Science.gov

Sample records for subsp tularensis clades

  1. Francisella tularensis subsp. tularensis Group A.I, United States

    PubMed Central

    Birdsell, Dawn N.; Johansson, Anders; Öhrman, Caroline; Kaufman, Emily; Molins, Claudia; Pearson, Talima; Gyuranecz, Miklós; Naumann, Amber; Vogler, Amy J.; Myrtennäs, Kerstin; Larsson, Pär; Forsman, Mats; Sjödin, Andreas; Gillece, John D.; Schupp, James; Petersen, Jeannine M.; Keim, Paul

    2014-01-01

    We used whole-genome analysis and subsequent characterization of geographically diverse strains using new genetic signatures to identify distinct subgroups within Francisella tularensis subsp. tularensis group A.I: A.I.3, A.I.8, and A.I.12. These subgroups exhibit complex phylogeographic patterns within North America. The widest distribution was observed for A.I.12, which suggests an adaptive advantage. PMID:24755401

  2. Glycosylation of DsbA in Francisella tularensis subsp. tularensis.

    PubMed

    Thomas, Rebecca M; Twine, Susan M; Fulton, Kelly M; Tessier, Luc; Kilmury, Sara L N; Ding, Wen; Harmer, Nicholas; Michell, Stephen L; Oyston, Petra C F; Titball, Richard W; Prior, Joann L

    2011-10-01

    In Francisella tularensis subsp. tularensis, DsbA has been shown to be an essential virulence factor and has been observed to migrate to multiple protein spots on two-dimensional electrophoresis gels. In this work, we show that the protein is modified with a 1,156-Da glycan moiety in O-linkage. The results of mass spectrometry studies suggest that the glycan is a hexasaccharide, comprised of N-acetylhexosamines, hexoses, and an unknown monosaccharide. Disruption of two genes within the FTT0789-FTT0800 putative polysaccharide locus, including a galE homologue (FTT0791) and a putative glycosyltransferase (FTT0798), resulted in loss of glycan modification of DsbA. The F. tularensis subsp. tularensis ΔFTT0798 and ΔFTT0791::Cm mutants remained virulent in the murine model of subcutaneous tularemia. This indicates that glycosylation of DsbA does not play a major role in virulence under these conditions. This is the first report of the detailed characterization of the DsbA glycan and putative role of the FTT0789-FTT0800 gene cluster in glycan biosynthesis. PMID:21803994

  3. Construction of targeted insertion mutations in Francisella tularensis subsp. novicida.

    PubMed

    Liu, Jirong; Zogaj, Xhavit; Barker, Jeffrey R; Klose, Karl E

    2007-10-01

    Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study of this mysterious bacterium and facilitate vaccine development against tularemia. Moreover the general strategy of gene disruption by PCR-based antibiotic resistance cassette insertion is broadly applicable to many bacterial species. PMID:18019340

  4. Characterization of the Francisella tularensis subsp. novicida type IV pilus.

    PubMed

    Zogaj, Xhavit; Chakraborty, Subhra; Liu, Jirong; Thanassi, David G; Klose, Karl E

    2008-07-01

    Francisella tularensis causes the disease tularaemia. Type IV pili (Tfp) genes are present in the genomes of all F. tularensis subspecies. We show that the wild-type F. tularensis subsp. novicida expresses pilus fibres on its surface, and mutations in the Tfp genes pilF and pilT disrupt pilus biogenesis. Mutations in other Tfp genes (pilQ and pilG) do not eliminate pilus expression. A mutation in pilE4 eliminates pilus expression, whereas mutations in the other pilin subunits pilE1-3 and pilE5 do not, suggesting that pilE4 is the major pilus structural subunit. The virulence regulator MglA is required for pilus expression, and it regulates the transcription of a putative Tfp glycosylation gene (FTN0431). However, MglA does not regulate transcription of pilF, pilT or pilE4, and a strain lacking FTN0431 still expresses pili; thus, it is unclear how MglA regulates pilus expression. Only pilF was also required for protein secretion, while pilE4 and pilT were not, indicating that there is very little overlap of the protein secretion/Tfp functions of the pil genes. The protein secretion component pilE1 was more important for in vitro intramacrophage growth and mouse virulence than the Tfp component pilE4. Our results provide the first genetic characterization of the novel Tfp system of F. tularensis. PMID:18599841

  5. Glycosylation of DsbA in Francisella tularensis subsp. tularensis▿†

    PubMed Central

    Thomas, Rebecca M.; Twine, Susan M.; Fulton, Kelly M.; Tessier, Luc; Kilmury, Sara L. N.; Ding, Wen; Harmer, Nicholas; Michell, Stephen L.; Oyston, Petra C. F.; Titball, Richard W.; Prior, Joann L.

    2011-01-01

    In Francisella tularensis subsp. tularensis, DsbA has been shown to be an essential virulence factor and has been observed to migrate to multiple protein spots on two-dimensional electrophoresis gels. In this work, we show that the protein is modified with a 1,156-Da glycan moiety in O-linkage. The results of mass spectrometry studies suggest that the glycan is a hexasaccharide, comprised of N-acetylhexosamines, hexoses, and an unknown monosaccharide. Disruption of two genes within the FTT0789-FTT0800 putative polysaccharide locus, including a galE homologue (FTT0791) and a putative glycosyltransferase (FTT0798), resulted in loss of glycan modification of DsbA. The F. tularensis subsp. tularensis ΔFTT0798 and ΔFTT0791::Cm mutants remained virulent in the murine model of subcutaneous tularemia. This indicates that glycosylation of DsbA does not play a major role in virulence under these conditions. This is the first report of the detailed characterization of the DsbA glycan and putative role of the FTT0789-FTT0800 gene cluster in glycan biosynthesis. PMID:21803994

  6. Hare-to-human transmission of Francisella tularensis subsp. holarctica, Germany.

    PubMed

    Otto, Peter; Kohlmann, Rebekka; Müller, Wolfgang; Julich, Sandra; Geis, Gabriele; Gatermann, Sören G; Peters, Martin; Wolf, Peter Johannes; Karlsson, Edvin; Forsman, Mats; Myrtennäs, Kerstin; Tomaso, Herbert

    2015-01-01

    In November 2012, a group of 7 persons who participated in a hare hunt in North Rhine-Westphalia, Germany, acquired tularemia. Two F. tularensis subsp. holarctica isolates were cultivated from human and hare biopsy material. Both isolates belonged to the FTN002-00 genetic subclade (derived for single nucleotide polymorphisms B.10 and B.18), thus indicating likely hare-to-human transmission. PMID:25531286

  7. Draft genome sequence of Francisella tularensis subsp. holarctica BD11-00177

    PubMed Central

    Coolen, Jordy P. M.; Sjödin, Andreas; Maraha, Boulos; Hajer, Gerard F.; Forsman, Mats; Verspui, Ellen; Frenay, Hendrina M.E.; Notermans, Daan W.; de Vries, Maaike C.; Reubsaet, Frans A.G.; Paauw, Armand

    2013-01-01

    Francisella tularensis is a facultative intracellular bacterium in the class Gammaproteobacteria. This strain is of interest because it is the etiologic agent of tularemia and a highly virulent category A biothreat agent. Here we describe the draft genome sequence and annotation of Francisella tularensis subsp. holarctica BD11-00177, isolated from the first case of indigenous tularemia detected in The Netherlands since 1953. Whole genome DNA sequence analysis assigned this isolate to the genomic group B.FTNF002–00, which previously has been exclusively reported from Spain, France, Italy, Switzerland and Germany. Automatic annotation of the 1,813,372 bp draft genome revealed 2,103 protein-coding and 46 RNA genes. PMID:24501637

  8. Comparison of bacterial culture and polymerase chain reaction (PCR) for the detection of F. tularensis subsp. holarctica in wild animals.

    PubMed

    Sting, Reinhard; Runge, Martin; Eisenberg, Tobias; Braune, Silke; Müller, Wolfgang; Otto, Peter

    2013-01-01

    Detection of the zoonotic pathogen Francisella tularensis subsp. holarctica (EF tularensis) in wild animals with culture techniques as well as polymerase chain reaction were compared and discussed on the basis of the investigation of 60 animals. The samples originated from 55 European brown hares (Lepus europaeus), two red foxes (Vulpes vulpes) and one each from a wild rabbit (Oryctolagus cuniculus), a European beaver (Castor fiber), and a lemur (Lemur catta). When comparing the growth of 28 F. tularensis isolates on the cysteine blood agar and the modified Martin-Lewis-agar used in this study, cultivation was successful for 26 isolates on both media, but for two isolates only on the cysteine blood agar. Out of 43 carcasses 19 tested positive in bacteriological culture and PCR. Two culture positive samples of tonsils originating from foxes could not be confirmed by PCR, although PCR was positive in 22 samples that missed growth of F. tularensis. Comparative studies on cultural detection of E. tularensis were performed on samples of 16 hares from lung, spleen, liver and gut and in one case with a peritoneal swab. In at least one of these localizations cultivation of the pathogen was successful. Detection rate was reduced to 94% (15 of 16 hares) considering only the results of the cultures of the lungs and spleens. For a sensitive and rapid detection of F. tularensis subsp. holarctica, the PCR is a suitable method thereby avoiding hazardous multiplying of the pathogen. However, cultivation of F. tularensis is often a prerequisite for further studies on antibiotic resistance patterns of the pathogen, molecular epidemiological and pathological analyses of tularaemia. PMID:23901583

  9. 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

  10. MglA Regulates Francisella tularensis subsp. novicida (Francisella novicida) Response to Starvation and Oxidative Stress▿ †

    PubMed Central

    Guina, Tina; Radulovic, Dragan; Bahrami, Arya J.; Bolton, Diana L.; Rohmer, Laurence; Jones-Isaac, Kendan A.; Chen, Jinzy; Gallagher, Larry A.; Gallis, Byron; Ryu, Soyoung; Taylor, Greg K.; Brittnacher, Mitchell J.; Manoil, Colin; Goodlett, David R.

    2007-01-01

    MglA is a transcriptional regulator of genes that contribute to the virulence of Francisella tularensis, a highly infectious pathogen and the causative agent of tularemia. This study used a label-free shotgun proteomics method to determine the F. tularensis subsp. novicida (F. novicida) proteins that are regulated by MglA. The differences in relative protein amounts between wild-type F. novicida and the mglA mutant were derived directly from the average peptide precursor ion intensity values measured with the mass spectrometer by using a suite of mathematical algorithms. Among the proteins whose relative amounts changed in an F. novicida mglA mutant were homologs of oxidative and general stress response proteins. The F. novicida mglA mutant exhibited decreased survival during stationary-phase growth and increased susceptibility to killing by superoxide generated by the redox-cycling agent paraquat. The F. novicida mglA mutant also showed increased survival upon exposure to hydrogen peroxide, likely due to increased amounts of the catalase KatG. Our results suggested that MglA coordinates the stress response of F. tularensis and is likely essential for bacterial survival in harsh environments. PMID:17644593

  11. Phylogeographical pattern of Francisella tularensis in a nationwide outbreak of tularaemia in Norway, 2011.

    PubMed

    Afset, J E; Larssen, K W; Bergh, K; Larkeryd, A; Sjodin, A; Johansson, A; Forsman, M

    2015-01-01

    In 2011, a nationwide outbreak of tularaemia occurred in Norway with 180 recorded cases. It was associated with the largest peak in lemming density seen in 40 years. Francisella tularensis was isolated from 18 patients. To study the geographical distribution of F.tularensis genotypes in Norway and correlate genotype with epidemiology and clinical presentation,we performed whole genome sequencing of patient isolates. All 18 genomes from the outbreak carried genetic signatures of F. tularensis subsp. holarctica and were assigned to genetic clades using canonical single nucleotide polymorphisms. Ten isolates were assigned to major genetic clade B.6 (subclade B.7),seven to clade B.12, and one to clade B.4. The B.6 subclade B.7 was most common in southern and central Norway, while clade B.12 was evenly distributed between the southern, central and northern parts of the country. There was no association between genotype and clinical presentation of tularaemia, time of year or specimen type. We found extensive sequence similarity with F. tularensis subsp. holarctica genomes from high-endemic tularaemia areas in Sweden.Finding nearly identical genomes across large geographical distances in Norway and Sweden imply a life cycle of the bacterium without replication between the outbreaks and raise new questions about long-range migration mechanisms. PMID:25990357

  12. Francisella tularensis subsp. tularensis Induces a Unique Pulmonary Inflammatory Response: Role of Bacterial Gene Expression in Temporal Regulation of Host Defense Responses

    PubMed Central

    Walters, Kathie-Anne; Olsufka, Rachael; Kuestner, Rolf E.; Cho, Ji Hoon; Li, Hong; Zornetzer, Gregory A.; Wang, Kai; Skerrett, Shawn J.; Ozinsky, Adrian

    2013-01-01

    Pulmonary exposure to Francisella tularensis is associated with severe lung pathology and a high mortality rate. The lack of induction of classical inflammatory mediators, including IL1-β and TNF-α, during early infection has led to the suggestion that F. tularensis evades detection by host innate immune surveillance and/or actively suppresses inflammation. To gain more insight into the host response to Francisella infection during the acute stage, transcriptomic analysis was performed on lung tissue from mice exposed to virulent (Francisella tularensis ssp tularensis SchuS4). Despite an extensive transcriptional response in the lungs of animals as early as 4 hrs post-exposure, Francisella tularensis was associated with an almost complete lack of induction of immune-related genes during the initial 24 hrs post-exposure. This broad subversion of innate immune responses was particularly evident when compared to the pulmonary inflammatory response induced by other lethal (Yersinia pestis) and non-lethal (Legionella pneumophila, Pseudomonas aeruginosa) pulmonary infections. However, the unique induction of a subset of inflammation-related genes suggests a role for dysregulation of lymphocyte function and anti-inflammatory pathways in the extreme virulence of Francisella. Subsequent activation of a classical inflammatory response 48 hrs post-exposure was associated with altered abundance of Francisella-specific transcripts, including those associated with bacterial surface components. In summary, virulent Francisella induces a unique pulmonary inflammatory response characterized by temporal regulation of innate immune pathways correlating with altered bacterial gene expression patterns. This study represents the first simultaneous measurement of both host and Francisella transcriptome changes that occur during in vivo infection and identifies potential bacterial virulence factors responsible for regulation of host inflammatory pathways. PMID:23690939

  13. Comparative Genomic Characterization of Francisella tularensis Strains Belonging to Low and High Virulence Subspecies

    PubMed Central

    Nix, Eli B.; Nano, Francis E.; Keim, Paul; Kodira, Chinnappa D.; Borowsky, Mark; Young, Sarah; Koehrsen, Michael; Engels, Reinhard; Pearson, Matthew; Howarth, Clint; Larson, Lisa; White, Jared; Alvarado, Lucia; Forsman, Mats; Bearden, Scott W.; Sjöstedt, Anders; Titball, Richard; Michell, Stephen L.; Birren, Bruce; Galagan, James

    2009-01-01

    Tularemia is a geographically widespread, severely debilitating, and occasionally lethal disease in humans. It is caused by infection by a gram-negative bacterium, Francisella tularensis. In order to better understand its potency as an etiological agent as well as its potential as a biological weapon, we have completed draft assemblies and report the first complete genomic characterization of five strains belonging to the following different Francisella subspecies (subsp.): the F. tularensis subsp. tularensis FSC033, F. tularensis subsp. holarctica FSC257 and FSC022, and F. tularensis subsp. novicida GA99-3548 and GA99-3549 strains. Here, we report the sequencing of these strains and comparative genomic analysis with recently available public Francisella sequences, including the rare F. tularensis subsp. mediasiatica FSC147 strain isolate from the Central Asian Region. We report evidence for the occurrence of large-scale rearrangement events in strains of the holarctica subspecies, supporting previous proposals that further phylogenetic subdivisions of the Type B clade are likely. We also find a significant enrichment of disrupted or absent ORFs proximal to predicted breakpoints in the FSC022 strain, including a genetic component of the Type I restriction-modification defense system. Many of the pseudogenes identified are also disrupted in the closely related rarely human pathogenic F. tularensis subsp. mediasiatica FSC147 strain, including modulator of drug activity B (mdaB) (FTT0961), which encodes a known NADPH quinone reductase involved in oxidative stress resistance. We have also identified genes exhibiting sequence similarity to effectors of the Type III (T3SS) and components of the Type IV secretion systems (T4SS). One of the genes, msrA2 (FTT1797c), is disrupted in F. tularensis subsp. mediasiatica and has recently been shown to mediate bacterial pathogen survival in host organisms. Our findings suggest that in addition to the duplication of the Francisella

  14. Genetic manipulation of francisella tularensis.

    PubMed

    Zogaj, Xhavit; Klose, Karl E

    2010-01-01

    Francisella tularensis is a facultative intracellular pathogen that causes the disease tularemia. F. tularensis subsp. tularensis causes the most severe disease in humans and has been classified as a Category A select agent and potential bioweapon. There is currently no vaccine approved for human use, making genetic manipulation of this organism critical to unraveling the genetic basis of pathogenesis and developing countermeasures against tularemia. The development of genetic techniques applicable to F. tularensis have lagged behind those routinely used for other bacteria, primarily due to lack of research and the restricted nature of the biocontainment required for studying this pathogen. However, in recent years, genetic techniques, such as transposon mutagenesis and targeted gene disruption, have been developed, that have had a dramatic impact on our understanding of the genetic basis of F. tularensis virulence. In this review, we describe some of the methods developed for genetic manipulation of F. tularensis. PMID:21607086

  15. Genetic Manipulation of Francisella Tularensis

    PubMed Central

    Zogaj, Xhavit; Klose, Karl E.

    2011-01-01

    Francisella tularensis is a facultative intracellular pathogen that causes the disease tularemia. F. tularensis subsp. tularensis causes the most severe disease in humans and has been classified as a Category A select agent and potential bioweapon. There is currently no vaccine approved for human use, making genetic manipulation of this organism critical to unraveling the genetic basis of pathogenesis and developing countermeasures against tularemia. The development of genetic techniques applicable to F. tularensis have lagged behind those routinely used for other bacteria, primarily due to lack of research and the restricted nature of the biocontainment required for studying this pathogen. However, in recent years, genetic techniques, such as transposon mutagenesis and targeted gene disruption, have been developed, that have had a dramatic impact on our understanding of the genetic basis of F. tularensis virulence. In this review, we describe some of the methods developed for genetic manipulation of F. tularensis. PMID:21607086

  16. Whole genome single nucleotide polymorphism based phylogeny of Francisella tularensis and its application to the development of a strain typing assay

    PubMed Central

    2009-01-01

    Background A low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates. Results We have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip® resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2). Conclusion Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for

  17. Francisella tularensis Peritonitis in Stomach Cancer Patient

    PubMed Central

    Ho, Linus X.; Safdar, Amar

    2004-01-01

    Tularemia with peritonitis developed in a 50-year-old man soon after diagnosis of stomach cancer with metastasis. The ascites grew Francisella tularensis subsp. holarctica, which was identified by sequencing analysis of the 16S rDNA. The infection resolved with antimicrobial treatment. Antibodies detected 4 weeks after onset disappeared after chemotherapy-associated lymphopenia. PMID:15663872

  18. Detection of Francisella tularensis in voles in Finland.

    PubMed

    Rossow, Heidi; Sissonen, Susanna; Koskela, Katja A; Kinnunen, Paula M; Hemmilä, Heidi; Niemimaa, Jukka; Huitu, Otso; Kuusi, Markku; Vapalahti, Olli; Henttonen, Heikki; Nikkari, Simo

    2014-03-01

    Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis-infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals. PMID:24575824

  19. Detection of Francisella tularensis in Voles in Finland

    PubMed Central

    Sissonen, Susanna; Koskela, Katja A.; Kinnunen, Paula M.; Hemmilä, Heidi; Niemimaa, Jukka; Huitu, Otso; Kuusi, Markku; Vapalahti, Olli; Henttonen, Heikki; Nikkari, Simo

    2014-01-01

    Abstract Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis–infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals. PMID:24575824

  20. German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity

    PubMed Central

    2013-01-01

    Background Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis. Results Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level. Conclusions F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among

  1. A 14.7 kDa Protein from Francisella tularensis subsp. novicida (Named FTN_1133), Involved in the Response to Oxidative Stress Induced by Organic Peroxides, Is Not Endowed with Thiol-Dependent Peroxidase Activity

    PubMed Central

    Meireles, Diogo de Abreu; Alegria, Thiago Geronimo Pires; Alves, Simone Vidigal; Arantes, Carla Rani Rocha; Netto, Luis Eduardo Soares

    2014-01-01

    Francisella genus comprises Gram-negative facultative intracellular bacteria that are among the most infectious human pathogens. A protein of 14.7 KDa named as FTN_1133 was previously described as a novel hydroperoxide resistance protein in F. tularensis subsp. novicida, implicated in organic peroxide detoxification and virulence. Here, we describe a structural and biochemical characterization of FTN_1133. Contrary to previous assumptions, multiple amino acid sequence alignment analyses revealed that FTN_1133 does not share significant similarity with proteins of the Ohr/OsmC family or any other Cys-based, thiol dependent peroxidase, including conserved motifs around reactive cysteine residues. Circular dichroism analyses were consistent with the in silico prediction of an all-α-helix secondary structure. The pKa of its single cysteine residue, determined by a monobromobimane alkylation method, was shown to be 8.0±0.1, value that is elevated when compared with other Cys-based peroxidases, such as peroxiredoxins and Ohr/OsmC proteins. Attempts to determine a thiol peroxidase activity for FTN_1133 failed, using both dithiols (DTT, thioredoxin and lipoamide) and monothiols (glutathione or 2-mercaptoethanol) as reducing agents. Heterologous expression of FTN_1133 gene in ahpC and oxyR mutants of E. coli showed no complementation. Furthermore, analysis of FTN_1133 protein by non-reducing SDS-PAGE showed that an inter-molecular disulfide bond (not detected in Ohr proteins) can be generated under hydroperoxide treatment, but the observed rates were not comparable to those observed for other thiol-dependent peroxidases. All the biochemical and structural data taken together indicated that FTN_1133 displayed distinct characteristics from other thiol dependent peroxidases and, therefore, suggested that FTN_1133 is not directly involved in hydroperoxide detoxification. PMID:24959833

  2. Rapid dissemination of Francisella tularensis and the effect of route of infection

    PubMed Central

    Ojeda, Sandra S; Wang, Zheng J; Mares, Chris A; Chang, Tingtung A; Li, Qun; Morris, Elizabeth G; Jerabek, Paul A; Teale, Judy M

    2008-01-01

    Background Francisella tularensis subsp. tularensis is classified as a Category A bioweapon that is capable of establishing a lethal infection in humans upon inhalation of very few organisms. However, the virulence mechanisms of this organism are not well characterized. Francisella tularensis subsp. novicida, which is an equally virulent subspecies in mice, was used in concert with a microPET scanner to better understand its temporal dissemination in vivo upon intranasal infection and how such dissemination compares with other routes of infection. Adult mice were inoculated intranasally with F. tularensis subsp. novicida radiolabeled with 64Cu and imaged by microPET at 0.25, 2 and 20 hours post-infection. Results 64Cu labeled F. tularensis subsp. novicida administered intranasally or intratracheally were visualized in the respiratory tract and stomach at 0.25 hours post infection. By 20 hours, there was significant tropism to the lung compared with other tissues. In contrast, the images of radiolabeled F. tularensis subsp. novicida when administered intragastrically, intradermally, intraperitoneally and intravenouslly were more generally limited to the gastrointestinal system, site of inoculation, liver and spleen respectively. MicroPET images correlated with the biodistribution of isotope and bacterial burdens in analyzed tissues. Conclusion Our findings suggest that Francisella has a differential tissue tropism depending on the route of entry and that the virulence of Francisella by the pulmonary route is associated with a rapid bacteremia and an early preferential tropism to the lung. In addition, the use of the microPET device allowed us to identify the cecum as a novel site of colonization of Francisella tularensis subsp. novicida in mice. PMID:19068128

  3. Molecular bases of proliferation of Francisella tularensis in Arthropod vectors

    PubMed Central

    Asare, Rexford; Akimana, Christine; Jones, Snake; Kwaik, Yousef Abu

    2010-01-01

    Summary Arthropod vectors are important vehicles for transmission of Francisella tularensis between mammals, but very little is known about the F. tularensis-arthropod vector interaction. Drosophila melanogaster has been recently developed as an arthropod vector model for F. tularensis. We have shown that intracellular trafficking of F. tularensis within human monocytes-derived macrophages and D. melanogaster-derived S2 cells is very similar. Within both evolutionarily distant host cells, the Francisella-containing phagosome matures to a late endosome-like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol where the bacterial proliferate. To decipher the molecular bases of intracellular proliferation of F. tularensis within arthropod-derived cells, we screened a comprehensive library of mutants of F. tularensis subsp novicida for their defect in intracellular proliferation within D. melanogaster-derived S2 cells. Our data show that 394 genes, representing 22% of the genome, are required for intracellular proliferation within D. melanogaster-derived S2 cells, including many of the Francisella Pathogenicity Island (FPI) genes that are also required for proliferation within mammalian macrophages. Functional gene classes that exhibit growth defect include metabolic (25%), FPI (2%), Type IV pili (1%), transport (16%) and DNA modification (5%). Among 168 most defective mutants in intracellular proliferation in S2 cells, 80 are defective in lethality and proliferation within adult D. melanogaster. The observation that only 135 of the 394 mutants that are defective in S2 cells are also defective in human macrophages indicates that F. tularensis utilize common as well as distinct mechanisms to proliferate within mammalian and arthropod cells. Our studies will facilitate deciphering the molecular aspects of F. tularensis-arthropod vector interaction and its patho-adaptation to infect mammals. PMID:20482589

  4. Comparative Transcriptional Analyses of Francisella tularensis and Francisella novicida.

    PubMed

    Sarva, Siva T; Waldo, Robert H; Belland, Robert J; Klose, Karl E

    2016-01-01

    Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of

  5. Comparative Transcriptional Analyses of Francisella tularensis and Francisella novicida

    PubMed Central

    Waldo, Robert H.; Belland, Robert J.; Klose, Karl E.

