct measurement in a functioning junction. PMID:23782345

  5. Adaptive virulence evolution: the good old fitness-based approach.

    PubMed

    Alizon, Samuel; Michalakis, Yannis

    2015-05-01

    Infectious diseases could be expected to evolve towards complete avirulence to their hosts if given enough time. However, this is not the case. Often, virulence is maintained because it is linked to adaptive advantages to the parasite, a situation that is often associated with the hypothesis known as the transmission-virulence trade-off hypothesis. Here, we argue that this hypothesis has three limitations, which are related to how virulence is defined, the possibility of multiple trade-offs, and the difficulty of testing the hypothesis empirically. By adopting a fitness-based approach, where the relation between virulence and the fitness of the parasite throughout its life cycle is directly assessed, it is possible to address these limitations and to determine directly whether virulence is adaptive. PMID:25837917

  6. Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion

    PubMed Central

    Tenor, Jennifer L.; Oehlers, Stefan H.; Yang, Jialu L.

    2015-01-01

    ABSTRACT The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. PMID:26419880

  7. Fitness, Stress Resistance, and Extraintestinal Virulence in Escherichia coli

    PubMed Central

    Bleibtreu, Alexandre; Gros, Pierre-Alexis; Laouénan, Cédric; Clermont, Olivier; Le Nagard, Hervé; Picard, Bertrand; Tenaillon, Olivier

    2013-01-01

    The extraintestinal virulence of Escherichia coli is dependent on numerous virulence genes. However, there is growing evidence for a role of the metabolic properties and stress responses of strains in pathogenesis. We assessed the respective roles of these factors in strain virulence by developing phenotypic assays for measuring in vitro individual and competitive fitness and the general stress response, which we applied to 82 commensal and extraintestinal pathogenic E. coli strains previously tested in a mouse model of sepsis. Individual fitness properties, in terms of maximum growth rates in various media (Luria-Bertani broth with and without iron chelator, minimal medium supplemented with gluconate, and human urine) and competitive fitness properties, estimated as the mean relative growth rate per generation in mixed cultures with a reference fluorescent E. coli strain, were highly diverse between strains. The activity of the main general stress response regulator, RpoS, as determined by iodine staining of the colonies, H2O2 resistance, and rpoS sequencing, was also highly variable. No correlation between strain fitness and stress resistance and virulence in the mouse model was found, except that the maximum growth rate in urine was higher for virulent strains. Multivariate analysis showed that the number of virulence factors was the only independent factor explaining the virulence in mice. At the species level, growth capacity and stress resistance are heterogeneous properties that do not contribute significantly to the intrinsic virulence of the strains. PMID:23690401

  8. Structural and functional analysis of RopB: a major virulence regulator in S treptococcus pyogenes

    PubMed Central

    Makthal, Nishanth; Gavagan, Maire; Do, Hackwon; Olsen, Randall J.; Musser, James M.

    2016-01-01

    Summary Group A Streptococcus (GAS) is an exclusive human pathogen that causes significant disease burden. Global regulator RopB of GAS controls the expression of several major virulence factors including secreted protease SpeB during high cell density. However, the molecular mechanism for RopB‐dependent speB expression remains unclear. To understand the mechanism of transcription activation by RopB, we determined the crystal structure of the C‐terminal domain of RopB. RopB‐CTD has the TPR motif, a signature motif involved in protein–peptide interactions and shares significant structural homology with the quorum sensing RRNPP family regulators. Characterization of the high cell density‐specific cell‐free growth medium demonstrated the presence of a low molecular weight proteinaceous secreted factor that upregulates RopB‐dependent speB expression. Together, these results suggest that RopB and its cognate peptide signals constitute an intercellular signalling machinery that controls the virulence gene expression in concert with population density. Structure‐guided mutational analyses of RopB dimer interface demonstrated that single alanine substitutions at this critical interface significantly altered RopB‐dependent speB expression and attenuated GAS virulence. Results presented here suggested that a properly aligned RopB dimer interface is important for GAS pathogenesis and highlighted the dimerization interactions as a plausible therapeutic target for the development of novel antimicrobials. PMID:26714274

  9. Molecular identification, characterization and assessment of virulence of non-culturable biofilm isolates of L. monocytogenes isolated from chronic infections of turkeys: Implications for product and plant contamination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have hypothesized that sub-clinical, stress-induced infection of turkeys may be an unrecognized source of processing plant contamination with Listeria monocytogenes (Lm) biofilm. The objectives of this study were to determine the effects of early cold stress and concurrent environmental challenge...

  10. Virulence and molecular diversity of the Puccinia striiformis f. sp. tritici population in Xinjiang in relation to other regions of western China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In recent years, wheat stripe rust caused severe yield losses in western China, especially the Xinjiang Autonomous Region. The population of the stripe rust fungus, Puccinia striiformis f. sp. tritici (Pst), in the vast region had not been well studied. To determine the population structure and comp...

  11. Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers

    PubMed Central

    Grosso-Becerra, María Victoria; Croda-García, Gerardo; Merino, Enrique; Servín-González, Luis; Mojica-Espinosa, Raúl; Soberón-Chávez, Gloria

    2014-01-01

    In a number of bacterial pathogens, the production of virulence factors is induced at 37 °C; this effect is often regulated by mRNA structures formed in the 5′ untranslated region (UTR) that block translation initiation of genes at environmental temperatures. At 37 °C, the RNA structures become unstable and ribosomes gain access to their binding sites in the mRNAs. Pseudomonas aeruginosa is an important opportunistic pathogen and the expression of many of its virulence-associated traits is regulated by the quorum-sensing (QS) response, but the effect of temperature on virulence-factor expression is not well-understood. The aim of this work is the characterization of the molecular mechanism involved in thermoregulation of QS-dependent virulence-factor production. We demonstrate that traits that are dependent on the QS transcriptional regulator RhlR have a higher expression at 37 °C, correlating with a higher RhlR concentration as measured by Western blot. We also determined, using gene fusions and point mutations, that RhlR thermoregulation is a posttranscriptional effect dependent on an RNA thermometer of the ROSE (Repression Of heat-Shock gene Expression) family. This RNA element regulates the expression of the rhlAB operon, involved in rhamnolipid production, and of the downstream rhlR gene. We also identified a second functional thermometer in the 5′ UTR of the lasI gene. We confirmed that these RNA thermometers are the main mechanism of thermoregulation of QS-dependent gene expression in P. aeruginosa using quantitative real-time PCR. This is the first description, to our knowledge, of a ROSE element regulating the expression of virulence traits and of an RNA thermometer controlling multiple genes in an operon through a polar effect. PMID:25313031

  12. Mycobacterium tuberculosis virulence: insights and impact on vaccine development.

    PubMed

    Delogu, Giovanni; Provvedi, Roberta; Sali, Michela; Manganelli, Riccardo

    2015-01-01

    The existing TB vaccine, the attenuated Mycobacterium bovis strain BCG, is effective in protecting infants from severe forms of the disease, while its efficacy in protecting adults from pulmonary TB is poor. In the last two decades, a renewed interest in TB resulted in the development of several candidate vaccines that are now entering clinical trials. However, most of these vaccines are based on a common rationale and aim to induce a strong T-cell response against Mycobacterium tuberculosis. Recent advancements in the understanding of M. tuberculosis virulence determinants and associated pathogenic strategies are opening a new and broader view of the complex interaction between this remarkable pathogen and the human host, providing insights at molecular level that could lead to a new rationale for the design of novel antitubercular vaccines. A vaccination strategy that simultaneously targets different steps in TB pathogenesis may result in improved protection and reduced TB transmission. PMID:26119086

  13. Mutations of residues 249 and 256 in VP2 are involved in the replication and virulence of infectious Bursal disease virus.

    PubMed

    Qi, Xiaole; Zhang, Lizhou; Chen, Yuming; Gao, Li; Wu, Guan; Qin, Liting; Wang, Yongqiang; Ren, Xiangang; Gao, Yulong; Gao, Honglei; Wang, Xiaomei

    2013-01-01

    Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. Although the PDE and PFG domains of the capsid protein VP2 contribute significantly to virulence and fitness, the detailed molecular basis for the pathogenicity of IBDV is still not fully understood. Because residues 253 and 284 of VP2 are not the sole determinants of virulence, we hypothesized that other residues involved in virulence and fitness might exist in the PDE and PFG domains of VP2. To test this, five amino acid changes selected by sequence comparison of the PDE and PFG domains of VP2 were introduced individually using a reverse genetics system into the virulent strain (rGx-F9VP2). Then reverse mutations of the selected residues 249 and 256 were introduced individually into the attenuated strain (rGt). Seven modified viruses were generated and evaluated in vitro (CEF cells) and in vivo (SPF chicken). For residue 249, Q249R could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while R249Q could reduce in vitro and elevate in vivo the replication of rGt; meanwhile Q249R reduced the virulence of rGx-F9VP2 while R249Q increased the virulence of rGt, which indicated that residue 249 significantly contributed to the replication and virulence of IBDV. For residue 256, I256V could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while V256I could reduce in vitro but didn't change in vivo the replication of rGt; although V256I didn't increase the virulence of rGt, I256V obviously reduced the virulence of virulent IBDV. The present results demonstrate for the first time, to different extent, residues 249 and 256 of VP2 are involved in the replication efficiency and virulence of IBDV; this is not only beneficial to further understanding of pathogenic mechanism but also to the design of newly tailored vaccines against IBDV. PMID:23923037

  14. Conversion Problems: How (Not) to Determine Molecular Masses in Dwarf Galaxies (Oral Contribution)

    NASA Astrophysics Data System (ADS)

    Hüttemeister, S.

    The determination of molecular gas masses in star forming dwarf irregular galaxies is crucial to assess the star formation process in these objects. But the derivation of the molecular gas content of dwarf galaxies has been a long-standing problem. CO, as the only practical tracer of cold molecular gas, has been (and to some extent still is) notoriously diffucult to detect. Yet, star formation clearly takes place in many dwarf irregulars. This conference contribution contrasts a number of methods commonly used to derive the molecular gas contents of dwarf galaxies based on CO observations: Procedures based on the virial theorem and those relying on radiative transfer arguments. It is shown that both classes of methods have serious drawbacks. Still, examples show that there seem to be real differences in the `correct' conversion factor both between and within star forming dwarf irregular galaxies.

  15. Determination of glyphosate in foodstuff by one novel chemiluminescence-molecular imprinting sensor

    NASA Astrophysics Data System (ADS)

    Zhao, Peini; Yan, Mei; Zhang, Congcong; Peng, Ruixue; Ma, Dongsheng; Yu, Jinghua

    2011-05-01

    A novel chemiluminescence (CL) sensor for the determination of glyphosate (GLY) was made up based on molecularly imprinted polymer (MIP). The molecularly imprinted microspheres (MIMs) with a small dimension which possess extremely high surface-to-volume ratio were synthesized using precipitation polymerization with GLY as template. And then the MIMs were modified on glass sheets, which were placed at the bottom of wells of microplate as the recognizer. Subsequently, a highly selective and high throughput chemiluminescence (CL)-molecular imprinting (MI) sensor for detection of GLY was achieved. Influencing factors were investigated and optimized in detail. The method can perform 96 independent measurements sequentially in 10 min and the limit of detection (LOD) for GLY was 0.046 μg mL -1. The relative standard deviation (RSD) for 11 parallel measurements of GLY was 4.68%. The results show that CL-MI sensor can become a useful analytical technology for quick molecular recognition.

  16. Determination of molecular weights of humic substances by analytical (UV scanning) ultracentrifugation

    NASA Astrophysics Data System (ADS)

    Reid, Patrick M.; Wilkinson, Alan E.; Tipping, Edward; Jones, Malcolm N.

    1990-01-01

    Samples of peat humic acid (PHA) and surface water humic (WBHA) and fulvic (WBFA) acids have been extracted from Whitray Beck in North Yorkshire, U.K. The molecular weights of the extracts have been investigated by sedimentation equilibrium using an analytical ultracentrifuge equipped with a UV scanning system. The system allows measurements to be made at low concentrations of humic substances, comparable to those existing in natural humic-rich water. A method is described for correcting UV scanning data for changes in the optical properties of the materials with changing molecular weight. Measurements have also been made on reference samples of Suwannee river humic (SRHA) and fulvic (SRFA) acids from the International Humic Substances Society (IHSS). The weight-average molecular weights of the extracted samples range from approximately 2000 to 17000 and follow a series PHA > WBHA > WBFA. Apparent specific volumes of these materials were in a range from 0.45 to 0.58 cm 3 g -1 as measured by digital densimetry. All the samples studied were analysed by gel filtration, but the molecular weights determined by this method based on a globular protein calibration are not in good accord with the absolute determinations by the sedimentation-equilibrium technique. The molecular weight of the SRHA determined by sedimentation equilibrium is in good agreement with that reported by BECKETT (1987) et al., based on flow field-flow fractionation.

  17. Identification of a nucleotide in 5' untranslated region contributing to virus replication and virulence of Coxsackievirus A16.

    PubMed

    Li, Zhaolong; Liu, Xin; Wang, Shaohua; Li, Jingliang; Hou, Min; Liu, Guanchen; Zhang, Wenyan; Yu, Xiao-Fang

    2016-01-01

    Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are two main causative pathogens of hand, foot and mouth disease (HFMD). Unlike EV71, virulence determinants of CA16, particularly within 5' untranslated region (5'UTR), have not been investigated until now. Here, a series of nucleotides present in 5'UTR of lethal but not in non-lethal CA16 strains were screened by aligning nucleotide sequences of lethal circulating Changchun CA16 and the prototype G10 as well as non-lethal SHZH05 strains. A representative infectious clone based on a lethal Changchun024 sequence and infectious mutants with various nucleotide alterations in 5'UTR were constructed and further investigated by assessing virus replication in vitro and virulence in neonatal mice. Compared to the lethal infectious clone, the M2 mutant with a change from cytosine to uracil at nucleotide 104 showed weaker virulence and lower replication capacity. The predicted secondary structure of the 5'UTR of CA16 RNA showed that M2 mutant located between the cloverleaf and stem-loop II, affected interactions between the 5'UTR and the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and A1 (hnRNP A1) that are important for translational activity. Thus, our research determined a virulence-associated site in the 5'UTR of CA16, providing a crucial molecular target for antiviral drug development. PMID:26861413

  18. Identification of a nucleotide in 5′ untranslated region contributing to virus replication and virulence of Coxsackievirus A16

    PubMed Central

    Li, Zhaolong; Liu, Xin; Wang, Shaohua; Li, Jingliang; Hou, Min; Liu, Guanchen; Zhang, Wenyan; Yu, Xiao-Fang

    2016-01-01

    Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are two main causative pathogens of hand, foot and mouth disease (HFMD). Unlike EV71, virulence determinants of CA16, particularly within 5′ untranslated region (5′UTR), have not been investigated until now. Here, a series of nucleotides present in 5′UTR of lethal but not in non-lethal CA16 strains were screened by aligning nucleotide sequences of lethal circulating Changchun CA16 and the prototype G10 as well as non-lethal SHZH05 strains. A representative infectious clone based on a lethal Changchun024 sequence and infectious mutants with various nucleotide alterations in 5′UTR were constructed and further investigated by assessing virus replication in vitro and virulence in neonatal mice. Compared to the lethal infectious clone, the M2 mutant with a change from cytosine to uracil at nucleotide 104 showed weaker virulence and lower replication capacity. The predicted secondary structure of the 5′UTR of CA16 RNA showed that M2 mutant located between the cloverleaf and stem-loop II, affected interactions between the 5′UTR and the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and A1 (hnRNP A1) that are important for translational activity. Thus, our research determined a virulence-associated site in the 5′UTR of CA16, providing a crucial molecular target for antiviral drug development. PMID:26861413

  19. Molecular evolution of Dmrt1 accompanies change of sex-determining mechanisms in reptilia

    PubMed Central

    Janes, Daniel E.; Organ, Christopher L.; Stiglec, Rami; O'Meally, Denis; Sarre, Stephen D.; Georges, Arthur; Graves, Jennifer A. M.; Valenzuela, Nicole; Literman, Robert A.; Rutherford, Kim; Gemmell, Neil; Iverson, John B.; Tamplin, Jeffrey W.; Edwards, Scott V.; Ezaz, Tariq

    2014-01-01

    In reptiles, sex-determining mechanisms have evolved repeatedly and reversibly between genotypic and temperature-dependent sex determination. The gene Dmrt1 directs male determination in chicken (and presumably other birds), and regulates sex differentiation in animals as distantly related as fruit flies, nematodes and humans. Here, we show a consistent molecular difference in Dmrt1 between reptiles with genotypic and temperature-dependent sex determination. Among 34 non-avian reptiles, a convergently evolved pair of amino acids encoded by sequence within exon 2 near the DM-binding domain of Dmrt1 distinguishes species with either type of sex determination. We suggest that this amino acid shift accompanied the evolution of genotypic sex determination from an ancestral condition of temperature-dependent sex determination at least three times among reptiles, as evident in turtles, birds and squamates. This novel hypothesis describes the evolution of sex-determining mechanisms as turnover events accompanied by one or two small mutations. PMID:25540158

  20. Microfluidics Meets Dilute Solution Viscometry: An Undergraduate Laboratory to Determine Polymer Molecular Weight Using a Microviscometer

    ERIC Educational Resources Information Center

    Pety, Stephen J.; Lu, Hang; Thio, Yonathan S.

    2011-01-01

    This paper describes a student laboratory experiment to determine the molecular weight of a polymer sample by measuring the viscosity of dilute polymer solutions in a PDMS microfluidic viscometer. Sample data are given for aqueous solutions of poly(ethylene oxide) (PEO). A demonstration of shear thinning behavior using the microviscometer is…

  1. Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2007-01-01

    SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

  2. Using molecular recognition of beta-cyclodextrin to determine molecular weights of low-molecular-weight explosives by MALDI-TOF mass spectrometry.

    PubMed

    Zhang, Min; Shi, Zhen; Bai, Yinjuan; Gao, Yong; Hu, Rongzu; Zhao, Fenqi

    2006-02-01

    This study presents a novel method for determining the molecular weights of low molecular weight (MW) energetic compounds through their complexes of beta-cyclodextrin (beta-CD) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in a mass range of 500 to 1700 Da, avoiding matrix interference. The MWs of one composite explosive composed of 2,6-DNT, TNT, and RDX, one propellant with unknown components, and 14 single-compound explosives (RDX, HMX, 3,4-DNT, 2,6-DNT, 2,5-DNT, 2,4,6-TNT, TNAZ, DNI, BTTN, NG, TO, NTO, NP, and 662) were measured. The molecular recognition and inclusion behavior of beta-CD to energetic materials (EMs) were investigated. The results show that (1) the established method is sensitive, simple, accurate, and suitable for determining the MWs of low-MW single-compound explosives and energetic components in composite explosives and propellants; and (2) beta-CD has good inclusion and modular recognition abilities to the above EMs. PMID:16406809

  3. Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell.

    PubMed Central

    Couderc, T; Hogle, J; Le Blay, H; Horaud, F; Blondel, B

    1993-01-01

    Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation

  4. Molecular Determinants of Co- and Post-translational N-glycosylation of Type I Transmembrane Peptides

    PubMed Central

    Malaby, Heidi L. H.; Kobertz, William R.

    2013-01-01

    Type I transmembrane peptides acquire N-linked glycans during and after protein synthesis to facilitate anterograde trafficking through the secretory pathway. Mutations in N-glycosylation consensus sites (NXT and NXS, where X ≠ P) that alter the kinetics of the initial N-glycan attachment have been associated with cardiac arrhythmias; however, the molecular determinants that define co- and post-translational consensus sites in proteins are not known. Here, we identified co- and post-translational consensus sites in the KCNE family of K+ channel regulatory subunits to uncover three determinants that favor co-translational N-glycosylation kinetics of type I transmembrane peptides that lack a cleavable signal sequence: threonine containing-consensus sites (NXT), multiple N-terminal consensus sites, and long C-termini. The identification of these three molecular determinants now makes it possible to predict co- and post-translational consensus sites in type I transmembrane peptides. PMID:23718681

  5. ALMOST: an all atom molecular simulation toolkit for protein structure determination.

    PubMed

    Fu, Biao; Sahakyan, Aleksandr B; Camilloni, Carlo; Tartaglia, Gian Gaetano; Paci, Emanuele; Caflisch, Amedeo; Vendruscolo, Michele; Cavalli, Andrea

    2014-05-30

    Almost (all atom molecular simulation toolkit) is an open source computational package for structure determination and analysis of complex molecular systems including proteins, and nucleic acids. Almost has been designed with two primary goals: to provide tools for molecular structure determination using various types of experimental measurements as conformational restraints, and to provide methods for the analysis and assessment of structural and dynamical properties of complex molecular systems. The methods incorporated in Almost include the determination of structural and dynamical features of proteins using distance restraints derived from nuclear Overhauser effect measurements, orientational restraints obtained from residual dipolar couplings and the structural restraints from chemical shifts. Here, we present the first public release of Almost, highlight the key aspects of its computational design and discuss the main features currently implemented. Almost is available for the most common Unix-based operating systems, including Linux and Mac OS X. Almost is distributed free of charge under the GNU Public License, and is available both as a source code and as a binary executable from the project web site at http://www.open-almost.org. Interested users can follow and contribute to the further development of Almost on http://sourceforge.net/projects/almost. PMID:24676684

  6. Determination of molecular weights of humic substances by analytical (UV scanning) ultracentrifugation

    SciTech Connect

    Reid, P.M.; Wilkinson, A.E.; Tipping, E.; Jones, M.N. Freshwater Biological Association, Ambleside, Cumbria )

    1990-01-01

    Samples of peat humic acid (PHA) and surface water humic (WBHA) and fulvic (WBFA) acids have been extracted from Whitray Beck in North Yorkshire, U.K. The molecular weights of the extracts have been investigated by sedimentation equilibrium using an analytical ultracentrifuge equipped with a UV scanning system. The system allows measurements to be made at low concentrations of humic substances, comparable to those existing in natural humic-rich water. A method is described for correcting UV scanning data for changes in the optical properties of the materials with changing molecular weight. Measurements have also been made on reference samples of Suwannee river humic (SRHA) and fulvic (SRFA) acids from the International Humic Substances Society (IHSS). The weight-average moleuclar weights of the extracted samples range from approximately 2,000 to 17,000 and follow a series PHA > WBHA > WBFA. Apparent specific volumes of these materials were in a range from 0.45 to 0.58 cm{sup 3} g{sup {minus}1} as measured by digital densimetry. Al the samples studied were analysed by gel filtration, but the molecular weights determined by this method based on a globular protein calibration are not in good accord with the absolute determinations by the sedimentation-equilibrium technique. The molecular weight of the SRHA determined by sedimentation equilibrium is in good agreement with that reported by BECKETT (1987) et al., based on flow field-flow fractionation.

  7. A molecular-gap device for specific determination of mercury ions

    NASA Astrophysics Data System (ADS)

    Guo, Zheng; Liu, Zhong-Gang; Yao, Xian-Zhi; Zhang, Kai-Sheng; Chen, Xing; Liu, Jin-Huai; Huang, Xing-Jiu

    2013-11-01

    Specific determination/monitoring of trace mercury ions (Hg2+) in environmental water is of significant importance for drinking safety. Complementarily to conventional inductively coupled plasma mass spectrometry and atomic emission/absorption spectroscopy, several methods, i.e., electrochemical, fluorescent, colorimetric, and surface enhanced Raman scattering approaches, have been developed recently. Despite great success, many inevitably encounter the interferences from other metal ions besides the complicated procedures and sophisticated equipments. Here we present a molecular-gap device for specific determination of trace Hg2+ in both standardized solutions and environmental samples based on conductivity-modulated glutathione dimer. Through a self-assembling technique, a thin film of glutathione monolayer capped Au nanoparticles is introduced into 2.5 μm-gap-electrodes, forming numerous double molecular layer gaps. Notably, the fabricated molecular-gap device shows a specific response toward Hg2+ with a low detection limit actually measured down to 1 nM. Theoretical calculations demonstrate that the specific sensing mechanism greatly depends on the electron transport ability of glutathione dimer bridged by heavy metal ions, which is determined by its frontier molecular orbital, not the binding energy.

  8. Virulence of Shigella flexneri Hybrids Expressing Escherichia coli Somatic Antigens

    PubMed Central

    Gemski, P.; Sheahan, D. G.; Washington, O.; Formal, S. B.

    1972-01-01

    The genes controlling either Escherichia coli somatic antigen 8 or 25 were conjugally transferred to virulent Shigella flexneri 2a recipients to determine whether the aquisition of these antigens would affect the virulence of the resulting hybrid. A high proportion of such hybrids were found to be rough and hence were avirulent. Some smooth S. flexneri hybrids which replaced their native group antigens with E. coli factor 25 were still virulent in the animal models employed. All S. flexneri O-8 hybrids were uniformly avirulent. Our finding, that S. flexneri hybrids with the chemically divergent E. coli O-8 repeat unit are avirulent whereas some hybrids with the chemically related O-25 repeat unit retain virulence, suggests that the chemical composition and structure of the O side chain of somatic antigens may represent one determining factor for bacterial penetration of mucosal epithelial cells, the primary step in the pathogenesis of bacillary dysentery. Images PMID:4569915

  9. Incidence and Virulence Determinants of Verocytotoxin-Producing Escherichia coli Infections in the Brussels-Capital Region, Belgium, in 2008–2010

    PubMed Central

    De Gheldre, Yves; Dediste, Anne; de Moreau, Anne-Isabelle; Mascart, Georges; Simon, Anne; Allemeersch, Daniël; Scheutz, Flemming; Lauwers, Sabine; Piérard, Denis

    2012-01-01

    The incidence of verocytotoxin-producing Escherichia coli (VTEC) was investigated by PCR in all human stools from Universitair Ziekenhuis Brussel (UZB) and in selected stools from six other hospital laboratories in the Brussels-Capital Region, Belgium, collected between April 2008 and October 2010. The stools selected to be included in this study were those from patients with hemolytic-uremic syndrome (HUS), patients with a history of bloody diarrhea, patients linked to clusters of diarrhea, children up to the age of 6 years, and stools containing macroscopic blood. Verocytotoxin genes (vtx) were detected significantly more frequently in stools from patients with the selected conditions (2.04%) than in unselected stools from UZB (1.20%) (P = 0.001). VTEC was detected most frequently in patients with HUS (35.3%), a history of bloody diarrhea (5.15%), or stools containing macroscopic blood (1.85%). Stools from patients up to the age of 17 years were significantly more frequently vtx positive than those from adult patients between the ages of 18 and 65 years (P = 0.022). Although stools from patients older than 65 years were also more frequently positive for vtx than those from patients between 18 and 65 years, this trend was not significant. VTEC was isolated from 140 (67.9%) vtx-positive stools. One sample yielded two different serotypes; thus, 141 isolates could be characterized. Sixty different O:H serotypes harboring 85 different virulence profiles were identified. Serotypes O157:H7/H− (n = 34), O26:H11/H− (n = 21), O63:H6 (n = 8), O111:H8/H− (n = 7), and O146:H21/H− (n = 6) accounted for 53.9% of isolates. All O157 isolates carried vtx2, eae, and a complete O island 122 (COI-122); 15 also carried vtx1. Non-O157 isolates (n = 107), however, accounted for the bulk (75.9%) of isolates. Fifty-nine (55.1%) isolates were positive for vtx1, 36 (33.6%) were positive for vtx2, and 12 (11.2%) carried both vtx1 and vtx2. Pulsed-field gel electrophoresis revealed

  10. Toxin-Independent Virulence of Bacillus anthracis in Rabbits

    PubMed Central

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

    2014-01-01

    The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP) model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV) of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC) administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation. PMID:24416317

  11. Molecular Simulation of the Free Energy for the Accurate Determination of Phase Transition Properties of Molecular Solids

    NASA Astrophysics Data System (ADS)

    Sellers, Michael; Lisal, Martin; Brennan, John

    2015-06-01

    Investigating the ability of a molecular model to accurately represent a real material is crucial to model development and use. When the model simulates materials in extreme conditions, one such property worth evaluating is the phase transition point. However, phase transitions are often overlooked or approximated because of difficulty or inaccuracy when simulating them. Techniques such as super-heating or super-squeezing a material to induce a phase change suffer from inherent timescale limitations leading to ``over-driving,'' and dual-phase simulations require many long-time runs to seek out what frequently results in an inexact location of phase-coexistence. We present a compilation of methods for the determination of solid-solid and solid-liquid phase transition points through the accurate calculation of the chemical potential. The methods are applied to the Smith-Bharadwaj atomistic potential's representation of cyclotrimethylene trinitramine (RDX) to accurately determine its melting point (Tm) and the alpha to gamma solid phase transition pressure. We also determine Tm for a coarse-grain model of RDX, and compare its value to experiment and atomistic counterpart. All methods are employed via the LAMMPS simulator, resulting in 60-70 simulations that total 30-50 ns. Approved for public release. Distribution is unlimited.

  12. Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid.

    PubMed Central

    Maurelli, A T; Blackmon, B; Curtiss, R

    1984-01-01

    In this study, we examined the relationship between the virulence of Shigella flexneri 2a and the ability of strains of S. flexneri 2a to absorb Congo red. Spontaneous nonpigmented (i.e., unable to bind Congo red [Pcr-]) derivatives of a virulent, pigmented (Pcr+) strain of S. flexneri 2a were isolated and assayed for virulence as determined by their ability to invade epithelial cells. All Pcr- mutants examined lost the ability to invade epithelial cells and were thus avirulent. Agarose gel electrophoresis of plasmid DNA from these avirulent, Pcr- mutants showed that the majority of these strains had lost a plasmid band corresponding to a virulence-associated plasmid, pSf2a140. In many of the mutants, concomitant loss of pigmentation, virulence, and pSf2a140 was accompanied by the appearance of a new plasmid, smaller than pSf2a140. We believe these new plasmids to be deletion derivatives of pSf2a140 and that loss of pigmentation and loss of virulence are associated with deletions in pSf2a140. We transduced Pcr- mutants to Pcr+ and isolated transductants which suppressed the Pcr- phenotype. None of the Pcr+ transductants regained the ability to invade epithelial cells. Several suppressors of the Pcr- phenotype were identified as mutations in cell wall biosynthesis. These results support our belief that although pigmentation is usually associated with virulence, genetic determinants unrelated to virulence can also affect the ability of the cell to bind Congo red. Therefore, the ability of S. flexneri 2a to bind Congo red does not necessarily imply the ability to invade epithelial cells. However, loss of ability to bind Congo red is accompanied by loss of virulence. Images PMID:6360906

  13. Identification of disulfide bond isomerase substrates reveals bacterial virulence factors

    PubMed Central

    Ren, Guoping; Champion, Matthew M.; Huntley, Jason F.

    2014-01-01

    Summary Bacterial pathogens are exposed to toxic molecules inside the host and require efficient systems to form and maintain correct disulfide bonds for protein stability and function. The intracellular pathogen Francisella tularensis encodes a disulfide bond formation protein ortholog, DsbA, which previously was reported to be required for infection of macrophages and mice. However, the molecular mechanisms by which F. tularensis DsbA contributes to virulence are unknown. Here, we demonstrate that F. tularensis DsbA is a bifunctional protein that oxidizes and, more importantly, isomerizes complex disulfide connectivity in substrates. A single amino acid in the conserved cis-proline loop of the DsbA thioredoxin domain was shown to modulate both isomerase activity and F. tularensis virulence. Trapping experiments in F. tularensis identified over 50 F. tularensis DsbA substrates, including outer membrane proteins, virulence factors, and many hypothetical proteins. Six of these hypothetical proteins were randomly selected and deleted, revealing two novel proteins, FTL_1548 and FTL_1709, which are required for F. tularensis virulence. We propose that the extreme virulence of F. tularensis is partially due to the bifunctional nature of DsbA, that many of the newly-identified substrates are required for virulence, and that the development of future DsbA inhibitors could have broad anti-bacterial implications. PMID:25257164

  14. Poxviruses and the Evolution of Host Range and Virulence

    PubMed Central

    Haller, Sherry L.; Peng, Chen; McFadden, Grant; Rothenburg, Stefan

    2013-01-01

    Poxviruses as a group can infect a large number of animals. However, at the level of individual viruses, even closely related poxviruses display highly diverse host ranges and virulence. For example, variola virus, the causative agent of smallpox, is human-specific and highly virulent only to humans, whereas related cowpox viruses naturally infect a broad spectrum of animals and only cause relatively mild disease in humans. The successful replication of poxviruses depends on their effective manipulation of the host antiviral responses, at the cellular-, tissue- and species-specific levels, which constitutes a molecular basis for differences in poxvirus host range and virulence. A number of poxvirus genes have been identified that possess host range function in experimental settings, and many of these host range genes target specific antiviral host pathways. Herein, we review the biology of poxviruses with a focus on host range, zoonotic infections, virulence, genomics and host range genes as well as the current knowledge about the function of poxvirus host range factors and how their interaction with the host innate immune system contributes to poxvirus host range and virulence. We further discuss the evolution of host range and virulence in poxviruses as well as host switches and potential poxvirus threats for human and animal health. PMID:24161410

  15. Genes Similar to the Vibrio parahaemolyticus Virulence-Related Genes tdh, tlh, and vscC2 Occur in Other Vibrionaceae Species Isolated from a Pristine Estuary

    PubMed Central

    Klein, Savannah L.; Gutierrez West, Casandra K.; Mejia, Diana M.

    2014-01-01

    Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2α gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections. PMID:24212573

  16. Brucella, nitrogen and virulence.

    PubMed

    Ronneau, Severin; Moussa, Simon; Barbier, Thibault; Conde-Álvarez, Raquel; Zuniga-Ripa, Amaia; Moriyon, Ignacio; Letesson, Jean-Jacques

    2016-08-01

    The brucellae are α-Proteobacteria causing brucellosis, an important zoonosis. Although multiplying in endoplasmic reticulum-derived vacuoles, they cause no cell death, suggesting subtle but efficient use of host resources. Brucellae are amino-acid prototrophs able to grow with ammonium or use glutamate as the sole carbon-nitrogen source in vitro. They contain more than twice amino acid/peptide/polyamine uptake genes than the amino-acid auxotroph Legionella pneumophila, which multiplies in a similar vacuole, suggesting a different nutritional strategy. During these two last decades, many mutants of key actors in nitrogen metabolism (transporters, enzymes, regulators, etc.) have been described to be essential for full virulence of brucellae. Here, we review the genomic and experimental data on Brucella nitrogen metabolism and its connection with virulence. An analysis of various aspects of this metabolism (transport, assimilation, biosynthesis, catabolism, respiration and regulation) has highlighted differences and similarities in nitrogen metabolism with other α-Proteobacteria. Together, these data suggest that, during their intracellular life cycle, the brucellae use various nitrogen sources for biosynthesis, catabolism and respiration following a strategy that requires prototrophy and a tight regulation of nitrogen use. PMID:25471320

  17. Virulence of enterococci.

    PubMed Central

    Jett, B D; Huycke, M M; Gilmore, M S

    1994-01-01

    Enterococci are commensal organisms well suited to survival in intestinal and vaginal tracts and the oral cavity. However, as for most bacteria described as causing human disease, enterococci also possess properties that can be ascribed roles in pathogenesis. The natural ability of enterococci to readily acquire, accumulate, and share extrachromosomal elements encoding virulence traits or antibiotic resistance genes lends advantages to their survival under unusual environmental stresses and in part explains their increasing importance as nosocomial pathogens. This review discusses the current understanding of enterococcal virulence relating to (i) adherence to host tissues, (ii) invasion and abscess formation, (iii) factors potentially relevant to modulation of host inflammatory responses, and (iv) potentially toxic secreted products. Aggregation substance, surface carbohydrates, or fibronectin-binding moieties may facilitate adherence to host tissues. Enterococcus faecalis appears to have the capacity to translocate across intact intestinal mucosa in models of antibiotic-induced superinfection. Extracellular toxins such as cytolysin can induce tissue damage as shown in an endophthalmitis model, increase mortality in combination with aggregation substance in an endocarditis model, and cause systemic toxicity in a murine peritonitis model. Finally, lipoteichoic acid, superoxide production, or pheromones and corresponding peptide inhibitors each may modulate local inflammatory reactions. Images PMID:7834601

  18. Hyaluronic Acid Molecular Weight Determines Lung Clearance and Biodistribution after Instillation.

    PubMed

    Kuehl, Christopher; Zhang, Ti; Kaminskas, Lisa M; Porter, Christopher J H; Davies, Neal M; Forrest, Laird; Berkland, Cory

    2016-06-01

    Hyaluronic acid (HA) has emerged as a versatile polymer for drug delivery. Multiple commercial products utilize HA, it can be obtained in a variety of molecular weights, and it offers chemical handles for cross-linkers, drugs, or imaging agents. Previous studies have investigated multiple administration routes, but the absorption, biodistribution, and pharmacokinetics of HA after delivery to the lung is relatively unknown. Here, pharmacokinetic parameters were investigated by delivering different molecular weights of HA (between 7 and 741 kDa) to the lungs of mice. HA was labeled with either a near-infrared dye or with iodine-125 conjugated to HA using a tyrosine linker. In initial studies, dye-labeled HA was instilled into the lungs and fluorescent images of organs were collected at 1, 8, and 24 h post administration. Data suggested longer lung persistence of higher molecular weight HA, but signal diminished for all molecular weights at 8 h. To better quantitate pharmacokinetic parameters, different molecular weights of iodine-125 labeled HA were instilled and organ radioactivity was determined after 1, 2, 4, 6, and 8 h. The data showed that, after instillation, the lungs contained the highest levels of HA, as expected, followed by the gastrointestinal tract. Smaller molecular weights of HA showed more rapid systemic distribution, while 67 and 215 kDa HA showed longer persistence in the lungs. Lung exposure appeared to be optimum in this size range due to the rapid absorption of <67 kDa HA and the poor lung penetration and mucociliary clearance of viscous solutions of HA > 215 kDa. The versatility of HA molecular weight and conjugation chemistries may, therefore, provide new opportunities to extend pulmonary drug exposure and potentially facilitate access to lymph nodes draining the pulmonary bed. PMID:27157508

  19. Electrochemical Molecular Imprinted Sensors Based on Electrospun Nanofiber and Determination of Ascorbic Acid.

    PubMed

    Zhai, Yunyun; Wang, Dandan; Liu, Haiqing; Zeng, Yanbo; Yin, Zhengzhi; Li, Lei

    2015-01-01

    In this study, electrochemical molecularly imprinted sensors were fabricated and used for the determination of ascorbic acid (AA). Nanofiber membranes of cellulose acetate (CA)/multi-walled carbon nanotubes (MWCNTs)/polyvinylpyrrolidone (PVP) (CA/MWCNTs/PVP) were prepared by electrospinning technique. After being transferred to a glass carbon electrode (GC), the nanofiber interface was further polymerized with pyrrole through electrochemical cyclic voltammetry (CV) technique. Meanwhile, target molecules (such as AA) were embedded into the polypyrrole through the hydrogen bond. The effects of monomer concentration (pyrrole), the number of scan cycles and scan rates of polymerization were optimized. Differential pulse voltammetry (DPV) tests indicated that the oxidation current of AA (the selected target) were higher than that of the structural analogues, which illustrated the selective recognition of AA by molecularly imprinted sensors. Simultaneously, the molecularly imprinted sensors had larger oxidation current of AA than non-imprinted sensors in the processes of rebinding. The electrochemical measurements showed that the molecularly imprinted sensors demonstrated good identification behavior for the detection of AA with a linear range of 10.0 - 1000 μM, a low detection limit down to 3 μM (S/N = 3), and a recovery rate range from 94.0 to 108.8%. Therefore, the electrochemical molecularly imprinted sensors can be used for the recognition and detection of AA without any time-consuming elution. The method presented here demonstrates the great potential for electrospun nanofibers and MWCNTs to construct electrochemical sensors. PMID:26256603

  20. From the H II Region to the Molecular Cloud: Determining Physical Conditions in Star Forming Regions

    NASA Astrophysics Data System (ADS)

    Abel, N. P.