    2016-01-01

    Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of

  6. Phylogeography of Francisella tularensis subspecies holarctica from the country of Georgia

    PubMed Central

    2011-01-01

    Background Francisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context. Results We identified a new genetic lineage of F. tularensis subsp. holarctica from Georgia that belongs to the B.Br.013 group. This new lineage is genetically and geographically distinct from lineages previously described from the B.Br.013 group from Central-Eastern Europe. Importantly, this new lineage is basal within the B.Br.013 group, indicating the Georgian lineage diverged before the diversification of the other known B.Br.013 lineages. Although two isolates from the Georgian lineage were collected nearby in the Ukrainian region of Crimea, all other global isolates assigned to this lineage were collected in Georgia. This restricted geographic distribution, as well as the high levels of genetic diversity within the lineage, is consistent with a relatively older origin and localized differentiation. Conclusions We identified a new lineage of F. tularensis subsp. holarctica from Georgia that appears to have an older origin than any other diversified lineages previously described from

  7. Case Report of Low Virulence Francisella tularensis Presented as Severe Bacteremic Pneumonia

    PubMed Central

    Su, Ting-Yi; Shie, Shian-Sen; Chia, Ju-Hsin; Huang, Ching-Tai

    2016-01-01

    Abstract Tularemia is a zoonotic infection seen primarily in the Northern Hemisphere. It is caused by the bacteria Francisella tularensis. Although the ulceroglandular form of the disease is the more common manifestation of infection, F tularensis is known to cause pneumonia. F tularensis has two predominant subspecies, namely subsp. tularensis (type A) and subsp. holarctica (type B). Type B tularemia is considered to be much less virulent than type A and barely caused lethal disease and pneumonia. We reported a case with a 68-year-old man immune-compromised patient diagnosed with bacteremic pneumonia engendered by type B tularemia with initial presentation of high fever, pneumonia with pleural effusion; the diagnosis was performed using 16S rRNA gene sequence analysis. The patient's fever, pneumonia, and pleural effusion were resolved with appropriate antibiotics for tularemia. This case involving severe bacteremic pneumonia in an immune-compromised patient is rare. This case suggests that low virulence F tularensis should be included in the differential diagnoses of bacteremic pneumonia for endemic tularemia. PMID:27175638

  8. Role of the wbt locus of Francisella tularensis in lipopolysaccharide O-antigen biogenesis and pathogenicity.

    PubMed

    Raynaud, Catherine; Meibom, Karin L; Lety, Marie-Annick; Dubail, Iharilalao; Candela, Thomas; Frapy, Eric; Charbit, Alain

    2007-01-01

    Francisella tularensis is a highly infectious bacterial pathogen, responsible for the zoonotic disease tularemia. We screened a bank of transposon insertion mutants of F. tularensis subsp. holarctica LVS for colony morphology alterations and selected a mutant with a transposon insertion in wbtA, the first gene of the predicted lipopolysaccharide O-antigen gene cluster. Inactivation of wbtA led to the complete loss of O antigen, conferred serum sensitivity, impaired intracellular replication, and severely attenuated virulence in the mouse model. Notably, this mutant afforded protection against a challenge against virulent LVS. PMID:17030571

  9. Enhancement of Vaccine Efficacy by Expression of a TLR5 Ligand in the Defined Live Attenuated Francisella tularensis subsp. novicida Strain U112▲iglB::fljB

    PubMed Central

    Cunningham, Aimee L.; Dang, Kim Minh; Yu, Jieh-Juen; Guentzel, M. Neal; Heidner, Hans; Klose, Karl E.; Arulanandam, Bernard P.

    2014-01-01

    Oral vaccination with the defined live attenuated Francisella novicida vaccine strain U112▲iglB has been demonstrated to induce protective immunity against pulmonary challenge with the highly human virulent F. tularensis strain SCHU S4. However, this vaccination regimen requires a booster dose in mice and exhibits 50% protective efficacy in the Fischer 344 rat model. To enhance the efficacy of this vaccine strain, we engineered U112▲iglB to express the Salmonella typhimurium FljB flagellin D1 domain, a TLR5 agonist. The U112▲iglB::fljB strain was highly attenuated for intracellular macrophage replication, and although the FljB protein was expressed within the cytosol, it exhibited TLR5 activation in a TLR5-expressing HEK cell line. Additionally, infection of splenocytes and lymphocytes with U112▲iglB::fljB induced significantly greater TNF-α production than infection with U112▲iglB. Oral vaccination with U112▲iglB::fljB also induced significantly greater protection than U112ΔiglB against pulmonary SCHU S4 challenge in rats. The enhanced protection was accompanied by higher IgG2a production and serum-mediated reduction of Francisella infectivity. Thus, the U112▲iglB::fljB strain may serve as a potential vaccine candidate against pneumonic tularemia. PMID:25050972

  10. 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

  11. A new dye uptake assay to test the activity of antibiotics against intracellular Francisella tularensis

    PubMed Central

    Sutera, Vivien; Caspar, Yvan; Boisset, Sandrine; Maurin, Max

    2014-01-01

    Francisella tularensis, a facultative intracellular bacterium, is the aetiological agent of tularaemia. Antibiotic treatment of this zoonosis is based on the administration of a fluoroquinolone or a tetracycline for cases with mild to moderate severity, whereas an aminoglycoside (streptomycin or gentamicin) is advocated for severe cases. However, treatment failures and relapses remain frequent, especially in patients suffering from chronic lymph node suppuration. Therefore, new treatment alternatives are needed. We have developed a dye uptake assay for determination of minimal inhibitory extracellular concentrations (MIECs) of antibiotics against intracellular F. tularensis, and validated the method by comparing the results obtained using a CFU-enumerating method. We also compared MIECs with MICs of the same compounds determined using a CLSI broth microdilution method. We tested the activity of 11 antibiotics against two clinical strains of F. tularensis subsp. holarctica isolated in France. Both strains displayed low MICs (≤1 μg/mL) to fluoroquinolones (ciprofloxacin, levofloxacin and moxifloxacin), gentamicin, doxycycline and rifampicin. Higher MICs (≥8 μg/mL) were found for carbapenems (imipenem and meropenem), daptomycin and linezolid. Erythromycin MICs were 4.0 and 16.0 μg/mL, respectively, for the two clinical strains. MIECs were almost the same with the two methods used. They were concordant with MICs, except for erythromycin and linezolid (respectively, four and eight times more active against intracellular F. tularensis) and gentamicin (four to eight times less active against intracellular F. tularensis). This study validated the dye uptake assay as a new tool for determination of the activity of a large panel of antibiotics against intracellular F. tularensis. This test confirmed the intracellular activity of first-line antibiotics used for tularaemia treatment, but also revealed significant activity of linezolid against intracellular F. tularensis

  12. Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis

    PubMed Central

    Cuccui, Jon; Thomas, Rebecca M.; Moule, Madeleine G.; D'Elia, Riccardo V.; Laws, Thomas R.; Mills, Dominic C.; Williamson, Diane; Atkins, Timothy P.; Prior, Joann L.; Wren, Brendan W.

    2013-01-01

    Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l−1 of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines. PMID:23697804

  13. Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis.

    PubMed

    Cuccui, Jon; Thomas, Rebecca M; Moule, Madeleine G; D'Elia, Riccardo V; Laws, Thomas R; Mills, Dominic C; Williamson, Diane; Atkins, Timothy P; Prior, Joann L; Wren, Brendan W

    2013-05-01

    Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l(-1) of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines. PMID:23697804

  14. Alpha-1 antitrypsin is markedly decreased following pulmonary F. tularensis challenge.

    PubMed

    Chambers, James P; Yu, Jieh-Juen; Jupelli, Madhulika; Weintraub, Susan T; Lopez-Ribot, Jose L; Valdes, James J; Arulanandam, Bernard P

    2011-01-01

    Neutrophils form the first line of defense during infection and are indispensable in this function. The neutrophil elastase is a key effector molecule of the innate immune system with potent antimicrobial activity against Gram-negative bacteria, spirochaetes, and fungi. However, the release of neutrophil elastase during bacterial infection must be checked otherwise its release in the extracellular milieu will result in damage to surrounding tissues. Alpha-1 antitrypsin is a small glycoprotein clade A serpine serine protease inhibitor and has been shown to increase in humans following bacterial and viral infection. Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of tularemia. Type A strains are the most virulent with an infectious dose as low as 10 colony forming units and a mortality rate of 30-60% among untreated cases of pneumonic tularemia. We report here significant reduction of this major inhibitor of the neutrophil elastase in plasma of F. tularensis LVS and F. tularensis (type A) SCHU S4 infected animals following pulmonary challenge. Associated with an imbalance of protease-antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresivity. Consistent with a competent acute phase response following F. tularensis LVS and F. tularensis (type A) SCHU S4 pulmonary challenge and proposed up-regulation of plasma haptoglobin during the course of the acute phase reaction, haptoglobin was observed significantly increased. These data suggest that unchecked neutrophil serine protease activity may arise from F. tularensis targeted reduction of plasma α(1)-antitrysin promoting lung tissue damage facilitating increased dissemination of this bacterium in infected animals. PMID:22919586

  15. Rapid diagnosis and quantification of Francisella tularensis in organs of naturally infected common squirrel monkeys (Saimiri sciureus).

    PubMed

    Abril, Carlos; Nimmervoll, Helena; Pilo, Paola; Brodard, Isabelle; Korczak, Bozena; Markus, Seiler; Miserez, Raymond; Frey, Joachim

    2008-02-01

    Francisella tularensis, a small Gram-negative facultative intracellular bacterium, is the causative agent of tularaemia, a severe zoonotic disease transmitted to humans mostly by vectors such as ticks, flies and mosquitoes. The disease is endemic in many parts of the northern hemisphere. Among animals, the most affected species belong to rodents and lagomorphs, in particular hares. However, in the recent years, many cases of tularaemia among small monkeys in zoos were reported. We have developed a real-time PCR that allows to quantify F. tularensis in tissue samples. Using this method, we identified the spleen and the kidney as the most heavily infected organ containing up to 400 F. tularensis bacteria per simian host cell in two common squirrel monkeys (Saimiri sciureus) from a zoo that died of tularaemia. In other organs such as the brain, F. tularensis was detected at much lower titres. The strain that caused the infection was identified as F. tularensis subsp. holarctica biovar I, which is susceptible to erythromycin. The high number of F. tularensis present in soft organs such as spleen, liver and kidney represents a high risk for persons handling such carcasses and explains the transmission of the disease to a pathologist during post-mortem analysis. Herein, we show that real-time PCR allows a reliable and rapid diagnosis of F. tularensis directly from tissue samples of infected animals, which is crucial in order to attempt accurate prophylactic measures, especially in cases where humans or other animals have been exposed to this highly contagious pathogen. PMID:17875369

  16. Francisella tularensis subspecies holarctica, Tasmania, Australia, 2011.

    PubMed

    Jackson, Justin; McGregor, Alistair; Cooley, Louise; Ng, Jimmy; Brown, Mitchell; Ong, Chong Wei; Darcy, Catharine; Sintchenko, Vitali

    2012-09-01

    We report a case of ulceroglandular tularemia that developed in a woman after she was bitten by a ringtail possum (Pseudocheirus peregrinus) in a forest in Tasmania, Australia. Francisella tularensis subspecies holarctica was identified. This case indicates the emergence of F. tularensis type B in the Southern Hemisphere. PMID:22931809

  17. Francisella tularensis Subspecies holarctica, Tasmania, Australia, 2011

    PubMed Central

    Jackson, Justin; McGregor, Alistair; Cooley, Louise; Ng, Jimmy; Brown, Mitchell; Ong, Chong Wei; Darcy, Catharine

    2012-01-01

    We report a case of ulceroglandular tularemia that developed in a woman after she was bitten by a ringtail possum (Pseudocheirus peregrinus) in a forest in Tasmania, Australia. Francisella tularensis subspecies holarctica was identified. This case indicates the emergence of F. tularensis type B in the Southern Hemisphere. PMID:22931809

  18. Substructure within Salmonella enterica subsp. enterica Isolates from Australian Wildlife▿

    PubMed Central

    Parsons, Sandra K.; Bull, C. Michael; Gordon, David M.

    2011-01-01

    Multilocus sequence typing of 56 Salmonella enterica subsp. enterica strains isolated from Australian wildlife hosts was performed. The results of population assignment algorithms revealed that the 56 strains could be subdivided into two distinct clades. Strains belonging to the two clades were further distinguished phenotypically, genotypically, and with respect to host distribution. PMID:21378038

  19. Metapopulation structure for perpetuation of Francisella tularensis tularensis

    PubMed Central

    2009-01-01

    Background Outbreaks of Type A tularemia due to Francisella tularensis tularensis are typically sporadic and unstable, greatly hindering identification of the determinants of perpetuation and human risk. Martha's Vineyard, Massachusetts has experienced an outbreak of Type A tularemia which has persisted for 9 years. This unique situation has allowed us to conduct long-term eco-epidemiologic studies there. Our hypothesis is that the agent of Type A tularemia is perpetuated as a metapopulation, with many small isolated natural foci of transmission. During times of increased transmission, the foci would merge and a larger scale epizootic would occur, with greater likelihood that humans become exposed. Methods We sampled questing dog ticks from two natural foci on the island and tested them for tularemia DNA. We determined whether the force of transmission differed between the two foci. In addition, we examined the population structure of F. tularensis from ticks by variable number tandem repeat (VNTR) analysis, which allowed estimates of diversity, linkage disequilibrium, and eBURST analysis. Results The prevalence of tularemia DNA in ticks from our two field sites was markedly different: one site was stable over the course of the study yielding as many as 5.6% positive ticks. In contrast, infected ticks from the comparison site markedly increased in prevalence, from 0.4% in 2003 to 3.9% in 2006. Using 4 VNTR loci, we documented 75 different haplotypes (diversity = 0.91). eBURST analysis indicates that the stable site was essentially clonal, but the comparison site contained multiple unrelated lineages. The general bacterial population is evolving clonally (multilocus disequilibrium) and the bacteria in the two sites are reproductively isolated. Conclusion Even within an isolated island, tularemia natural foci that are no more than 15 km apart are uniquely segregated. One of our sites has stable transmission and the other is emergent. The population structure at the

  20. 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

  1. Characterization of Tetratricopeptide Repeat-Like Proteins in Francisella tularensis and Identification of a Novel Locus Required for Virulence

    PubMed Central

    Dankova, Vera; Balonova, Lucie; Straskova, Adela; Spidlova, Petra; Putzova, Daniela; Kijek, Todd; Bozue, Joel; Cote, Christopher; Mou, Sherry; Worsham, Patricia; Szotakova, Barbora; Stulik, Jiri

    2014-01-01

    Francisella tularensis is a highly infectious bacterium that causes the potentially lethal disease tularemia. This extremely virulent bacterium is able to replicate in the cytosolic compartments of infected macrophages. To invade macrophages and to cope with their intracellular environment, Francisella requires multiple virulence factors, which are still being identified. Proteins containing tetratricopeptide repeat (TPR)-like domains seem to be promising targets to investigate, since these proteins have been reported to be directly involved in virulence-associated functions of bacterial pathogens. Here, we studied the role of the FTS_0201, FTS_0778, and FTS_1680 genes, which encode putative TPR-like proteins in Francisella tularensis subsp. holarctica FSC200. Mutants defective in protein expression were prepared by TargeTron insertion mutagenesis. We found that the locus FTS_1680 and its ortholog FTT_0166c in the highly virulent Francisella tularensis type A strain SchuS4 are required for proper intracellular replication, full virulence in mice, and heat stress tolerance. Additionally, the FTS_1680-encoded protein was identified as a membrane-associated protein required for full cytopathogenicity in macrophages. Our study thus identifies FTS_1680/FTT_0166c as a new virulence factor in Francisella tularensis. PMID:25245806

  2. The Early Phagosomal Stage of Francisella tularensis Determines Optimal Phagosomal Escape and Francisella Pathogenicity Island Protein Expression▿

    PubMed Central

    Chong, Audrey; Wehrly, Tara D.; Nair, Vinod; Fischer, Elizabeth R.; Barker, Jeffrey R.; Klose, Karl E.; Celli, Jean

    2008-01-01

    Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins. PMID:18852245

  3. Francisella tularensis: No Evidence for Transovarial Transmission in the Tularemia Tick Vectors Dermacentor reticulatus and Ixodes ricinus

    PubMed Central

    Genchi, Marco; Prati, Paola; Vicari, Nadia; Manfredini, Andrea; Sacchi, Luciano; Clementi, Emanuela; Bandi, Claudio; Epis, Sara; Fabbi, Massimo

    2015-01-01

    Background Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results. Objective The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus. Results Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes. Conclusions These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view. PMID:26244842

  4. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    SciTech Connect

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2006-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  5. Differential Mortality of Dog Tick Vectors Due to Infection by Diverse Francisella tularensis tularensis Genotypes

    PubMed Central

    Goethert, Heidi K.

    2011-01-01

    Abstract The factors involved in the long-term perpetuation of Francisella tularensis tularensis in nature are poorly understood. Martha's Vineyard, Massachusetts, has become a site of sustained transmission of Type A tularemia, with nearly 100 human cases reported from 2000 to 2010. We have identified a stable focus of F. tularensis transmission there, where the annual prevalence in host-seeking Dermacentor variabilis is about 3%, suggesting that this tick perpetuates the agent. However, laboratory studies have shown that infection with F. tularensis has a profound negative effect on dog tick mortality, presenting a paradox: how can a vector perpetuate an agent that negatively affects its fitness? It may be that experimental infection does not mimic that of natural transmission. Accordingly, we examined the effects that F. tularensis has on the longevity of field-derived ticks. Of 63 PCR-positive ticks collected in early summer, 89% were dead by December compared to 48% of 214 uninfected ticks collected at the same time and site. However, the quantum of F. tularensis DNA within each tick was not correlated with increased mortality. Instead, ticks with an uncommon genotype were more likely to die early than those with the common genotype. We conclude that the interaction between F. tularensis and its vector is complex and certain bacterial genotypes appear to be better adapted to their arthropod host. PMID:21612530

  6. [CONSTRUCTION AND PROPERTIES OF THE FRANCISELLA TULARENSIS VACCINE STRAIN WITHOUT ONE COPY OF THE IGLC GENE AND WITHOUT RECA GENE].

    PubMed

    Mokrievich, A N; Vakhrameeva, G M; Titareva, G M; Bakhteeva, I V; Mironova, R I; Kombarova, T I; Kravchenko, T B; Dyatlov, I A; Pavlov, V M

    2015-01-01

    The live vaccine based on the Francisella tularensis subsp. holarctica vaccine strain 15 NIIEG line is used in Russia against tularemia. This vaccine is highly effective, but fairly unstable. Therefore, development of stable live tularemia vaccine with minimal side effect is rather urgent. The method of allel removal in the F. tularensis vaccine strain was used to remove one copy of the iglC gene, which is required to provide intracellular production of the vaccine strain, as well as removal of the recA gene. The latter is crucial for homological recombination. pGM5 suicide vector based on pHV33 bireplicon plasmid was constructed to provide replacement of intact F. tularensis chromosome segments by modified segments. Modified chromosome segments contain F. Tularensis DNA fragment without iglC structural gene segment 545 p. b. (in pGMΔiglC plasmid), as well as DNA fragment containing no recA structural gene segment 1060 p.b. (pGMΔrecA plasmid). The constructed 15/23-1ΔrecA mutant, in contrast to the vaccine strain 15, was capable of reproducing in the macrophage-like cells J774A.1 line, whereas the efficiency of the reproduction was 8-10 times less. BALB/c mouse responded to immunization by the 15/23-1ΔrecA strain by smaller weight decrease (-2%) as compared to the strain 15 (-14%). Bacteria of the 15/23-1ΔrecA strain were virtually incapable of germinating from the BALB/c murine spleen 14 days after invasion, whereas bacteria of the strain 15 were found in the murine organs even after 21 days. The F. tularensis 15/23-1ΔrecA strain having smaller reaction ability can be used as a basis for construction of stable live safe tularemia vaccine. PMID:26665740

  7. Comparative review of Francisella tularensis and Francisella novicida

    PubMed Central

    Kingry, Luke C.; Petersen, Jeannine M.