    2004-12-01

    Infrared missions such as Spitzer offer new insights into the chemical evolution and star formation history of the universe. Although objects such as starburst galaxies, which are among the most luminous objects in the universe, are the primary focus, OMC-1, a lower luminosity region with superb spatial resolution, serves as a benchmark to test the physics of newly formed O stars interacting with the surrounding molecular environment. The classical approach in determining conditions in the ionized, photodissociated, and molecular regions is to treat each problem separately. In actuality, however, this is a single continuous phenomenon, linked through the transport of gas and radiation. Here we self-consistently calculate the physical conditions and emission from the hot HII region to the cold, molecular gas as a continuous hydrostatic layer. The ion states of the first 30 elements, along with the abundances of 70 molecules, are determined with the temperature and electron density. The grain physics is treated self-consistently, with grain charge transfer, single photon heating, and PAH effects all included. Additionally, level populations of all the rotational/vibrational levels of the ground electronic state of molecular hydrogen are determined (see the dissertation talk of Gargi Shaw). As a benchmark, we consider the physical conditions through OMC-1 1' west of the Trapezium, where emission-line observations of the HII region and the PDR/molecular cloud all exist. Accurately interpreting this spectrum will give us confidence that we can apply our calculations to more luminous and distant starburst galaxies. We predict the sometimes significant contribution of the HII region to important PDR emission-line diagnostics. This has consequences for the interpretation of IR observations, the deduced values of n(H) and G0 in PDRs, and hence the overall conditions in star forming regions. All calculations were developed with the spectral synthesis code Cloudy, which is

  1. Investigating the ?Trojan Horse? Mechanism of Yersinia pestis Virulence

    SciTech Connect

    McCutchen-Maloney, S L; Fitch, J P

    2005-02-08

    Yersinia pestis, the etiological agent of plague, is a Gram-negative, highly communicable, enteric bacterium that has been responsible for three historic plague pandemics. Currently, several thousand cases of plague are reported worldwide annually, and Y. pestis remains a considerable threat from a biodefense perspective. Y. pestis infection can manifest in three forms: bubonic, septicemic, and pneumonic plague. Of these three forms, pneumonic plague has the highest fatality rate ({approx}100% if left untreated), the shortest intervention time ({approx}24 hours), and is highly contagious. Currently, there are no rapid, widely available vaccines for plague and though plague may be treated with antibiotics, the emergence of both naturally occurring and potentially engineered antibiotic resistant strains makes the search for more effective therapies and vaccines for plague of pressing concern. The virulence mechanism of this deadly bacterium involves induction of a Type III secretion system, a syringe-like apparatus that facilitates the injection of virulence factors, termed Yersinia outer membrane proteins (Yops), into the host cell. These virulence factors inhibit phagocytosis and cytokine secretion, and trigger apoptosis of the host cell. Y. pestis virulence factors and the Type III secretion system are induced thermally, when the bacterium enters the mammalian host from the flea vector, and through host cell contact (or conditions of low Ca{sup 2+} in vitro). Apart from the temperature increase from 26 C to 37 C and host cell contact (or low Ca{sup 2+} conditions), other molecular mechanisms that influence virulence induction in Y. pestis are largely uncharacterized. This project focused on characterizing two novel mechanisms that regulate virulence factor induction in Y. pestis, immunoglobulin G (IgG) binding and quorum sensing, using a real-time reporter system to monitor induction of virulence. Incorporating a better understanding of the mechanisms of virulence

  2. Determining the Molecular Packing Arrangements on Protein Crystal Faces by Atomic Force Microscopy

    NASA Technical Reports Server (NTRS)

    Li, Huayu; Perozzo, Mary A.; Konnert, John H.; Nadarajan, Arunan; Pusey, Marc L.

    1998-01-01

    Periodic Bond Chain (PBC) analysis of the packing of tetragonal lysozyme crystals have revealed that there are two possible molecular packing arrangements for the crystal faces. The analysis also predicted that only one of these, involving the formation of helices about the 4(sub 3) axes, would prevail during crystal growth. In this study high resolution atomic force microscopy (AFM) was employed to verify these predictions for the (110) crystal face. A computer program was developed which constructs the expected AFM image for a given tip shape for each possible molecular packing arrangement. By comparing the actual AFM image with the predicted images the correct packing arrangement was determined. The prediction of an arrangement involving 4(sub 3) helices was confirmed in this manner,"while the alternate arrangement was not observed. The investigation also showed the protein molecules were packed slightly closer about the 4(sub 3) axes than in the crystallographic arrangement of the crystal interior. This study demonstrates a new approach for determining the molecular packing arrangements on protein crystal faces. It also shows the power of combining a theoretical PBC analysis with experimental high resolution AFM techniques in probing protein crystal growth processes at the molecular level.

  3. Variable Virulence Factors in Burkholderia pseudomallei (Melioidosis) Associated with Human Disease

    PubMed Central

    Webb, Jessica R.; Ward, Linda M.; Voutsinos, Marcos Y.; Tuanyok, Apichai; Mayo, Mark; Kaestli, Mirjam; Currie, Bart J.

    2014-01-01

    Burkholderia pseudomallei is a Gram-negative environmental bacterium that causes melioidosis, a potentially life-threatening infectious disease affecting mammals, including humans. Melioidosis symptoms are both protean and diverse, ranging from mild, localized skin infections to more severe and often fatal presentations including pneumonia, septic shock with multiple internal abscesses and occasionally neurological involvement. Several ubiquitous virulence determinants in B. pseudomallei have already been discovered. However, the molecular basis for differential pathogenesis has, until now, remained elusive. Using clinical data from 556 Australian melioidosis cases spanning more than 20 years, we identified a Burkholderia mallei-like actin polymerization bimABm gene that is strongly associated with neurological disease. We also report that a filamentous hemagglutinin gene, fhaB3, is associated with positive blood cultures but is negatively correlated with localized skin lesions without sepsis. We show, for the first time, that variably present virulence factors play an important role in the pathogenesis of melioidosis. Collectively, our study provides a framework for assessing other non-ubiquitous bacterial virulence factors and their association with disease, such as candidate loci identified from large-scale microbial genome-wide association studies. PMID:24618705

  4. Bacteriological and virulence study of a Mycobacterium chimaera isolate from a patient in China.

    PubMed

    Liu, Guan; Chen, Su-Ting; Yu, Xia; Li, Yu-Xun; Ling, Ying; Dong, Ling-Ling; Zheng, Su-Hua; Huang, Hai-Rong

    2015-04-01

    A clinical isolate from a patient was identified as Mycobacterium chimaera, a recently identified species of nontuberculous Mycobacteria. The biochemical and molecular identity, drug sensitivity and virulence of this isolated strain were investigated. 16S rRNA, the 16S-23S ITS, hsp65 and rpoB were amplified, and their sequence similarities with other mycobacteria were analyzed. The minimum inhibitory concentrations of 22 anti-microbial agents against this isolate were established, and the virulence of the isolate was evaluated by intravenous injection into C57BL/6 mice using Mycobacterium tuberculosis H37Rv as a control strain. Growth and morphological characteristics and mycolic acid profile analysis revealed that this isolated strain was a member of the Mycobacterium avium complex. BLAST analysis of the amplified sequences showed that the isolated strain was closely related to M. chimaera. Susceptibility testing showed that the isolate was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, imipenem and cefoxitin. Bacterial load determination and tissue histopathology of the infected mice indicated that the isolate was highly virulent. The first case of M. chimaera infection in China was evaluated. The information derived from this case may offer valuable guidance for clinical diagnosis and treatment. PMID:25763937

  5. Characterization of Antimicrobial Resistance Patterns and Detection of Virulence Genes in Campylobacter Isolates in Italy

    PubMed Central

    Di Giannatale, Elisabetta; Di Serafino, Gabriella; Zilli, Katiuscia; Alessiani, Alessandra; Sacchini, Lorena; Garofolo, Giuliano; Aprea, Giuseppe; Marotta, Francesca

    2014-01-01

    Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations. PMID:24556669

  6. Joint Transcriptional Control of Virulence and Resistance to Antibiotic and Environmental Stress in Acinetobacter baumannii

    PubMed Central

    Gallagher, Larry A.; Jacobson, Rachael K.; Usacheva, Elena A.; Peterson, Lance R.; Zurawski, Daniel V.; Shuman, Howard A.

    2015-01-01

    ABSTRACT The increasing emergence of antibiotic-resistant bacterial pathogens represents a serious risk to human health and the entire health care system. Many currently circulating strains of Acinetobacter baumannii exhibit resistance to multiple antibiotics. A key limitation in combating A. baumannii is that our understanding of the molecular mechanisms underlying the pathogenesis of A. baumannii is lacking. To identify potential virulence determinants of a contemporary multidrug-resistant isolate of A. baumannii, we used transposon insertion sequencing (TnSeq) of strain AB5075. A collection of 250,000 A. baumannii transposon mutants was analyzed for growth within Galleria mellonella larvae, an insect-based infection model. The screen identified 300 genes that were specifically required for survival and/or growth of A. baumannii inside G. mellonella larvae. These genes encompass both known, established virulence factors and several novel genes. Among these were more than 30 transcription factors required for growth in G. mellonella. A subset of the transcription factors was also found to be required for resistance to antibiotics and environmental stress. This work thus establishes a novel connection between virulence and resistance to both antibiotics and environmental stress in A. baumannii. PMID:26556274

  7. Virulence Factors of Aeromonas hydrophila: In the Wake of Reclassification.

    PubMed

    Rasmussen-Ivey, Cody R; Figueras, Maria J; McGarey, Donald; Liles, Mark R

    2016-01-01

    The ubiquitous "jack-of-all-trades," Aeromonas hydrophila, is a freshwater, Gram-negative bacterial pathogen under revision in regard to its phylogenetic and functional affiliation with other aeromonads. While virulence factors are expectedly diverse across A. hydrophila strains and closely related species, our mechanistic knowledge of the vast majority of these factors is based on the molecular characterization of the strains A. hydrophila AH-3 and SSU, which were reclassified as A. piscicola AH-3 in 2009 and A. dhakensis SSU in 2013. Individually, these reclassifications raise important questions involving the applicability of previous research on A. hydrophila virulence mechanisms; however, this issue is exacerbated by a lack of genomic data on other research strains. Collectively, these changes represent a fundamental gap in the literature on A. hydrophila and confirm the necessity of biochemical, molecular, and morphological techniques in the classification of research strains that are used as a foundation for future research. This review revisits what is known about virulence in A. hydrophila and the feasibility of using comparative genomics in light of this phylogenetic revision. Conflicting data between virulence factors, secretion systems, quorum sensing, and their effect on A. hydrophila pathogenicity appears to be an artifact of inappropriate taxonomic comparisons and/or be due to the fact that these properties are strain-specific. This review audits emerging data on dominant virulence factors that are present in both A. dhakensis and A. hydrophila in order to synthesize existing data with the aim of locating where future research is needed. PMID:27610107

  8. Size and number of DNA molecules from Chinese hamster ovary cells determined by molecular autoradiography

    SciTech Connect

    Todd, M.B.

    1980-06-01

    A new method for visualization of separable subunits of DNA is described. Autoradiography of tritium-labeled DNA from one or a few nuclei, lysed with detergent, moderate salt, and proteases, and gently deposited on a filter, allows determination of subunit molecular weight, size distribution, number per nucleus, and organization. The shape of the size distribution of CHO subunit images is similar to that of CHO mitotic chromosomes, and the numbers of subunits per nucleus supports a model of eight subunits per chromosome.

  9. Molecular Determinants of hERG Channel Block by Terfenadine and Cisapride

    PubMed Central

    Kamiya, Kaichiro; Niwa, Ryoko; Morishima, Mikio; Honjo, Haruo; Sanguinetti, Michael C.

    2010-01-01

    Block of cardiac hERG K+ channels by the antihistamine terfenadine and the prokinetic agent cisapride is associated with prolonged ventricular repolarization and an increased risk of ventricular arrhythmia. Here, we used a site-directed mutagenesis approach to determine the molecular determinants of hERG block by terfenadine and cisapride. Wild-type and mutant hERG channels were heterologously expressed in Xenopus laevis oocytes and characterized by measuring whole cell currents with two-microelectrode voltage clamp techniques. Mutation of T623, S624, Y652, or F656 to Ala reduced channel sensitivity to block by terfenadine. The same mutations reduced sensitivity to cisapride. These data confirm our previous findings that polar residues (T623, S624) located near the base of the pore helix and aromatic residues (Y652, F656) located in the S6 domain are key molecular determinants of the hERG drug binding site. Unlike methanesulfonanilides (dofetilide, MK-499, E-4031, ibutilide) or clofilium, mutation of V625, G648, or V659 did not alter the sensitivity of hERG channels to terfenadine or cisapride. As previously proposed by molecular modeling studies (Farid R, et al. Bioorg Med Chem. 2006;14:3160–3173), our findings suggest that different drugs can adopt distinct modes of binding to the central cavity of hERG. PMID:18987434

  10. Interactions between ether phospholipids and cholesterol as determined by scattering and molecular dynamics simulations.

    PubMed

    Pan, Jianjun; Cheng, Xiaolin; Heberle, Frederick A; Mostofian, Barmak; Kučerka, Norbert; Drazba, Paul; Katsaras, John

    2012-12-27

    Cholesterol and ether lipids are ubiquitous in mammalian cell membranes, and their interactions are crucial in ether lipid mediated cholesterol trafficking. We report on cholesterol's molecular interactions with ether lipids as determined using a combination of small-angle neutron and X-ray scattering, and all-atom molecular dynamics (MD) simulations. A scattering density profile model for an ether lipid bilayer was developed using MD simulations, which was then used to simultaneously fit the different experimental scattering data. From analysis of the data the various bilayer structural parameters were obtained. Surface area constrained MD simulations were also performed to reproduce the experimental data. This iterative analysis approach resulted in good agreement between the experimental and simulated form factors. The molecular interactions taking place between cholesterol and ether lipids were then determined from the validated MD simulations. We found that in ether membranes cholesterol primarily hydrogen bonds with the lipid headgroup phosphate oxygen, while in their ester membrane counterparts cholesterol hydrogen bonds with the backbone ester carbonyls. This different mode of interaction between ether lipids and cholesterol induces cholesterol to reside closer to the bilayer surface, dehydrating the headgroup's phosphate moiety. Moreover, the three-dimensional lipid chain spatial density distribution around cholesterol indicates anisotropic chain packing, causing cholesterol to tilt. These insights lend a better understanding of ether lipid-mediated cholesterol trafficking and the roles that the different lipid species have in determining the structural and dynamical properties of membrane associated biomolecules. PMID:23199292

  11. Interactions between Ether Phospholipids and Cholesterol as Determined by Scattering and Molecular Dynamics Simulations

    SciTech Connect

    Pan, Jianjun; Cheng, Xiaolin; Heberle, Frederick A; Mostofian, Barmak; Kucerka, Norbert; Drazba, Paul; Katsaras, John

    2012-01-01

    Cholesterol and ether lipids are ubiquitous in mammalian cell membranes, and their interactions are crucial in ether lipid mediated cholesterol trafficking. We report on cholesterol s molecular interactions with ether lipids as determined using a combination of small-angle neutron and Xray scattering, and all-atom molecular dynamics (MD) simulations. A scattering density profile model for an ether lipid bilayer was developed using MD simulations, which was then used to simultaneously fit the different experimental scattering data. From analysis of the data the various bilayer structural parameters were obtained. Surface area constrained MD simulations were also performed to reproduce the experimental data. This iterative analysis approach resulted in good agreement between the experimental and simulated form factors. The molecular interactions taking place between cholesterol and ether lipids were then determined from the validated MD simulations. We found that in ether membranes cholesterol primarily hydrogen bonds with the lipid headgroup phosphate oxygen, while in their ester membrane counterparts cholesterol hydrogen bonds with the backbone ester carbonyls. This different mode of interaction between ether lipids and cholesterol induces cholesterol to reside closer to the bilayer surface, dehydrating the headgroup s phosphate moiety. Moreover, the three-dimensional lipid chain spatial density distribution around cholesterol indicates anisotropic chain packing, causing cholesterol to tilt. These insights lend a better understanding of ether lipid-mediated cholesterol trafficking and the roles that the different lipid species have in determining the structural and dynamical properties of membrane associated biomolecules.

  12. 6-Gingerol reduces Pseudomonas aeruginosa biofilm formation and virulence via quorum sensing inhibition

    PubMed Central

    Kim, Han-Shin; Lee, Sang-Hoon; Byun, Youngjoo; Park, Hee-Deung

    2015-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors via quorum sensing (QS). Interfering with normal QS interactions between signal molecules and their cognate receptors is a developing strategy for attenuating its virulence. Here we tested the hypothesis that 6-gingerol, a pungent oil of fresh ginger, reduces biofilm formation and virulence by antagonistically binding to P. aeruginosa QS receptors. In silico studies demonstrated molecular binding occurs between 6-gingerol and the QS receptor LasR through hydrogen bonding and hydrophobic interactions. Experimentally 6-gingerol reduced biofilm formation, several virulence factors (e.g., exoprotease, rhamnolipid, and pyocyanin), and mice mortality. Further transcriptome analyses demonstrated that 6-gingerol successfully repressed QS-induced genes, specifically those related to the production of virulence factors. These results strongly support our hypothesis and offer insight into the molecular mechanism that caused QS gene repression. PMID:25728862

  13. Modified--Hill-determinant method for the hydrogen molecular-ion problem

    SciTech Connect

    Bhattacharjee, R.S.; Saxena, R.P.; Srivastava, P.K.; Sane, K.V.

    1983-10-01

    The problem of the H/sub 2/ molecular ion is reinvestigated with the use of a method of truncated Hill determinants. Modified Hill determinants are found such that the truncations thereof form a convergent sequence. The wave functions obtained from the present formulation are expected to be more reliable than those reported earlier and in fact are shown to be orthonormal to one part in 10/sup 6/. Equilibrium bond lengths (R/sub e/), stiffness constants, and the wave functions at R/sub e/ for various states are presented.

  14. Correlation between antimicrobial resistance and virulence in Klebsiella pneumoniae.

    PubMed

    Hennequin, C; Robin, F

    2016-03-01

    Klebsiella pneumoniae is responsible for a wide range of infections, including urinary tract infections, pneumonia, bacteremia, and liver abscesses. In addition to susceptible clinical isolates involved in nosocomial infections, multidrug-resistant (MDR) and hypervirulent (hvKP) strains have evolved separately in distinct clonal groups. The rapid geographic spread of these isolates is of particular concern. However, we still know little about the virulence of K. pneumoniae except for hvKP, whose secrets are beginning to be revealed. The treatment of K. pneumoniae infections is threatened by the emergence of antimicrobial resistance. The dissemination of resistance is associated with genetic mobile elements, such as plasmids that may also carry virulence determinants. A proficient pathogen should be virulent, resistant to antibiotics, and epidemic. However, the interplay between resistance and virulence is poorly understood. Here, we review current knowledge on the topic. PMID:26718943

  15. Biochemical and molecular characterization of the novobiocin and rifampicin resistant Aeromonas hydrophila vaccine strain AL09-71 N+R compared to its virulent parent strain AL90-71

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To understand the fitness cost of novobiocin- and rifampicin- resistance in an attenuated Aeromonas hydrophiila vaccine strain AL09-71 N+R compared to its virulent parent strain AL09-71, colony size, cell size, cell proliferation rate, chemotactic response, and the ability to invade catfish gill cel...

  16. Molecular epidemiology and virulence of Escherichia coli O16:H5-ST131: comparison with H30 and H30-Rx subclones of O25b:H4-ST131.

    PubMed

    Dahbi, Ghizlane; Mora, Azucena; Mamani, Rosalia; López, Cecilia; Alonso, María Pilar; Marzoa, Juan; Blanco, Miguel; Herrera, Alexandra; Viso, Susana; García-Garrote, Fernando; Tchesnokova, Veronika; Billig, Mariya; de la Cruz, Fernando; de Toro, María; González-López, Juan José; Prats, Guillermo; Chaves, Fernando; Martínez-Martínez, Luis; López-Cerezo, Lorena; Denamur, Erick; Blanco, Jorge

    2014-11-01

    The present study was carried out to evaluate the prevalence of the clonal subgroup O16:H5-ST131 and the H30 and H30-Rx subclones among E. coli isolates causing extraintestinal infections and to know their virulence potential. The ST131 clonal group accounted for 490 (16%) of the 2995 isolates obtained from clinical samples in five Spanish hospitals during the study period (2005-2012). Among those 490 ST131 isolates, 456 belonged to serotype O25b:H4, 27 to O16:H5 and seven were O-non-typeable:H4 (ONT:H4). All 27 O16:H5 isolates showed fimH41, whereas fimH30 and fimH22 alleles were the most frequently detected among O25b:H4 isolates. The majority (381/490; 78%) of ST131 isolates belonged to H30 subclone, and 302 of 381 (79%) H30 isolates belonged to the H30-Rx subclone. Of the 27 O16:H5 isolates, 48% produced CTX-M-14; however, none produced CTX-M-15. In contrast, 46% of O25b:H4 isolates produced CTX-M-15 while only 2% produced CTX-M-14. More than a half of the O16:H5 isolates (56%) showed the ExPEC status which was significantly more prevalent within O25b:H4 isolates (81%) (P<0.01), especially among H30-Rx (97%) isolates. In the present study, a modified virotype scheme was applied within which approximately half (52%) of the O16:H5 isolates showed the C1 specific virotype. Despite their low virulence-gene score (mean of virulence genes 6.4 versus 8.5 in O25b:H4 isolates), six out of the 10 O16:H5 isolates assayed showed high virulence in the mouse model of sepsis (killed 90-100% of mice challenged). Furthermore, four O16:H5 isolates of virotypes A and C1, carrying K2 variant of group II capsule, showed lethality at 24h. Thus, certain O16:H5 fimH41 isolates show a similar in vivo virulence to that reported with the highly virulent O25b:H4 H30-Rx isolates (Mora et al., PLOS ONE 2014, e87025), supporting their potential virulence for humans. PMID:25455219

  17. Proteomics Analysis Reveals Previously Uncharacterized Virulence Factors in Vibrio proteolyticus

    PubMed Central

    Ray, Ann; Kinch, Lisa N.; de Souza Santos, Marcela; Grishin, Nick V.