    2014-01-01

    Francisella tularensis is the causative agent of the acute disease tularemia. Due to its extreme infectivity and ability to cause disease upon inhalation, F. tularensis has been classified as a biothreat agent. Two subspecies of F. tularensis, tularensis and holarctica, are responsible for tularemia in humans. In comparison, the closely related species F. novicida very rarely causes human illness and cases that do occur are associated with patients who are immune compromised or have other underlying health problems. Virulence between F. tularensis and F. novicida also differs in laboratory animals. Despite this varying capacity to cause disease, the two species share ~97% nucleotide identity, with F. novicida commonly used as a laboratory surrogate for F. tularensis. As the F. novicida U112 strain is exempt from U.S. select agent regulations, research studies can be carried out in non-registered laboratories lacking specialized containment facilities required for work with virulent F. tularensis strains. This review is designed to highlight phenotypic (clinical, ecological, virulence, and pathogenic) and genomic differences between F. tularensis and F. novicida that warrant maintaining F. novicida and F. tularensis as separate species. Standardized nomenclature for F. novicida is critical for accurate interpretation of experimental results, limiting clinical confusion between F. novicida and F. tularensis and ensuring treatment efficacy studies utilize virulent F. tularensis strains. PMID:24660164

  8. Comparative review of Francisella tularensis and Francisella novicida.

    PubMed

    Kingry, Luke C; Petersen, Jeannine M

    2014-01-01

    Francisella tularensis is the causative agent of the acute disease tularemia. Due to its extreme infectivity and ability to cause disease upon inhalation, F. tularensis has been classified as a biothreat agent. Two subspecies of F. tularensis, tularensis and holarctica, are responsible for tularemia in humans. In comparison, the closely related species F. novicida very rarely causes human illness and cases that do occur are associated with patients who are immune compromised or have other underlying health problems. Virulence between F. tularensis and F. novicida also differs in laboratory animals. Despite this varying capacity to cause disease, the two species share ~97% nucleotide identity, with F. novicida commonly used as a laboratory surrogate for F. tularensis. As the F. novicida U112 strain is exempt from U.S. select agent regulations, research studies can be carried out in non-registered laboratories lacking specialized containment facilities required for work with virulent F. tularensis strains. This review is designed to highlight phenotypic (clinical, ecological, virulence, and pathogenic) and genomic differences between F. tularensis and F. novicida that warrant maintaining F. novicida and F. tularensis as separate species. Standardized nomenclature for F. novicida is critical for accurate interpretation of experimental results, limiting clinical confusion between F. novicida and F. tularensis and ensuring treatment efficacy studies utilize virulent F. tularensis strains. PMID:24660164

  9. Environmental Monitoring and Surveillance of Rodents and Vectors for Francisella tularensis Following Outbreaks of Human Tularemia in Georgia

    PubMed Central

    Elashvili, Eka; Kracalik, Ian; Burjanadze, Irma; Datukishvili, Sophio; Chanturia, Gvantsa; Tsertsvadze, Nikoloz; Beridze, Levan; Shavishvili, Merab; Dzneladze, Archil; Grdzelidze, Marina; Imnadze, Paata; Pearson, Andrew

    2015-01-01

    Abstract Tularemia is a re-emerging bacterial zoonosis, broadly distributed across the northern hemisphere. In Georgia, there is a history of human tularemia outbreaks dating back to the 1940s. In response to outbreaks, health officials initiated long-term field surveillance and environmental monitoring. The objective of our study was to obtain information from 57 years of field surveys to identify species that play a role in the occurrence Francisella tularensis subsp. holarctica in the environment in Georgia. We collected historical data on human outbreaks, field collections, population dynamics of the common vole (Microtus arvalis), and conducted surveys on small mammals and vectors from five regions in Georgia during 1956–2012. Bacterial isolation was conducted using standard culturing techniques, and isolation rates for species were obtained for a subset of years. We used a Spearman rank correlation to test for associations between the density of the common vole and isolation rates. From 1956 through 2012, there were four recorded outbreaks of human tularemia (362 cases). A total of 465 bacterial isolates of F. tularensis subsp. holarctica were obtained from 27 species and environmental samples. The number of isolations was highest in the common vole (M. arvalis; 149 isolates; 32%) and Dermacentor marginatus ticks (132 isolates; 28%); isolation rates ranged between 0–0.91% and 0–0.47%, respectively. Population dynamics of the common vole were not correlated with the isolation rate. Given the history of tularemia re-emergence in Georgia, continued field surveys and environmental monitoring may provide an early indication of outbreak risk in humans. In conclusion, our findings provide evidence of long-standing foci of F. tularensis subsp. holarctica that are likely maintained by the common vole–tick cycle. PMID:26394283

  10. Environmental Monitoring and Surveillance of Rodents and Vectors for Francisella tularensis Following Outbreaks of Human Tularemia in Georgia.

    PubMed

    Elashvili, Eka; Kracalik, Ian; Burjanadze, Irma; Datukishvili, Sophio; Chanturia, Gvantsa; Tsertsvadze, Nikoloz; Beridze, Levan; Shavishvili, Merab; Dzneladze, Archil; Grdzelidze, Marina; Imnadze, Paata; Pearson, Andrew; Blackburn, Jason K

    2015-10-01

    Tularemia is a re-emerging bacterial zoonosis, broadly distributed across the northern hemisphere. In Georgia, there is a history of human tularemia outbreaks dating back to the 1940s. In response to outbreaks, health officials initiated long-term field surveillance and environmental monitoring. The objective of our study was to obtain information from 57 years of field surveys to identify species that play a role in the occurrence Francisella tularensis subsp. holarctica in the environment in Georgia. We collected historical data on human outbreaks, field collections, population dynamics of the common vole (Microtus arvalis), and conducted surveys on small mammals and vectors from five regions in Georgia during 1956-2012. Bacterial isolation was conducted using standard culturing techniques, and isolation rates for species were obtained for a subset of years. We used a Spearman rank correlation to test for associations between the density of the common vole and isolation rates. From 1956 through 2012, there were four recorded outbreaks of human tularemia (362 cases). A total of 465 bacterial isolates of F. tularensis subsp. holarctica were obtained from 27 species and environmental samples. The number of isolations was highest in the common vole (M. arvalis; 149 isolates; 32%) and Dermacentor marginatus ticks (132 isolates; 28%); isolation rates ranged between 0-0.91% and 0-0.47%, respectively. Population dynamics of the common vole were not correlated with the isolation rate. Given the history of tularemia re-emergence in Georgia, continued field surveys and environmental monitoring may provide an early indication of outbreak risk in humans. In conclusion, our findings provide evidence of long-standing foci of F. tularensis subsp. holarctica that are likely maintained by the common vole-tick cycle. PMID:26394283

  11. Candida albicans clades.

    PubMed

    Soll, David R; Pujol, Claude

    2003-10-24

    DNA fingerprinting with the complex probe Ca3 has revealed the following five Candida albicans clades: group I, group II, group III, group SA and group E. These groups exhibit geographical specificity. Group SA is relatively specific (i.e., highly enriched) to South Africa, group E is relatively specific to Europe, and group II is absent in the Southwest USA and South America. The maintenance of deep-rooted clades side by side in the same geographical locale and the apparent absence of subclade structure suggest little recombination between clades, but higher rates of recombination within clades. Exclusive 5-fluorocytosine resistance in the majority of group I isolates reinforces the above conclusions on recombination, and demonstrates that clades differ phenotypically. The ramifications of these findings with regard to pathogenesis are discussed. In particular, these findings lay to rest the idea that one strain represents all strains of C. albicans, support the need for a worldwide analysis of population structure and clade-specific phenotypic characteristics, and demonstrate that in the future, pathogenic characteristics must be analyzed in representatives from all five clades. PMID:14556989

  12. Live Attenuated Francisella novicida Vaccine Protects against Francisella tularensis Pulmonary Challenge in Rats and Non-human Primates

    PubMed Central

    Chu, Ping; Cunningham, Aimee L.; Yu, Jieh-Juen; Nguyen, Jesse Q.; Barker, Jeffrey R.; Lyons, C. Rick; Wilder, Julie; Valderas, Michelle; Sherwood, Robert L.; Arulanandam, Bernard P.; Klose, Karl E.

    2014-01-01

    Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform. PMID:25340543

  13. Francisella tularensis type B ΔdsbA mutant protects against type A strain and induces strong inflammatory cytokine and Th1-like antibody response in vivo.

    PubMed

    Straskova, Adela; Spidlova, Petra; Mou, Sherry; Worsham, Patricia; Putzova, Daniela; Pavkova, Ivona; Stulik, Jiri

    2015-11-01

    Francisella tularensis subspecies tularensis is a highly virulent intracellular bacterial pathogen, causing the disease tularemia. However, a safe and effective vaccine for routine application against F. tularensis has not yet been developed. We have recently constructed the deletion mutants for the DsbA homolog protein (ΔdsbA/FSC200) and a hypothetical protein IglH (ΔiglH/FSC200) in the type B F. tularensis subsp. holarctica FSC200 strain, which exerted different protection capacity against parental virulent strain. In this study, we further investigated the immunological correlates for these different levels of protection provided by ΔdsbA/FSC200 and ΔiglH/FSC200 mutants. Our results show that ΔdsbA/FSC200 mutant, but not ΔiglH/FSC200 mutant, induces an early innate inflammatory response leading to strong Th1-like antibody response. Furthermore, vaccination with ΔdsbA/FSC200 mutant, but not with ΔiglH/FSC200, elicited protection against the subsequent challenge with type A SCHU S4 strain in mice. An immunoproteomic approach was used to map a spectrum of antigens targeted by Th1-like specific antibodies, and more than 80 bacterial antigens, including novel ones, were identified. Comparison of tularemic antigens recognized by the ΔdsbA/FSC200 post-vaccination and the SCHU S4 post-challenge sera then revealed the existence of 22 novel SCHU S4 specific antibody clones. PMID:26253078

  14. Regulation of apoptosis and anti-apoptosis signalling by Francisella tularensis

    PubMed Central

    Santic, Marina; Pavokovic, Gordana; Jones, Snake; Asare, Rexford; Kwaik, Yousef Abu

    2009-01-01

    Francisella tularensis induces apoptosis within macrophages but the temporal and spatial modulation through activation of caspase-1, caspase-3, and the anti-apoptosis nuclear transcription factor B (NF-κB) is not known. Whether escape of the bacteria into the cytosol is sufficient and/or essential for activation of NF-κB is not known. Our results show that F. tularensis subsp. novicida induces sustained nuclear translocation of NF-κB at early time points after infection of human monocytes derived macrophages (hMDMs). The sustained nuclear translocation of NF-κB is defective in the iglC mutant that fails to escape into the cytosol of macrophages. Nuclear translocation of NF-κB by the wild type strain is abolished upon treatment with the NF-κB inhibitor caffein acid phenyl ester. While the wild type strain triggers caspase-3 and caspase-1 activation by 6 h post-infection the iglC mutant is defective in triggering both caspases. In hMDMs treated with the apoptosis-inducing agent, staurosporin, there is an induction of cell death in the iglC mutant-infected macrophages despite reduced frequency of caspase-1 and caspase-3 activity. The wt-infected macrophages are resistant to cell death-induced agent. We conclude that although caspase-1 and capsase-3 are triggered within F. tularensis-infected hMDMs during early stages of infection, cell death is delayed, which is correlated with simultaneous activation of NF-κB. PMID:19925880

  15. Regulation of apoptosis and anti-apoptosis signalling by Francisella tularensis.

    PubMed

    Santic, Marina; Pavokovic, Gordana; Jones, Snake; Asare, Rexford; Kwaik, Yousef Abu

    2010-02-01

    Francisella tularensis induces apoptosis within macrophages but the temporal and spatial modulation through activation of caspase-1, caspase-3, and the anti-apoptosis nuclear transcription factor B (NF-kappaB) is not known. Whether escape of the bacteria into the cytosol is sufficient and/or essential for activation of NF-kappaB is not known. Our results show that F. tularensis subsp. novicida induces sustained nuclear translocation of NF-kappaB at early time points after infection of human monocytes derived macrophages (hMDMs). The sustained nuclear translocation of NF-kappaB is defective in the iglC mutant that fails to escape into the cytosol of macrophages. Nuclear translocation of NF-kappaB by the wild type strain is abolished upon treatment with the NF-kappaB inhibitor caffein acid phenyl ester. While the wild type strain triggers caspase-3 and caspase-1 activation by 6 h post-infection the iglC mutant is defective in triggering both caspases. In hMDMs treated with the apoptosis-inducing agent, staurosporin, there is an induction of cell death in the iglC mutant-infected macrophages despite reduced frequency of caspase-1 and caspase-3 activity. The wt-infected macrophages are resistant to cell death-induced agent. We conclude that although caspase-1 and capsase-3 are triggered within F. tularensis-infected hMDMs during early stages of infection, cell death is delayed, which is correlated with simultaneous activation of NF-kappaB. PMID:19925880

  16. Molecular Complexity Orchestrates Modulation of Phagosome Biogenesis and Escape to the Cytosol of macrophages by Francisella tularensis

    PubMed Central

    Asare, Rexford; Kwaik, Yousef Abu

    2010-01-01

    Upon entry of Francisella tularensis to macrophages, the Francisella-containing phagosome (FCP) is trafficked into an acidified late endosome-like phagosome with limited fusion to the lysosomes followed by rapid escape into the cytosol where the organism replicates. Although the Francisella Pathogenicity Island (FPI), which encodes a type VI-like secretion apparatus, is required for modulation of phagosome biogenesis and escape into the cytosol, the mechanisms involved are not known. To decipher the molecular bases of modulation of biogenesis of the FCP and bacterial escape into the macrophage cytosol, we have screened a comprehensive mutant library of F. tularensis subsp novicida for their defect in proliferation within human macrophages, followed by characterization of modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data show that at least 202 genes are required for intracellular proliferation within macrophages. Among the 125 most defective mutants in intracellular proliferation, we show that the FCP of at least 91 mutants co-localize persistently with the late endosomal/lysosomal marker LAMP-1 and fail to escape into the cytosol, as determined by fluorescence-based phagosome integrity assays and transmission electron microscopy. At least 34 genes are required for proliferation within the cytosol but do not play a detectable role in modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data indicate a tremendous adaptation and metabolic reprogramming by F. tularensis to adjust to the micro-environmental and nutritional cues within the FCP, and these adjustments play essential roles in modulation of phagosome biogenesis and escape into the cytosol of macrophages as well as proliferation in the cytosol. The plethora of the networks of genes that orchestrate F. tularensis-mediated modulation of phagosome biogenesis, phagosomal escape, and bacterial proliferation within the cytosol is novel, complex, and involves

  17. [A water-borne tularemia outbreak caused by Francisella tularensis subspecies holarctica in Central Anatolia region].

    PubMed

    Ulu Kılıç, Ayşegül; Kılıç, Selçuk; Sencan, Irfan; Ciçek Şentürk, Gönül; Gürbüz, Yunus; Tütüncü, Emin Ediz; Celebi, Bekir; Kıcıman, Özlem; Ergönül, Önder

    2011-04-01

    In this study, we investigated a waterborne tularemia outbreak occured in Kadiozu, a village of Cerkes county of Cankiri province (located in North-west part of central Anatolia, Turkey) between 18 November 2009-24 December 2009. Active surveillance was conducted to determine clinical characteristics and risk factors of cases after two patients from the same village had been diagnosed as oropharyngeal tularemia. All villagers were examined, and clinical specimens from cases and water samples which may be the source of outbreak in the field investigations were taken. Cases were in the form of oropharyngeal, glandular and pneumonic. Polymerase chain reaction (PCR) and cultures were conducted from lymph node aspirates, throat swabs taken from cases and samples from water sources of epidemic zone. All serum samples taken from the villagers were screened for F.tularensis antibodies with microagglutination test (MAT). Oropharyngeal tularemia was diagnosed in 11 patients, glandular form in 3 patients and pneumonic form in one patient according to clinical and laboratory results. Age of the patients ranged between 6-75 years old (mean age: 52.5 years) and thirty one of them (54.7%) were female. MAT titers ranged between 1/160 and 1/5120 in cases of tularemia. Causative agent was grown in the cultures of two patients (including a throat swab and a lymph node aspirate). F.tularensis DNA was shown by PCR in a throat swab and four lymph node aspirates. F.tularensis was also detected by PCR in the water sample obtained from one of the spring water commonly used by villagers. Only one of the lymph node samples obtained from two different patients, was positive by direct fluorescent antibody method. Causative agent was defined as F.tularensis subsp. holarctica by conventional and also molecular methods. Patients were treated with aminoglycoside (streptomycin, gentamicin, amikacin) or quinolone (ciprofloxacin, levofloxacin) antibiotics. Treatment failure was observed in five

  18. [An oropharyngeal tularemia case diagnosed by the isolation of Francisella tularensis on human blood agar].

    PubMed

    Ozel, Gönül; Arslan, Ilker Burak; Yeşilyurt, Murat; Celebi, Bekir; Kılıç, Selçuk

    2010-10-01

    subsp. holarctica by polymerase chain reaction. Microagglutination test performed on patient's serum yielded positive with an antibody titer of 1/5120. Gentamicin (5 mg/kg/day) was initiated, and the therapy was completed for two weeks. The patient recovered completely without sequela. This case was presented in order to call attention to the strain of F.tularensis which failed to demonstrate a requirement for cysteine and enriched medium on primary isolation, but grew well on conventional laboratory medium. Tularemia should be considered in the differential diagnosis of related infectious diseases since cases of tularemia have been reported from several parts of Turkey after the year 2004. PMID:21063979

  19. Identifying Francisella tularensis genes required for growth in host cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Technical Abstract: Francisella tularensis is a highly virulent Gram negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cel...

  20. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2007-02-06

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  1. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2009-02-24

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  2. Francisella tularensis as a potential agent of bioterrorism?

    PubMed

    Maurin, Max

    2015-02-01

    Francisella tularensis is a category A bioterrorism agent. It is the etiological agent of tularemia, a zoonotic disease found throughout the northern hemisphere. The intentional spread of F. tularensis aerosols would probably lead to severe and often fatal pneumonia cases, but also secondary cases from contaminated animals and environments. We are not ready to face such a situation. No vaccine is currently available. A few antibiotics are active against F. tularensis, but strains resistant to these antibiotics could be used in the context of bioterrorism. We need new therapeutic strategies to fight against category A bioterrorism agents, including development of new drugs inhibiting F. tularensis growth and/or virulence, or enhancing the host response to infection by this pathogen. PMID:25413334

  3. A novel nanoprobe for the sensitive detection of Francisella tularensis.

    PubMed

    Kim, Ji-eun; Seo, Youngmin; Jeong, Yoon; Hwang, Mintai P; Hwang, Jangsun; Choo, Jaebum; Hong, Jong Wook; Jeon, Jun Ho; Rhie, Gi-eun; Choi, Jonghoon

    2015-11-15

    Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens. PMID:26057442

  4. Long-Lasting Fever and Lymphadenitis: Think about F. tularensis

    PubMed Central

    Longo, Maria Vittoria; Jaton, Katia; Pilo, Paola; Chabanel, David; Erard, Véronique

    2015-01-01

    We report the case of glandular tularemia that developed in a man supposedly infected by a tick bite in Western Switzerland. Francisella tularensis (F. tularensis) was identified. In Europe tularemia most commonly manifests itself as ulcero-glandular or glandular disease; the diagnosis of tularemia may be delayed in glandular form where skin or mucous lesion is absent, particularly in areas which are assumed to have a low incidence of the disease. PMID:26612988

  5. Identification of a Small Molecule That Modifies MglA/SspA Interaction and Impairs Intramacrophage Survival of Francisella tularensis

    PubMed Central

    Wrench, Algevis P.; Gardner, Christopher L.; Gonzalez, Claudio F.; Lorca, Graciela L.

    2013-01-01

    The transcription factors MglA and SspA of Francisella tularensis form a heterodimer complex and interact with the RNA polymerase to regulate the expression of the Francisella pathogenicity island (FPI) genes. These genes are essential for this pathogen’s virulence and survival within host cells. In this study, we used a small molecule screening to identify quinacrine as a thermal stabilizing compound for F. tularensis SCHU S4 MglA and SspA. A bacterial two-hybrid system was used to analyze the in vivo effect of quinacrine on the heterodimer complex. The results show that quinacrine affects the interaction between MglA and SspA, indicated by decreased β-galactosidase activity. Further in vitro analyses, using size exclusion chromatography, indicated that quinacrine does not disrupt the heterodimer formation, however, changes in the alpha helix content were confirmed by circular dichroism. Structure-guided site-directed mutagenesis experiments indicated that quinacrine makes contact with amino acid residues Y63 in MglA, and K97 in SspA, both located in the “cleft” of the interacting surfaces. In F. tularensis subsp. novicida, quinacrine decreased the transcription of the FPI genes, iglA, iglD, pdpD and pdpA. As a consequence, the intramacrophage survival capabilities of the bacteria were affected. These results support use of the MglA/SspA interacting surface, and quinacrine’s chemical scaffold, for the design of high affinity molecules that will function as therapeutics for the treatment of Tularemia. PMID:23372736

  6. Phylogenomic analysis shows that Bacillus amyloliquefaciens subsp. plantarum is a later heterotypic synonym of Bacillus methylotrophicus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rhizosphere isolated bacteria belonging to the Bacillus amyloliquefaciens subsp. plantarum and Bacillus methylotrophicus clades are an important group of strains that are used as plant growth promoters and antagonists of plant pathogens. These properties have made these strains the focus of comm...

  7. Deletion of the Bacillus anthracis capB homologue in Francisella tularensis subspecies tularensis generates an attenuated strain that protects mice against virulent tularaemia.

    PubMed

    Michell, Stephen L; Dean, Rachel E; Eyles, Jim E; Hartley, Margaret Gill; Waters, Emma; Prior, Joann L; Titball, Richard W; Oyston, Petra C F

    2010-11-01

    As there is currently no licensed vaccine against Francisella tularensis, the causative agent of tularaemia, the bacterium is an agent of concern as a potential bioweapon. Although F. tularensis has a low infectious dose and high associated mortality, it possesses few classical virulence factors. An analysis of the F. tularensis subspecies tularensis genome sequence has revealed the presence of a region containing genes with low sequence homology to part of the capBCADE operon of Bacillus anthracis. We have generated an isogenic capB mutant of F. tularensis subspecies tularensis SchuS4 and shown it to be attenuated. Furthermore, using BALB/c mice, we have demonstrated that this capB strain affords protection against significant homologous challenge with the wild-type strain. These data have important implications for the development of a defined and efficacious tularaemia vaccine. PMID:20651039

  8. Symbiosis with Francisella tularensis provides resistance to pathogens in the silkworm

    PubMed Central

    Suzuki, Jin; Uda, Akihiko; Watanabe, Kenta; Shimizu, Takashi; Watarai, Masahisa

    2016-01-01

    Francisella tularensis, the causative agent of tularemia, is a highly virulent facultative intracellular pathogen found in a wide range of animals, including arthropods, and environments. This bacterium has been known for over 100 years, but the lifestyle of F. tularensis in natural reservoirs remains largely unknown. Thus, we established a novel natural host model for F. tularensis using the silkworm (Bombyx mori), which is an insect model for infection by pathogens. F. tularensis established a symbiosis with silkworms, and bacteria were observed in the hemolymph. After infection with F. tularensis, the induction of melanization and nodulation, which are immune responses to bacterial infection, were inhibited in silkworms. Pre-inoculation of silkworms with F. tularensis enhanced the expression of antimicrobial peptides and resistance to infection by pathogenic bacteria. These results suggest that silkworms acquire host resistance via their symbiosis with F. tularensis, which may have important fitness benefits in natural reservoirs. PMID:27507264

  9. Symbiosis with Francisella tularensis provides resistance to pathogens in the silkworm.

    PubMed

    Suzuki, Jin; Uda, Akihiko; Watanabe, Kenta; Shimizu, Takashi; Watarai, Masahisa

    2016-01-01

    Francisella tularensis, the causative agent of tularemia, is a highly virulent facultative intracellular pathogen found in a wide range of animals, including arthropods, and environments. This bacterium has been known for over 100 years, but the lifestyle of F. tularensis in natural reservoirs remains largely unknown. Thus, we established a novel natural host model for F. tularensis using the silkworm (Bombyx mori), which is an insect model for infection by pathogens. F. tularensis established a symbiosis with silkworms, and bacteria were observed in the hemolymph. After infection with F. tularensis, the induction of melanization and nodulation, which are immune responses to bacterial infection, were inhibited in silkworms. Pre-inoculation of silkworms with F. tularensis enhanced the expression of antimicrobial peptides and resistance to infection by pathogenic bacteria. These results suggest that silkworms acquire host resistance via their symbiosis with F. tularensis, which may have important fitness benefits in natural reservoirs. PMID:27507264

  10. Recombinant Attenuated Listeria monocytogenes Vaccine Expressing Francisella tularensis IglC Induces Protection in Mice Against Aerosolized Type A F. tularensis

    PubMed Central

    Jia, Qingmei; Lee, Bai-Yu; Clemens, Daniel L.; Bowen, Richard A.; Horwitz, Marcus A.

    2009-01-01

    Fransicella tularensis, the causative agent of tularemia, is in the top category (Category A) of potential agents of bioterrorism. To develop a safer vaccine against aerosolized F. tularensis, we have employed an attenuated Listeria monocytogenes, which shares with F. tularensis an intracellular and extraphagosomal lifestyle, as a delivery vehicle for F. tularensis antigens. We constructed recombinant L. monocytogenes (rLm) vaccines stably expressing 7 F. tularensis proteins including IglC (rLm/iglC), and tested their immunogenicity and protective efficacy against lethal F. tularensis challenge in mice. Mice immunized intradermally with rLm/iglC developed significant cellular immune responses to F. tularensis IglC as evidenced by lymphocyte proliferation and CD4+ and CD8+ T-cell intracellular expression of interferon gamma. Moreover, mice immunized with rLm/iglC were protected against lethal challenge with F. tularensis LVS administered by the intranasal route, a route chosen to mimic airborne infection, and, most importantly, against aerosol challenge with the highly virulent Type A F. tularensis SchuS4 strain. PMID:19126421

  11. Toward an integrated system of clade names.

    PubMed

    de Queiroz, Kevin

    2007-12-01

    Although the proposition that higher taxa should correspond to clades is widely accepted, current nomenclature does not distinguish clearly between different clades in nested series. In particular, the same name is often applied to a total clade, its crown clade, and clades originating with various nodes, branches, and apomorphies in between. An integrated system of clade names is described based on categories of clades defined with respect to lineages that have survived to the present time. In this system, the most widely known names are applied to crown clades, the names of total clades are formed by adding a standard prefix to the names of the corresponding crowns, and the names of apomorphy clades describe the specific apomorphies with which they originated. Relative to traditional approaches, this integrated approach to naming clades is both more precise concerning the associations of names with particular clades and more efficient with regard to the cognitive effort required to recognize the names of corresponding crown and total clades. It also seems preferable to five alternatives that could be used to make the same distinctions. The integrated system of clade names has several advantages, including the facilitation of communication among biologists who study distantly related clades, promoting a broader conceptualization of the origins of distinctive clades of extant organisms and emphasizing the continuous nature of evolution. PMID:18066930

  12. Preservation of viable Francisella tularensis for forensic analysis

    SciTech Connect

    Valentine, Nancy B.; Wunschel, Sharon C.; Valdez, Catherine O.; Kreuzer-Martin, Helen W.; Bartholomew, Rachel A.; Straub, Tim M.; Wahl, Karen L.

    2011-01-01

    As a preservation solution, (1%) ammonium chloride may be preferred over other conventionally used storage solutions because of its compatibility with analytical techniques such as Mass Spectrometry. In this study, ammonium chloride performed as well or better than phosphate buffered saline with Tween or Butterfields/Tween for preserving Francisella tularensis novicida.

  13. Uncovering the components of the Francisella tularensis virulence stealth strategy

    PubMed Central

    Jones, Bradley D.; Faron, Matthew; Rasmussen, Jed A.; Fletcher, Joshua R.

    2014-01-01

    Over the last decade, studies on the virulence of the highly pathogenic intracellular bacterial pathogen Francisella tularensis have increased dramatically. The organism produces an inert LPS, a capsule, escapes the phagosome to grow in the cytosol (FPI genes mediate phagosomal escape) of a variety of host cell types that include epithelial, endothelial, dendritic, macrophage, and neutrophil. This review focuses on the work that has identified and characterized individual virulence factors of this organism and we hope to highlight how these factors collectively function to produce the pathogenic strategy of this pathogen. In addition, several recent studies have been published characterizing F. tularensis mutants that induce host immune responses not observed in wild type F. tularensis strains that can induce protection against challenge with virulent F. tularensis. As more detailed studies with attenuated strains are performed, it will be possible to see how host models develop acquired immunity to Francisella. Collectively, detailed insights into the mechanisms of virulence of this pathogen are emerging that will allow the design of anti-infective strategies. PMID:24639953

  14. Water as Source of Francisella tularensis Infection in Humans, Turkey

    PubMed Central

    Kilic, Selcuk; Birdsell, Dawn N.; Karagöz, Alper; Çelebi, Bekir; Bakkaloglu, Zekiye; Arikan, Muzaffer; Sahl, Jason W.; Mitchell, Cedar; Rivera, Andrew; Maltinsky, Sara; Keim, Paul; Üstek, Duran; Durmaz, Rıza

    2015-01-01

    Francisella tularensis DNA extractions and isolates from the environment and humans were genetically characterized to elucidate environmental sources that cause human tularemia in Turkey. Extensive genetic diversity consistent with genotypes from human outbreaks was identified in environmental samples and confirmed water as a source of human tularemia in Turkey. PMID:26583383

  15. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  16. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  17. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  18. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  19. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  20. The use of Matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis.

    PubMed

    Karatuna, Onur; Celebi, Bekir; Can, Simge; Akyar, Isin; Kilic, Selcuk

    2016-01-01

    Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institute of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica due to RD1 subspecies-specific PCR result. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories. PMID:26773181

  1. The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis

    PubMed Central

    Karatuna, Onur; Çelebi, Bekir; Can, Simge; Akyar, Işın; Kiliç, Selçuk

    2016-01-01

    Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institution of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica according to region of difference 1 (RD1) subspecies-specific PCR results. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories. PMID:26773181

  2. A Combined Enrichment and Aptamer Pulldown Assay for Francisella tularensis Detection in Food and Environmental Matrices

    PubMed Central

    Enomoto, Shinichiro; Borewicz, Klaudyna; Abdallah, Ahmed; Isaacson, Richard E.; Sreevatsan, Srinand

    2014-01-01

    Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1–106 CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology. PMID:25536105

  3. Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

    PubMed Central

    Maggio, Savannah; Takeda, Kazuyo; Stark, Felicity; Meierovics, Anda I.; Yabe, Idalia; Cowley, Siobhan C.

    2015-01-01

    The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates. PMID:26379269

  4. New solid medium for enhanced growth of Pasteurella tularensis.

    PubMed

    GASPAR, A J; TRESSELT, H B; WARD, M K

    1961-10-01

    Gaspar, Andrew J. (Fort Detrick, Frederick, Md.), Hugh B. Tresselt, and Martha K. Ward. New solid medium for enhanced growth of Pasteurella tularensis. J. Bacteriol. 82:564-569. 1961.-The purpose of this work was to develop a solid medium for more rapid growth of Pasteurella tularensis, especially from small inocula comparable to those generally encountered in clinical materials. AFTER TITRATION OF VARIOUS INGREDIENTS, SEPARATELY AND IN COMBINATION, THE OPTIMAL BASE WAS FOUND TO BE: 2.6% tryptose broth (Difco) with thiamine, 0.5% cysteine-HCl, 0.2% sodium thioglycolate, 1.0% glucose, dissolved by mixing without heat, and adjusted to pH 7.2 +/- 0.03. To this base 1.0% agar (Difco) is added and dissolved by heating in flowing steam for about 5 min prior to autoclaving at 121 C for 20 min. After sterilization and cooling, 5.0% defibrinated rabbit blood is added aseptically. Plates of the completed medium are incubated at 37 C for 24 hr prior to use. Colonies of P. tularensis approximately 1.0 mm in diameter are obtained on this medium after 26 to 29 hr incubation if conditions of high relative humidity (90 to 100% saturation) are maintained.The mechanisms involved in the growth enhancement obtained on this medium are under study. The role of thioglycolate appears to be that of keeping in solution the relatively high concentration of cysteine-HCl required. The specific methods of preparation described, the incubation of plates prior to use, and final incubation under conditions of high humidity all are important for optimal results. This developmental work has employed only pure cultures. Attempts are being made to develop a selective medium, using this new preparation as base, for the direct isolation of P. tularensis from a variety of clinical materials. PMID:13897160

  5. [Multilocus VNTR-typing of Francisella tularensis strains].

    PubMed

    Vodop'ianov, A S; Vodop'ianov, S O; Pavlovich, N V; Mishan'kin, B N

    2004-01-01

    In the analysis of F. tularensis genome with the use of the specially developed program "DNA" a great number of loci containing tandem repeats were found. For analysis, 3 of them were selected and designated as FtA, FtB, FtC. The study of DNA of 40 F. tularensis strains in the polymerase chain reaction with specific primers to these loci a great variability in the number of repeats was established, the presence of 17 alleles being found in locus FtA, 5 alleles in locus FtB and 5 alleles in locus FtC. The strains under study formed 24 variants of genotypes, whose occurrence varied from 0.025 to 0.125. Taking into account the variability of the detected loci and a great number of potential loci VNTR in the genome, further development of this method will facilitate the creation of local and general data bases of the strains, thus ensuring more effective genetic typing of F. tularensis. PMID:15188553

  6. Francisella tularensis replicates within alveolar type II epithelial cells in vitro and in vivo following inhalation.

    PubMed

    Hall, Joshua D; Craven, Robin R; Fuller, James R; Pickles, Raymond J; Kawula, Thomas H

    2007-02-01

    Francisella tularensis replicates in macrophages and dendritic cells, but interactions with other cell types have not been well described. F. tularensis LVS invaded and replicated within alveolar epithelial cell lines. Following intranasal inoculation of C57BL/6 mice, Francisella localized to the alveolus and replicated within alveolar type II epithelial cells. PMID:17088343

  7. UV-C Inactivation of Francisella tularensis Utah-112 on agar surfaces, stainless steel, and foods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Francisella tularensis has been identified as a microorganism of concern in the field of food security. There is currently very little information on the ability to inactivate F. tularensis on foods using non-thermal processing technologies. The ability of ultraviolet light (UV-C) to inactivate F....

  8. Host–pathogen interactions and immune evasion strategies in Francisella tularensis pathogenicity

    PubMed Central

    Steiner, Don J; Furuya, Yoichi; Metzger, Dennis W

    2014-01-01

    Francisella tularensis is an intracellular Gram-negative bacterium that causes life-threatening tularemia. Although the prevalence of natural infection is low, F. tularensis remains a tier I priority pathogen due to its extreme virulence and ease of aerosol dissemination. F. tularensis can infect a host through multiple routes, including the intradermal and respiratory routes. Respiratory infection can result from a very small inoculum (ten organisms or fewer) and is the most lethal form of infection. Following infection, F. tularensis employs strategies for immune evasion that delay the immune response, permitting systemic distribution and induction of sepsis. In this review we summarize the current knowledge of F. tularensis in an immunological context, with emphasis on the host response and bacterial evasion of that response. PMID:25258544

  9. Evidence that clade A and clade B head lice live in sympatry and recombine in Algeria.

    PubMed

    Boutellis, A; Bitam, I; Fekir, K; Mana, N; Raoult, D

    2015-03-01

    Pediculus humanus L. (Psocodea: Pediculidae) can be characterized into three deeply divergent lineages (clades) based on mitochondrial DNA. Clade A consists of both head lice and clothing lice and is distributed worldwide. Clade B consists of head lice only and is mainly found in North and Central America, and in western Europe and Australia. Clade C, which consists only of head lice, is found in Ethiopia, Nepal and Senegal. Twenty-six head lice collected from pupils at different elementary schools in two localities in Algiers (Algeria) were analysed using molecular methods for genotyping lice (cytochrome b and the multi-spacer typing (MST) method. For the first time, we found clade B head lice in Africa living in sympatry with clade A head lice. The phylogenetic analysis of the concatenated sequences of these populations of head lice showed that clade A and clade B head lice had recombined, suggesting that interbreeding occurs when lice live in sympatry. PMID:25346378

  10. Genetic identification of unique immunological responses in mice infected with virulent and attenuated Francisella tularensis

    PubMed Central

    Kingry, Luke C.; Troyer, Ryan M.; Marlenee, Nicole L.; Bielefeldt-Ohmann, Helle; Bowen, Richard A.; Schenkel, Alan R.; Dow, Steven W.; Slayden, Richard A.

    2010-01-01

    Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in Francisella tularensis infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression profiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS. PMID:21070859

  11. The use of resazurin as a novel antimicrobial agent against Francisella tularensis.

    PubMed

    Schmitt, Deanna M; O'Dee, Dawn M; Cowan, Brianna N; Birch, James W-M; Mazzella, Leanne K; Nau, Gerard J; Horzempa, Joseph

    2013-01-01

    The highly infectious and deadly pathogen, Francisella tularensis, is classified by the CDC as a Category A bioterrorism agent. Inhalation of a single bacterium results in an acute pneumonia with a 30-60% mortality rate without treatment. Due to the prevalence of antibiotic resistance, there is a strong need for new types of antibacterial drugs. Resazurin is commonly used to measure bacterial and eukaryotic cell viability through its reduction to the fluorescent product resorufin. When tested on various bacterial taxa at the recommended concentration of 44 μM, a potent bactericidal effect was observed against various Francisella and Neisseria species, including the human pathogens type A F. tularensis (Schu S4) and N. gonorrhoeae. As low as 4.4 μM resazurin was sufficient for a 10-fold reduction in F. tularensis growth. In broth culture, resazurin was reduced to resorufin by F. tularensis. Resorufin also suppressed the growth of F. tularensis suggesting that this compound is the biologically active form responsible for decreasing the viability of F. tularensis LVS bacteria. Replication of F. tularensis in primary human macrophages and non-phagocytic cells was abolished following treatment with 44 μM resazurin indicating this compound could be an effective therapy for tularemia in vivo. PMID:24367766

  12. Role of mTOR Downstream Effector Signaling Molecules in Francisella Tularensis Internalization by Murine Macrophages

    PubMed Central

    Edwards, Michael W.; Aultman, James A.; Harber, Gregory; Bhatt, Jay M.; Sztul, Elizabeth; Xu, Qingan; Zhang, Ping; Michalek, Suzanne M.; Katz, Jannet

    2013-01-01

    Francisella tularensis is an infectious, gram-negative, intracellular microorganism, and the cause of tularemia. Invasion of host cells by intracellular pathogens like Francisella is initiated by their interaction with different host cell membrane receptors and the rapid phosphorylation of different downstream signaling molecules. PI3K and Syk have been shown to be involved in F. tularensis host cell entry, and both of these signaling molecules are associated with the master regulator serine/threonine kinase mTOR; yet the involvement of mTOR in F. tularensis invasion of host cells has not been assessed. Here, we report that infection of macrophages with F. tularensis triggers the phosphorylation of mTOR downstream effector molecules, and that signaling via TLR2 is necessary for these events. Inhibition of mTOR or of PI3K, ERK, or p38, but not Akt signaling, downregulates the levels of phosphorylation of mTOR downstream targets, and significantly reduces the number of F. tularensis cells invading macrophages. Moreover, while phosphorylation of mTOR downstream effectors occurs via the PI3K pathway, it also involves PLCγ1 and Ca2+ signaling. Furthermore, abrogation of PLC or Ca2+ signaling revealed their important role in the ability of F. tularensis to invade host cells. Together, these findings suggest that F. tularensis invasion of primary macrophages utilize a myriad of host signaling pathways to ensure effective cell entry. PMID:24312679

  13. Natural Selection in Virulence Genes of Francisella tularensis.

    PubMed

    Gunnell, Mark K; Robison, Richard A; Adams, Byron J

    2016-06-01

    A fundamental tenet of evolution is that alleles that are under negative selection are often deleterious and confer no evolutionary advantage. Negatively selected alleles are removed from the gene pool and are eventually extinguished from the population. Conversely, alleles under positive selection do confer an evolutionary advantage and lead to an increase in the overall fitness of the organism. These alleles increase in frequency until they eventually become fixed in the population. Francisella tularensis is a zoonotic pathogen and a potential biothreat agent. The most virulent type of F. tularensis, Type A, is distributed across North America with Type A.I occurring mainly in the east and Type A.II appearing mainly in the west. F. tularensis is thought to be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. To test this hypothesis, we sequenced and compared whole genomes of Type A.I and A.II isolates. We analyzed a subset of virulence and housekeeping genes from several F. tularensis subspecies genomes to ascertain the presence and extent of positive selection. Eleven previously identified virulence genes were screened for positive selection along with 10 housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection at loci implicated in cell surface structures and membrane proteins, metabolism and biosynthesis, transcription, translation and cell separation, and substrate binding and transport. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolution

  14. Exploitation of Host Cell Biology and Evasion of Immunity by Francisella Tularensis

    PubMed Central

    Asare, Rexford; Kwaik, Yousef Abu

    2011-01-01

    Francisella tularensis is an intracellular bacterium that infects humans and many small mammals. During infection, F. tularensis replicates predominantly in macrophages but also proliferate in other cell types. Entry into host cells is mediate by various receptors. Complement-opsonized F. tularensis enters into macrophages by looping phagocytosis. Uptake is mediated in part by Syk, which may activate actin rearrangement in the phagocytic cup resulting in the engulfment of F. tularensis in a lipid raft rich phagosome. Inside the host cells, F. tularensis resides transiently in an acidified late endosome-like compartment before disruption of the phagosomal membrane and escape into the cytosol, where bacterial proliferation occurs. Modulation of phagosome biogenesis and escape into the cytosol is mediated by the Francisella pathogenicity island-encoded type VI-like secretion system. Whilst inside the phagosome, F. tularensis temporarily induce proinflammatory cytokines in PI3K/Akt-dependent manner, which is counteracted by the induction of SHIP that negatively regulates PI3K/Akt activation and promotes bacterial escape into the cytosol. Interestingly, F. tularensis subverts CD4 T cells-mediated killing by inhibiting antigen presentation by activated macrophages through ubiquitin-dependent degradation of MHC II molecules on activated macrophages. In the cytosol, F. tularensis is recognized by the host cell inflammasome, which is down-regulated by F. tularensis that also inhibits caspase-1 and ASC activity. During late stages of intracellular proliferation, caspase-3 is activated but apoptosis is delayed through activation of NF-κB and Ras, which ensures cell viability. PMID:21687747

  15. What limits the morphological disparity of clades?

    PubMed

    Oyston, Jack W; Hughes, Martin; Wagner, Peter J; Gerber, Sylvain; Wills, Matthew A

    2015-12-01

    The morphological disparity of species within major clades shows a variety of trajectory patterns through evolutionary time. However, there is a significant tendency for groups to reach their maximum disparity relatively early in their histories, even while their species richness or diversity is comparatively low. This pattern of early high-disparity suggests that there are internal constraints (e.g. developmental pleiotropy) or external restrictions (e.g. ecological competition) upon the variety of morphologies that can subsequently evolve. It has also been demonstrated that the rate of evolution of new character states decreases in most clades through time (character saturation), as does the rate of origination of novel bodyplans and higher taxa. Here, we tested whether there was a simple relationship between the level or rate of character state exhaustion and the shape of a clade's disparity profile: specifically, its centre of gravity (CG). In a sample of 93 extinct major clades, most showed some degree of exhaustion, but all continued to evolve new states up until their extinction. Projection of states/steps curves suggested that clades realized an average of 60% of their inferred maximum numbers of states. Despite a weak but significant correlation between overall levels of homoplasy and the CG of clade disparity profiles, there were no significant relationships between any of our indices of exhaustion curve shape and the clade disparity CG. Clades showing early high-disparity were no more likely to have early character saturation than those with maximum disparity late in their evolution. PMID:26640649

  16. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp plantarum and ‘Bacillus oryzicola’ are later heterotypic synonyms of Bacillus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rhizosphere isolated bacteria belonging to the Bacillus amyloliquefaciens subsp. plantarum and Bacillus methylotrophicus clades are an important group of strains that are used as plant growth promoters and antagonists of plant pathogens. These properties have made these strains the focus of comm...

  17. Cranial base evolution within the hominin clade

    PubMed Central

    Nevell, L; Wood, B

    2008-01-01

    The base of the cranium (i.e. the basioccipital, the sphenoid and the temporal bones) is of particular interest because it undergoes significant morphological change within the hominin clade, and because basicranial morphology features in several hominin species diagnoses. We use a parsimony analysis of published cranial and dental data to predict the cranial base morphology expected in the hypothetical last common ancestor of the Pan–Homo clade. We also predict the primitive condition of the cranial base for the hominin clade, and document the evolution of the cranial base within the major subclades within the hominin clade. This analysis suggests that cranial base morphology has continued to evolve in the hominin clade, both before and after the emergence of the genus Homo. PMID:18380865

  18. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  19. Identification of immunoreactive antigens in membrane proteins enriched fraction from Francisella tularensis LVS.

    PubMed

    Janovská, Sylva; Pávková, Ivona; Hubálek, Martin; Lenco, Juraj; Macela, Ales; Stulík, Jirí

    2007-02-15

    Francisella tularensis is a Gram-negative, facultative intracellular bacterium causing disease in many mammalian species. The low infectious dose of F. tularensis and the ease of air-borne transmission are the main features responsible for the classification of this bacterium as a potential biological weapon. The live attenuated strain of F. tularensis live vaccine strain (LVS) is currently only effective vaccine against tularemia, however, this type of vaccine has not been approved for human use. In the presented study, sub-immunoproteome analysis was performed to search for new immunogenic proteins of Francisella tularensis LVS grown under different conditions. By this approach 35 immunoreactive antigens were identified, 19 of them showed to be novel immunogens. In conclusion, sub-immunoproteome analysis resulted in successful identification of novel immunoreactive proteins. PMID:17241671

  20. The Genetic Diversity and Evolution of Francisella tularensis with Comments on Detection by PCR.

    PubMed

    Gunnell, Mark K; Adams, Byron J; Robison, Richard A

    2016-01-01

    Francisella tularensis has been the focus of much research over the last two decades mainly because of its potential use as an agent of bioterrorism. F. tularensis is the causative agent of zoonotic tularemia and has a worldwide distribution. The different subspecies of F. tularensis vary in their biogeography and virulence, making early detection and diagnosis important in both the biodefense and public health sectors. Recent genome sequencing efforts reveal aspects of genetic diversity, evolution and phylogeography previously unknown for this relatively small organism, and highlight a role for detection by various PCR assays. This review explores the advances made in understanding the evolution and genetic diversity of F. tularensis and how these advances have led to better PCR assays for detection and identification of the subspecies. PMID:26336102

  1. ECO-EPIZOOTIOLOGIC STUDY OF FRANCISELLA TULARENSIS, THE AGENT OF TULAREMIA, IN QUÉBEC WILDLIFE.

    PubMed

    Gabriele-Rivet, Vanessa; Ogden, Nicholas; Massé, Ariane; Antonation, Kym; Corbett, Cindi; Dibernardo, Antonia; Lindsay, L Robbin; Leighton, Patrick A; Arsenault, Julie

    2016-04-28

    In Canada, Francisella tularensis , the zoonotic bacterial agent of tularemia, affects mostly snowshoe hares ( Lepus americanus ), muskrats ( Ondatra zibethicus ), and beavers ( Castor canadensis ). Despite numerous studies, the ecologic cycle and natural reservoirs of F. tularensis are not clearly defined. We conducted a cross-sectional study to estimate the prevalence of F. tularensis in snowshoe hares, muskrats, and coyotes ( Canis latrans ) in four regions of Québec, Canada, and to describe the risk of infection in relation to host and environmental characteristics at three spatial scales. Between October 2012 and April 2013, trappers captured 345 snowshoe hares, 411 muskrats, and 385 coyotes. Blood samples were tested by microagglutination tests, and DNA extracts of liver, kidney, lung, and spleen of snowshoe hares and muskrats were tested by real-time PCR to detect past and active infection to F. tularensis , respectively. Individual host characteristics, including body condition, age, and sex, were evaluated as risk factors of infection, along with ecologic characteristics of the location of capture extracted from geographic databases. Prevalences of antibody to F. tularensis and 95% confidence intervals were 2.9% (1.4-5.1%) in coyotes, 0.6% (0.1-2.1%) in hares, and 0% (0.0-0.9%) in muskrats. Francisella tularensis DNA was not detected by real-time PCR in the pools of four organs from muskrats and hares, but F. tularensis type AI was detected during testing of the individual organs of two antibody-positive hares. Exact logistic regression analyses showed that age was a significant predictor of antibody detection in coyotes, as were the proportion of forest and the proportion of area considered as suitable habitat for hares in the environment around the location of capture of the coyotes. Our results suggest a terrestrial cycle of F. tularensis in the regions studied. PMID:26967133

  2. From the Outside-In: The Francisella tularensis Envelope and Virulence

    PubMed Central

    Rowe, Hannah M.; Huntley, Jason F.