    2016-01-01

    ABSTRACT Members of the genus Vibrio include many pathogens of humans and marine animals that share genetic information via horizontal gene transfer. Hence, the Vibrio pan-genome carries the potential to establish new pathogenic strains by sharing virulence determinants, many of which have yet to be characterized. Here, we investigated the virulence properties of Vibrio proteolyticus, a Gram-negative marine bacterium previously identified as part of the Vibrio consortium isolated from diseased corals. We found that V. proteolyticus causes actin cytoskeleton rearrangements followed by cell lysis in HeLa cells in a contact-independent manner. In search of the responsible virulence factor involved, we determined the V. proteolyticus secretome. This proteomics approach revealed various putative virulence factors, including active type VI secretion systems and effectors with virulence toxin domains; however, these type VI secretion systems were not responsible for the observed cytotoxic effects. Further examination of the V. proteolyticus secretome led us to hypothesize and subsequently demonstrate that a secreted hemolysin, belonging to a previously uncharacterized clan of the leukocidin superfamily, was the toxin responsible for the V. proteolyticus-mediated cytotoxicity in both HeLa cells and macrophages. Clearly, there remains an armory of yet-to-be-discovered virulence factors in the Vibrio pan-genome that will undoubtedly provide a wealth of knowledge on how a pathogen can manipulate host cells. PMID:27460800

  18. Molecular species composition of plant cardiolipin determined by liquid chromatography mass spectrometry.

    PubMed

    Zhou, Yonghong; Peisker, Helga; Dörmann, Peter

    2016-07-01

    Cardiolipin (CL), an anionic phospholipid of the inner mitochondrial membrane, provides essential functions for stabilizing respiratory complexes and is involved in mitochondrial morphogenesis and programmed cell death in animals. The role of CL and its metabolism in plants are less well understood. The measurement of CL in plants, including its molecular species composition, is hampered by the fact that CL is of extremely low abundance, and that plants contain large amounts of interfering compounds including galactolipids, neutral lipids, and pigments. We used solid phase extraction by anion exchange chromatography to purify CL from crude plant lipid extracts. LC/MS was used to determine the content and molecular species composition of CL. Thus, up to 23 different molecular species of CL were detected in different plant species, including Arabidopsis, mung bean, spinach, barley, and tobacco. Similar to animals, plant CL is dominated by highly unsaturated species, mostly containing linoleic and linolenic acid. During phosphate deprivation or exposure to an extended dark period, the amount of CL decreased in Arabidopsis, accompanied with an increased degree in unsaturation. The mechanism of CL remodeling during stress, and the function of highly unsaturated CL molecular species, remains to be defined. PMID:27179363

  19. Phylogeny and virulence divergency analyses of Toxoplasma gondii isolates from China

    PubMed Central

    2014-01-01

    Background Toxoplasma gondii (T. gondii) is a very successful parasite that can infect virtually all warm blooded animals with a worldwide distribution. It causes a large range of clinical manifestations in both humans and domesticated animals. In addition, marked biological differences exist among T. gondii strains in the pathogenicity and geographical distribution. Molecular epidemiology studies primarily based on restriction fragment length polymorphism (RFLP) method revealed that three main types are predominant in North America and Europe, whereas other diverse genotypes are found in other parts of the world. Microsatellite (MS) as a type of genetic marker has been widely used in many organisms. Limited MS genotyping, however, to fingerprint T. gondii isolates has been reported and little is known about the MS data of the strains predominantly prevalent in China. Methods Genotyping of twenty-eight Chinese T. gondii isolates were performed using 15 MS markers located on 12 different chromosomes. Results were analyzed in terms of population structure by a Bayesian statistical approach. Phylogenetic analysis was obtained from a Neighbor-Net phylogenetic network. The virulence analyses of some representative isolates were determined by inoculation of mice and cell invasion assays. The gene expressions of some virulence-associated factors (VFs) were performed by quantitative real-time PCR (qRT- PCR). Results Three haplogroups were clustered among the 28 isolates although minor genetic differences were found within haplogroups. The majority of strains belong to one haplogroup corresponding to the previously described Chinese 1 type (ToxoDB#9). Phylogenetic networks uncovered a limited diversity of T. gondii strains and the virulence differs in the strains sharing the same genotype. No remarkable difference, however, was noted in the tested VFs except for dense granule protein3 (GRA3), which was found to have a higher expression in low virulent TgCtwh6 (Wh6) strain

  20. Occurrence of virulent genes among environmental isolates of Legionella pneumophila serogroup 1 strains from various parts of peninsular Malaysia.

    PubMed

    Arushothy, Revathy; Ahmad, Norazah

    2008-12-01

    Legionella pneumophila are intracellular pathogens, associated with human disease, attributed to the presence and absence of certain virulent genes. In this study, virulent gene loci (lvh and rtxA regions) associated with human disease were determined. Thirty-three cooling tower water isolates, isolated between 2004 to 2006, were analyzed for the presence of these genes by PCR method. Results showed that 19 of 33 (57.5%) of the L. pneumophila serogroup 1 isolates have both the genes. Six (18.2%) of the isolates have only the lvh gene and 2 (6.1%) of the isolates have only the rtxA gene. However, both genes were absent in 6 (18.2%) of the L. pneumophila isolates. The result of our study provides some insight into the presence of the disease causing L. pneumophila serogroup 1 in the environment. Molecular epidemiological studies will provide better understanding of the prevalence of the disease in Malaysia. PMID:19287368

  1. Regulation of Francisella Tularensis Virulence

    PubMed Central

    Dai, Shipan; Mohapatra, Nrusingh P.; Schlesinger, Larry S.; Gunn, John S.

    2011-01-01

    Francisella tularensis is one of the most virulent bacteria known and a Centers for Disease Control and Prevention Category A select agent. It is able to infect a variety of animals and insects and can persist in the environment, thus Francisella spp. must be able to survive in diverse environmental niches. However, F. tularensis has a surprising dearth of sensory and regulatory factors. Recent advancements in the field have identified new functions of encoded transcription factors and greatly expanded our understanding of virulence gene regulation. Here we review the current knowledge of environmental adaptation by F. tularensis, its transcriptional regulators and their relationship to animal virulence. PMID:21687801

  2. Molecular Characterization of Multidrug Resistant Hospital Isolates Using the Antimicrobial Resistance Determinant Microarray

    PubMed Central

    Leski, Tomasz A.; Vora, Gary J.; Barrows, Brian R.; Pimentel, Guillermo; House, Brent L.; Nicklasson, Matilda; Wasfy, Momtaz; Abdel-Maksoud, Mohamed; Taitt, Chris Rowe

    2013-01-01

    Molecular methods that enable the detection of antimicrobial resistance determinants are critical surveillance tools that are necessary to aid in curbing the spread of antibiotic resistance. In this study, we describe the use of the Antimicrobial Resistance Determinant Microarray (ARDM) that targets 239 unique genes that confer resistance to 12 classes of antimicrobial compounds, quaternary amines and streptothricin for the determination of multidrug resistance (MDR) gene profiles. Fourteen reference MDR strains, which either were genome, sequenced or possessed well characterized drug resistance profiles were used to optimize detection algorithms and threshold criteria to ensure the microarray's effectiveness for unbiased characterization of antimicrobial resistance determinants in MDR strains. The subsequent testing of Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae hospital isolates revealed the presence of several antibiotic resistance genes [e.g. belonging to TEM, SHV, OXA and CTX-M classes (and OXA and CTX-M subfamilies) of β-lactamases] and their assemblages which were confirmed by PCR and DNA sequence analysis. When combined with results from the reference strains, ∼25% of the ARDM content was confirmed as effective for representing allelic content from both Gram-positive and –negative species. Taken together, the ARDM identified MDR assemblages containing six to 18 unique resistance genes in each strain tested, demonstrating its utility as a powerful tool for molecular epidemiological investigations of antimicrobial resistance in clinically relevant bacterial pathogens. PMID:23936031

  3. Sexual Cycle of Cryptococcus neoformans var. grubii and Virulence of Congenic a and α Isolates

    PubMed Central

    Nielsen, Kirsten; Cox, Gary M.; Wang, Ping; Toffaletti, Dena L.; Perfect, John R.; Heitman, Joseph

    2003-01-01

    Cryptococcus neoformans is a human-pathogenic fungus that has evolved into three distinct varieties that infect most prominently the central nervous system. A sexual cycle involving haploid cells of a and α mating types has been reported for two varieties (C. neoformans var. neoformans, serotype D, and C. neoformans var. gattii, serotypes B and C), yet the vast majority of infections involve a distinct variety (C. neoformans var. grubii, serotype A) that has been thought to be clonal and restricted to the α mating type. We recently identified the first serotype A isolate of the a mating type which had been thought to be extinct (strain 125.91). Here we report that this unusual strain can mate with a subset of pathogenic serotype A strains to produce a filamentous dikaryon with fused clamp connections, basidia, and viable recombinant basidiospores. One meiotic segregant mated poorly with the serotype A reference strain H99 but robustly with a crg1 mutant that lacks a regulator of G protein signaling and is hyperresponsive to mating pheromone. This meiotic segregant was used to create congenic a and α mating type serotype A strains. Virulence tests with rabbit and murine models of cryptococcal meningitis showed that the serotype A congenic a and α mating type strains had equivalent virulence in animal models, in contrast to previous studies linking the α mating type to increased virulence in congenic serotype D strains. Our studies highlight a role for sexual recombination in the evolution of a human fungal pathogen and provide a robust genetic platform to establish the molecular determinants of virulence. PMID:12933823

  4. Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene.

    PubMed Central

    Fletcher, H M; Schenkein, H A; Morgan, R M; Bailey, K A; Berry, C R; Macrina, F L

    1995-01-01

    In a previous study we cloned and determined the nucleotide sequence of the prtH gene from Porphyromonas gingivalis W83. This gene specifies a 97-kDa protease which is normally found in the membrane vesicles produced by P. gingivalis and which cleaves the C3 complement protein under defined conditions. We developed a novel ermF-ermAM antibiotic resistance gene cassette, which was used with the cloned prtH gene to prepare an insertionally inactivated allele of this gene. This genetic construct was introduced by electroporation into P. gingivalis W83 in order to create a protease-deficient mutant by recombinational allelic exchange. The mutant strain, designated V2296, was compared with the parent strain W83 for proteolytic activity and virulence. Extracellular protein preparations from V2296 showed decreased proteolytic activity compared with preparations from W83. Casein substrate zymography revealed that the 97-kDa proteolytic component as well as a 45-kDa protease was missing in the mutant. In in vivo experiments using a mouse model, V2296 was dramatically reduced in virulence compared with the wild-type W83 strain. A molecular survey of several clinical isolates of P. gingivalis using the prtH gene as a probe suggested that prtH gene sequences were conserved and that they may have been present in multiple copies. Two of 10 isolates did not hybridize with the prtH gene probe. These strains, like the V2296 mutant, also displayed decreased virulence in the mouse model. Taken together, these results suggest an important role for P. gingivalis proteases in soft tissue infections and specifically indicate that the prtH gene product is a virulence factor. PMID:7890419

  5. Determination of Quantum Chemistry Based Force Fields for Molecular Dynamics Simulations of Aromatic Polymers

    NASA Technical Reports Server (NTRS)

    Jaffe, Richard; Langhoff, Stephen R. (Technical Monitor)

    1995-01-01

    Ab initio quantum chemistry calculations for model molecules can be used to parameterize force fields for molecular dynamics simulations of polymers. Emphasis in our research group is on using quantum chemistry-based force fields for molecular dynamics simulations of organic polymers in the melt and glassy states, but the methodology is applicable to simulations of small molecules, multicomponent systems and solutions. Special attention is paid to deriving reliable descriptions of the non-bonded and electrostatic interactions. Several procedures have been developed for deriving and calibrating these parameters. Our force fields for aromatic polyimide simulations will be described. In this application, the intermolecular interactions are the critical factor in determining many properties of the polymer (including its color).

  6. Determination of molecular weight distributions of large water soluble macromolecules using dynamic light scattering

    SciTech Connect

    Mettille, M.J.; Hester, R.D.

    1988-05-01

    Characterization of polymer molecular weight is an extremely important aspect of polymer research, and a vast number of analytical techniques has been used to determine molecular weights. One method is dynamic light scattering (DLS). DLS is also referred to as photon correlation spectroscopy (PCS), quasi-elastic light scattering (QLS), and may other appellations. The phenomenon that gives rise to the DLS technique was first observed in the early 1930's. In the mid 1950's, measurement techniques similar to modern dynamic light scattering were developed. Two major technical developments have greatly enhanced the use of DLS. The first was the development of the laser. This provided a light source with very high intensity at a single frequency. Also, major advances in digital electronics have allowed better data acquisition and faster data analysis than were previously available.

  7. Affinity and competition for TBP are molecular determinants of gene expression noise

    PubMed Central

    Ravarani, Charles N. J.; Chalancon, Guilhem; Breker, Michal; de Groot, Natalia Sanchez; Babu, M. Madan

    2016-01-01

    Cell-to-cell variation in gene expression levels (noise) generates phenotypic diversity and is an important phenomenon in evolution, development and disease. TATA-box binding protein (TBP) is an essential factor that is required at virtually every eukaryotic promoter to initiate transcription. While the presence of a TATA-box motif in the promoter has been strongly linked with noise, the molecular mechanism driving this relationship is less well understood. Through an integrated analysis of multiple large-scale data sets, computer simulation and experimental validation in yeast, we provide molecular insights into how noise arises as an emergent property of variable binding affinity of TBP for different promoter sequences, competition between interaction partners to bind the same surface on TBP (to either promote or disrupt transcription initiation) and variable residence times of TBP complexes at a promoter. These determinants may be fine-tuned under different conditions and during evolution to modulate eukaryotic gene expression noise. PMID:26832815

  8. Determination of ractopamine in pork using a magnetic molecularly imprinted polymer as adsorbent followed by HPLC.

    PubMed

    Tang, Yiwei; Gao, Jingwen; Liu, Xiuying; Lan, Jianxing; Gao, Xue; Ma, Yong; Li, Min; Li, Jianrong

    2016-06-15

    A new magnetic molecularly imprinted polymers (MMIPs) for separation and concentration of ractopamine (RAC) were prepared using surface molecular imprinting technique with methacryloyl chloride as functional monomer and RAC as template. The MMIPs were characterized using transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and vibrating sample magnetometer. The results of re-binding experiments indicated that the MMIPs had fast adsorption kinetics and could reach binding equilibrium within 20 min, and the adsorption capacity of the MMIPs was 2.87-fold higher than that of the corresponding non-imprinted polymer. The selectivity of the MMIPs was evaluated according to its recognition to RAC and its analogues. The synthesized MMIPs were successfully applied to extraction, followed by high performance liquid chromatography to determine RAC in real food samples. Spiked recoveries ranged from 73.60% to 94.5%, with relative standard deviations of <11.17%. PMID:26868550

  9. Conformational analysis of environmental agents: use of X-ray crystallographic data to determine molecular reactivity.

    PubMed Central

    Cody, V

    1985-01-01

    This paper explores the use of crystallographic techniques as an aid in understanding the molecular reactivities of a number of agents that are of concern to pharmacologists and toxicologists. The selected examples demonstrate the role of structural data in the determination of absolute configuration, configurational flexibility and active-site topology for a reactive species. For example, the role of absolute stereochemistry in understanding synthetic pyrethroid structure-activity relationships is shown from analysis of their crystal structures; conformational flexibility among DDT analogues, and the importance of conformational and electronic properties in phenylalkanoic acid herbicides are shown from systematic analysis of their crystal structures; and interpretation of active-site stereochemistry is made by study of computer modeling of enzyme inhibitors in the active sites of related protein crystal structures. Thus, the observed patterns in conformational flexibility and their resultant effects on substrate pharmacological profile can be interpreted in understanding the molecular level events that influence biological reactivity. PMID:3905372

  10. The role of a small-scale cutoff in determining molecular layers at fluid interfaces.

    PubMed

    Sega, Marcello

    2016-08-17

    The existence of molecular layers at liquid/vapour interfaces has been a long debated issue. More than ten years ago it was shown, using computer simulations, that correlations at the liquid/vapour interface resemble those of bulk liquids, even though they can be detected in experiments only in a few cases, where they are so strong that they cannot be concealed by the geometrical smearing of capillary fluctuations. The results of the intrinsic analysis techniques used in computer experiments, however, are still often questioned because of their dependence on a free parameter that usually represents a small-scale cutoff used to determine the interface. In this work I show that there is only one value of the cutoff that can ensure a quantitative explanation of the intrinsic density correlation peaks in terms of successive layer contributions. The value of the cutoff coincides, with a high accuracy, with the molecular diameter. PMID:27499039

  11. Virulence factors of the family Legionellaceae.

    PubMed Central

    Dowling, J N; Saha, A K; Glew, R H

    1992-01-01

    Whereas bacteria in the genus Legionella have emerged as relatively frequent causes of pneumonia, the mechanisms underlying their pathogenicity are obscure. The legionellae are facultative intracellular pathogens which multiply within the phagosome of mononuclear phagocytes and are not killed efficiently by polymorphonuclear leukocytes. The functional defects that might permit the intracellular survival of the legionellae have remained an enigma until recently. Phagosome-lysosome fusion is inhibited by a single strain (Philadelphia 1) of Legionella pneumophila serogroup 1, but not by other strains of L. pneumophila or other species. It has been found that following the ingestion of Legionella organisms, the subsequent activation of neutrophils and monocytes in response to both soluble and particulate stimuli is profoundly impaired and the bactericidal activity of these cells is attenuated, suggesting that Legionella bacterial cell-associated factors have an inhibitory effect on phagocyte activation. Two factors elaborated by the legionellae which inhibit phagocyte activation have been described. First, the Legionella (cyto)toxin blocks neutrophil oxidative metabolism in response to various agonists by an unknown mechanism. Second, L. micdadei bacterial cells contain a phosphatase which blocks superoxide anion production by stimulated neutrophils. The Legionella phosphatase disrupts the formation of critical intracellular second messengers in neutrophils. In addition to the toxin and phosphatase, several other moieties that may serve as virulence factors by promoting cell invasion or intracellular survival and multiplication are elaborated by the legionellae. Molecular biological studies show that a cell surface protein named Mip is necessary for the efficient invasion of monocytes. A possible role for a Legionella phospholipase C as a virulence factor is still largely theoretical. L. micdadei contains an unusual protein kinase which catalyzes the phosphorylation of

  12. Effects of zinc transporters on Cryptococcus gattii virulence

    PubMed Central

    Schneider, Rafael de Oliveira; Diehl, Camila; dos Santos, Francine Melise; Piffer, Alícia Corbellini; Garcia, Ane Wichine Acosta; Kulmann, Marcos Iuri Roos; Schrank, Augusto; Kmetzsch, Lívia; Vainstein, Marilene Henning; Staats, Charley C.