    2015-01-01

    Francisella tularensis is a highly-infectious bacterium that causes the rapid, and often lethal disease, tularemia. Many studies have been performed to identify and characterize the virulence factors that F. tularensis uses to infect a wide variety of hosts and host cell types, evade immune defenses, and induce severe disease and death. This review focuses on the virulence factors that are present in the F. tularensis envelope, including capsule, LPS, outer membrane, periplasm, inner membrane, secretion systems, and various molecules in each of aforementioned sub-compartments. Whereas, no single bacterial molecule or molecular complex single-handedly controls F. tularensis virulence, we review here how diverse bacterial systems work in conjunction to subvert the immune system, attach to and invade host cells, alter phagosome/lysosome maturation pathways, replicate in host cells without being detected, inhibit apoptosis, and induce host cell death for bacterial release and infection of adjacent cells. Given that the F. tularensis envelope is the outermost layer of the bacterium, we highlight herein how many of these molecules directly interact with the host to promote infection and disease. These and future envelope studies are important to advance our collective understanding of F. tularensis virulence mechanisms and offer targets for future vaccine development efforts. PMID:26779445

  3. Temperature-Dependent Gentamicin Resistance of Francisella tularensis is Mediated by Uptake Modulation.

    PubMed

    Loughman, Kathleen; Hall, Jesse; Knowlton, Samantha; Sindeldecker, Devin; Gilson, Tricia; Schmitt, Deanna M; Birch, James W-M; Gajtka, Tara; Kobe, Brianna N; Florjanczyk, Aleksandr; Ingram, Jenna; Bakshi, Chandra S; Horzempa, Joseph

    2016-01-01

    Gentamicin (Gm) is an aminoglycoside commonly used to treat bacterial infections such as tularemia - the disease caused by Francisella tularensis. In addition to being pathogenic, F. tularensis is found in environmental niches such as soil where this bacterium likely encounters Gm producers (Micromonospora sp.). Here we show that F. tularensis exhibits increased resistance to Gm at ambient temperature (26°C) compared to mammalian body temperature (37°C). To evaluate whether F. tularensis was less permeable to Gm at 26°C, a fluorescent marker [Texas Red (Tr)] was conjugated with Gm, yielding Tr-Gm. Bacteria incubated at 26°C showed reduced fluorescence compared to those at 37°C when exposed to Tr-Gm suggesting that uptake of Gm was reduced at 26°C. Unconjugated Gm competitively inhibited uptake of Tr-Gm, demonstrating that this fluorescent compound was taken up similarly to unconjugated Gm. Lysates of F. tularensis bacteria incubated with Gm at 37°C inhibited the growth of Escherichia coli significantly more than lysates from bacteria incubated at 26°C, further indicating reduced uptake at this lower temperature. Other facultative pathogens (Listeria monocytogenes and Klebsiella pneumoniae) exhibited increased resistance to Gm at 26°C suggesting that the results generated using F. tularensis may be generalizable to diverse bacteria. Regulation of the uptake of antibiotics provides a mechanism by which facultative pathogens survive alongside antibiotic-producing microbes in nature. PMID:26858709

  4. Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection.

    PubMed

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-08-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-kappaB activation. ATII cells pretreated with an NF-kappaB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  5. Role of Primary Human Alveolar Epithelial Cells in Host Defense against Francisella tularensis Infection▿

    PubMed Central

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B.; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L.; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-01-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-κB activation. ATII cells pretreated with an NF-κB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  6. Temperature-Dependent Gentamicin Resistance of Francisella tularensis is Mediated by Uptake Modulation

    PubMed Central

    Loughman, Kathleen; Hall, Jesse; Knowlton, Samantha; Sindeldecker, Devin; Gilson, Tricia; Schmitt, Deanna M.; Birch, James W.-M.; Gajtka, Tara; Kobe, Brianna N.; Florjanczyk, Aleksandr; Ingram, Jenna; Bakshi, Chandra S.; Horzempa, Joseph

    2016-01-01

    Gentamicin (Gm) is an aminoglycoside commonly used to treat bacterial infections such as tularemia – the disease caused by Francisella tularensis. In addition to being pathogenic, F. tularensis is found in environmental niches such as soil where this bacterium likely encounters Gm producers (Micromonospora sp.). Here we show that F. tularensis exhibits increased resistance to Gm at ambient temperature (26°C) compared to mammalian body temperature (37°C). To evaluate whether F. tularensis was less permeable to Gm at 26°C, a fluorescent marker [Texas Red (Tr)] was conjugated with Gm, yielding Tr-Gm. Bacteria incubated at 26°C showed reduced fluorescence compared to those at 37°C when exposed to Tr-Gm suggesting that uptake of Gm was reduced at 26°C. Unconjugated Gm competitively inhibited uptake of Tr-Gm, demonstrating that this fluorescent compound was taken up similarly to unconjugated Gm. Lysates of F. tularensis bacteria incubated with Gm at 37°C inhibited the growth of Escherichia coli significantly more than lysates from bacteria incubated at 26°C, further indicating reduced uptake at this lower temperature. Other facultative pathogens (Listeria monocytogenes and Klebsiella pneumoniae) exhibited increased resistance to Gm at 26°C suggesting that the results generated using F. tularensis may be generalizable to diverse bacteria. Regulation of the uptake of antibiotics provides a mechanism by which facultative pathogens survive alongside antibiotic-producing microbes in nature. PMID:26858709

  7. Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion*

    PubMed Central

    Feld, Geoffrey K.; El-Etr, Sahar; Corzett, Michele H.; Hunter, Mark S.; Belhocine, Kamila; Monack, Denise M.; Frank, Matthias; Segelke, Brent W.; Rasley, Amy

    2014-01-01

    Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen's long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1′ recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells. PMID:25231992

  8. Population Structure of Francisella tularensis†

    PubMed Central

    Nübel, Ulrich; Reissbrodt, Rolf; Weller, Annette; Grunow, Roland; Porsch-Özcürümez, Mustafa; Tomaso, Herbert; Hofer, Erwin; Splettstoesser, Wolf; Finke, Ernst-Jürgen; Tschäpe, Helmut; Witte, Wolfgang

    2006-01-01

    We have sequenced fragments of five metabolic housekeeping genes and two genes encoding outer membrane proteins from 81 isolates of Francisella tularensis, representing all four subspecies. Phylogenetic clustering of gene sequences from F. tularensis subsp. tularensis and F. tularensis subsp. holarctica aligned well with subspecies affiliations. In contrast, F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica were indicated to be phylogenetically incoherent taxa. Incongruent gene trees and mosaic structures of housekeeping genes provided evidence for genetic recombination in F. tularensis. PMID:16816208

  9. Disruption of Francisella tularensis Schu S4 iglI, iglJ, and pdpC Genes Results in Attenuation for Growth in Human Macrophages and In Vivo Virulence in Mice and Reveals a Unique Phenotype for pdpC

    PubMed Central

    Long, Matthew E.; Lindemann, Stephen R.; Rasmussen, Jed A.; Jones, Bradley D.

    2013-01-01

    Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 108 CFU iglJ- or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype. PMID:23275090

  10. Disruption of Francisella tularensis Schu S4 iglI, iglJ, and pdpC genes results in attenuation for growth in human macrophages and in vivo virulence in mice and reveals a unique phenotype for pdpC.

    PubMed

    Long, Matthew E; Lindemann, Stephen R; Rasmussen, Jed A; Jones, Bradley D; Allen, Lee-Ann H

    2013-03-01

    Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10(8) CFU iglJ- or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype. PMID:23275090

  11. Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

    PubMed Central

    Lindgren, Helena

    2015-01-01

    The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo. PMID:26503658

  12. Francisella tularensis Subtype A.II Genomic Plasticity in Comparison with Subtype A.I

    PubMed Central

    Larson, Marilynn A.; Nalbantoglu, Ufuk; Sayood, Khalid; Zentz, Emily B.; Bartling, Amanda M.; Francesconi, Stephen C.; Fey, Paul D.; Dempsey, Michael P.; Hinrichs, Steven H.

    2015-01-01

    Although Francisella tularensis is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the tularensis subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed F. tularensis type A genomes. In contrast with the F. tularensis A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced F. tularensis A.II strain (WY-00W4114) and the only other publically available A.II strain (WY96-3418). Genome differences between WY-00W4114 and WY96-3418 included three major chromosomal translocations, 1580 indels, and 286 nucleotide substitutions of which 159 were observed in predicted open reading frames and 127 were located in intergenic regions. The majority of WY-00W4114 nucleotide deletions occurred in intergenic regions, whereas most of the insertions and substitutions occurred in predicted genes. Of the nucleotide substitutions, 48 (30%) were synonymous and 111 (70%) were nonsynonymous. WY-00W4114 and WY96-3418 nucleotide polymorphisms were predominantly G/C to A/T allelic mutations, with WY-00W4114 having more A+T enrichment. In addition, the A.II genomes contained a considerably higher number of intact genes and longer repetitive sequences, including transposon remnants than the A.I genomes. Together these findings support the premise that F. tularensis A.II may have a fitness advantage compared to the A.I subtype due to the higher abundance of functional genes and repeated chromosomal sequences. A better understanding of the selective forces driving F. tularensis genetic diversity and plasticity is needed. PMID:25918839

  13. The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host.

    PubMed

    He, Lihong; Nair, Manoj Kumar Mohan; Chen, Yuling; Liu, Xue; Zhang, Mengyun; Hazlett, Karsten R O; Deng, Haiteng; Zhang, Jing-Ren

    2016-05-01

    Francisella tularensis is the causative agent of tularemia and a category A potential agent of bioterrorism, but the pathogenic mechanisms of F. tularensis are largely unknown. Our previous transposon mutagenesis screen identified 95 lung infectivity-associated F. tularensis genes, including those encoding the Lon and ClpP proteases. The present study validates the importance of Lon and ClpP in intramacrophage growth and infection of the mammalian host by using unmarked deletion mutants of the F. tularensis live vaccine strain (LVS). Further experiments revealed that lon and clpP are also required for F. tularensis tolerance to stressful conditions. A quantitative proteomic comparison between heat-stressed LVS and the isogenic Lon-deficient mutant identified 29 putative Lon substrate proteins. The follow-up protein degradation experiments identified five substrates of the F. tularensis Lon protease (FTL578, FTL663, FTL1217, FTL1228, and FTL1957). FTL578 (ornithine cyclodeaminase), FTL663 (heat shock protein), and FTL1228 (iron-sulfur activator complex subunit SufD) have been previously described as virulence-associated factors in F. tularensis Identification of these Lon substrates has thus provided important clues for further understanding of the F. tularensis stress response and pathogenesis. The high-throughput approach developed in this study can be used for systematic identification of the Lon substrates in other prokaryotic and eukaryotic organisms. PMID:26902724

  14. Dermacentor andersoni transmission of Francisella tularensis subsp. novicida reflects bacterial colonization, dissemination and replication coordinated with tick feeding.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ticks serve as biological vectors for a wide variety of bacterial pathogens which must be able to efficiently colonize specific tick tissues prior to transmission. The bacterial determinants of tick colonization are largely unknown, a knowledge gap attributed in large part to the paucity of tools t...

  15. Proteomic analysis of bronchoalveolar lavage fluid proteins from mice infected with Francisella tularensis ssp novicida

    SciTech Connect

    Varnum, Susan M.; Webb-Robertson, Bobbie-Jo M.; Pounds, Joel G.; Moore, Ronald J.; Smith, Richard D.; Frevert, Charles; Skerret, Shawn J.; Wunschel, David S.

    2012-07-06

    Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA); and Pseudomonas aeruginosa. The composition of BALF proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system, however the timing of their induction varied. Francisella tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection, however within 24 hours they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response, however this response is dimished by 24 hours. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection.

  16. A variety of novel lipid A structures obtained from Francisella tularensis live vaccine strain.

    PubMed

    Beasley, Ashley S; Cotter, Robert J; Vogel, Stefanie N; Inzana, Thomas J; Qureshi, Asaf A; Qureshi, Nilofer

    2012-04-01

    F. tularensis is a Gram-negative coccobacillus that causes tularemia. Its LPS has nominal biological activity. Currently, there is controversy regarding the structure of the lipid A obtained from F. tularensis live vaccine strain (LVS). Therefore, to resolve this controversy, the purification and structural identification of this LPS was crucial. To achieve this, LPS from F. tularensis LVS was acid hydrolyzed to obtain crude lipid A that was methylated and purified by HPLC and the fractions were analyzed by MALDI-TOF MS. The structure of the major lipid A species was composed of a glucosamine disaccharide backbone substituted with four fatty acyl groups and a phosphate (1-position) with a molecular mass of 1505. The major lipid A component contained 18:0[3-O(16:0)] in the distal subunit and two 18:0(3-OH) fatty acyl chains at the 2- or 3-positions of the reducing subunit. Additional variations in the lipid A species include: heterogeneity in fatty acyl groups, a phosphate or a phosphoryl galactosamine at the 1-position, and a hexose at the 4' or 6' position, some of which have not been previously described for F. tularensis LVS. This analysis revealed that lipid A from F. tularensis LVS is far more complex than originally believed. PMID:21709054

  17. Aerosol survival of Pasteurella tularensis and the influence of relative humidity.

    PubMed

    Cox, C S; Goldberg, L J

    1972-01-01

    The aerosol survival in air was determined for Pasteurella tularensis live vaccine strain (LVS) as a function of relative humidity (RH). Three different preparations of bacteria were used: (i) liquid suspension of P. tularensis LVS in spent culture medium; (ii) powders of P. tularensis LVS freeze-dried in spent culture fluid; (iii) P. tularensis LVS freeze-dried in spent culture fluid and then reconstituted with distilled water and disseminated as a liquid suspension. Preparation (i) gave greatest survival at high RH and lowest survival at intermediate RH. Preparation (ii), in contrast, gave greatest survival at low RH and minimum survival at 81% RH. Preparation (iii) was the same as preparation (i), i.e., the process of freeze-drying and reconstituting with distilled water before aerosol formation had little or no effect upon aerosol survival as a function of RH. Hence, control of aerosol survival appears to be through the water content of P. tularensis LVS at the moment of aerosol generation rather than the water content of the bacteria in the aerosol phase. PMID:4551041

  18. Proteomic analysis of bronchoalveolar lavage fluid proteins from mice infected with Francisella tularensis ssp novicida

    PubMed Central

    Varnum, Susan M.; Webb-Robertson, Bobbie-Jo M.; Pounds, Joel G.; Moore, Ronald J.; Smith, Richard D.; Frevert, Charles W.; Skerrett, Shawn J.; Wunschel, David

    2012-01-01

    Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA); and Pseudomonas aeruginosa. The composition of BALF proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system, however the timing of their induction varied. Francisella tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection, however within 24 hours they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response, however this response is dimished by 24 hours. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection. PMID:22663564

  19. Testing the hypothesis that a clade has adaptively radiated: iguanid lizard clades as a case study.

    PubMed

    Losos, Jonathan B; Miles, Donald B

    2002-08-01

    The study of adaptive radiations has played a fundamental role in understanding mechanisms of evolution. A recent resurgence in the study of adaptive radiations highlights a gap in our knowledge about determining whether a clade constitutes adaptive diversification. Specifically, no objective criteria exist to judge whether a clade constitutes an adaptive radiation. Most clades, given enough time, will diversify adaptively to some extent; therefore, we argue that the term "adaptive radiation" should be reserved for those clades that are exceptionally diverse in terms of the range of habitats occupied and attendant morphological adaptations. Making such a definition operational, however, requires a comparative analysis of many clades. Only by comparing clades can one distinguish those that are exceptionally diverse (or nondiverse) from those exhibiting a normal degree of adaptive disparity. We propose such a test, focusing on disparity in the ecological morphology of monophyletic groups within the lizard family Iguanidae. We find that two clades, the Polychrotinae and Phrynosomatinae, are exceptionally diverse and that two others, the Crotaphytinae and Oplurinae, are exceptionally nondiverse. Potential explanations for differences in diversity are discussed, as are caveats and future extensions of our approach. PMID:18707482

  20. Symbiodinium Clade Affects Coral Skeletal Isotopic Ratio

    NASA Astrophysics Data System (ADS)

    Carilli, J.; Charles, C. D.; Garren, M.; McField, M.; Norris, R. D.

    2011-12-01

    The influence of different physiologies of Symbiodinium dinoflagellate symbiont clades on the skeletal chemistry of associated coral hosts has not previously been investigated. This is an important issue because coral skeletons are routinely used for tropical paleoclimatic reconstructions. We analyzed coral skeletal samples collected simultaneously from neighboring colonies off Belize and found that those harboring different clades of Symbiodinium displayed significantly different skeletal oxygen isotopic compositions. We also found evidence for mean shifts in skeletal oxygen isotopic composition after coral bleaching (the loss and potential exchange of symbionts) in two of four longer coral cores from the Mesoamerican Reef, though all experienced similar climatic conditions. Thus, we suggest that symbiont clade identity leaves a signature in the coral skeletal archive and that this influence must be considered for quantitative environmental reconstruction. In addition, we suggest that the skeletal isotopic signature may be used to identify changes in the dominant symbiont clade that have occurred in the past, to identify how common and widespread this phenomenon is--a potential adaptation to climate change.

  1. Evolutionary Ecology of the Marine Roseobacter Clade

    PubMed Central

    Luo, Haiwei

    2014-01-01

    SUMMARY Members of the Roseobacter clade are equipped with a tremendous diversity of metabolic capabilities, which in part explains their success in so many different marine habitats. Ideas on how this diversity evolved and is maintained are reviewed, focusing on recent evolutionary studies exploring the timing and mechanisms of Roseobacter ecological diversification. PMID:25428935

  2. Experimental Infection of Voles with Francisella tularensis Indicates Their Amplification Role in Tularemia Outbreaks

    PubMed Central

    Rossow, Heidi; Forbes, Kristian M.; Tarkka, Eveliina; Kinnunen, Paula M.; Hemmilä, Heidi; Huitu, Otso; Nikkari, Simo; Henttonen, Heikki; Kipar, Anja; Vapalahti, Olli

    2014-01-01

    Tularemia outbreaks in humans have been linked to fluctuations in rodent population density, but the mode of bacterial maintenance in nature is unclear. Here we report on an experiment to investigate the pathogenesis of Francisella tularensis infection in wild rodents, and thereby assess their potential to spread the bacterium. We infected 20 field voles (Microtus agrestis) and 12 bank voles (Myodes glareolus) with a strain of F. tularensis ssp. holarctica isolated from a human patient. Upon euthanasia or death, voles were necropsied and specimens collected for histological assessment and identification of bacteria by immunohistology and PCR. Bacterial excretion and a rapid lethal clinical course with pathological changes consistent with bacteremia and tissue necrosis were observed in infected animals. The results support a role for voles as an amplification host of F. tularensis, as excreta and, in particular, carcasses with high bacterial burden could serve as a source for environmental contamination. PMID:25271640

  3. Experimental Infection of voles with Francisella tularensis indicates their amplification role in tularemia outbreaks.

    PubMed

    Rossow, Heidi; Forbes, Kristian M; Tarkka, Eveliina; Kinnunen, Paula M; Hemmilä, Heidi; Huitu, Otso; Nikkari, Simo; Henttonen, Heikki; Kipar, Anja; Vapalahti, Olli

    2014-01-01

    Tularemia outbreaks in humans have been linked to fluctuations in rodent population density, but the mode of bacterial maintenance in nature is unclear. Here we report on an experiment to investigate the pathogenesis of Francisella tularensis infection in wild rodents, and thereby assess their potential to spread the bacterium. We infected 20 field voles (Microtus agrestis) and 12 bank voles (Myodes glareolus) with a strain of F. tularensis ssp. holarctica isolated from a human patient. Upon euthanasia or death, voles were necropsied and specimens collected for histological assessment and identification of bacteria by immunohistology and PCR. Bacterial excretion and a rapid lethal clinical course with pathological changes consistent with bacteremia and tissue necrosis were observed in infected animals. The results support a role for voles as an amplification host of F. tularensis, as excreta and, in particular, carcasses with high bacterial burden could serve as a source for environmental contamination. PMID:25271640

  4. Lactobacillus plantarum subsp. argentoratensis subsp. nov., isolated from vegetable matrices.

    PubMed

    Bringel, Françoise; Castioni, Anna; Olukoya, Daniel K; Felis, Giovanna E; Torriani, Sandra; Dellaglio, Franco

    2005-07-01

    Fourteen strains isolated from vegetable sources and identified as belonging to Lactobacillus plantarum presented an atypical pattern of amplification with a species-specific multiplex-PCR assay. Phylogenetic analysis of two protein-encoding genes, recA (encoding the recombinase A protein) and cpn60 (encoding the GroEL chaperonin), as well as phenotypic and genomic traits revealed a homogeneous group of very closely related strains for which subspecies status is proposed, with the name Lactobacillus plantarum subsp. argentoratensis. The type strain is DKO 22(T) (=CIP 108320(T)=DSM 16365(T)). PMID:16014493

  5. Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19

    PubMed Central

    Fink, Avner; Hassan, Musa A.; Okan, Nihal A.; Sheffer, Michal; Camejo, Ana; Saeij, Jeroen P. J.

    2016-01-01

    ABSTRACT Differences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background. PMID:26980837

  6. Antioxidant Defenses of Francisella tularensis Modulate Macrophage Function and Production of Proinflammatory Cytokines.

    PubMed

    Rabadi, Seham M; Sanchez, Belkys C; Varanat, Mrudula; Ma, Zhuo; Catlett, Sally V; Melendez, Juan Andres; Malik, Meenakshi; Bakshi, Chandra Shekhar

    2016-03-01

    Francisella tularensis, the causative agent of a fatal human disease known as tularemia, has been used in the bioweapon programs of several countries in the past, and now it is considered a potential bioterror agent. Extreme infectivity and virulence of F. tularensis is due to its ability to evade immune detection and to suppress the host's innate immune responses. However, Francisella-encoded factors and mechanisms responsible for causing immune suppression are not completely understood. Macrophages and neutrophils generate reactive oxygen species (ROS)/reactive nitrogen species as a defense mechanism for the clearance of phagocytosed microorganisms. ROS serve a dual role; at high concentrations they act as microbicidal effector molecules that destroy intracellular pathogens, and at low concentrations they serve as secondary signaling messengers that regulate the expression of various inflammatory mediators. We hypothesized that the antioxidant defenses of F. tularensis maintain redox homeostasis in infected macrophages to prevent activation of redox-sensitive signaling components that ultimately result in suppression of pro-inflammatory cytokine production and macrophage microbicidal activity. We demonstrate that antioxidant enzymes of F. tularensis prevent the activation of redox-sensitive MAPK signaling components, NF-κB signaling, and the production of pro-inflammatory cytokines by inhibiting the accumulation of ROS in infected macrophages. We also report that F. tularensis inhibits ROS-dependent autophagy to promote its intramacrophage survival. Collectively, this study reveals novel pathogenic mechanisms adopted by F. tularensis to modulate macrophage innate immune functions to create an environment permissive for its intracellular survival and growth. PMID:26644475

  7. Inhibitors of Ribosome Rescue Arrest Growth of Francisella tularensis at All Stages of Intracellular Replication.

    PubMed

    Goralski, Tyler D P; Dewan, Kalyan K; Alumasa, John N; Avanzato, Victoria; Place, David E; Markley, Rachel L; Katkere, Bhuvana; Rabadi, Seham M; Bakshi, Chandra Shekhar; Keiler, Kenneth C; Kirimanjeswara, Girish S

    2016-06-01

    Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans-translation, which is conserved in >99% of sequenced bacterial genomes. Some species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans-translation activity. Small-molecule inhibitors of ribosome rescue have broad-spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against the tier 1 select agent Francisella tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis Neither KKL-10 nor KKL-40 was cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle and suggest that KKL-10 and KKL-40 are good lead compounds for antibiotic development. PMID:26953190

  8. The Francisella Tularensis Proteome and its Recognition by Antibodies

    PubMed Central

    Kilmury, Sara L. N.; Twine, Susan M.

    2011-01-01

    Francisella tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. The extreme virulence of the pathogen in humans, combined with the low infectious dose and the ease of dissemination by aerosol have led to concerns about its abuse as a bioweapon. Until recently, nothing was known about the virulence mechanisms and even now, there is still a relatively poor understanding of pathogen virulence. Completion of increasing numbers of Francisella genome sequences, combined with comparative genomics and proteomics studies, are contributing to the knowledge in this area. Tularemia may be treated with antibiotics, but there is currently no licensed vaccine. An attenuated strain, the Live Vaccine Strain (LVS) has been used to vaccinate military and at risk laboratory personnel, but safety concerns mean that it is unlikely to be licensed by the FDA for general use. Little is known about the protective immunity induced by vaccination with LVS, in humans or animal models. Immunoproteomics studies with sera from infected humans or vaccinated mouse strains, are being used in gel-based or proteome microarray approaches to give insight into the humoral immune response. In addition, these data have the potential to be exploited in the identification of new diagnostic or protective antigens, the design of next generation live vaccine strains, and the development of subunit vaccines. Herein, we briefly review the current knowledge from Francisella comparative proteomics studies and then focus upon the findings from immunoproteomics approaches. PMID:21687770

  9. Keep an Ear Out for Francisella tularensis: Otomastoiditis Cases after Canyoneering

    PubMed Central

    Guerpillon, Brice; Boibieux, Andre; Guenne, Clemence; Ploton, Christine; Ferry, Tristan; Maurin, Max; Forestier, Emmanuel; Dauwalder, Olivier; Manipoud, Patrick; Ltaïef-Boudrigua, Aicha; Gürkov, Robert; Vandenesch, Francois; Bouchiat, Coralie

    2016-01-01

    We report here three unusual cases of otomastoiditis due to Francisella tularensis, complicated by cervical abscesses and persistent hearing loss, plus facial paralysis for one patient. Intriguingly, the three patients had practiced canyoneering independently in the same French river, between 2009 and 2014, several days before clinical symptoms onset. The results point out that fresh water exposure may be a potential contamination route for tularemia. Besides, due to the frequent complications and sequelae, we believe that F. tularensis should be considered as a possible etiology in case of otitis media, failure of the conventional antibiotic treatment, and suspicious exposure of the bacteria. PMID:26973838

  10. Dissecting the taxonomic heterogeneity within Propionibacterium acnes: proposal for Propionibacterium acnes subsp. acnes subsp. nov. and Propionibacterium acnes subsp. elongatum subsp. nov.

    PubMed

    Dekio, Itaru; Culak, Renata; Misra, Raju; Gaulton, Tom; Fang, Min; Sakamoto, Mitsuo; Ohkuma, Moriya; Oshima, Kenshiro; Hattori, Masahira; Klenk, Hans-Peter; Rajendram, Dunstan; Gharbia, Saheer E; Shah, Haroun N

    2015-12-01

    Propionibacterium acnes subsp. acnes subsp. nov. and Propionibacterium acnes subsp. elongatum subsp. nov. are described. These emanate from the three known phylotypes of P. acnes, designated types I, II and III. Electron microscopy confirmed the filamentous cell shape of type III, showing a striking difference from types I/II, which were short rods. Biochemical tests indicated that, in types I/II, either the pyruvate, l-pyrrolidonyl arylamidase or d-ribose 2 test was positive, whereas all of these were negative among type III strains. Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra, which profile mainly their ribosomal proteins, were different between these two groups. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) spectra of all phylotypes revealed a specific protein biomarker that was overexpressed in type III strains compared with types I/II only when grown aerobically. Reference strains had high whole-genome similarity between types I (>91 %) and II (>75 %), but a considerably lower level of 72 % similarity with type III. recA and gyrB sequence dendrograms confirmed the distant relatedness of type III, indicating the presence of two distinct centres of variation within the species P. acnes. On the other hand, cellular fatty acid profiles and 16S rRNA gene sequence relatedness (>99.3 %) circumscribed the species. Thus, we propose two subspecies, Propionibacterium acnes subsp. acnes subsp. nov. for types I/II and Propionibacterium acnes subsp. elongatum subsp. nov. for type III. The type strain of Propionibacterium acnes subsp. acnes is NCTC 737T ( = ATCC 6919T = JCM 6425T = DSM 1897T = CCUG 1794T), while the type strain of Propionibacterium acnes subsp. elongatum is K124T ( = NCTC 13655T = JCM 18919T). PMID:26432704

  11. Identification of Mycobacterium avium subsp. hominissuis Isolated From Drinking Water

    EPA Science Inventory

    Mycobacterium avium (MA) is divided into four subspecies based primarily on host-range and consists of MA subsp. avium (birds), MA subsp. silvaticum (wood pigeons), MA subsp. paratuberculosis (broad, poorly-defined host range), and the recently described MA subsp. hominissuis (hu...

  12. Members of the Francisella tularensis Phagosomal Transporter Subfamily of Major Facilitator Superfamily Transporters Are Critical for Pathogenesis

    PubMed Central

    Marohn, Mark E.; Santiago, Araceli E.; Shirey, Kari Ann; Lipsky, Michael; Vogel, Stefanie N.

    2012-01-01

    Francisella tularensis is the causative agent of tularemia. Due to its aerosolizable nature and low infectious dose, F. tularensis is classified as a category A select agent and, therefore, is a priority for vaccine development. Survival and replication in macrophages and other cell types are critical to F. tularensis pathogenesis, and impaired intracellular survival has been linked to a reduction in virulence. The F. tularensis genome is predicted to encode 31 major facilitator superfamily (MFS) transporters, and the nine-member Francisella phagosomal transporter (Fpt) subfamily possesses homology with virulence factors in other intracellular pathogens. We hypothesized that these MFS transporters may play an important role in F. tularensis pathogenesis and serve as good targets for attenuation and vaccine development. Here we show altered intracellular replication kinetics and attenuation of virulence in mice infected with three of the nine Fpt mutant strains compared with wild-type (WT) F. tularensis LVS. The vaccination of mice with these mutant strains was protective against a lethal intraperitoneal challenge. Additionally, we observed pronounced differences in cytokine profiles in the livers of mutant-infected mice, suggesting that alterations in in vivo cytokine responses are a major contributor to the attenuation observed for these mutant strains. These results confirm that this subset of MFS transporters plays an important role in the pathogenesis of F. tularensis and suggest that a focus on the development of attenuated Fpt subfamily MFS transporter mutants is a viable strategy toward the development of an efficacious vaccine. PMID:22508856

  13. Herbivory increases diversification across insect clades

    PubMed Central

    Wiens, John J.; Lapoint, Richard T.; Whiteman, Noah K.

    2015-01-01

    Insects contain more than half of all living species, but the causes of their remarkable diversity remain poorly understood. Many authors have suggested that herbivory has accelerated diversification in many insect clades. However, others have questioned the role of herbivory in insect diversification. Here, we test the relationships between herbivory and insect diversification across multiple scales. We find a strong, positive relationship between herbivory and diversification among insect orders. However, herbivory explains less variation in diversification within some orders (Diptera, Hemiptera) or shows no significant relationship with diversification in others (Coleoptera, Hymenoptera, Orthoptera). Thus, we support the overall importance of herbivory for insect diversification, but also show that its impacts can vary across scales and clades. In summary, our results illuminate the causes of species richness patterns in a group containing most living species, and show the importance of ecological impacts on diversification in explaining the diversity of life. PMID:26399434

  14. Iron Acquisition in Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Wang, Joyce; Moolji, Jalal; Dufort, Alex; Staffa, Alfredo; Domenech, Pilar; Reed, Michael B.

    2015-01-01

    ABSTRACT Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSPP15. We obtained a transposon (Tn) mutant with a disruption in the LSPP15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSPP15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen. IMPORTANCE Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth

  15. Lipase production by diverse phylogenetic clades of Aureobasidium pullulans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2-0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains i...

  16. Multifaceted effects of Francisella tularensis on human neutrophil function and lifespan.

    PubMed

    Kinkead, Lauren C; Allen, Lee-Ann H

    2016-09-01

    Francisella tularensis in an intracellular bacterial pathogen that causes a potentially lethal disease called tularemia. Studies performed nearly 100 years ago revealed that neutrophil accumulation in infected tissues correlates directly with the extent of necrotic damage during F. tularensis infection. However, the dynamics and details of bacteria-neutrophil interactions have only recently been studied in detail. Herein, we review current understanding regarding the mechanisms that recruit neutrophils to F. tularensis-infected lungs, opsonization and phagocytosis, evasion and inhibition of neutrophil defense mechanisms, as well as the ability of F. tularensis to prolong neutrophil lifespan. In addition, we discuss distinctive features of the bacterium, including its ability to act at a distance to alter overall neutrophil responsiveness to exogenous stimuli, and the evidence which suggests that macrophages and neutrophils play distinct roles in tularemia pathogenesis, such that macrophages are major vehicles for intracellular growth and dissemination, whereas neutrophils drive tissue destruction by dysregulation of the inflammatory response. PMID:27558340

  17. Liposome Encapsulation of Ciprofloxacin Improves Protection against Highly Virulent Francisella tularensis Strain Schu S4

    PubMed Central

    Armstrong, Stuart J.; Barnes, Kay B.; Davies, Carwyn; Wong, Jonathan P.; Blanchard, James D.; Harding, Sarah V.; Simpson, Andrew J. H.; Atkins, Helen S.

    2014-01-01

    Liposome-encapsulated ciprofloxacin for inhalation (CFI) was investigated as a putative postexposure therapeutic for two strains of Francisella tularensis. The efficacies of oral ciprofloxacin and intranasally instilled CFI could not be distinguished in a mouse model of infection with the F. tularensis live vaccine strain (LVS), where a single dose of either formulation offered full protection against a lethal challenge. However, mouse studies with the more virulent Schu S4 strain of F. tularensis demonstrated that a higher level of protection against a lethal aerosol infection is provided by CFI than by oral ciprofloxacin. In addition, using this infection model, it was possible to discriminate the efficacy of intranasally instilled CFI from that of aerosolized CFI, with aerosolized CFI providing full protection after just a single dose. The improved efficacy of CFI compared to oral ciprofloxacin is likely due to the high sustained concentrations of ciprofloxacin in the lung. In summary, CFI may be a promising therapy, perhaps enabling the prophylactic regimen to be shortened, for use in the event of a deliberate release of F. tularensis. The prophylactic efficacy of CFI against other biological warfare (BW) threat agents also warrants investigation. PMID:24637682

  18. [Real time PCR hybridization for the rapid and specific identification of Francisella tularensis].

    PubMed

    Bielawska-Drózd, Agata; Niemcewicz, Marcin; Gaweł, Jerzy; Bartoszcze, Michał; Graniak, Grzegorz; Joniec, Justyna; Kołodziej, Marcin

    2010-01-01

    Tularemia is highly infectious and fatal zoonotic disease caused by Gram negative bacteria Francisella tularensis. The necessity to undergo medical treatment in early phase of illness in humans and possibility of making use of bacterial aerosol by terrorists in an attack create an urgent need to implement a rapid and effective method which enables to identify the agent. In our study two primers FopA F/R and hybridization probes FopA S1/S2 designed from fopA gene sequence, were tested for their potential applicability to identify F. tularensis. In this research 50 strains of F. tularensis were used and the test gave positive results. Reaction specificity was confirmed by using of non-Francisella tularensis bacterial species. The results obtained in the real-time PCR reaction with primers Tul4 F/R and hybridization probes Tul4 S1/S2, designed from tul4 gene, were comparable to the results from previous experiment with fopA - primers set. Investigation of fopA and tul4 primers and hybridization probes properties revealed characteristic Tm (melting temperature) value of the products--61 degrees C and 60 degrees C, respectively. Detection sensitivity was remarkably higher when fopA primers set was used 1 fg/microl, and for tul4 primers set, minimal detectable concentration is 10 fg/microl. PMID:21473100

  19. Inactivation of F.tularensis Utah-112 on food and food contact surfaces by ultraviolet light

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Francisella tularensis is the causative agent of tularemia, a plague-like illness that affects animals and humans, and has caused large illness pandemics in the last century. It has also been used as a biological warfare agent, and tularemia can be contracted through consumption of contaminated food...

  20. Metabolic Reprogramming of Host Cells by Virulent Francisella tularensis for Optimal Replication and Modulation of Inflammation.

    PubMed

    Wyatt, Elliott V; Diaz, Karina; Griffin, Amanda J; Rasmussen, Jed A; Crane, Deborah D; Jones, Bradley D; Bosio, Catharine M

    2016-05-15

    A shift in macrophage metabolism from oxidative phosphorylation to aerobic glycolysis is a requirement for activation to effectively combat invading pathogens. Francisella tularensis is a facultative intracellular bacterium that causes an acute, fatal disease called tularemia. Its primary mechanism of virulence is its ability to evade and suppress inflammatory responses while replicating in the cytosol of macrophages. The means by which F. tularensis modulates macrophage activation are not fully elucidated. In this study, we demonstrate that virulent F. tularensis impairs production of inflammatory cytokines in primary macrophages by preventing their shift to aerobic glycolysis, as evidenced by the downregulation of hypoxia inducible factor 1α and failure to upregulate pfkfb3 We also show that Francisella capsule is required for this process. In addition to modulating inflammatory responses, inhibition of glycolysis in host cells is also required for early replication of virulent Francisella Taken together, our data demonstrate that metabolic reprogramming of host cells by F. tularensis is a key component of both inhibition of host defense mechanisms and replication of the bacterium. PMID:27029588

  1. Mast cells inhibit intramacrophage Francisella tularensis replication via contact and secreted products including IL-4

    PubMed Central

    Ketavarapu, Jyothi M.; Rodriguez, Annette R.; Yu, Jieh-Juen; Cong, Yu; Murthy, Ashlesh K.; Forsthuber, Thomas G.; Guentzel, M. Neal; Klose, Karl E.; Berton, Michael T.; Arulanandam, Bernard P.

    2008-01-01

    Francisella tularensis is an intracellular, Gram-negative bacterium that is the causative agent of pulmonary tularemia. The pathogenesis and mechanisms related to innate resistance against F. tularensis are not completely understood. Mast cells are strategically positioned within mucosal tissues, the major interface with the external environment, to initiate innate responses at the site of infection. Mast cell numbers in the cervical lymph nodes and the lungs progressively increased as early as 48 h after intranasal F. tularensis live vaccine strain (LVS) challenge. We established a primary bone marrow-derived mast cell–macrophage coculture system and found that mast cells significantly inhibit F. tularensis LVS uptake and growth within macrophages. Importantly, mice deficient in either mast cells or IL-4 receptor displayed greater susceptibility to the infection when compared with corresponding wild-type animals. Contact-dependent events and secreted products including IL-4 from mast cells, and IL-4 production from other cellular sources, appear to mediate the observed protective effects. These results demonstrate a previously unrecognized role for mast cells and IL-4 and provide a new dimension to our understanding of the innate immune mechanisms involved in controlling intramacrophage Francisella replication. PMID:18591675

  2. A response regulator promotes Francisella tularensis intramacrophage growth by repressing an anti-virulence factor.

    PubMed

    Ramsey, Kathryn M; Dove, Simon L

    2016-08-01

    The orphan response regulator PmrA is essential for the intramacrophage growth and survival of Francisella tularensis. PmrA was thought to promote intramacrophage growth by binding directly to promoters on the Francisella Pathogenicity Island (FPI) and positively regulating the expression of FPI genes, which encode a Type VI secretion system required for intramacrophage growth. Using both ChIP-Seq and RNA-Seq we identify those regions of the F. tularensis chromosome occupied by PmrA and those genes that are regulated by PmrA. We find that PmrA associates with 252 distinct regions of the F. tularensis chromosome, but exerts regulatory effects at only a few of these locations. Rather than by functioning directly as an activator of FPI gene expression we present evidence that PmrA promotes intramacrophage growth by repressing the expression of a single target gene we refer to as priM (PmrA-repressed inhibitor of intramacrophage growth). Our findings thus indicate that the role of PmrA in facilitating intracellular growth is to repress a previously unknown anti-virulence factor. PriM is the first bacterially encoded factor to be described that can interfere with the intramacrophage growth and survival of F. tularensis. PMID:27169554

  3. PATHOGENESIS AND IMMUNE RESPONSES OF FRANCISELLA TULARENSIS STRAINS IN WILD-CAUGHT COTTONTAIL RABBITS (SYLVILAGUS SPP.).

    PubMed

    Brown, Vienna R; Adney, Danielle R; Bielefeldt-Ohmann, Helle; Gordy, Paul W; Felix, Todd A; Olea-Popelka, Francisco J; Bowen, Richard A

    2015-07-01

    Francisella tularensis is a highly virulent, zoonotic bacterium that causes significant natural disease and is of concern as an organism for bioterrorism. Serologic testing of wildlife is frequently used to monitor spatial patterns of infection and to quantify exposure. Cottontail rabbits (Sylvilagus spp.) are a natural reservoir for F. tularensis in the US, although very little work has been done experimentally to determine how these animals respond to infection; thus, information gathered from field samples can be difficult to interpret. We characterized clinical disease, bacteremia, pathology, and antibody kinetics of North American cottontail rabbits experimentally infected with five strains of F. tularensis. Rabbits were infected with four field strains, including MA00-2987 (type A1b), WY96-3418 (type A2), KY99-3387, and OR96-0246 (type B), and with SchuS4 (type A1a), a widely used, virulent laboratory strain. Infection with the different strains of the bacterium resulted in varied patterns of clinical disease, gross pathology, and histopathology. Each of the type A strains were highly virulent, with rabbits succumbing to infection 3-13 d after infection. At necropsy, numerous microabscesses were observed in the livers and spleens of most rabbits, associated with high bacterial organ burdens. In contrast, most rabbits infected with type B strains developed mild fever and became lethargic, but the disease was infrequently lethal. Those rabbits infected with type B strains that survived past 14 d developed a robust humoral immune response, and F. tularensis was not isolated from liver, spleen, or lung of those animals. Understanding F. tularensis infection in a natural reservoir species can guide serosurveillance and generate new insights into environmental maintenance of this pathogen. PMID:25984770

  4. Contribution of Citrulline Ureidase to Francisella tularensis Strain Schu S4 Pathogenesis▿

    PubMed Central

    Mahawar, Manish; Kirimanjeswara, Girish S.; Metzger, Dennis W.; Bakshi, Chandra Shekhar

    2009-01-01

    The citrulline ureidase (CTU) activity has been shown to be associated with highly virulent Francisella tularensis strains, including Schu S4, while it is absent in avirulent or less virulent strains. A definitive role of the ctu gene in virulence and pathogenesis of F. tularensis Schu S4 has not been assessed; thus, an understanding of the significance of this phenotype is long overdue. CTU is a carbon-nitrogen hydrolase encoded by the citrulline ureidase (ctu) gene (FTT0435) on the F. tularensis Schu S4 genome. In the present study, we evaluated the contribution of the ctu gene in the virulence of category A agent F. tularensis Schu S4 by generating a nonpolar deletion mutant, the Δctu mutant. The deletion of the ctu gene resulted in loss of CTU activity, which was restored by transcomplementing the ctu gene. The Δctu mutant did not exhibit any growth defect under acellular growth conditions; however, it was impaired for intramacrophage growth in resting as well as gamma interferon-stimulated macrophages. The Δctu mutant was further tested for its virulence attributes in a mouse model of respiratory tularemia. Mice infected intranasally with the Δctu mutant showed significantly reduced bacterial burden in the lungs, liver, and spleen compared to wild-type (WT) Schu S4-infected mice. The reduced bacterial burden in mice infected with the Δctu mutant was also associated with significantly lower histopathological scores in the lungs. Mice infected with the Δctu mutant succumbed to infection, but they survived longer and showed significantly extended median time to death compared to that shown by WT Schu S4-infected mice. To conclude, this study demonstrates that ctu contributes to intracellular survival, in vivo growth, and pathogenesis. However, ctu is not an absolute requirement for the virulence of F. tularensis Schu S4 in mice. PMID:19502406

  5. Nucleolin, a Shuttle Protein Promoting Infection of Human Monocytes by Francisella tularensis

    PubMed Central

    Barel, Monique; Meibom, Karin; Charbit, Alain

    2010-01-01

    Background Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells. Methodology/Principal Findings Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant. Conclusions/Significance We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages. PMID:21152024

  6. Thermal resistance of Francisella tularensis in infant formula and fruit juices.

    PubMed

    Day, J B; Trujillo, S; Hao, Y Y D; Whiting, R C

    2008-11-01

    Francisella tularensis is a gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, little information exists on the thermal stability of this organism in food matrices. In the present study, the thermal resistance of the live vaccine strain of F. tularensis in four food products (liquid infant formula, apple juice, mango juice, and orange juice) was investigated. D-values ranged from 12 s (57.5 degrees C) to 580 s (50 degrees C) in infant formula with a z-value of 4.37 degrees C. D-values in apple juice ranged from 8 s (57.5 degrees C) to 59 s (50 degrees C) with a z-value of 9.17 degrees C. The live vaccine strain did not survive at temperatures above 55 degrees C in mango juice and orange juice (>6-log inactivation). D-values at 55 to 47.5 degrees C were 15 to 59 s in mango juice and 16 to 105 s in orange juice with z-values of 9.28 and 12.30 degrees C, respectively. These results indicate that current pasteurization parameters used for destroying common foodborne bacterial pathogens are adequate for eliminating F. tularensis in the four foods tested. This study is the first to determine thermal inactivation of F. tularensis in specific foods and will permit comparisons with the thermal inactivation data of other more traditional foodborne pathogens. PMID:19044262

  7. Inactivation of Yersinia pseudotuberculosis 197 and Francisella tularensis LVS in beverages by high pressure processing.

    PubMed

    Schlesser, Joseph E; Parisi, Brian

    2009-01-01

    In 2003, the U.S. Department of Health and Human Services announced a new research program to develop technologies and strategies to prevent and minimize potential food safety and security threats. The threat of terrorist attacks against the nation's food supplies has created the need to study microorganisms not typically associated with foodborne illness. High-pressure processing has been proposed as a treatment to reduce Yersinia pestis and Francisella tularensis LVS levels in beverages. The objectives of this work were to determine the pressure resistance of Y. pseudotuberculosis 197 (surrogate for Y. pestis) and F. tularensis LVS (vaccine strain). For each bacterium, samples of ultrahigh-temperature pasteurized skim milk and pasteurized reduced-acid orange juice (pH ca. 4.2) were inoculated at a minimum level of 5 log CFU/ml. Ten-milliliter samples of the inoculated product were vacuum sealed in polyester pouches and subjected to pressures of 300 and 500 MPa for holding times ranging from 30 s to 6 min. One set of trials was performed at an initial temperature of 10 degrees C and another at 25 degrees C. Processed samples were immediately plated and enumerated. A pressure treatment of 300 MPa at 25 degrees C for less than 6 min was not sufficient to achieve a 5-log reduction of Y. pseudotuberculosis 197 or F. tularensis LVS in milk. However, a pressure treatment of 500 MPa was effective at hold times as low as 30 s. Overall, F. tularensis LVS demonstrated less pressure resistance than Y. pseudotuberculosis 197. Based on these findings, a high-pressure process designed to inactivate 5 log CFU of Y. pseudotuberculosis 197 per ml and F. tularensis LVS in orange juice or milk should be set at or above 500 MPa with a hold time of 2 min or greater. PMID:19205479

  8. Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis.

    PubMed

    Wilkinson, Rachael C; Batten, Laura E; Wells, Neil J; Oyston, Petra C F; Roach, Peter L

    2015-01-01

    The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the 'alarmones' (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5',3'-dibisphosphate guanosine) with an EC50 of 60±1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia. PMID:26450927

  9. Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis

    PubMed Central

    Wilkinson, Rachael C.; Batten, Laura E.; Wells, Neil J.; Oyston, Petra C.F.; Roach, Peter L.