    2015-01-01

    Zinc is an essential nutrient for all living organisms because it is a co-factor of several important proteins. Furthermore, zinc may play an essential role in the infectiousness of microorganisms. Previously, we determined that functional zinc metabolism is associated with Cryptococcus gattii virulence. Here, we characterized the ZIP zinc transporters in this human pathogen. Transcriptional profiling revealed that zinc levels regulated the expression of the ZIP1, ZIP2 and ZIP3 genes, although only the C. gattii zinc transporter Zip1 was required for yeast growth under zinc-limiting conditions. To associate zinc uptake defects with virulence, the most studied cryptococcal virulence factors (i.e., capsule, melanin and growth at 37 °C) were assessed in ZIP mutant strains; however, no differences were detected in these classical virulence-associated traits among the mutant and WT strains. Interestingly, higher levels of reactive oxygen species were detected in the zip1Δ and in the zip1Δ zip2Δ double mutants. In line with these phenotypic alterations, the zip1Δ zip2Δ double mutant displayed attenuated virulence in a murine model of cryptococcosis. Together, these results indicate that adequate zinc uptake is necessary for cryptococcal fitness and virulence. PMID:25951314

  13. Advanced Structural Determination of Diterpene Esters Using Molecular Modeling and NMR Spectroscopy.

    PubMed

    Nothias-Scaglia, Louis-Félix; Gallard, Jean-François; Dumontet, Vincent; Roussi, Fanny; Costa, Jean; Iorga, Bogdan I; Paolini, Julien; Litaudon, Marc

    2015-10-23

    Three new jatrophane esters (1-3) were isolated from Euphorbia amygdaloides ssp. semiperfoliata, including an unprecedented macrocyclic jatrophane ester bearing a hemiketal substructure, named jatrohemiketal (3). The chemical structures of compounds 1-3 and their relative configurations were determined by spectroscopic analysis. The absolute configuration of compound 3 was determined unambiguously through an original strategy combining NMR spectroscopy and molecular modeling. Conformational search calculations were performed for the four possible diastereomers 3a-3d differing in their C-6 and C-9 stereocenters, and the lowest energy conformer was used as input structure for geometry optimization. The prediction of NMR parameters ((1)H and (13)C chemical shifts and (1)H-(1)H coupling constants) by density functional theory (DFT) calculations allowed identifying the most plausible diastereomer. Finally, the stereostructure of 3 was solved by comparison of the structural features obtained by molecular modeling for 3a-3d with NMR-derived data (the values of dihedral angles deduced from the vicinal proton-proton coupling constants ((3)JHH) and interproton distances determined by ROESY). The methodology described herein provides an efficient way to solve or confirm structural elucidation of new macrocyclic diterpene esters, in particular when no crystal structure is available. PMID:26431312

  14. Molecular Machines Determining the Fate of Endocytosed Synaptic Vesicles in Nerve Terminals

    PubMed Central

    Fassio, Anna; Fadda, Manuela; Benfenati, Fabio

    2016-01-01

    The cycle of a synaptic vesicle (SV) within the nerve terminal is a step-by-step journey with the final goal of ensuring the proper synaptic strength under changing environmental conditions. The SV cycle is a precisely regulated membrane traffic event in cells and, because of this, a plethora of membrane-bound and cytosolic proteins are devoted to assist SVs in each step of the journey. The cycling fate of endocytosed SVs determines both the availability for subsequent rounds of release and the lifetime of SVs in the terminal and is therefore crucial for synaptic function and plasticity. Molecular players that determine the destiny of SVs in nerve terminals after a round of exo-endocytosis are largely unknown. Here we review the functional role in SV fate of phosphorylation/dephosphorylation of SV proteins and of small GTPases acting on membrane trafficking at the synapse, as they are emerging as key molecules in determining the recycling route of SVs within the nerve terminal. In particular, we focus on: (i) the cyclin-dependent kinase-5 (cdk5) and calcineurin (CN) control of the recycling pool of SVs; (ii) the role of small GTPases of the Rab and ADP-ribosylation factor (Arf) families in defining the route followed by SV in their nerve terminal cycle. These regulatory proteins together with their synaptic regulators and effectors, are molecular nanomachines mediating homeostatic responses in synaptic plasticity and potential targets of drugs modulating the efficiency of synaptic transmission. PMID:27242505

  15. Photoresponsive hollow molecularly imprinted polymer for the determination of trace bisphenol A in water.

    PubMed

    Gong, Cheng-Bin; Yang, Yu-Zhu; Yang, Yue-Hong; Zheng, An-Xun; Liu, Song; Tang, Qian

    2016-11-01

    A photoresponsive hollow molecularly imprinted polymer (PHMIP) was fabricated for photoresponsive recognition and determination of trace bisphenol A (BPA) in aqueous media using a water-soluble azo compound as the functional monomer. The PHMIP was prepared on sacrificial silica microspheres by surface imprinting and subsequent removal of the silica core. The PHMIP displayed photocontrolled recognition for BPA. SEM, TEM, FT-IR, TGA and N2 adsorption-desorption analyses confirmed successful formation of the hollow structure. The PHMIP displayed higher binding capacity, a larger specific area, and faster mass transfer rate than its corresponding surface molecularly imprinted polymer. The PHMIP was used to determine trace BPA in real samples with a limit of detection of 0.5ppm. For samples spiked at 0-10ppm, the BPA recoveries were in the range of 93.0%-99.0%. This PHMIP-based method provides convenient and inexpensive detection method for trace BPA in environmental samples. This method is especially suitable for determining materials that do not possess specific spectroscopic or luminescent properties. PMID:27478978

  16. Molecular Machines Determining the Fate of Endocytosed Synaptic Vesicles in Nerve Terminals.

    PubMed

    Fassio, Anna; Fadda, Manuela; Benfenati, Fabio

    2016-01-01

    The cycle of a synaptic vesicle (SV) within the nerve terminal is a step-by-step journey with the final goal of ensuring the proper synaptic strength under changing environmental conditions. The SV cycle is a precisely regulated membrane traffic event in cells and, because of this, a plethora of membrane-bound and cytosolic proteins are devoted to assist SVs in each step of the journey. The cycling fate of endocytosed SVs determines both the availability for subsequent rounds of release and the lifetime of SVs in the terminal and is therefore crucial for synaptic function and plasticity. Molecular players that determine the destiny of SVs in nerve terminals after a round of exo-endocytosis are largely unknown. Here we review the functional role in SV fate of phosphorylation/dephosphorylation of SV proteins and of small GTPases acting on membrane trafficking at the synapse, as they are emerging as key molecules in determining the recycling route of SVs within the nerve terminal. In particular, we focus on: (i) the cyclin-dependent kinase-5 (cdk5) and calcineurin (CN) control of the recycling pool of SVs; (ii) the role of small GTPases of the Rab and ADP-ribosylation factor (Arf) families in defining the route followed by SV in their nerve terminal cycle. These regulatory proteins together with their synaptic regulators and effectors, are molecular nanomachines mediating homeostatic responses in synaptic plasticity and potential targets of drugs modulating the efficiency of synaptic transmission. PMID:27242505

  17. Tracking bacterial virulence: global modulators as indicators.

    PubMed

    Prieto, Alejandro; Urcola, Imanol; Blanco, Jorge; Dahbi, Ghizlane; Muniesa, Maite; Quirós, Pablo; Falgenhauer, Linda; Chakraborty, Trinad; Hüttener, Mário; Juárez, Antonio

    2016-01-01

    The genomes of Gram-negative bacteria encode paralogues and/or orthologues of global modulators. The nucleoid-associated H-NS and Hha proteins are an example: several enterobacteria such as Escherichia coli or Salmonella harbor H-NS, Hha and their corresponding paralogues, StpA and YdgT proteins, respectively. Remarkably, the genome of the pathogenic enteroaggregative E. coli strain 042 encodes, in addition to the hha and ydgT genes, two additional hha paralogues, hha2 and hha3. We show in this report that there exists a strong correlation between the presence of these paralogues and the virulence phenotype of several E. coli strains. hha2 and hha3 predominate in some groups of intestinal pathogenic E. coli strains (enteroaggregative and shiga toxin-producing isolates), as well as in the widely distributed extraintestinal ST131 isolates. Because of the relationship between the presence of hha2/hha3 and some virulence factors, we have been able to provide evidence for Hha2/Hha3 modulating the expression of the antigen 43 pathogenic determinants. We show that tracking global modulators or their paralogues/orthologues can be a new strategy to identify bacterial pathogenic clones and propose PCR amplification of hha2 and hha3 as a virulence indicator in environmental and clinical E. coli isolates. PMID:27169404

  18. Tracking bacterial virulence: global modulators as indicators

    PubMed Central

    Prieto, Alejandro; Urcola, Imanol; Blanco, Jorge; Dahbi, Ghizlane; Muniesa, Maite; Quirós, Pablo; Falgenhauer, Linda; Chakraborty, Trinad; Hüttener, Mário; Juárez, Antonio

    2016-01-01

    The genomes of Gram-negative bacteria encode paralogues and/or orthologues of global modulators. The nucleoid-associated H-NS and Hha proteins are an example: several enterobacteria such as Escherichia coli or Salmonella harbor H-NS, Hha and their corresponding paralogues, StpA and YdgT proteins, respectively. Remarkably, the genome of the pathogenic enteroaggregative E. coli strain 042 encodes, in addition to the hha and ydgT genes, two additional hha paralogues, hha2 and hha3. We show in this report that there exists a strong correlation between the presence of these paralogues and the virulence phenotype of several E. coli strains. hha2 and hha3 predominate in some groups of intestinal pathogenic E. coli strains (enteroaggregative and shiga toxin-producing isolates), as well as in the widely distributed extraintestinal ST131 isolates. Because of the relationship between the presence of hha2/hha3 and some virulence factors, we have been able to provide evidence for Hha2/Hha3 modulating the expression of the antigen 43 pathogenic determinants. We show that tracking global modulators or their paralogues/orthologues can be a new strategy to identify bacterial pathogenic clones and propose PCR amplification of hha2 and hha3 as a virulence indicator in environmental and clinical E. coli isolates. PMID:27169404

  19. The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa

    PubMed Central

    Nadal Jimenez, Pol; Koch, Gudrun; Thompson, Jessica A.; Xavier, Karina B.; Cool, Robbert H.

    2012-01-01

    Summary: Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce the selective pressures for development of resistance. We provide here an up-to-date overview of the molecular basis and physiological implications of cell-to-cell signaling systems in Gram-negative bacteria, focusing on the well-studied bacterium Pseudomonas aeruginosa. All of the known cell-to-cell signaling systems in this bacterium are described, from the most-studied systems, i.e., N-acyl homoserine lactones (AHLs), the 4-quinolones, the global activator of antibiotic and cyanide synthesis (GAC), the cyclic di-GMP (c-di-GMP) and cyclic AMP (cAMP) systems, and the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), to less-well-studied signaling molecules, including diketopiperazines, fatty acids (diffusible signal factor [DSF]-like factors), pyoverdine, and pyocyanin. This overview clearly illustrates that bacterial communication is far more complex than initially thought and delivers a clear distinction between signals that are quorum sensing dependent and those relying on alternative factors for their production. PMID:22390972

  20. Virulence Factors of Erwinia amylovora: A Review.

    PubMed

    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

  1. 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

  2. Proposed Molecular Beam Determination of Energy Partition in the Photodissociation of Polyatomic Molecules

    DOE R&D Accomplishments Database

    Zare, P. N.; Herschbach, D. R.

    1964-01-29

    Conventional photochemical experiments give no information about the partitioning of energy between translational recoil and internal excitation of the fragment molecules formed in photodissociation of a polyatomic molecule. In a molecular beam experiment, it becomes possible to determine the energy partition from the form of the laboratory angular distribution of one of the photodissociation products. A general kinematic analysis is worked out in detail, and the uncertainty introduced by the finite angular resolution of the apparatus and the velocity spread in the parent beam is examined. The experimental requirements are evaluated for he photolysis of methyl iodide by the 2537 angstrom Hg line.

  3. Pathogenicity and virulence: another view.

    PubMed Central

    Isenberg, H D

    1988-01-01

    The concepts of pathogenicity and virulence have governed our perception of microbial harmfulness since the time of Pasteur and Koch. These concepts resulted in the recognition and identification of numerous etiological agents and provided natural and synthetic agents effective in therapy and prevention of diseases. However, Koch's postulates--the premier product of this view--place the onus of harmfulness solely on the microbial world. Our recent experiences with polymicrobic and nosocomial infections, legionellosis, and acquired immunodeficiency syndrome point to the host as the major determinant of disease. The principles of parasitism, enunciated by Theobold Smith, approximate more accurately the disturbances of the host-parasite equilibrium we designate as infection. Many complex attributes of microbial anatomy and physiology have been obscured by our dependency on the pure-culture technique. For example, bacterial attachment organelles and the production of exopolysaccharides enable microorganisms to interact with mammalian glycocalyces and specific receptors. In addition, selection, through the use of therapeutic agents, aids in the progression of environmental organisms to members of the intimate human biosphere, with the potential to complicate the recovery of patients. These factors emphasize further the pivotal significance of host reactions in infections. Parasitism, in its negative aspects, explains the emergence of "new" infections that involve harm to more than host organs and cells: we may encounter subtler infections that reveal parasitic and host cell nucleic acid interactions in a form of genomic parasitism. PMID:3060244

  4. Geometry-independent Determination of Radial Density Distributions in Molecular Cloud Cores and Other Astronomical Objects

    NASA Astrophysics Data System (ADS)

    Krčo, Marko; Goldsmith, Paul F.

    2016-05-01

    We present a geometry-independent method for determining the shapes of radial volume density profiles of astronomical objects whose geometries are unknown, based on a single column density map. Such profiles are often critical to understand the physics and chemistry of molecular cloud cores, in which star formation takes place. The method presented here does not assume any geometry for the object being studied, thus removing a significant source of bias. Instead, it exploits contour self-similarity in column density maps, which appears to be common in data for astronomical objects. Our method may be applied to many types of astronomical objects and observable quantities so long as they satisfy a limited set of conditions, which we describe in detail. We derive the method analytically, test it numerically, and illustrate its utility using 2MASS-derived dust extinction in molecular cloud cores. While not having made an extensive comparison of different density profiles, we find that the overall radial density distribution within molecular cloud cores is adequately described by an attenuated power law.

  5. Thickness determination of molecularly thin lubricant films by angle-dependent X-ray photoelectron spectroscopy

    NASA Astrophysics Data System (ADS)

    Pang, Chongjun; Bai, Mingwu

    2007-03-01

    An angle-dependent X-ray photoelectron spectroscopy (XPS) method used to measure the thickness of molecularly thin lubricants was developed. The method was built based on an island model of patched overlayer on a flat substrate by using the photoemission signal solely from the lubricant film. Typical molecularly thin Zdol films on the CHx overcoat of unused commercial magnetic disks were measured to verify the metrology. The lubricant thickness determined by the metrology was equal to the recent result by thermostatic high vacuum atomic force microscopy. The measured deduction in the thickness of the molecularly thin lubricant films, successively irradiated by the monochromatic source operated at 14 kV/250 W, was as low as 1 Ǻ during the first irradiation hour. XPS spectra showed that no hydrocarbons, water or oxygen were adsorbed over the Zdol outer surfaces in the tested XPS conditions. The inelastic mean free path (IMFP) of C 1s in Zdol or in CHx was found to be independent of take off angle (TOA) when TOA < 40°. The IMFP of C 1s in Zdol was ˜63.5 Ǻ and the lubricant island thickness was ˜35 Ǻ.

  6. Determination of domoic acid in shellfish extracted by molecularly imprinted polymers.

    PubMed

    Lin, Zhengzhong; Wang, Dan; Peng, Aihong; Huang, Zhiyong; Lin, Yuhui

    2016-08-01

    A selective sample cleanup method using molecularly imprinted polymers was developed for the separation of domoic acid (a shellfish toxin) from shellfish samples. The molecularly imprinted polymers for domoic acid was prepared by emulsion polymerization using 1,3,5-pentanetricarboxylic acid as the template molecule, 4-vinyl pyridine as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, and Span80/Tween-80 (1:1 v/v) as the composite emulsifiers. The molecularly imprinted polymer showed high affinity to domoic acid with a dissociation constant of 13.5 μg/mL and apparent maximum adsorption capacity of 1249 μg/g. They were used as a selective sorbent for the detection of domoic acid from seafood samples coupled with high-performance liquid chromatography. The detection limit of 0.17 μg/g was lower than the maximum level permitted by several authorities. The mean recoveries of domoic acid from clam samples were 93.0-98.7%. It was demonstrated that the proposed method could be applied to the determination of domoic acid from shellfish samples. PMID:27311699

  7. Rapid Virulence Annotation (RVA): Identification of virulence factors using a bacterial genome library and multiple invertebrate hosts

    PubMed Central

    Waterfield, Nicholas R.; Sanchez-Contreras, Maria; Eleftherianos, Ioannis; Dowling, Andrea; Yang, Guowei; Wilkinson, Paul; Parkhill, Julian; Thomson, Nicholas; Reynolds, Stuart E.; Bode, Helge B.; Dorus, Steven; ffrench-Constant, Richard H.

    2008-01-01

    Current sequence databases now contain numerous whole genome sequences of pathogenic bacteria. However, many of the predicted genes lack any functional annotation. We describe an assumption-free approach, Rapid Virulence Annotation (RVA), for the high-throughput parallel screening of genomic libraries against four different taxa: insects, nematodes, amoeba, and mammalian macrophages. These hosts represent different aspects of both the vertebrate and invertebrate immune system. Here, we apply RVA to the emerging human pathogen Photorhabdus asymbiotica using “gain of toxicity” assays of recombinant Escherichia coli clones. We describe a wealth of potential virulence loci and attribute biological function to several putative genomic islands, which may then be further characterized using conventional molecular techniques. The application of RVA to other pathogen genomes promises to ascribe biological function to otherwise uncharacterized virulence genes. PMID:18838673

  8. Respiratory Syncytial Virus Increases the Virulence of Streptococcus pneumoniae by Binding to Penicillin Binding Protein 1a. A New Paradigm in Respiratory Infection

    PubMed Central

    Smith, Claire M.; Sandrini, Sara; Datta, Sumit; Freestone, Primrose; Shafeeq, Sulman; Radhakrishnan, Priya; Williams, Gwyneth; Glenn, Sarah M.; Kuipers, Oscar P.; Hirst, Robert A.; Easton, Andrew J.; Andrew, Peter W.

    2014-01-01

    Rationale: Respiratory syncytial virus (RSV) and Streptococcus pneumoniae are major respiratory pathogens. Coinfection with RSV and S. pneumoniae is associated with severe and often fatal pneumonia but the molecular basis for this remains unclear. Objectives: To determine if interaction between RSV and pneumococci enhances pneumococcal virulence. Methods: We used confocal microscopy and Western blot to identify the receptors involved in direct binding of RSV and pneumococci, the effects of which were studied in both in vivo and in vitro models of infection. Human ciliated respiratory epithelial cell cultures were infected with RSV for 72 hours and then challenged with pneumococci. Pneumococci were collected after 2 hours exposure and changes in gene expression determined using quantitative real-time polymerase chain reaction. Measurements and Main Results: Following incubation with RSV or purified G protein, pneumococci demonstrated a significant increase in the inflammatory response and bacterial adherence to human ciliated epithelial cultures and markedly increased virulence in a pneumonia model in mice. This was associated with extensive changes in the pneumococcal transcriptome and significant up-regulation in the expression of key pneumococcal virulence genes, including the gene for the pneumococcal toxin, pneumolysin. We show that mechanistically this is caused by RSV G glycoprotein binding penicillin binding protein 1a. Conclusions: The direct interaction between a respiratory virus protein and the pneumococcus resulting in increased bacterial virulence and worsening disease outcome is a new paradigm in respiratory infection. PMID:24941423

  9. Determinants of molecular reactivity as criteria for predicting toxicity: problems and approaches.

    PubMed Central

    Weinstein, H; Rabinowitz, J; Liebman, M N; Osman, R

    1985-01-01

    We discuss the physicochemical basis for mechanisms of action of toxic chemicals and theoretical methods that can be used to understand the relation to the structure of these chemicals. Molecular properties that determine the chemical reactivity of the compounds are proposed as parameters in the analysis of such structure-activity relationships and as criteria for predicting potential toxicity. The theoretical approaches include quantitative methods for structural superposition of molecules and for superposition of their reactivity characteristics. Applications to polychlorinated hydrocarbons are used to illustrate both rigid superposition methods, and methods that take advantage of structural flexibility. These approaches and their results are discussed and compared with methods that afford quantitative structural comparisons without direct superposition, with special emphasis on the need for efficient automated methods suitable for rapid scans of large structural data bases. Quantum mechanical methods for the calculation of molecular properties that can serve as reactivity criteria are presented and illustrated. Special attention is given to the electrostatic properties of the molecules such as the molecular electrostatic potential, the electric fields, and the polarizability terms calculated from perturbation expansions. The practical considerations related to the rapid calculation of these properties on relevant molecular surfaces (e.g., solvent- or reagent-accessible surfaces) are discussed and exemplified, stressing the special problems posed by the structural variety of toxic substances and the paucity of information on their mechanisms of action. The discussion leads to a rationale for the use of the combination of theoretical methods to reveal discriminant criteria for toxicity and to analyze the initial steps in the metabolic processes that could yield toxic products. PMID:3905371

  10. Final Report: Tolerance to phytoalexins and its implication for the evolution of host specific virulence traits, July 1, 1996 - June 30, 1998

    SciTech Connect

    VanEtten, Hans

    1998-06-30

    This research focused on determining the importance of non-degradative tolerance (NDT) to pisatin for the virulence of N. haematococca MPVI on pea. An attempt was also made to determine the importance of pda for virulence of F. oxysporum f.sp.pisi,A. pisi and M. pinodes. This research aided in refining the author's understanding of those characteristics of the pda genes that contribute to virulence and the role of non-degradative tolerance in the virulence of other pea pathogens.

  11. Molecularly imprinted polymer-based bulk optode for the determination of itopride hydrochloride in physiological fluids.