    2015-01-01

    The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the ‘alarmones’ (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5′,3′-dibisphosphate guanosine) with an EC50 of 60±1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia. PMID:26450927

  10. Re-emergence of tularemia in Germany: Presence of Francisella tularensis in different rodent species in endemic areas

    PubMed Central

    Kaysser, Philipp; Seibold, Erik; Mätz-Rensing, Kerstin; Pfeffer, Martin; Essbauer, Sandra; Splettstoesser, Wolf D

    2008-01-01

    Background Tularemia re-emerged in Germany starting in 2004 (with 39 human cases from 2004 to 2007) after over 40 years of only sporadic human infections. The reasons for this rise in case numbers are unknown as is the possible reservoir of the etiologic agent Francisella (F.) tularensis. No systematic study on the reservoir situation of F. tularensis has been published for Germany so far. Methods We investigated three areas six to ten months after the initial tularemia outbreaks for the presence of F. tularensis among small mammals, ticks/fleas and water. The investigations consisted of animal live-trapping, serologic testing, screening by real-time-PCR and cultivation. Results A total of 386 small mammals were trapped. F. tularensis was detected in five different rodent species with carrier rates of 2.04, 6.94 and 10.87% per trapping area. None of the ticks or fleas (n = 432) tested positive for F. tularensis. We were able to demonstrate F. tularensis-specific DNA in one of 28 water samples taken in one of the outbreak areas. Conclusion The findings of our study stress the need for long-term surveillance of natural foci in order to get a better understanding of the reasons for the temporal and spatial patterns of tularemia in Germany. PMID:19014635

  11. Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia

    PubMed Central

    Lu, Zhaohua; Madico, Guillermo; Roche, Marly I; Wang, Qi; Hui, Julia H; Perkins, Hillary M; Zaia, Joseph; Costello, Catherine E; Sharon, Jacqueline

    2012-01-01

    Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg. PMID:22486311

  12. Molecular Evolution of Mycoplasma capricolum subsp. capripneumoniae Strains, Based on Polymorphisms in the 16S rRNA Genes

    PubMed Central

    Pettersson, Bertil; Bölske, Göran; Thiaucourt, François; Uhlén, Mathias; Johansson, Karl-Erik

    1998-01-01

    Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP. PMID:9573185

  13. Francisella tularensis type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks post Infection

    SciTech Connect

    El-Etr, S H; Margolis, J; Monack, D; Robison, R; Cohen, M; Moore, E; Rasley, A

    2009-07-28

    Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown, and the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype (REP) is caused by factor(s) secreted by amoebae and/or F. tularensis into the co-culture media. Further, our results indicate that in contrast to LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks post infection and that induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that the interactions between F. tularensis strains and amoeba may play a role in the environmental persistence of F. tularensis.

  14. Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis

    PubMed Central

    Takimoto, Kazuhiro; Deyu, Tian; Koyama, Yuuki; Park, Eun-sil; Fujita, Osamu; Hotta, Akitoyo; Morikawa, Shigeru

    2016-01-01

    Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo. PMID:27448164

  15. Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis.

    PubMed

    Uda, Akihiko; Sharma, Neekun; Takimoto, Kazuhiro; Deyu, Tian; Koyama, Yuuki; Park, Eun-Sil; Fujita, Osamu; Hotta, Akitoyo; Morikawa, Shigeru

    2016-01-01

    Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo. PMID:27448164

  16. Leucobacter musarum subsp. musarum sp. nov., subsp. nov., Leucobacter musarum subsp. japonicus subsp. nov., and Leucobacter celer subsp. astrifaciens subsp. nov., three nematopathogenic bacteria isolated from Caenorhabditis, with an emended description of Leucobacter celer

    PubMed Central

    Hodgkin, Jonathan

    2015-01-01

    Three Gram-stain-positive, irregular-rod-shaped, non-motile, non-spore-forming bacteria were isolated from nematodes collected from Santa Antao, Cabo Verde (CBX151T, CBX152T) and Kakegawa, Japan (CBX130T). Based on 16S rRNA gene sequence similarity, strains CBX130T, CBX151T and CBX152T were shown to belong to the genus Leucobacter. This affiliation was supported by chemotaxonomic data (2,4-diaminobutyric acid in the cell wall; major respiratory quinones MK-10 and MK-11; major polar lipids phosphatidylglycerol and diphosphatidylglycerol; major fatty acids anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0). Strains CBX130T and CBX152T were found to share salient characteristics. Based on morphological, physiological, chemotaxonomic and biochemical analysis, strain CBX152T represents a novel species of the genus Leucobacter, for which the name Leucobacter musarum sp. nov. (type strain CBX152T = DSM 27160T = CIP 110721T) is proposed. Two subspecies of Leucobacter musarum sp. nov. are proposed: Leucobacter musarum sp. nov. subsp. musarum subsp. nov. (type strain CBX152T = DSM 27160T = CIP 110721T) and Leucobacter musarum sp. nov. subsp. japonicus subsp. nov. (type strain CBX130T = DSM 27158T = CIP 110719T). The third novel strain, CBX151T, showed genetic similarities with Leucobacter celer NAL101T indicating that these strains belong to the same species. Based on morphological, physiological, chemotaxonomic and biochemical differences it is proposed to split the species Leucobacter celer into two novel subspecies, Leucobacter celer subsp. celer subsp. nov. (type strain NAL101T = KACC 14220T = JCM 16465T) and Leucobacter celer subsp. astrifaciens subsp. nov. (type strain CBX151T = DSM 27159T = CIP 110720T), and to emend the description of Leucobacter celer Shin et al. 2011. PMID:26275616

  17. Nutritional features of Bacteroides fragilis subsp. fragilis.

    PubMed

    Varel, V H; Bryant, M P

    1974-08-01

    Studies of three reference strains of Bacteroides fragilis subsp. fragilis showed that they grow well in a minimal defined medium containing glucose, hemin, vitamin B(12), minerals, bicarbonate-carbon dioxide buffer, NH(4)Cl, and sulfide. The vitamin B(12) requirement of 0.1 ng/ml was replaced with 7.5 mug of methionine. Cysteine or sulfide was an excellent source of sulfur, thioglycolate was a poor source, and thiosulfate, methionine, beta-mercaptoethanol, dithiothreitol, sulfate, or sulfite did not serve as sole sources of sulfur. Neither single amino acids, nitrate, urea, nor a complex mixture of L-amino acids or peptides effectively replaced ammonia as the nitrogen source. Comparative studies with a few strains of other subspecies of B. fragilis including B. fragilis subsp. vulgatus, B. fragilis subsp. thetaiotaomicron, and B. fragilis subsp. distasonis indicate that they exhibit similar growth responses in the minimal medium. A single strain of B. fragilis subsp. ovatus required other materials. The results indicate the great biosynthetic ability of these organisms and suggest that, in their ecological niche within the large intestine, many nutrients such as amino acids are in very low supply, whereas materials such as ammonia, heme, and vitamin B(12), or related compounds, must be available during much of the time. PMID:4853401

  18. Mucosal Immunization with Live Attenuated Francisella novicida U112ΔiglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model

    PubMed Central

    Signarovitz, Aimee L.; Ray, Heather J.; Yu, Jieh-Juen; Guentzel, M. N.; Chambers, James P.; Klose, Karl E.; Arulanandam, Bernard P.

    2012-01-01

    The need for an efficacious vaccine against Francisella tularensis is a consequence of its low infectious dose and high mortality rate if left untreated. This study sought to characterize a live attenuated subspecies novicida-based vaccine strain (U112ΔiglB) in an established second rodent model of pulmonary tularemia, namely the Fischer 344 rat using two distinct routes of vaccination (intratracheal [i.t.] and oral). Attenuation was verified by comparing replication of U112ΔiglB with wild type parental strain U112 in F344 primary alveolar macrophages. U112ΔiglB exhibited an LD50>107 CFU compared to the wild type (LD50 = 5×106 CFU i.t.). Immunization with 107 CFU U112ΔiglB by i.t. and oral routes induced antigen-specific IFN-γ and potent humoral responses both systemically (IgG2a>IgG1 in serum) and at the site of mucosal vaccination (respiratory/intestinal compartment). Importantly, vaccination with U112ΔiglB by either i.t. or oral routes provided equivalent levels of protection (50% survival) in F344 rats against a subsequent pulmonary challenge with ∼25 LD50 (1.25×104 CFU) of the highly human virulent strain SCHU S4. Collectively, these results provide further evidence on the utility of a mucosal vaccination platform with a defined subsp. novicida U112ΔiglB vaccine strain in conferring protective immunity against pulmonary tularemia. PMID:23118885

  19. Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

    PubMed

    Shakerley, Nicole L; Chandrasekaran, Akshaya; Trebak, Mohamed; Miller, Barbara A; Melendez, J Andrés

    2016-02-19

    As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function. PMID:26679996

  20. Francisella tularensis Bacteria Associated with Feline Tularemia in the United States

    PubMed Central

    Fey, Paul D.; Hinrichs, Steven H.; Iwen, Peter C.

    2014-01-01

    Tularemia in the United States was examined by reviewing 106 Francisella tularensis isolates, mostly from Nebraska, collected during 1998–2012: 48% of Nebraska cases were cat-associated; 7/8 human cases were caused by subtype A.I. A vaccine is needed to reduce feline-associated tularemia, and cat owners should protect against bites/scratches and limit their pet’s outdoor access. PMID:25424732

  1. Use of Temperature for Standardizing the Progression of Francisella tularensis in Mice

    PubMed Central

    Molins, Claudia R.; Delorey, Mark J.; Young, John W.; Yockey, Brook M.; Belisle, John T.; Schriefer, Martin E.; Petersen, Jeannine M.

    2012-01-01

    The study of infectious agents, their pathogenesis, the host response and the evaluation of newly developed countermeasures often requires the use of a living system. Murine models are frequently used to undertake such investigations with the caveat that non-biased measurements to assess the progression of infection are underutilized. Instead, murine models predominantly rely on symptomology exhibited by the animal to evaluate the state of the animal's health and to determine when euthanasia should be performed. In this study, we used subcutaneous temperature as a non-subjective measurement to follow and compare infection in mice inoculated with Francisella tularensis, a Gram-negative pathogen that produces an acute and fatal illness in mice. A reproducible temperature pattern defined by three temperature phases (normal, febrile and hypothermic) was identified in all mice infected with F. tularensis, regardless of the infecting strain. More importantly and for the first time a non-subjective, ethical, and easily determined surrogate endpoint for death based on a temperature, termed drop point, was identified and validated with statistical models. In comparative survival curve analyses for F. tularensis strains with differing virulence, the drop point temperature yielded the same results as those obtained using observed time to death. Incorporation of temperature measurements to evaluate F. tularensis was standardized based on statistical models to provide a new level of robustness for comparative analyses in mice. These findings should be generally applicable to other pathogens that produce acute febrile disease in animal models and offers an important tool for understanding and following the infection process. PMID:23028924

  2. Identification of Genes Contributing to the Virulence of Francisella tularensis SCHU S4 in a Mouse Intradermal Infection Model

    PubMed Central

    Golovliov, Igor; Bolanowski, Mark; Shen, Hua; Conlan, Wayne; Sjöstedt, Anders

    2009-01-01

    Background Francisella tularensis is a highly virulent human pathogen. The most virulent strains belong to subspecies tularensis and these strains cause a sometimes fatal disease. Despite an intense recent research effort, there is very limited information available that explains the unique features of subspecies tularensis strains that distinguish them from other F. tularensis strains and that explain their high virulence. Here we report the use of targeted mutagenesis to investigate the roles of various genes or pathways for the virulence of strain SCHU S4, the type strain of subspecies tularensis. Methodology/Principal Findings The virulence of SCHU S4 mutants was assessed by following the outcome of infection after intradermal administration of graded doses of bacteria. By this route, the LD50 of the SCHU S4 strain is one CFU. The virulence of 20 in-frame deletion mutants and 37 transposon mutants was assessed. A majority of the mutants did not show increased prolonged time to death, among them notably ΔpyrB and ΔrecA. Of the remaining, mutations in six unique targets, tolC, rep, FTT0609, FTT1149c, ahpC, and hfq resulted in significantly prolonged time to death and mutations in nine targets, rplA, wbtI, iglB, iglD, purL, purF, ggt, kdtA, and glpX, led to marked attenuation with an LD50 of >103 CFU. In fact, the latter seven mutants showed very marked attenuation with an LD50 of ≥107 CFU. Conclusions/Significance The results demonstrate that the characterization of targeted mutants yielded important information about essential virulence determinants that will help to identify the so far little understood extreme virulence of F. tularensis subspecies tularensis. PMID:19424499

  3. Complete Genome Sequences of Campylobacter hyointestinalis subsp. hyointestinalis Strain LMG 9260 and C. hyointestinalis subsp. lawsonii Strain LMG 15993

    PubMed Central

    Yee, Emma; Chapman, Mary H.

    2016-01-01

    Campylobacter hyointestinalis is isolated primarily from ruminants and swine, but is also occasionally isolated from humans. C. hyointestinalis is currently divided into two subspecies, C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii. This study describes the first closed whole-genome sequences of C. hyointestinalis subsp. hyointestinalis isolate LMG 9260 and C. hyointestinalis subsp. lawsonii isolate LMG 15993. PMID:27417840

  4. Complete Genome Sequences of Campylobacter hyointestinalis subsp. hyointestinalis Strain LMG 9260 and C. hyointestinalis subsp. lawsonii Strain LMG 15993.

    PubMed

    Miller, William G; Yee, Emma; Chapman, Mary H

    2016-01-01

    Campylobacter hyointestinalis is isolated primarily from ruminants and swine, but is also occasionally isolated from humans. C. hyointestinalis is currently divided into two subspecies, C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii This study describes the first closed whole-genome sequences of C. hyointestinalis subsp. hyointestinalis isolate LMG 9260 and C. hyointestinalis subsp. lawsonii isolate LMG 15993. PMID:27417840

  5. Delineation of the Molecular Mechanisms of Francisella tularensis-Induced Apoptosis in Murine Macrophages

    PubMed Central

    Lai, Xin-He; Sjöstedt, Anders

    2003-01-01

    Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. Here we analyzed the pathway leading to apoptosis in the murine macrophage-like cell line J774A.1 after infection with F. tularensis strain LVS (named LVS for live vaccine strain). We obtained evidence that the infection affected the mitochondria of the macrophages, since it induced release of the mitochondrial molecule cytochrome c into the cytosol and changed the potential over the mitochondrial membrane. Moreover, activation of caspase 9 and the executioner caspase 3 was also observed in the LVS-infected J774A.1 macrophages. The activated caspase 3 degraded poly(ADP-ribose) polymerase (PARP). All of these events were observed within 9 to 12 h after the initiation of infection, and maximum degradation of a synthetic caspase 3 substrate occurred at 18 h. The internucleosomal fragmentation and PARP degradation resulting from activation of this apoptotic pathway was prevented by the caspase 3 inhibitor Z-DEVD-fmk. No involvement of caspase 1, caspase 8, Bcl-2, or Bid was observed. Thus, the F. tularensis infection induces macrophage apoptosis through a pathway partly resembling the intrinsic apoptotic pathway. PMID:12874344

  6. Examination of in vitro epithelial cell lines as models for Francisella tularensis non-phagocytic infections.

    PubMed

    Lo, Karen Yi-Shyuan; Chua, Michael Dominic; Abdulla, Salima; Law, H T; Guttman, Julian Andrew

    2013-05-01

    Francisella tularensis (F. tularensis), the causative agent of tularemia, has long been known to invade and occupy non-phagocytic epithelial cells. Many epithelial cell infection models have been developed to study this process; however, due to the lack of consensus on infection methods and precise experimental procedures to evaluate invasion and replication, selection of appropriate models to use based on the literature is challenging. To evaluate in vitro non-phagocytic cell infection models, we chose 8 epithelial cultured cell lines from published models to infect with F. tularensis subspecies novicida (F. novicida) and compared the results to a recently developed model that used the mouse hepatocyte BNL CL.2 cell line. We utilized classical gentamicin-based invasion assays to determine total intracellular bacterial loads and employed microscopic examination with staining techniques that distinguished between intracellular and extracellular bacteria to provide an accurate assessment of the proportion of invaded host cells and the degree of bacterial replication. We found that COS-7 cells exhibited the greatest invasion rates; CMT-93 cells contained the largest intracellular bacterial loads; ad HEK-293s were capable of invasion and replication rates at high levels, but required shorter infection incubation times. Although COS-7, CMT-93 and HEK-293 cell lines may be suited to study certain aspects of invasion or replication, we found that BNL CL.2 cells appeared the most appropriate to study the overall pathogenesis of F. novicida when examined in toto. PMID:23523968

  7. A multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism

    PubMed Central

    Balonova, Lucie; Hernychova, Lenka; Mann, Benjamin F.; Link, Marek; Bilkova, Zuzana; Novotny, Milos V.; Stulik, Jiri

    2010-01-01

    It appears that most glycoproteins found in pathogenic bacteria are associated with virulence. Despite the recent identification of novel virulence factors, the mechanisms of virulence in Francisella tularensis are poorly understood. In spite of its importance, questions about glycosylation of proteins in this bacterium and its potential connection with bacterial virulence have not been answered yet. In the present study, several putative Francisella tularensis glycoproteins were characterized through the combination of carbohydrate-specific detection and lectin affinity with highly sensitive mass spectrometry utilizing the bottom-up proteomic approach. The protein PilA that was recently found as being possibly glycosylated, as well as other proteins with designation as novel factors of virulence, were among the proteins identified in this study. The reported data compile the list of potential glycoproteins that may serve as a take-off platform for a further definition of proteins modified by glycans, faciliting a better understanding of the function of protein glycosylation in pathogenicity of Francisella tularensis. PMID:20175567

  8. Evidence of Sympatry of Clade A and Clade B Head Lice in a Pre-Columbian Chilean Mummy from Camarones

    PubMed Central

    Boutellis, Amina; Drali, Rezak; Rivera, Mario A.; Mumcuoglu, Kosta Y.; Raoult, Didier

    2013-01-01

    Three different lineages of head lice are known to parasitize humans. Clade A, which is currently worldwide in distribution, was previously demonstrated to be present in the Americas before the time of Columbus. The two other types of head lice are geographically restricted to America and Australia for clade B and to Africa and Asia for clade C. In this study, we tested two operculated nits from a 4,000-year-old Chilean mummy of Camarones for the presence of the partial Cytb mitochondrial gene (270 bp). Our finding shows that clade B head lice were present in America before the arrival of the European colonists. PMID:24204678

  9. [Development of a novel Francisella tularensis antigen stained with tetrazolium-blue for tularemia microagglutination test].

    PubMed

    Celebi, Bekir; Kılıç, Selçuk

    2013-07-01

    The isolation of Francisella tularensis in cultures is the reference method for the laboratory diagnosis of tularemia. However, due to the limitations such as the low sensitivity and need for high safety level and equipped laboratories, serologic methods are frequently used as diagnostic tools. F.tularensis-specific antibodies may be demonstrated by several methods, however microagglutination test (MA) remains the most common method with its high sensitivity and specificity. The aim of this study was to develop a novel MA test antigen prepared from whole F.tularensis cells and stained with tetrazolium-blue for more clear and easier evaluation. F.tularensis NCTC 10857 strain was cultured on the cysteine heart agar supplemented with 9% sheep blood and bacterial cells were harvested by scraping, collected in physiological saline (PS) and centrifuged at 1500 rpm for 10 minutes. For preparing stock antigen suspension cell concentration was adjusted to OD600=1.5, spectrophotometrically. Tetrazolium-blue solution (BTC [3,3'-(3,3'-Dimethoxy[1,1'-biphenyl]-4,4'-diyl) bis [2,5-diphenyl-2H-tetrazolium dichloride], T4375 Sigma-Aldrich) at the final concentration of 1% was added to cell suspension and incubated at 37°C for 5 hours for absorption. Then, the living cells were chemically inactivated by formaldehyde. Repeating centrifugations were performed to discard excess dye and formaline, then 0.4% formaline saline was added on the sediment. Optimum concentration of this novel antigen (BTC-Ag) for MA test was determined by plate titration method by using standard serum sample with a known MA titer (1/128). The performance of BTC-Ag in MA test was evaluated by using 100 patient sera positive for F.tularensis antibodies, and 100 tularemia negative patient sera (of them 50 were seropositive for brucellosis). All of the results were compared with standard MA test in which safranin-O stained antigen (SO-Ag) was used. There was 100% agreement between the two tests performed with

  10. The Francisella tularensis Pathogenicity Island Encodes a Secretion System that is required for Phagosome Escape and Virulence

    PubMed Central

    Barker, Jeffrey R.; Chong, Audrey; Wehrly, Tara D.; Yu, Jieh-Juen; Rodriguez, Stephen A.; Liu, Jirong; Celli, Jean; Arulanandam, Bernard P.; Klose, Karl E.

    2009-01-01

    Summary Francisella tularensis causes the human disease tularemia. F. tularensis is able to survive and replicate within macrophages, a trait that has been correlated with its high virulence, but it is unclear the exact mechanism(s) this organism uses to escape killing within this hostile environment. F. tularensis virulence is dependent upon the Francisella Pathogenicity Island (FPI), a cluster of genes that we show here shares homology with Type VI secretion gene clusters in Vibrio cholerae and Pseudomonas aeruginosa. We demonstrate that two FPI proteins, VgrG and IglI, are secreted into the cytosol of infected macrophages. VgrG and IglI are required for F. tularensis phagosomal escape, intramacrophage growth, inflammasome activation, and virulence in mice. Interestingly, VgrG secretion does not require the other FPI genes. However, VgrG and other FPI genes, including PdpB (an IcmF homologue), are required for the secretion of IglI into the macrophage cytosol, suggesting VgrG and other FPI factors are components of a secretion system. This is the first report of F. tularensis FPI virulence proteins required for intramacrophage growth that are translocated into the macrophage. PMID:20054881

  11. Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio; Tanabayashi, Kiyoshi

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  12. Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  13. Molecular Investigation of Francisella-Like Endosymbiont in Ticks and Francisella tularensis in Ixodid Ticks and Mosquitoes in Turkey.

    PubMed

    Duzlu, Onder; Yildirim, Alparslan; Inci, Abdullah; Gumussoy, Kadir Semih; Ciloglu, Arif; Onder, Zuhal

    2016-01-01

    This study was carried out to investigate the molecular prevalence of Francisella-like endosymbionts (FLEs) and Francisella tularensis in ticks (Acari: Ixodidae) and mosquitoes in Turkey. Genomic DNA pools were constructed from a total of 1477 adult hard ticks of Rhipicephalus (Rh.) annulatus, Rh. turanicus, Rh. sanguineus, Rh. bursa, Haemaphysalis (Hae.) parva, Hae. sulcata, Hyalomma marginatum marginatum, H. anatolicum anatolicum, H. anatolicum excavatum, H. detritum detritum, H. dromedarii, Dermacentor marginatus, and Ixodes ricinus species, which were collected from several barns, cattle, and people. Genomic DNA was also extracted from pools consisting of 6203 adult female mosquito species belonging to Aedes vexans, Culex (Cx.) pipiens, Cx. hortensis, Cx. theileri, Culiseta annulata, and Anopheles maculipennis species. Conventional PCR and TaqMan probe-based real- time PCR targeting the 16S rRNA gene for FLEs and the lpnA gene for F. tularensis, respectively, were performed on the DNA isolates obtained. FLEs and F. tularensis were not found in any genomic DNA pools constructed from ixodid ticks and mosquitos. This study represents the first investigation of F. tularensis and FLEs in potential vector ticks and mosquitoes by molecular methods in Turkey. The present study provides useful insights into the molecular epidemiology of F. tularensis and FLEs. One of the major conclusions of the study is that tularemia outbreaks may be essentially due to direct transmission from the environment (especially from water) in Turkey and not to vector-borne transmission. PMID:26741324

  14. Bioproducts from diverse phylogenetic clades of Aureobasidium pullulans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    More than 90 isolates of the fungus A. pullulans from tropical and temperate climates were classified into 13 phylogenetic clades using multilocus sequence analyses (ITS, IGS, BT2, RPB2, and EF-1a). Tropical isolates appeared to exhibit the greatest genetic diversity. Representatives of each clade...

  15. The Longibrachiatum Clade of Trichoderma: a revision with new species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Longibrachiatum Clade of Trichoderma is revised. Eight new species are described (T. aethiopicum, T. capillare, T. flagellatum, T. gillesii, T. gracile, T. pinnatum, T. saturnisporopsis, T. solani). The twenty-one species known to belong to the Longibrachiatum Clade are included in a synoptic ke...