    PubMed

    Abdel-Haleem, F M; Madbouly, Adel; El Nashar, R M; Abdel-Ghani, N T

    2016-11-15

    We report here for the first time on the use of Molecularly Imprinted Polymers as modifiers in bulk optodes, Miptode, for the determination of a pharmaceutical compound, itopride hydrochloride as an example in a concentration range of 1×10(-1)-1×10(-4)molL(-1). In comparison to the optode containing the ion exchanger only (Miptode 3), the optode containing the ion exchanger and the MIP particles (Miptode 2) showed improved selectivity over the most lipophilic species, Na(+) and K(+), by more than two orders of magnitude. For instance, the optical selectivity coefficients using Miptode 2, [Formula: see text] , were as follow: NH4(+)˂-6; Na(+)=-4.0, which were greatly enhanced in comparison with that obtained by Miptode 3. This work opens a new avenue for using miptodes for the determination of all the pharmaceutical preparations without the need for the development of new ionophores. PMID:27266658

  12. Direct determination of three-phase contact line properties on nearly molecular scale

    NASA Astrophysics Data System (ADS)

    Winkler, P. M.; McGraw, R. L.; Bauer, P. S.; Rentenberger, C.; Wagner, P. E.

    2016-05-01

    Wetting phenomena in multi-phase systems govern the shape of the contact line which separates the different phases. For liquids in contact with solid surfaces wetting is typically described in terms of contact angle. While in macroscopic systems the contact angle can be determined experimentally, on the molecular scale contact angles are hardly accessible. Here we report the first direct experimental determination of contact angles as well as contact line curvature on a scale of the order of 1nm. For water nucleating heterogeneously on Ag nanoparticles we find contact angles around 15 degrees compared to 90 degrees for the corresponding macroscopically measured equilibrium angle. The obtained microscopic contact angles can be attributed to negative line tension in the order of ‑10‑10 J/m that becomes increasingly dominant with increasing curvature of the contact line. These results enable a consistent theoretical description of heterogeneous nucleation and provide firm insight to the wetting of nanosized objects.

  13. Direct determination of three-phase contact line properties on nearly molecular scale.

    PubMed

    Winkler, P M; McGraw, R L; Bauer, P S; Rentenberger, C; Wagner, P E

    2016-01-01

    Wetting phenomena in multi-phase systems govern the shape of the contact line which separates the different phases. For liquids in contact with solid surfaces wetting is typically described in terms of contact angle. While in macroscopic systems the contact angle can be determined experimentally, on the molecular scale contact angles are hardly accessible. Here we report the first direct experimental determination of contact angles as well as contact line curvature on a scale of the order of 1nm. For water nucleating heterogeneously on Ag nanoparticles we find contact angles around 15 degrees compared to 90 degrees for the corresponding macroscopically measured equilibrium angle. The obtained microscopic contact angles can be attributed to negative line tension in the order of -10(-10) J/m that becomes increasingly dominant with increasing curvature of the contact line. These results enable a consistent theoretical description of heterogeneous nucleation and provide firm insight to the wetting of nanosized objects. PMID:27183880

  14. Direct determination of three-phase contact line properties on nearly molecular scale

    PubMed Central

    Winkler, P. M.; McGraw, R. L.; Bauer, P. S.; Rentenberger, C.; Wagner, P. E.

    2016-01-01

    Wetting phenomena in multi-phase systems govern the shape of the contact line which separates the different phases. For liquids in contact with solid surfaces wetting is typically described in terms of contact angle. While in macroscopic systems the contact angle can be determined experimentally, on the molecular scale contact angles are hardly accessible. Here we report the first direct experimental determination of contact angles as well as contact line curvature on a scale of the order of 1nm. For water nucleating heterogeneously on Ag nanoparticles we find contact angles around 15 degrees compared to 90 degrees for the corresponding macroscopically measured equilibrium angle. The obtained microscopic contact angles can be attributed to negative line tension in the order of −10−10 J/m that becomes increasingly dominant with increasing curvature of the contact line. These results enable a consistent theoretical description of heterogeneous nucleation and provide firm insight to the wetting of nanosized objects. PMID:27183880

  15. Characterization of molecular determinants for nucleocytoplasmic shuttling of PRV UL54

    SciTech Connect

    Li Meili; Wang Shuai; Cai Mingsheng; Guo Hong; Zheng Chunfu

    2011-09-01

    The pseudorabies virus (PRV) early protein UL54 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein and essential for HSV-1 infection. To determine if UL54 might shuttle between the nucleus and cytoplasm, as has been shown for its homologues in human herpesviruses, the molecular determinants for its nucleocytoplasmic shuttling were investigated. Heterokaryon assays demonstrated that UL54 was a nucleocytoplasmic shuttling protein and this property could not be blocked by leptomycin B, an inhibitor of chromosome region maintenance 1 (CRM1). However, TAP/NXF1 promoted the nuclear export of UL54 and interacted with UL54, suggesting that UL54 shuttles between the nucleus and the cytoplasm via a TAP/NXF1, but not CRM1, dependent nuclear export pathway. Furthermore, UL54 was demonstrated to target to the nucleus through a classic Ran-, importin {beta}1- and {alpha}5-dependent nuclear import mechanism.

  16. A molecular imprinted SPR biosensor for sensitive determination of citrinin in red yeast rice.

    PubMed

    Atar, Necip; Eren, Tanju; Yola, Mehmet Lütfi

    2015-10-01

    A novel and sensitive molecular imprinted surface plasmon resonance (SPR) biosensor was developed for selective determination of citrinin (CIT) in red yeast rice. Firstly, the gold surface of SPR chip was modified with allyl mercaptane. Then, CIT-imprinted poly(2-hydroxyethyl methacrylate-methacryloylamidoglutamic acid) (p(HEMA-MAGA)) film was generated on the gold surface modified with allyl mercaptane. The unmodified and imprinted surfaces were characterized by Fourier transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM) and contact angle measurements. The linearity range and the detection limit were obtained as 0.005-1.0 ng/mL and 0.0017 ng/mL, respectively. The SPR biosensor was applied to determination of CIT in red yeast rice sample. PMID:25872420

  17. In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles.

    PubMed

    Caspers, P J; Lucassen, G W; Carter, E A; Bruining, H A; Puppels, G J

    2001-03-01

    Confocal Raman spectroscopy is introduced as a noninvasive in vivo optical method to measure molecular concentration profiles in the skin. It is shown how it can be applied to determine the water concentration in the stratum corneum as a function of distance to the skin surface, with a depth resolution of 5 microm. The resulting in vivo concentration profiles are in qualitative and quantitative agreement with published data, obtained by in vitro X-ray microanalysis of skin samples. Semi-quantitative concentration profiles were determined for the major constituents of natural moisturizing factor (serine, glycine, pyrrolidone-5-carboxylic acid, arginine, ornithine, citrulline, alanine, histidine, urocanic acid) and for the sweat constituents lactate and urea. A detailed description is given of the signal analysis methodology that enables the extraction of this information from the skin Raman spectra. No other noninvasive in vivo method exists that enables an analysis of skin molecular composition as a function of distance to the skin surface with similar detail and spatial resolution. Therefore, it may be expected that in vivo confocal Raman spectroscopy will find many applications in basic and applied dermatologic research. PMID:11231318

  18. Determination of lipoprotein(a) concentrations and apolipoprotein(a) molecular weights in diabetic patients.

    PubMed

    Ribault, A; Durou, M R; Letellier, C; Wojcik, F; Poirier, J Y; Ruelland, A

    2000-04-01

    Lipoprotein(a) (Lp(a)) with atherogenic and thrombotic properties has been frequently studied in diabetes, because a high cardiovascular risk has been reported both in type 1 and type 2 diabetes. Few studies have considered genetic factors, especially the isoforms of apolipoprotein(a). The aim of this work is to determine the distribution of apo(a) phenotypes in the serum of 148 diabetic patients (59 type 1, 89 type 2) with or without vascular complications. Apo(a) phenotypes are determined using 4-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting (PhastSystem - Pharmacia). An inverse relationship is observed between Lp(a) serum concentration and the apparent molecular mass of apo(a) isoforms: type 1 r=- 0.61, p<0.01; type 2 r=- 0.55, p<0.01. The frequency of apo(a) isoforms is significantly different between type 1 and type 2 diabetes mellitus. A higher prevalence of isoforms of low molecular weight was observed in the type 2 diabetic population. PMID:10804324

  19. Structure determination of an 11-subunit exosome in complex with RNA by molecular replacement

    SciTech Connect

    Makino, Debora Lika Conti, Elena

    2013-11-01

    The crystallographic steps towards the structure determination of a complete eukaryotic exosome complex bound to RNA are presented. Phasing of this 11-protein subunit complex was carried out via molecular replacement. The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3′ degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.

  20. Utility of morphological and molecular techniques for determination of paternity in two subspecies of Diabrotica undecimpunctata (Coleoptera: Chrysomelidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An experiment was conducted to determine the paternity of F1 progeny using morphological and molecular methods in Diabrotica (Coleoptera: Chrysomelidae) subspecies: Diabrotica undecimpunctata howardi Barber, also known as spotted cucumber beetle and D. undecimpunctata undecimpunctata Mannerheim, als...

  1. Molecular size of the canine and human brain D2 dopamine receptor as determined by radiation inactivation

    SciTech Connect

    Lilly, L.; Fraser, C.M.; Jung, C.Y.; Seeman, P.; Venter, J.C.

    1983-07-01

    Target-size analysis (radiation inactivation) has been utilized for determination of the molecular size of the striatal D2 dopamine receptor of both canine and human membranes. The dog and human receptors were found to have a molecular size of 123,000 daltons. The identity of molecular size values is consistent with available pharmacological and biochemical evidence supporting D2 dopamine receptor identity in canine and human tissues. These data suggest that the canine receptor may be a valid model for molecular and structural investigation of the human D2 dopamine receptor.

  2. Structural characterisation of the virulence-associated protein VapG from the horse pathogen Rhodococcus equi

    PubMed Central

    Okoko, Tebekeme; Blagova, Elena V.; Whittingham, Jean L.; Dover, Lynn G.; Wilkinson, Anthony J.

    2015-01-01

    Virulence and host range in Rhodococcus equi depends on the variable pathogenicity island of their virulence plasmids. Notable gene products are a family of small secreted virulence-associated proteins (Vaps) that are critical to intramacrophagic proliferation. Equine-adapted strains, which cause severe pyogranulomatous pneumonia in foals, produce a cell-associated VapA that is necessary for virulence, alongside five other secreted homologues. In the absence of biochemical insight, attention has turned to the structures of these proteins to develop a functional hypothesis. Recent studies have described crystal structures for VapD and a truncate of the VapA orthologue of porcine-adapted strains, VapB. Here, we crystallised the full-length VapG and determined its structure by molecular replacement. Electron density corresponding to the N-terminal domain was not visible suggesting that it is disordered. The protein core adopted a compact elliptical, anti-parallel β-barrel fold with β1–β2–β3–β8–β5–β6–β7–β4 topology decorated by a single peripheral α-helix unique to this family. The high glycine content of the protein allows close packing of secondary structural elements. Topologically, the surface has no indentations that indicate a nexus for molecular interactions. The distribution of polar and apolar groups on the surface of VapG is markedly uneven. One-third of the surface is dominated by exposed apolar side-chains, with no ionisable and only four polar side-chains exposed, giving rise to an expansive flat hydrophobic surface. Other surface regions are more polar, especially on or near the α-helix and a belt around the centre of the β-barrel. Possible functional significance of these recent structures is discussed. PMID:25746683

  3. Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization

    PubMed Central

    Chiu, Elaine; Hijnen, Marcel; Bunker, Richard D.; Boudes, Marion; Rajendran, Chitra; Aizel, Kaheina; Oliéric, Vincent; Schulze-Briese, Clemens; Mitsuhashi, Wataru; Young, Vivienne; Ward, Vernon K.; Bergoin, Max; Metcalf, Peter; Coulibaly, Fasséli

    2015-01-01

    The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides. PMID:25787255

  4. Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization.

    PubMed

    Chiu, Elaine; Hijnen, Marcel; Bunker, Richard D; Boudes, Marion; Rajendran, Chitra; Aizel, Kaheina; Oliéric, Vincent; Schulze-Briese, Clemens; Mitsuhashi, Wataru; Young, Vivienne; Ward, Vernon K; Bergoin, Max; Metcalf, Peter; Coulibaly, Fasséli

    2015-03-31

    The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides. PMID:25787255

  5. When experimental selection for virulence leads to loss of virulence.

    PubMed

    Rafaluk, Charlotte; Jansen, Gunther; Schulenburg, Hinrich; Joop, Gerrit

    2015-09-01

    Information on virulence evolution is critical for understanding disease dynamics. Theory predicts that under certain evolutionary conditions virulence should increase; for example, during host-parasite coevolution. Although these theoretical predictions are supported by natural observations, tests of these hypotheses using experimental evolution have yielded confounding and contradictory results, with discrepancies often being seen among experiments. Here we provide a critical overview of experimental tests of hypotheses regarding virulence evolution and provide potential explanations for the contradictory results. We emphasise the key role of parasite transmission mechanisms that can explain many of the observed discrepancies among evolution experiments. Finally, we make suggestions for how evolution experiments could be conducted in the future to avoid potentially confounding factors. PMID:26166750

  6. New directions in testicular cancer; molecular determinants of oncogenesis and treatment success.

    PubMed

    Jones, R H; Vasey, P A

    2003-01-01

    Metastatic testicular cancer is highly curable with conventional cytotoxic drugs. This is in contrast to most other metastatic solid tumours which can only be palliated with chemotherapy achieving only a modest impact on overall survival. If we could understand at the molecular level why chemotherapy is so effective in the treatment of testicular cancer, we may be better able to move other forms of metastatic cancer into the curable bracket. Most cytotoxic drugs appear to induce cell death by activating intracellular apoptotic mechanisms. Thus, the ability of a cancer to activate and execute such mechanisms in response to treatment is paramount in determining the effectiveness of chemotherapy. The basic study of cancer molecular biology is providing some insight into the proteins involved in this process and the ability to apply this information to actual human tumours is essential to rationalise clinical treatment failures at a molecular level. Testicular cancer provides an excellent model system in this analysis. Whereas there are large numbers of patients that are cured by chemotherapy, there are some whose cancers become resistant to treatment. An understanding of testicular cancer molecular biology may allow the identification of the genes regulating such a crucial behavioural switch. It may then be possible to manipulate specific signalling pathways to overcome drug resistance. This review focuses on recent developments in our understanding of the molecular biology of testicular cancer. A number of key players have been implicated including p53, pRb, cyclin D2, p INK proteins, c-kit and the bcl-2 family of proteins. The exact manner by which cellular transformation occurs has still not been established, but it is clear that many of the above proteins also have important roles in normal spermatogenesis. This is a developmental phase when the generation of genetic diversity is at a premium, but when selective apoptotic mechanisms are paramount. We discuss why

  7. Molecular determinants of human T-lymphotropic virus type 1 transmission and spread.

    PubMed

    Lairmore, Michael D; Anupam, Rajaneesh; Bowden, Nadine; Haines, Robyn; Haynes, Rashade A H; Ratner, Lee; Green, Patrick L

    2011-07-01

    Human T-lymphotrophic virus type-1 (HTLV-1) infects approximately 15 to 20 million people worldwide, with endemic areas in Japan, the Caribbean, and Africa. The virus is spread through contact with bodily fluids containing infected cells, most often from mother to child through breast milk or via blood transfusion. After prolonged latency periods, approximately 3 to 5% of HTLV-1 infected individuals will develop either adult T-cell leukemia/lymphoma (ATL), or other lymphocyte-mediated disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The genome of this complex retrovirus contains typical gag, pol, and env genes, but also unique nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo such as, p30, p12, p13 and the antisense encoded HBZ. While progress has been made in the understanding of viral determinants of cell transformation and host immune responses, host and viral determinants of HTLV-1 transmission and spread during the early phases of infection are unclear. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the early events of HTLV-1 infection. This review will focus on studies that test HTLV-1 determinants in context to full length infectious clones of the virus providing insights into the mechanisms of transmission and spread of HTLV-1. PMID:21994774

  8. Determining Structural and Mechanical Properties from Molecular Dynamics Simulations of Lipid Vesicles

    PubMed Central

    2015-01-01

    We have developed an algorithm to determine membrane structure, area per lipid, and bending rigidity from molecular dynamics simulations of lipid vesicles. Current methods to extract structure from vesicle simulations define densities relative to the global center of mass of the vesicle. This approach ignores the long-wavelength fluctuations (undulations) that develop across the sphere and broaden the underlying structure. Our method establishes a local reference frame by defining a radially undulating reference surface (URS) and thereby removes the broadening effect of the undulations. Using an arc-length low-pass filter, we render the URS by defining the bilayer midplane on an equi-angular θ, ϕ-grid (colatitude, longitude). This surface is then expanded onto a truncated series of spherical harmonics. The spherical harmonic coefficients characterize the long-wavelength fluctuations that define both the local reference frame—used to determine the bilayer’s structure—and the area per lipid (AL) along the undulating surface. Additionally, the resulting power spectrum of spherical harmonic coefficients can be fit to a Helfrich continuum model for membrane bending in spherical geometry to extract bending rigidity (kc). kc values determined for both DMPC and DMPC + cholesterol (30 mol %) vesicles are consistent with values from corresponding flat-patch systems determined using an independent, previously published spectral method. These new tools to accurately extract structure, AL, and kc should prove invaluable in evaluating the construction and equilibration of lipid vesicle simulations. PMID:25221448

  9. Determining Structural and Mechanical Properties from Molecular Dynamics Simulations of Lipid Vesicles.

    PubMed

    Braun, Anthony R; Sachs, Jonathan N

    2014-09-01

    We have developed an algorithm to determine membrane structure, area per lipid, and bending rigidity from molecular dynamics simulations of lipid vesicles. Current methods to extract structure from vesicle simulations define densities relative to the global center of mass of the vesicle. This approach ignores the long-wavelength fluctuations (undulations) that develop across the sphere and broaden the underlying structure. Our method establishes a local reference frame by defining a radially undulating reference surface (URS) and thereby removes the broadening effect of the undulations. Using an arc-length low-pass filter, we render the URS by defining the bilayer midplane on an equi-angular θ, ϕ-grid (colatitude, longitude). This surface is then expanded onto a truncated series of spherical harmonics. The spherical harmonic coefficients characterize the long-wavelength fluctuations that define both the local reference frame-used to determine the bilayer's structure-and the area per lipid (A L) along the undulating surface. Additionally, the resulting power spectrum of spherical harmonic coefficients can be fit to a Helfrich continuum model for membrane bending in spherical geometry to extract bending rigidity (k c). k c values determined for both DMPC and DMPC + cholesterol (30 mol %) vesicles are consistent with values from corresponding flat-patch systems determined using an independent, previously published spectral method. These new tools to accurately extract structure, A L, and k c should prove invaluable in evaluating the construction and equilibration of lipid vesicle simulations. PMID:25221448

  10. Virulence Plasmids of Spore-Forming Bacteria.

    PubMed

    Adams, Vicki; Li, Jihong; Wisniewski, Jessica A; Uzal, Francisco A; Moore, Robert J; McClane, Bruce A; Rood, Julian I

    2014-12-01

    Plasmid-encoded virulence factors are important in the pathogenesis of diseases caused by spore-forming bacteria. Unlike many other bacteria, the most common virulence factors encoded by plasmids in Clostridium and Bacillus species are protein toxins. Clostridium perfringens causes several histotoxic and enterotoxin diseases in both humans and animals and produces a broad range of toxins, including many pore-forming toxins such as C. perfringens enterotoxin, epsilon-toxin, beta-toxin, and NetB. Genetic studies have led to the determination of the role of these toxins in disease pathogenesis. The genes for these toxins are generally carried on large conjugative plasmids that have common core replication, maintenance, and conjugation regions. There is considerable functional information available about the unique tcp conjugation locus carried by these plasmids, but less is known about plasmid maintenance. The latter is intriguing because many C. perfringens isolates stably maintain up to four different, but closely related, toxin plasmids. Toxin genes may also be plasmid-encoded in the neurotoxic clostridia. The tetanus toxin gene is located on a plasmid in Clostridium tetani, but the botulinum toxin genes may be chromosomal, plasmid-determined, or located on bacteriophages in Clostridium botulinum. In Bacillus anthracis it is well established that virulence is plasmid determined, with anthrax toxin genes located on pXO1 and capsule genes on a separate plasmid, pXO2. Orthologs of these plasmids are also found in other members of the Bacillus cereus group such as B. cereus and Bacillus thuringiensis. In B. thuringiensis these plasmids may carry genes encoding one or more insecticidal toxins. PMID:26104459

  11. Characterization of Virulent West Nile Virus Kunjin Strain, Australia, 2011

    PubMed Central

    Frost, Melinda J.; Zhang, Jing; Edmonds, Judith H.; Prow, Natalie A.; Gu, Xingnian; Davis, Rodney; Hornitzky, Christine; Arzey, Kathleen E.; Finlaison, Deborah; Hick, Paul; Read, Andrew; Hobson-Peters, Jody; May, Fiona J.; Doggett, Stephen L.; Haniotis, John; Russell, Richard C.; Hall, Roy A.; Khromykh, Alexander A.

    2012-01-01

    To determine the cause of an unprecedented outbreak of encephalitis among horses in New South Wales, Australia, in 2011, we performed genomic sequencing of viruses isolated from affected horses and mosquitoes. Results showed that most of the cases were caused by a variant West Nile virus (WNV) strain, WNVNSW2011, that is most closely related to WNV Kunjin (WNVKUN), the indigenous WNV strain in Australia. Studies in mouse models for WNV pathogenesis showed that WNVNSW2011 is substantially more neuroinvasive than the prototype WNVKUN strain. In WNVNSW2011, this apparent increase in virulence over that of the prototype strain correlated with at least 2 known markers of WNV virulence that are not found in WNVKUN. Additional studies are needed to determine the relationship of the WNVNSW2011 strain to currently and previously circulating WNVKUN strains and to confirm the cause of the increased virulence of this emerging WNV strain. PMID:22516173

  12. Towards the elucidation of molecular determinants of cooperativity in the liver bile acid binding protein.

    PubMed

    Pedò, Massimo; D'Onofrio, Mariapina; Ferranti, Pasquale; Molinari, Henriette; Assfalg, Michael

    2009-11-15

    Bile acid binding proteins (BABPs) are cytosolic lipid chaperones contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Liver BABPs act in parallel with ileal transporters to ensure vectorial transport of bile salts in hepatocytes and enterocytes, respectively. We describe the investigation of ligand binding to liver BABP, an essential step in the understanding of intracellular bile salt transport. Binding site occupancies were monitored in NMR titration experiments using (15)N-labelled ligand, while the relative populations of differently bound BABP forms were assessed by mass spectrometry. This site-specific information allowed the determination of intrinsic thermodynamic parameters and the identification of an extremely high cooperativity between two binding sites. Protein-observed NMR experiments revealed a global structural rearrangement which suggests an allosteric mechanism at the basis of the observed cooperativity. The view of a molecular tool capable of buffering against significant concentrations of free bile salts in a large range of solution conditions emerges from the observed pH-dependence of binding. We set to determine the molecular determinants of cooperativity by analysing the binding properties of a protein containing a mutated internal histidine. Both mass spectrometry and NMR experiments are consistent with an overall decreased binding affinity of the mutant, while the measured diffusion coefficients of ligand species reveal that the affinity loss concerns essentially one of the two binding sites. We therefore identified a mutation able to disrupt energetic communication functional to efficient binding and conclude that the buried histidine establishes contacts that stabilize the ternary complex. PMID:19603488

  13. Determination of sulfur in food by high resolution continuum source flame molecular absorption spectrometry

    NASA Astrophysics Data System (ADS)

    Zambrzycka, Elżbieta; Godlewska-Żyłkiewicz, Beata

    2014-11-01

    In the present work, a fast, simple and sensitive analytical method for determination of sulfur in food and beverages by high resolution continuum source flame molecular absorption spectrometry was developed. The determination was performed via molecular absorption of carbon monosulfide, CS. Different CS rotational lines (257.959 nm, 258.033 nm, 258.055 nm), number of pixels and types of standard solution of sulfur, namely: sulfuric acid, sodium sulfate, ammonium sulfate, sodium sulfite, sodium sulfide, DL-cysteine, and L-cystine, were studied in terms of sensitivity, repeatability of results as well as limit of detection and limit of quantification. The best results were obtained for measurements of absorption of the CS molecule at 258.055 nm at the wavelength range covering 3 pixels and DL-cysteine in 0.2 mol L- 1 HNO3 solution as a calibration standard. Under optimized conditions the limit of detection and the limit of quantification achieved for sulfur were 10.9 mg L- 1 and 36.4 mg L- 1, respectively. The repeatability of the results expressed as relative standard deviation was typically < 5%. The accuracy of the method was tested by analysis of digested biological certified reference materials (soya bean flour, corn flour and herbs) and recovery experiment for beverage samples with added known amount of sulfur standard. The recovery of analyte from such samples was in the range of 93-105% with the repeatability in the range of 4.1-5.0%. The developed method was applied for the determination of sulfur in milk (194 ± 10 mg kg- 1), egg white (2188 ± 29 mg kg- 1), mineral water (31.0 ± 0.9 mg L- 1), white wine (260 ± 4 mg L- 1) and red wine (82 ± 2 mg L- 1), as well as in sample rich in ions, such as bitter mineral water (6900 ± 100 mg L- 1).

  14. Importance of Molecular Methods to Determine Whether a Probiotic is the Source of Lactobacillus Bacteremia.

    PubMed

    Aroutcheva, Alla; Auclair, Julie; Frappier, Martin; Millette, Mathieu; Lolans, Karen; de Montigny, Danielle; Carrière, Serge; Sokalski, Stephen; Trick, William E; Weinstein, Robert A

    2016-03-01

    There has been an increasing interest in the use of probiotic products for the prevention of Clostridium difficile infection (CDI). Bio-K+(®) is a commercial probiotic product comprising three strains of lactobacilli--Lactobacillus acidophilus CL1285(®), Lact. casei LBC80R(®) and Lact. rhamnosus CLR2(®)--that have been applied to prevent CDI. Generally considered as safe, lactobacilli have potential to cause bacteremia, endocarditis and other infections. The source of Lactobacillus bacteremia can be normal human flora or lactobacilli-containing probiotic. The aim of this study was to assess whether probiotic lactobacilli caused bacteremia and to show the value of molecular identification and typing techniques to determine probiotic and patient strain relatedness. We report an episode of Lactobacillus bacteremia in a 69-year-old man admitted to a hospital with severe congestive heart failure. During his hospitalization, he required long-term antibiotic therapy. Additionally, the patient received Bio-K+(®) probiotic as part of a quality improvement project to prevent CDI. Subsequently, Lactobacillus bacteremia occurred. Two independent blinded laboratory evaluations, using pulse field gel electrophoresis, 16S rRNA gene sequencing and DNA fingerprint analysis (rep-PCR), were performed to determine whether the recovered Lact. acidophilus originated from the probiotic product. Ultimately, the patient strain was identified as Lact. casei and both laboratories found no genetic relation between the patient's strain and any of the probiotic lactobacilli. This clinical case of lactobacillus bacteremia in the setting of probiotic exposure demonstrates the value of using discriminatory molecular methods to clearly determine whether there were a link between the patient's isolate and the probiotic strains. PMID:26915093

  15. Pseudomonas aeruginosa Virulence and Therapy: Evolving Translational Strategies

    PubMed Central

    Veesenmeyer, Jeffrey L.; Lisboa, Thiago; Rello, Jordi

    2009-01-01

    Structured abstract Objective Although most reviews of Pseudomonas aeruginosa therapeutics focus on antibiotics currently in use or in the pipeline, we review evolving translational strategies aimed at using virulence factor antagonists as adjuvant therapies. Data Source Current literature regarding P. aeruginosa virulence determinants and approaches that target them, with an emphasis on type III secretion, quorum-sensing, biofilms, and flagella. Data Extraction and Synthesis P. aeruginosa remains one of the most important pathogens in nosocomial infections, with high associated morbidity and mortality. Its predilection to develop resistance to antibiotics and expression of multiple virulence factors contributes to the frequent ineffectiveness of current therapies. Among the many P. aeruginosa virulence determinants that impact infections, type III secretion, quorum sensing, biofilm formation, and flagella have been the focus of much recent investigation. Here we review how increased understanding of these important bacterial structures and processes has enabled the development of novel approaches to inhibit each. These promising translational strategies may lead to the development of adjuvant therapies capable of improving outcomes. Conclusions Adjuvant therapies directed against virulence factors have the potential to improve outcomes in P. aeruginosa infections. PMID:19325463

  16. Capsule depolymerase overexpression reduces Bacillus anthracis virulence.

    PubMed

    Scorpio, Angelo; Chabot, Donald J; Day, William A; Hoover, Timothy A; Friedlander, Arthur M

    2010-05-01

    Capsule depolymerase (CapD) is a gamma-glutamyl transpeptidase and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and to capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-type B. anthracis Ames but was detected in strains engineered with a consensus ribosome-binding site for capD. Strains overexpressing capD at amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence. PMID:20110296

  17. Molecular Determinants Defining the Triggering Range of Prefusion F Complexes of Canine Distemper Virus

    PubMed Central

    Avila, Mislay; Alves, Lisa; Khosravi, Mojtaba; Ader-Ebert, Nadine; Origgi, Francesco; Schneider-Schaulies, Jürgen; Zurbriggen, Andreas; Plemper, Richard K.

    2014-01-01

    ABSTRACT The morbillivirus cell entry machinery consists of a fusion (F) protein trimer that refolds to mediate membrane fusion following receptor-induced conformational changes in its binding partner, the tetrameric attachment (H) protein. To identify molecular determinants that control F refolding, we generated F chimeras between measles virus (MeV) and canine distemper virus (CDV). We located a central pocket in the globular head domain of CDV F that regulates the stability of the metastable, prefusion conformational state of the F trimer. Most mutations introduced into this “pocket'” appeared to mediate a destabilizing effect, a phenotype associated with enhanced membrane fusion activity. Strikingly, under specific triggering conditions (i.e., variation of receptor type and H protein origin), some F mutants also exhibited resistance to a potent morbillivirus entry inhibitor, which is known to block F triggering by enhancing the stability of prefusion F trimers. Our data reveal that the molecular nature of the F stimulus and the intrinsic stability of metastable prefusion F both regulate the efficiency of F refolding and escape from small-molecule refolding blockers. IMPORTANCE With the aim to better characterize the thermodynamic basis of morbillivirus membrane fusion for cell entry and spread, we report here that the activation energy barrier of prefusion F trimers together with the molecular nature of the triggering “stimulus” (attachment protein and receptor types) define a “triggering range,” which governs the initiation of the membrane fusion process. A central “pocket” microdomain in the globular F head contributes substantially to the regulation of the conformational stability of the prefusion complexes. The triggering range also defines the mechanism of viral escape from entry inhibitors and describes how the cellular environment can affect membrane fusion efficiency. PMID:24371057

  18. Determination of theophylline in serum by molecularly imprinted solid-phase extraction with pulsed elution.

    PubMed

    Mullett, W M; Lai, E P

    1998-09-01

    The technique of molecular imprinting is used to produce an extensively cross-linked poly(methacrylic acid-co-ethylene dimethacrylate) material that contains theophylline as a print molecule. After Soxhlet extraction of the theophylline, binding sites are formed in the polymer with complementary size, shape, and positioning of chemical functionalities. The molecularly imprinted polymer's (MIP) high theophylline selectivity, chemical stability, and physically robust nature make it an ideal stationary-phase material in columns for HPLC separation of theophylline from other structurally related drug compounds. Mobile-phase tests confirm that a retention mechanism typical of normal-phase chromatography governs the separation, and selectivity of the MIP column can be controlled by a combination of the mobile phase and the sample solvent. Under optimal conditions, the MIP column functions like a solid-phase sorbent for theophylline extraction. Rapid elution of the bound theophylline can be accomplished in a pulsed format through injection of 20 μL of a solvent that has the proper polarity and protic nature to disrupt the electrostatic interactions and hydrogen bonding between theophylline and binding sites. A concentration detection limit of 120 ng/mL is obtained using direct UV absorption detection at 270 nm, which corresponds to a mass detection limit of 2.4 ng. This new technique, molecularly imprinted solid-phase extraction with pulsed elution (MISPE-PE), permits on-line preconcentration of theophylline from a large volume of dilute sample solution. Using a sample volume of 300 μL, a 40 ng/mL standard solution produces a detectable peak signal. Application of MISPE-PE in serum analysis further demonstrates the high capability of the MIP column to selectively isolate theophylline from other matrix components for fast, accurate determination. PMID:21644709

  19. PrfA regulation offsets the cost of L isteria virulence outside the host

    PubMed Central

    Vasanthakrishnan, Radhakrishnan B.; de las Heras, Aitor; Scortti, Mariela; Deshayes, Caroline; Colegrave, Nick

    2015-01-01

    Summary Virulence traits are essential for pathogen fitness, but whether they affect microbial performance in the environment, where they are not needed, remains experimentally unconfirmed. We investigated this question with the facultative pathogen L isteria monocytogenes and its PrfA virulence regulon. PrfA‐regulated genes are activated intracellularly (PrfA ‘ON’) but shut down outside the host (PrfA ‘OFF’). Using a mutant PrfA regulator locked ON (PrfA*) and thus causing PrfA‐controlled genes to be constitutively activated, we show that virulence gene expression significantly impairs the listerial growth rate (μ) and maximum growth (A) in rich medium. Deletion analysis of the PrfA regulon and complementation of a L. monocytogenes mutant lacking all PrfA‐regulated genes with PrfA* indicated that the growth reduction was specifically due to the unneeded virulence determinants and not to pleiotropic regulatory effects of PrfA ON. No PrfA*‐associated fitness disadvantage was observed in infected eukaryotic cells, where PrfA‐regulated virulence gene expression is critical for survival. Microcosm experiments demonstrated that the constitutively virulent state strongly impaired L . monocytogenes performance in soil, the natural habitat of these bacteria. Our findings provide empirical proof that virulence carries a significant cost to the pathogen. They also experimentally substantiate the assumed, although not proven, key role of virulence gene regulation systems in suppressing the cost of bacterial virulence outside the host. PMID:26178789

  20. Systems analysis of multiple regulator perturbations allows discovery of virulence factors in Salmonella

    SciTech Connect

    Yoon, Hyunjin; Ansong, Charles; McDermott, Jason E.; Gritsenko, Marina A.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.

    2011-06-28

    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 of 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.

  1. Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae.

    PubMed

    Karki, Hari S; Shrestha, Bishnu K; Han, Jae Woo; Groth, Donald E; Barphagha, Inderjit K; Rush, Milton C; Melanson, Rebecca A; Kim, Beom Seok; Ham, Jong Hyun

    2012-01-01

    Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR. PMID:23028972

  2. 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

  3. Determination of cyproheptadine in feeds using molecularly imprinted solid-phase extraction coupled with HPLC.

    PubMed

    Yang, Jianwen; Wang, Zongnan; Zhou, Tong; Song, Xuqin; Liu, Qingyong; Zhang, Yuman; He, Limin

    2015-05-15

    A novel method was developed for the determination of cyproheptadine in feeds using molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography. The polymers were prepared using cyproheptadine as a template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent, and dichloromethane as a solvent by bulk polymerization. Under the optimum solid-phase extraction conditions, the molecular imprinting cartridge can selectively extract and enrich cyproheptadine from a variety of feeds. Mean recoveries of cyproheptadine from four kinds of feeds spiked at 0.1, 1.0 and 10mgkg(-1) ranged from 85.5% to 96.2%, with intra-day and inter-day relative standard deviation less than 10%. The calibration curve of cyproheptadine was good linear relationship (r>0.9993) within the range of 0.1-50μgmL(-1). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.04 and 0.1mgkg(-1), respectively. PMID:25855316

  4. Molecular determinants of tetramerization in the KcsA cytoplasmic domain

    PubMed Central

    Kamnesky, Guy; Hirschhorn, Orel; Shaked, Hadassa; Chen, Jingfei; Yao, Lishan; Chill, Jordan H

    2014-01-01

    The cytoplasmic C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four-fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD-derived 34-residue peptide associates into a tetramer in a pH-dependent manner (Kamnesky et al., JMB 2012;418:237-247). Here we further investigate the molecular determinants of tetramer formation in the CTD by characterizing the kinetics of monomer-tetramer equilibrium for 10 alanine mutants using NMR, sedimentation equilibrium (SE) and molecular dynamics simulation. NMR and SE concur in finding single-residue contributions to tetramer stability to be in the 0.5 to 3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than electrostatic interactions between residues R147, D149 and E152. In particular, alanine replacement of residue R147, a key contributor to inter-subunit salt bridges, resulted in only a minor effect on tetramer dissociation. Mutations outside of the inter-subunit interface also influenced tetramer stability by affecting the tetramerization on-rate, possibly by changing the inherent helical propensity of the peptide. These findings are interpreted in the context of established paradigms of protein-protein interactions and protein folding, and lay the groundwork for further studies of the CTD in full-length KcsA channels. PMID:25042120

  5. Molecular weight determination of an active photosystem I preparation from a thermophilic cyanobacterium, Synechococcus elongatus

    SciTech Connect

    Schafheutle, M.E.; Setlikova, E.; Timmins, P.A.; Johner, H.; Gutgesell, P.; Setlik, I.; Welte, W. )

    1990-02-06

    An active photosystem I (PSI) complex was isolated from the thermophilic cyanobacterium Synechococcus elongatus by a procedure consisting of three steps: First, extraction of photosystem II from the thylakoids by a sulfobetaine detergent yields PSI-enriched membranes. Second, the latter are treated with Triton X-100 to extract PSI particles, which are further purified by preparative isoelectric focusing. Third, anion-exchange chromatography is used to remove contaminating phycobilisome polypeptides. The purified particles show three major bands in sodium dodecyl sulfate gel electrophoresis of apparent molecular mass of 110, 15, and 10 kDa. Charge separation was monitored by the kinetics of flash-induced absorption changes at 820 nm. A chlorophyll/P700 ratio of 60 was found. When the particles are stored at 4 degrees C, charge separation was stable for weeks. The molecular mass of the PSI particles, determined by measurement of zero-angle neutron scattering intensity, was 217,000 Da. The PSI particles thus consist of one heterodimer of the 60-80-kDa polypeptides and presumably one copy of the 15- and 10-kDa polypeptides, respectively.

  6. Molecular Determinants of Cardiac Transient Outward Potassium Current (Ito) Expression and Regulation

    PubMed Central

    Niwa, Noriko; Nerbonne, Jeanne M.

    2009-01-01

    Rapidly activating and inactivating cardiac transient outward K+ currents, Ito, are expressed in most mammalian cardiomyocytes, and contribute importantly to the early phase of action potential repolarization and to plateau potentials. The rapidly recovering (Ito,f) and slowly recovering (Ito,s) components are differentially expressed in the myocardium, contributing to regional heterogeneities in action potential waveforms. Consistent with the marked differences in biophysical properties, distinct pore-forming (α) subunits underlie the two Ito components: Kv4.3/Kv4.2 subunits encode Ito,f, whereas Kv1.4 encodes Ito,s, channels. It has also become increasingly clear that cardiac Ito channels function as components of macromolecular protein complexes, comprising (four) Kv α subunits and a variety of accessory subunits and regulatory proteins that influence channel expression, biophysical properties and interactions with the actin cytoskeleton, and contribute to the generation of normal cardiac rhythms. Derangements in the expression or the regulation of Ito channels in inherited or acquired cardiac diseases would be expected to increase the risk of potentially life-threatening cardiac arrhythmias. Indeed, a recently identified Brugada syndrome mutation in KCNE3 (MiRP2) has been suggested to result in increased Ito,f densities. Continued focus in this area seems certain to provide new and fundamentally important insights into the molecular determinants of functional Ito channels and into the molecular mechanisms involved in the dynamic regulation of Ito channel functioning in the normal and diseased myocardium. PMID:19619557

  7. Preparation, Purification, and Secondary Structure Determination of Bacillus Circulans Xylanase. A Molecular Laboratory Incorporating Aspects of Molecular Biology, Biochemistry, and Biophysical Chemistry

    ERIC Educational Resources Information Center

    Russo, Sal; Gentile, Lisa

    2006-01-01

    A project module designed for biochemistry or cellular and molecular biology student which involves determining the secondary structure of Bacillus circulans xylanase (BCX) by circular dichroism (CD) spectroscopy under conditions that compromise its stabilizing intramolecular forces is described. The lab model enhanced students knowledge of the…

  8. Determination of melamine in powdered milk by molecularly imprinted stir bar sorptive extraction coupled with HPLC.

    PubMed

    Zhu, Ling; Xu, Guanhong; Wei, Fangdi; Yang, Jing; Hu, Qin

    2015-09-15

    A novel molecularly imprinted stir bar (MIP-SB) was developed with melamine (MA) as the template molecule in this study. The sorptive capacity of MIP-SB was nearly three times over that of non-imprinted stir bar (NIP-SB). The MIP-SB presented much better selectivity than NIP-SB when used to absorb MA and its analogues. An analytical method to determine MA in powdered milk was established by combining MIP-SB sorptive extraction with HPLC. The liner range was 0.0631-12.6ng/mL with good correlation coefficient of 0.9983, and the limit of detection was 0.0127ng/mL based on three times ratio of signal to noise. This method was successfully applied to the determination of MA in powdered milk with satisfactory results. The method built is simple and suitable for the determination of MA in milk products which is of great significance for quality control of milk products. PMID:26001132

  9. Molecular determinants of plaque size as an indicator of dengue virus attenuation.

    PubMed

    Goh, Kenneth Choon Meng; Tang, Choon Kit; Norton, Diana Catherine; Gan, Esther Shuyi; Tan, Hwee Cheng; Sun, Bo; Syenina, Ayesa; Yousuf, Amjad; Ong, Xin Mei; Kamaraj, Uma Sangumathi; Cheung, Yin Bun; Gubler, Duane J; Davidson, Andrew; St John, Ashley Lauren; Sessions, October Michael; Ooi, Eng Eong

    2016-01-01

    The development of live viral vaccines relies on empirically derived phenotypic criteria, especially small plaque sizes, to indicate attenuation. However, while some candidate vaccines successfully translated into licensed applications, others have failed safety trials, placing vaccine development on a hit-or-miss trajectory. We examined the determinants of small plaque phenotype in two dengue virus (DENV) vaccine candidates, DENV-3 PGMK30FRhL3, which produced acute febrile illness in vaccine recipients, and DENV-2 PDK53, which has a good clinical safety profile. The reasons behind the failure of PGMK30FRhL3 during phase 1 clinical trial, despite meeting the empirically derived criteria of attenuation, have never been systematically investigated. Using in vitro, in vivo and functional genomics approaches, we examined infections by the vaccine and wild-type DENVs, in order to ascertain the different determinants of plaque size. We show that PGMK30FRhL3 produces small plaques on BHK-21 cells due to its slow in vitro growth rate. In contrast, PDK53 replicates rapidly, but is unable to evade antiviral responses that constrain its spread hence also giving rise to small plaques. Therefore, at least two different molecular mechanisms govern the plaque phenotype; determining which mechanism operates to constrain plaque size may be more informative on the safety of live-attenuated vaccines. PMID:27185466

  10. Determination of malachite green in fish based on magnetic molecularly imprinted polymer extraction followed by electrochemiluminescence.

    PubMed

    Huang, Baomei; Zhou, Xibin; Chen, Jing; Wu, Guofan; Lu, Xiaoquan

    2015-09-01

    A novel procedure for selective extraction of malachite green (MG) from fish samples was set up by using magnetic molecularly imprinted polymers (MMIP) as the solid phase extraction material followed by electrochemiluminescence (ECL) determination. MMIP was prepared by using Fe3O4 magnetite as magnetic component, MG as template molecule, methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as crosslinking agent. MMIP was characterized by SEM, TEM, FT-IR, VSM and XRD. Leucomalachite green (LMG) was oxidized in situ to MG by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). And then MMIP was successfully used to selectively enrich MG from fish samples. Adsorbed MG was desorbed and determined by ECL. Under the optimal conditions, calibration curve was good linear in the range of 0.29-290 μg/kg and the limit of detection (LOD) was 7.3 ng/kg (S/N=3). The recoveries of MMIP extraction were 77.1-101.2%. In addition, MMIP could be regenerated. To the best of our knowledge, MMIP coupling with ECL quenching of Ru(bpy)3(2+)/TPA for the determination of MG has not yet been developed. PMID:26003716

  11. Molecular determinants of plaque size as an indicator of dengue virus attenuation

    PubMed Central

    Goh, Kenneth Choon Meng; Tang, Choon Kit; Norton, Diana Catherine; Gan, Esther Shuyi; Tan, Hwee Cheng; Sun, Bo; Syenina, Ayesa; Yousuf, Amjad; Ong, Xin Mei; Kamaraj, Uma Sangumathi; Cheung, Yin Bun; Gubler, Duane J; Davidson, Andrew; St John, Ashley Lauren; Sessions, October Michael; Ooi, Eng Eong

    2016-01-01

    The development of live viral vaccines relies on empirically derived phenotypic criteria, especially small plaque sizes, to indicate attenuation. However, while some candidate vaccines successfully translated into licensed applications, others have failed safety trials, placing vaccine development on a hit-or-miss trajectory. We examined the determinants of small plaque phenotype in two dengue virus (DENV) vaccine candidates, DENV-3 PGMK30FRhL3, which produced acute febrile illness in vaccine recipients, and DENV-2 PDK53, which has a good clinical safety profile. The reasons behind the failure of PGMK30FRhL3 during phase 1 clinical trial, despite meeting the empirically derived criteria of attenuation, have never been systematically investigated. Using in vitro, in vivo and functional genomics approaches, we examined infections by the vaccine and wild-type DENVs, in order to ascertain the different determinants of plaque size. We show that PGMK30FRhL3 produces small plaques on BHK-21 cells due to its slow in vitro growth rate. In contrast, PDK53 replicates rapidly, but is unable to evade antiviral responses that constrain its spread hence also giving rise to small plaques. Therefore, at least two different molecular mechanisms govern the plaque phenotype; determining which mechanism operates to constrain plaque size may be more informative on the safety of live-attenuated vaccines. PMID:27185466

  12. Determination of sulphur in various vegetables by solid sampling high-resolution electrothermal molecular absorption spectrometry.

    PubMed

    Gunduz, Sema; Akman, Suleyman

    2015-04-01

    Sulphur was determined in various vegetables via molecular absorption of carbon monosulphide (CS) at 258.056 nm using a solid sampling high resolution continuum source electrothermal atomic absorption spectrometer (SS HR-CS ETAAS). Samples were dried, ground and directly introduced into the ruthenium coated graphite furnace as 0.05 to 0.50mg. All determinations were performed using palladium+citric acid modifier and applying a pyrolysis temperature of 1000 °C and a volatilisation temperature of 2400 °C. The results were in good agreement with certified sulphur concentrations of various vegetal CRM samples applying linear calibration technique prepared from thioacetamide. The limit of detection and characteristic mass of the method were 7.5 and 8.7 ng of S, respectively. The concentrations of S in various spinach, leek, lettuce, radish, Brussels sprouts, zucchini and chard samples were determined. It was showed that distribution of sulphur in CRM and grinded food samples were homogeneous even in micro-scale. PMID:25442545

  13. An elemental mercury diffusion coefficient for natural waters determined by molecular dynamics simulation.

    PubMed

    Kuss, Joachim; Holzmann, Jörg; Ludwig, Ralf

    2009-05-01

    Mercury is a priority pollutant as its mobility between the hydrosphere and the atmosphere threatens the biosphere globally. The air-water gas transfer of elemental mercury (Hg0) is controlled by its diffusion through the water-side boundary layer and thus by its diffusion coefficient, D(Hg), the value of which, however, has not been established. Here, the diffusion of Hg0 in water was modeled by molecular dynamics (MD) simulation and the diffusion coefficient subsequently determined. Therefore the movement of either Hg(0) or xenon and 1000 model water molecules (TIP4P-Ew) were traced for time spans of 50 ns. The modeled D(Xe) of the monatomic noble gas agreed well with measured data; thus, MD simulation was assumed to be a reliable approach to determine D(Hg) for monatomic Hg(0) as well. Accordingly, Hg(0) diffusion was then simulated for freshwater and seawater, and the data were well-described by the equation of Eyring. The activation energies for the diffusion of Hg0 in freshwater was 17.0 kJ mol(-1) and in seawater 17.8 kJ mol(-1). The newly determined D(Hg) is clearly lower than the one previously used for an oceanic mercury budget. Thus, its incorporation into the model should lead to lower estimates of global ocean mercury emissions. PMID:19534132

  14. Repeat Size Determination by Two Molecular Rulers in the Type I-E CRISPR Array.

    PubMed

    Goren, Moran G; Doron, Shany; Globus, Rea; Amitai, Gil; Sorek, Rotem; Qimron, Udi

    2016-09-13

    Prokaryotic adaptive immune systems are composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. These systems adapt to new threats by integrating short nucleic acids, termed spacers, into the CRISPR array. The functional motifs in the repeat and the mechanism by which a constant repeat size is maintained are still elusive. Here, through a series of mutations within the repeat of the CRISPR-Cas type I-E, we identify motifs that are crucial for adaptation and show that they serve as anchor sites for two molecular rulers determining the size of the new repeat. Adaptation products from various repeat mutants support a model in which two motifs in the repeat bind to two different sites in the adaptation complex that are 8 and 16 bp away from the active site. This model significantly extends our understanding of the adaptation process and broadens the scope of its applications. PMID:27626652

  15. Voltammetric Determination of Flunixin on Molecularly Imprinted Polypyrrole Modified Glassy Carbon Electrode

    PubMed Central

    Radi, Abd-Elgawad; Abd El-Ghany, Nadia; Wahdan, Tarek

    2016-01-01

    A novel electrochemical sensing approach, based on electropolymerization of a molecularly imprinted polypyrrole (MIPpy) film onto a glassy carbon electrode (GCE) surface, was developed for the detection of flunixin (FXN). The sensing conditions and the performance of the constructed sensor were assessed by cyclic, differential pulse and (DPV) square wave voltammetry (SWV). The sensor exhibited high sensitivity, with linear responses in the range of 5.0 to 50.0 µM with detection limits of 1.5 and 1.0 µM for DPV and SWV, respectively. In addition, the sensor showed high selectivity towards FXN in comparison to other interferents. The sensor was successfully utilized for the direct determination of FXN in buffalo raw milk samples. PMID:27242945

  16. A novel approach for determining the minimum feed in nanochannels processing via molecular dynamics simulation

    NASA Astrophysics Data System (ADS)

    Ren, Jiaqi; Dong, Zeguang; Zhao, Jinsheng; Liu, Pinkuan

    2016-04-01

    A novel approach based on molecular dynamics (MD) simulation has been proposed for the first time with the focus on quantifying the minimum feed (MF) in atomic force microscope (AFM) based nanochannel fabrication. This approach involves a coarse-to-fine criterion to determine MF so that regular nanochannel patterns can be obtained. The method is first introduced step by step and then confirmatory test is performed to demonstrate the capability of this contour-based method. MF judging studies are also performed systematically in which they vary in the aspects of scratching depth, tip angles, and tip shapes. Dislocations generation, surface quality, and scratching forces in the initial and subsequent scratches are investigated in detail. This method can overcome the drawbacks of high cost and low efficiency in experimental studies. Furthermore, our method sheds light on the manufacturing technique of nanochannels, which can help to obtain the surface morphologies with higher quality than traditional approaches.

  17. The prolyl isomerase Pin1 as a molecular switch to determine the fate of phosphoproteins

    PubMed Central

    Liou, Yih-Cherng; Zhou, Xiao Zhen; Lu, Kun Ping

    2011-01-01

    Pin1 is a highly conserved enzyme that only isomerizes specific phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Such conformational changes represent a novel and tightly controlled signaling mechanism regulating a spectrum of protein activities in physiology and disease, often through phosphorylation-dependent, ubiquitin-mediated proteasomal degradation. In this review, we summarize recent advances in elucidating the role and regulation of Pin1 in controlling protein stability. We also propose a mechanism by which Pin1 functions as a molecular switch to control the fates of phosphoproteins. We finally stress the need to develop tools to directly visualize Pin1-catalyzed protein conformational changes as a way to determine their roles in the development and treatment of human diseases. PMID:21852138

  18. Tom70 serves as a molecular switch to determine pathological cardiac hypertrophy

    PubMed Central

    Li, Jun; Qi, Man; Li, Changming; Shi, Dan; Zhang, Dasheng; Xie, Duanyang; Yuan, Tianyou; Feng, Jing; Liu, Yi; Liang, Dandan; Xu, Xinran; Chen, Jinjin; Xu, Liang; Zhang, Hong; Ye, Jiangchuan; Lv, Fei; Huang, Jian; Peng, Luying; Chen, Yi-Han

    2014-01-01

    Pathological cardiac hypertrophy is an inevitable forerunner of heart failure. Regardless of the etiology of cardiac hypertrophy, cardiomyocyte mitochondrial alterations are always observed in this context. The translocases of mitochondrial outer membrane (Tom) complex governs the import of mitochondrial precursor proteins to maintain mitochondrial function under pathophysiological conditions; however, its role in the development of pathological cardiac hypertrophy remains unclear. Here, we showed that Tom70 was downregulated in pathological hypertrophic hearts from humans and experimental animals. The reduction in Tom70 expression produced distinct pathological cardiomyocyte hypertrophy both in vivo and in vitro. The defective mitochondrial import of Tom70-targeted optic atrophy-1 triggered intracellular oxidative stress, which led to a pathological cellular response. Importantly, increased Tom70 levels provided cardiomyocytes with full resistance to diverse pro-hypertrophic insults. Together, these results reveal that Tom70 acts as a molecular switch that orchestrates hypertrophic stresses and mitochondrial responses to determine pathological cardiac hypertrophy. PMID:25022898

  19. Determination of the water vapor continuum absorption by THz-TDS and Molecular Response Theory.

    PubMed

    Yang, Yihong; Mandehgar, Mahboubeh; Grischkowsky, D

    2014-02-24

    Determination of the water vapor continuum absorption from 0.35 to 1 THz is reported. The THz pulses propagate though a 137 m long humidity-controlled chamber and are measured by THz time-domain spectroscopy (THz-TDS). The average relative humidity along the entire THz path is precisely obtained by measuring the difference between transit times of the sample and reference THz pulses to an accuracy of 0.1 ps. Using the measured total absorption and the calculated resonance line absorption with the Molecular Response Theory lineshape, based on physical principles and measurements, an accurate continuum absorption is obtained within four THz absorption windows, that agrees well with the empirical theory. The absorption is significantly smaller than that obtained using the van Vleck-Weisskopf lineshape with a 750 GHz cut-off. PMID:24663762

  20. FvBck1, a component of cell wall integrity MAP kinase pathway, is required for virulence and oxidative stress response in sugarcane Pokkah Boeng pathogen.

    PubMed

    Zhang, Chengkang; Wang, Jianqiang; Tao, Hong; Dang, Xie; Wang, Yang; Chen, Miaoping; Zhai, Zhenzhen; Yu, Wenying; Xu, Liping; Shim, Won-Bo; Lu, Guodong; Wang, Zonghua

    2015-01-01

    Fusarium verticillioides (formerly F. moniliforme) is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a possibly carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane. PMID:26500635

  1. FvBck1, a component of cell wall integrity MAP kinase pathway, is required for virulence and oxidative stress response in sugarcane Pokkah Boeng pathogen

    PubMed Central

    Zhang, Chengkang; Wang, Jianqiang; Tao, Hong; Dang, Xie; Wang, Yang; Chen, Miaoping; Zhai, Zhenzhen; Yu, Wenying; Xu, Liping; Shim, Won-Bo; Lu, Guodong; Wang, Zonghua

    2015-01-01

    Fusarium verticillioides (formerly F. moniliforme) is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a possibly carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane. PMID:26500635

  2. Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles

    PubMed Central

    Ellis, Terri N.; Kuehn, Meta J.

    2010-01-01

    Summary: Outer membrane (OM) vesicles are ubiquitously produced by Gram-negative bacteria during all stages of bacterial growth. OM vesicles are naturally secreted by both pathogenic and nonpathogenic bacteria. Strong experimental evidence exists to categorize OM vesicle production as a type of Gram-negative bacterial virulence factor. A growing body of data demonstrates an association of active virulence factors and toxins with vesicles, suggesting that they play a role in pathogenesis. One of the most popular and best-studied pathogenic functions for membrane vesicles is to serve as natural vehicles for the intercellular transport of virulence factors and other materials directly into host cells. The production of OM vesicles has been identified as an independent bacterial stress response pathway that is activated when bacteria encounter environmental stress, such as what might be experienced during the colonization of host tissues. Their detection in infected human tissues reinforces this theory. Various other virulence factors are also associated with OM vesicles, including adhesins and degradative enzymes. As a result, OM vesicles are heavily laden with pathogen-associated molecular patterns (PAMPs), virulence factors, and other OM components that can impact the course of infection by having toxigenic effects or by the activation of the innate immune response. However, infected hosts can also benefit from OM vesicle production by stimulating their ability to mount an effective defense. Vesicles display antigens and can elicit potent inflammatory and immune responses. In sum, OM vesicles are likely to play a significant role in the virulence of Gram-negative bacterial pathogens. PMID:20197500

  3. Complete DNA Sequence and Analysis of the Large Virulence Plasmid of Shigella flexneri

    PubMed Central

    Venkatesan, Malabi M.; Goldberg, Marcia B.; Rose, Debra J.; Grotbeck, Erik J.; Burland, Valerie; Blattner, Frederick R.

    2001-01-01

    The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined. Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known Shigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles of ipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigella virulence determinants. PMID:11292750

  4. Determinants of Reactivity and Selectivity in Soluble Epoxide Hydrolase from Quantum Mechanics/Molecular Mechanics Modeling

    PubMed Central

    2012-01-01

    Soluble epoxide hydrolase (sEH) is an enzyme involved in drug metabolism that catalyzes the hydrolysis of epoxides to form their corresponding diols. sEH has a broad substrate range and shows high regio- and enantioselectivity for nucleophilic ring opening by Asp333. Epoxide hydrolases therefore have potential synthetic applications. We have used combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations (at the AM1/CHARMM22 level) and high-level ab initio (SCS-MP2) QM/MM calculations to analyze the reactions, and determinants of selectivity, for two substrates: trans-stilbene oxide (t-SO) and trans-diphenylpropene oxide (t-DPPO). The calculated free energy barriers from the QM/MM (AM1/CHARMM22) umbrella sampling MD simulations show a lower barrier for phenyl attack in t-DPPO, compared with that for benzylic attack, in agreement with experiment. Activation barriers in agreement with experimental rate constants are obtained only with the highest level of QM theory (SCS-MP2) used. Our results show that the selectivity of the ring-opening reaction is influenced by several factors, including proximity to the nucleophile, electronic stabilization of the transition state, and hydrogen bonding to two active site tyrosine residues. The protonation state of His523 during nucleophilic attack has also been investigated, and our results show that the protonated form is most consistent with experimental findings. The work presented here illustrates how determinants of selectivity can be identified from QM/MM simulations. These insights may also provide useful information for the design of novel catalysts for use in the synthesis of enantiopure compounds. PMID:22280021

  5. Determination of phosphorus using high-resolution diphosphorus molecular absorption spectra produced in the graphite furnace

    NASA Astrophysics Data System (ADS)

    Huang, Mao Dong; Becker-Ross, Helmut; Okruss, Michael; Geisler, Sebastian; Florek, Stefan

    2016-01-01

    Molecular absorption of diphosphorus was produced in a graphite furnace and evaluated in view of its suitability for phosphorus determination. Measurements were performed with two different high-resolution continuum source absorption spectrometers. The first system is a newly in-house developed simultaneous broad-range spectrograph, which was mainly used for recording overview absorption spectra of P2 between 193 nm and 245 nm. The region covers the main part of the C 1Σu+ ← X 1Σg+ electronic transition and shows a complex structure with many vibrational bands, each consisting of a multitude of sharp rotational lines. With the help of molecular data available for P2, an assignment of the vibrational bands was possible and the rotational structure could be compared with simulated spectra. The second system is a commercial sequential continuum source spectrometer, which was used for the basic analytical measurements. The P2 rotational line at 204.205 nm was selected and systematically evaluated with regard to phosphorus determination. The conditions for P2 generation were optimized and it was found that the combination of a ZrC modified graphite tube and borate as a chemical modifier were essential for a good production of P2. Serious interferences were found in the case of nitrate and sulfuric acid, although the nitrate interference can be eliminated by a higher pyrolysis temperature. The reliability of the method was proved by analysis of certified samples. Using standard tubes, a characteristic mass of 10 ng and a limit of detection of 7 ng were found. The values could further be improved by a factor of ten using a miniaturized tube with an internal diameter of 2 mm. Compared to the conventional method based on the phosphorus absorption line at 213.618 nm, the advantages of using P2 are the gentle temperature conditions and the potential of performing a simultaneous multi-line evaluation to further improve the limit of detection.

  6. A temperature-responsive network links cell shape and virulence traits in a primary fungal pathogen.

    PubMed

    Beyhan, Sinem; Gutierrez, Matias; Voorhies, Mark; Sil, Anita

    2013-07-01

    Survival at host temperature is a critical trait for pathogenic microbes of humans. Thermally dimorphic fungal pathogens, including Histoplasma capsulatum, are soil fungi that undergo dramatic changes in cell shape and virulence gene expression in response to host temperature. How these organisms link changes in temperature to both morphologic development and expression of virulence traits is unknown. Here we elucidate a temperature-responsive transcriptional network in H. capsulatum, which switches from a filamentous form in the environment to a pathogenic yeast form at body temperature. The circuit is driven by three highly conserved factors, Ryp1, Ryp2, and Ryp3, that are required for yeast-phase growth at 37°C. Ryp factors belong to distinct families of proteins that control developmental transitions in fungi: Ryp1 is a member of the WOPR family of transcription factors, and Ryp2 and Ryp3 are both members of the Velvet family of proteins whose molecular function is unknown. Here we provide the first evidence that these WOPR and Velvet proteins inte