  16. Two new species of Lactarius associated with Alnus acuminata subsp. arguta in Mexico.

    PubMed

    Montoya, Leticia; Bandala, Victor M; Garay, Edith

    2014-01-01

    In pure stands of Alnus acuminata subsp. arguta trees from Sierra Norte de Puebla (central Mexico) two undescribed ectomycorrhizal species of Lactarius were discovered. Distinction of the two new species is based on morphological characters and supported with phylogenetic analyses of the nuclear ribosomal DNA ITS region and part of the gene that encodes for the second largest subunit of RNA polymerase II (rpb2). The phylogenies inferred recovered the two species in different clades strongly supported by posterior probabilities and bootstrap values. The new Lactarius species are recognized as part of the assemblage of ectomycorrhizal fungi associated with Alnus acuminata. Information about these taxa includes the morphological variation achieved along 16 monitories 2010-2013. Descriptions are provided. They are accompanied by photos including SEM photomicrographs of basidiospores and information on differences between them and other related taxa from Europe and the United States. PMID:24895428

  17. Streptococcus equi subsp. zooepidemicus meningitis in Peru

    PubMed Central

    Guevara, Jose M.; Tilley, Drake H.; Briceno, Jesus A.; Zunt, Joseph R.; Montano, Silvia M.

    2013-01-01

    A 59-year-old man with a history of fever, unsteadiness, hemiparesis, motor aphasia and consciousness disturbance was hospitalized for Streptococcus equi subsp. zooepidemicus meningitis. He denied contact with farm animals, but had a practice of consuming unpasteurized goats’ cheese from an uncertain source. PMID:23105024

  18. Campylobacter fetus subsp. fetus in homosexual males.

    PubMed Central

    Devlin, H R; McIntyre, L

    1983-01-01

    Campylobacter fetus subsp. fetus was isolated from the stools of two homosexual males. One was asymptomatic at the time of isolation. The other presented with diarrhea. Both isolates were initially grown at 42 degrees C. This organism should be included among the list of organisms that are found in homosexual males. PMID:6630480

  19. Phylogenetic Analysis and Polyphasic Characterization of Clavibacter michiganensis Strains Isolated from Tomato Seeds Reveal that Nonpathogenic Strains Are Distinct from C. michiganensis subsp. michiganensis

    PubMed Central

    Durand, Karine; Orgeur, Geoffrey; Balidas, Samuel; Fricot, Céline; Bonneau, Sophie; Quillévéré, Anne; Audusseau, Corinne; Olivier, Valérie; Grimault, Valérie; Mathis, René

    2012-01-01

    The genus Clavibacter comprises one species and five subspecies of plant-pathogenic bacteria, four of which are classified as quarantine organisms due to the high economic threat they pose. Clavibacter michiganensis subsp. michiganensis is one of the most important pathogens of tomato, but the recommended diagnostic tools are not satisfactory due to false-negative and/or -positive results. To provide a robust analysis of the genetic relatedness among a worldwide collection of C. michiganensis subsp. michiganensis strains, relatives (strains from the four other C. michiganensis subspecies), and nonpathogenic Clavibacter-like strains isolated from tomato, we performed multilocus sequence-based analysis and typing (MLSA and MLST) based on six housekeeping genes (atpD, dnaK, gyrB, ppK, recA, and rpoB). We compared this “framework” with phenotypic and genotypic characteristics such as pathogenicity on tomato, reaction to two antisera by immunofluorescence and to five PCR identification tests, and the presence of four genes encoding the main C. michiganensis subsp. michiganensis pathogenicity determinants. We showed that C. michiganensis subsp. michiganensis is monophyletic and is distinct from its closest taxonomic neighbors. The nonpathogenic Clavibacter-like strains were identified as C. michiganensis using 16S rRNA gene sequencing. These strains, while cross-reacting with C. michiganensis subsp. michiganensis identification tools, are phylogenetically distinct from the pathogenic strains but belong to the C. michiganensis clade. C. michiganensis subsp. michiganensis clonal complexes linked strains from highly diverse geographical origins and also strains isolated over long periods of time in the same location. This illustrates the importance of seed transmission in the worldwide dispersion of this pathogen and its survival and adaptation abilities in a new environment once introduced. PMID:23001675

  20. Rapid High Resolution Genotyping of Francisella tularensis by Whole Genome Sequence Comparison of Annotated Genes (“MLST+”)

    PubMed Central

    Mellmann, Alexander; Höppner, Sebastian; Splettstoesser, Wolf D.; Harmsen, Dag

    2015-01-01

    The zoonotic disease tularemia is caused by the bacterium Francisella tularensis. This pathogen is considered as a category A select agent with potential to be misused in bioterrorism. Molecular typing based on DNA-sequence like canSNP-typing or MLVA has become the accepted standard for this organism. Due to the organism’s highly clonal nature, the current typing methods have reached their limit of discrimination for classifying closely related subpopulations within the subspecies F. tularensis ssp. holarctica. We introduce a new gene-by-gene approach, MLST+, based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains and apply this approach to investigate an epidemic of lethal tularemia among non-human primates in two animal facilities in Germany. Due to the high resolution of MLST+ we are able to demonstrate that three independent clones of this highly infectious pathogen were responsible for these spatially and temporally restricted outbreaks. PMID:25856198

  1. An approach to the identification of T cell epitopes in the genomic era: application to Francisella tularensis

    PubMed Central

    Valentino, Michael; Frelinger, John

    2016-01-01

    The identification and characterization of epitopes is essential for modern immunologic studies. Here we describe a novel methodology we have developed to identify T cell epitopes exploiting the phenomenon of cross presentation. Particulate antigens, in the form of beads, are very effective in delivering exogenous antigen to both the class I and class II pathways. We will review our efforts to screen entire genomes of pathogens for T cell epitopes taking advantage of the advances in genomics using Francisella tularensis as a model. By automating aspects of this technology we will be able to functionally screen the entire genome of F. tularensis for T cell epitopes. This technology should be applicable not only to F. tularensis, but also to many other pathogens as well. PMID:19212707

  2. Disparate Host Immunity to Mycobacterium avium subsp. paratuberculosis Antigens in Calves Inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii, and M. bovis

    PubMed Central

    Waters, W. R.; Bannantine, J. P.; Palmer, M. V.

    2013-01-01

    The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratuberculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with either M. avium subsp. paratuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO+ CD25+ T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting

  3. The first closed genome sequence of Campylobacter fetus subsp. venerealis biovar intermedius

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter fetus venerealis biovar intermedius is a variant of Campylobacter fetus subsp. venerealis, the causative agent of Bovine Genital Campylobacteriosis. In contrast to Campylobacter fetus subsp. venerealis which is restricted to the genital tract of cattle, Campylobacter fetus subsp. vener...

  4. Mouse Models of Aerosol-Acquired Tularemia Caused by Francisella tularensis Types A and B

    PubMed Central

    Fritz, David L; England, Marilyn J; Miller, Lynda; Waag, David M

    2014-01-01

    After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans. PMID:25402174

  5. Nonrandom Distribution of Vector Ticks (Dermacentor variabilis) Infected by Francisella tularensis

    PubMed Central

    Goethert, Heidi K.; Telford, Sam R.

    2009-01-01

    The island of Martha's Vineyard, Massachusetts, is the site of a sustained outbreak of tularemia due to Francisella tularensis tularensis. Dog ticks, Dermacentor variabilis, appear to be critical in the perpetuation of the agent there. Tularemia has long been characterized as an agent of natural focality, stably persisting in characteristic sites of transmission, but this suggestion has never been rigorously tested. Accordingly, we sought to identify a natural focus of transmission of the agent of tularemia by mapping the distribution of PCR-positive ticks. From 2004 to 2007, questing D. variabilis were collected from 85 individual waypoints along a 1.5 km transect in a field site on Martha's Vineyard. The positions of PCR-positive ticks were then mapped using ArcGIS. Cluster analysis identified an area approximately 290 meters in diameter, 9 waypoints, that was significantly more likely to yield PCR-positive ticks (relative risk 3.3, P = 0.001) than the rest of the field site. Genotyping of F. tularensis using variable number tandem repeat (VNTR) analysis on PCR-positive ticks yielded 13 different haplotypes, the vast majority of which was one dominant haplotype. Positive ticks collected in the cluster were 3.4 times (relative risk = 3.4, P<0.0001) more likely to have an uncommon haplotype than those collected elsewhere from the transect. We conclude that we have identified a microfocus where the agent of tularemia stably perpetuates and that this area is where genetic diversity is generated. PMID:19247435

  6. A Conserved and Immunodominant Lipoprotein of Francisella tularensis is Proinflammatory but not Essential for Virulence

    PubMed Central

    Forestal, Colin A.; Gil, Horacio; Monfett, Michael; Noah, Courtney E.; Platz, Gabrielle J.; Thanassi, David G.; Benach, Jorge L.; Furie, Martha B.

    2008-01-01

    Francisella tularensis is a highly virulent bacterium that causes tularemia, a disease that is often fatal if untreated. A live vaccine strain (LVS) of this bacterium is attenuated for virulence in humans but produces lethal disease in mice. F. tularensis has been classified as a Category A agent of bioterrorism. Despite this categorization, little is known about the components of the organism that are responsible for causing disease in its hosts. Here, we report the deletion of a well-characterized lipoprotein of F. tularensis, designated LpnA (also known as Tul4), in the LVS. An LpnA deletion mutant was comparable to the wild-type strain in its ability to grow intracellularly and cause lethal disease in mice. Additionally, mice inoculated with a sublethal dose of the mutant strain were afforded the same protection against a subsequent lethal challenge with the LVS as were mice initially administered a sublethal dose of the wild-type bacterium. The LpnA-deficient strain showed an equivalent ability to promote secretion of chemokines by human monocyte-derived macrophages as its wild-type counterpart. However, recombinant LpnA potently stimulated primary cultures of human macrophages in a Toll-like receptor 2-dependent manner. Although human endothelial cells also were activated by recombinant LpnA, their response was relatively modest. LpnA is clearly unnecessary for multiple functions of the LVS, but its inflammatory capacity implicates it and other Francisella lipoproteins as potentially important to the pathogenesis of tularemia. PMID:18304778

  7. Automated microfluidically controlled electrochemical biosensor for the rapid and highly sensitive detection of Francisella tularensis.

    PubMed

    Dulay, Samuel B; Gransee, Rainer; Julich, Sandra; Tomaso, Herbert; O'Sullivan, Ciara K

    2014-09-15

    Tularemia is a highly infectious zoonotic disease caused by a Gram-negative coccoid rod bacterium, Francisella tularensis. Tularemia is considered as a life-threatening potential biological warfare agent due to its high virulence, transmission, mortality and simplicity of cultivation. In the work reported here, different electrochemical immunosensor formats for the detection of whole F. tularensis bacteria were developed and their performance compared. An anti-Francisella antibody (FB11) was used for the detection that recognises the lipopolysaccharide found in the outer membrane of the bacteria. In the first approach, gold-supported self-assembled monolayers of a carboxyl terminated bipodal alkanethiol were used to covalently cross-link with the FB11 antibody. In an alternative second approach F(ab) fragments of the FB11 antibody were generated and directly chemisorbed onto the gold electrode surface. The second approach resulted in an increased capture efficiency and higher sensitivity. Detection limits of 4.5 ng/mL for the lipopolysaccharide antigen and 31 bacteria/mL for the F. tularensis bacteria were achieved. Having demonstrated the functionality of the immunosensor, an electrode array was functionalised with the antibody fragment and integrated with microfluidics and housed in a tester set-up that facilitated complete automation of the assay. The only end-user intervention is sample addition, requiring less than one-minute hands-on time. The use of the automated microfluidic set-up not only required much lower reagent volumes but also the required incubation time was considerably reduced and a notable increase of 3-fold in assay sensitivity was achieved with a total assay time from sample addition to read-out of less than 20 min. PMID:24747573

  8. Ubiquitous Promoter-Localization of Essential Virulence Regulators in Francisella tularensis

    PubMed Central

    Ramsey, Kathryn M.; Osborne, Melisa L.; Vvedenskaya, Irina O.; Su, Cathy; Nickels, Bryce E.; Dove, Simon L.

    2015-01-01

    Francisella tularensis is a Gram-negative bacterium whose ability to replicate within macrophages and cause disease is strictly dependent upon the coordinate activities of three transcription regulators called MglA, SspA, and PigR. MglA and SspA form a complex that associates with RNA polymerase (RNAP), whereas PigR is a putative DNA-binding protein that functions by contacting the MglA-SspA complex. Most transcription activators that bind the DNA are thought to occupy only those promoters whose activities they regulate. Here we show using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq) that PigR, MglA, and SspA are found at virtually all promoters in F. tularensis and not just those of regulated genes. Furthermore, we find that the ability of PigR to associate with promoters is dependent upon the presence of MglA, suggesting that interaction with the RNAP-associated MglA-SspA complex is what directs PigR to promoters in F. tularensis. Finally, we present evidence that the ability of PigR (and thus MglA and SspA) to positively control the expression of genes is dictated by a specific 7 base pair sequence element that is present in the promoters of regulated genes. The three principal regulators of virulence gene expression in F. tularensis therefore function in a non-classical manner with PigR interacting with the RNAP-associated MglA-SspA complex at the majority of promoters but only activating transcription from those that contain a specific sequence element. Our findings reveal how transcription factors can exert regulatory effects at a restricted set of promoters despite being associated with most or all. This distinction between occupancy and regulatory effect uncovered by our data may be relevant to the study of RNAP-associated transcription regulators in other pathogenic bacteria. PMID:25830507

  9. Staphylococcus petrasii subsp. pragensis subsp. nov., occurring in human clinical material.

    PubMed

    Švec, Pavel; De Bel, Annelies; Sedláček, Ivo; Petráš, Petr; Gelbíčová, Tereza; Černohlávková, Jitka; Mašlanˇová, Ivana; Cnockaert, Margo; Varbanovová, Ivana; Echahidi, Fedoua; Vandamme, Peter; Pantuček, Roman

    2015-07-01

    Seven coagulase-negative, oxidase-negative and novobiocin-susceptible staphylococci assigned tentatively as Staphylococcus petrasii were investigated in this study in order to elucidate their taxonomic position. All strains were initially shown to form a genetically homogeneous group separated from remaining species of the genus Staphylococcus by using a repetitive sequence-based PCR fingerprinting with the (GTG)5 primer. Phylogenetic analysis based on 16S rRNA gene, hsp60, rpoB, dnaJ, gap and tuf sequences showed that the group is closely related to Staphylococcus petrasii but separated from the three hitherto known subspecies, S. petrasii subsp. petrasii, S. petrasii subsp. croceilyticus and S. petrasii subsp. jettensis. Further investigation using automated ribotyping, MALDI-TOF mass spectrometry, fatty acid methyl ester analysis, DNA-DNA hybridization and extensive biotyping confirmed that the analysed group represents a novel subspecies within S. petrasii, for which the name Staphylococcus petrasii subsp. pragensis subsp. nov. is proposed. The type strain is NRL/St 12/356(T) ( = CCM 8529(T) = LMG 28327(T)). PMID:25829332

  10. Complete genome sequence of Bifidobacterium animalis subsp. lactis BLC1.

    PubMed

    Bottacini, Francesca; Dal Bello, Fabio; Turroni, Francesca; Milani, Christian; Duranti, Sabrina; Foroni, Elena; Viappiani, Alice; Strati, Francesco; Mora, Diego; van Sinderen, Douwe; Ventura, Marco

    2011-11-01

    Bifidobacterium animalis subsp. lactis BLC1 is a probiotic bacterium that is widely exploited by food industries as the active ingredient of various functional foods. Here we report the complete genome sequence of B. animalis subsp. lactis BLC1, which is expected to provide insights into the biology of this health-promoting microorganism and improve our understanding of its phylogenetic relatedness with other members of the B. animalis subsp. lactis taxon. PMID:22038957

  11. Lipase production by diverse phylogenetic clades of Aureobasidium pullulans.

    PubMed

    Leathers, Timothy D; Rich, Joseph O; Anderson, Amber M; Manitchotpisit, Pennapa

    2013-10-01

    Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2-0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains in clade 9, which exhibit a dark olivaceous pigment, produced the highest levels of lipase, with strain NRRL 62034 yielding 0.57 U lipase/ml. By comparison, Candida cylindracea strain NRRL Y-17506 produced 0.05 U lipase/ml under identical conditions. A. pullulans strain NRRL 62034 reached maximal lipase levels in 5 days on lipase induction medium, while A. pullulans strain NRRL Y-2311-1 and strains in clades 4 and 10 were highest after 6 days. A. pullulans strain NRRL Y-2311-1 and strains in clade 9 produced two extracellular proteins in common, at >50 and <37 kDa. PMID:23801121

  12. Gastrointestinal Tract Colonization Dynamics by Different Enterococcus faecium Clades.

    PubMed

    Montealegre, Maria Camila; Singh, Kavindra V; Murray, Barbara E

    2016-06-15

    Colonization of the gastrointestinal tract (GIT) generally precedes infection with antibiotic-resistant Enterococcus faecium We used a mouse GIT colonization model to test differences in the colonization levels by strains from different E. faecium lineages: clade B, part of the healthy human microbiota; subclade A1, associated with infections; and subclade A2, primarily associated with animals. After mono-inoculation, there was no significant difference in colonization (measured as the geometric mean number of colony-forming units per gram) by the E. faecium clades at any time point (P > .05). However, in competition assays, with 6 of the 7 pairs, clade B strains outcompeted clade A strains in their ability to persist in the GIT; this difference was significant in some pairs by day 2 and in all pairs by day 14 (P < .0008-.0283). This observation may explain the predominance of clade B in the community and why antibiotic-resistant hospital-associated E. faecium are often replaced by clade B strains once patients leave the hospital. PMID:26671890

  13. The hominin fossil record: taxa, grades and clades

    PubMed Central

    Wood, Bernard; Lonergan, Nicholas

    2008-01-01

    This paper begins by reviewing the fossil evidence for human evolution. It presents summaries of each of the taxa recognized in a relatively speciose hominin taxonomy. These taxa are grouped in grades, namely possible and probable hominins, archaic hominins, megadont archaic hominins, transitional hominins, pre-modern Homo and anatomically modern Homo. The second part of this contribution considers some of the controversies that surround hominin taxonomy and systematics. The first is the vexed question of how you tell an early hominin from an early panin, or from taxa belonging to an extinct clade closely related to the Pan-Homo clade. Secondly, we consider how many species should be recognized within the hominin fossil record, and review the philosophies and methods used to identify taxa within the hominin fossil record. Thirdly, we examine how relationships within the hominin clade are investigated, including descriptions of the methods used to break down an integrated structure into tractable analytical units, and then how cladograms are generated and compared. We then review the internal structure of the hominin clade, including the problem of how many subclades should be recognized within the hominin clade, and we examine the reliability of hominin cladistic hypotheses. The last part of the paper reviews the concepts of a genus, including the criteria that should be used for recognizing genera within the hominin clade. PMID:18380861

  14. Antibacterial Activity of Alkyl Gallates against Xanthomonas citri subsp. citri

    PubMed Central

    Silva, I. C.; Regasini, L. O.; Petrônio, M. S.; Silva, D. H. S.; Bolzani, V. S.; Belasque, J.; Sacramento, L. V. S.

    2013-01-01

    The plant-pathogenic bacterium Xanthomonas citri subsp. citri is the causal agent of Asiatic citrus canker, a serious disease that affects all the cultivars of citrus in subtropical citrus-producing areas worldwide. There is no curative treatment for citrus canker; thus, the eradication of infected plants constitutes the only effective control of the spread of X. citri subsp. citri. Since the eradication program in the state of São Paulo, Brazil, is under threat, there is a clear risk of X. citri subsp. citri becoming endemic in the main orange-producing area in the world. Here we evaluated the potential use of alkyl gallates to prevent X. citri subsp. citri growth. These esters displayed a potent anti-X. citri subsp. citri activity similar to that of kanamycin (positive control), as evaluated by the resazurin microtiter assay (REMA). The treatment of X. citri subsp. citri cells with these compounds induced altered cell morphology, and investigations of the possible intracellular targets using X. citri subsp. citri strains labeled for the septum and centromere pointed to a common target involved in chromosome segregation and cell division. Finally, the artificial inoculation of citrus with X. citri subsp. citri cells pretreated with alkyl gallates showed that the bacterium loses the ability to colonize its host, which indicates the potential of these esters to protect citrus plants against X. citri subsp. citri infection. PMID:23104804

  15. Aeromonas hydrophila subsp. ranae subsp. nov., isolated from septicaemic farmed frogs in Thailand.

    PubMed

    Huys, Geert; Pearson, Marianne; Kämpfer, Peter; Denys, Rik; Cnockaert, Margo; Inglis, Valerie; Swings, Jean

    2003-05-01

    A group of seven sucrose-negative Aeromonas strains (referred to as group Au) isolated from the internal organs of septicaemic farmed frogs (Rana rugulosa) in Thailand was subjected to a polyphasic taxonomic study including fluorescent amplified fragment length polymorphism (FAFLP) and ERIC-PCR fingerprinting, 16S rDNA sequencing, microplate DNA-DNA hybridizations and extensive phenotypic characterization. Comparison of FAFLP and ERIC-PCR fingerprints indicated that the group Au isolates belonged to the species Aeromonas hydrophila DNA hybridization group (HG) 1 in which they represent a genotypic subgroup closely affiliated to A. hydrophila subsp. hydrophila and subsp. dhakensis. One representative of the Au group exhibited > or = 99.0% 16S rDNA sequence similarity with the type strains of the two A. hydrophila subspecies. DNA-DNA hybridization with type and reference strains of all known Aeromonas taxa revealed that the Au group represented a homogeneous taxon that exhibited the highest relatedness with members of the two A. hydrophila subspecies, ranging from 75 to 93%. Phenotypic characterization on the basis of 152 features further revealed that the Au group isolates differed from A. hydrophila subsp. hydrophila or subsp. dhakensis in a total of 13 biochemical properties. Of these, assimilation of L-glycine and isobutyrate as sole carbon source, acid production from salicin and D-sucrose, and aesculin hydrolysis were of diagnostic value. From the results of this study, it can be concluded that the Aeromonas frog isolates of the Au group represent a new subspecies of A. hydrophila, for which the name Aeromonas hydrophila subsp. ranae subsp. nov. is proposed. Its type strain is Au-1D12(T) (=LMG 19707(T) = CCUG 46211(T)). PMID:12807217

  16. IL-10 Restrains IL-17 to Limit Lung Pathology Characteristics following Pulmonary Infection with Francisella tularensis Live Vaccine Strain

    PubMed Central

    Slight, Samantha R.; Monin, Leticia; Gopal, Radha; Avery, Lyndsay; Davis, Marci; Cleveland, Hillary; Oury, Tim D.; Rangel-Moreno, Javier; Khader, Shabaana A.

    2014-01-01

    IL-10 production during intracellular bacterial infections is generally thought to be detrimental because of its role in suppressing protective T-helper cell 1 (Th1) responses. Francisella tularensis is a facultative intracellular bacterium that activates both Th1 and Th17 protective immune responses. Herein, we report that IL-10–deficient mice (Il10−/−), despite having increased Th1 and Th17 responses, exhibit increased mortality after pulmonary infection with F. tularensis live vaccine strain. We demonstrate that the increased mortality observed in Il10−/−-infected mice is due to exacerbated IL-17 production that causes increased neutrophil recruitment and associated lung pathology. Thus, although IL-17 is required for protective immunity against pulmonary infection with F. tularensis live vaccine strain, its production is tightly regulated by IL-10 to generate efficient induction of protective immunity without mediating pathology. These data suggest a critical role for IL-10 in maintaining the delicate balance between host immunity and pathology during pulmonary infection with F. tularensis live vaccine strain. PMID:24007881

  17. Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

    PubMed

    Abdelbaqi, Suha; Deslouches, Berthony; Steckbeck, Jonathan; Montelaro, Ronald; Reed, Douglas S

    2016-02-01

    Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections. PMID:26673248

  18. Differential Induction of Interleukin-10 in Monocytes by HIV-1 Clade B and Clade C Tat Proteins*

    PubMed Central

    Wong, Justine K.; Campbell, Grant R.; Spector, Stephen A.

    2010-01-01

    The clade B human immunodeficiency virus, type 1 (HIV-1) Tat (trans-acting regulatory protein) induces interleukin-10 (IL-10) production in monocytes. IL-10, an anti-inflammatory cytokine, down-regulates proinflammatory cytokines and suppresses the immune response, leading to a rapid progression from HIV-1 infection to AIDS. Nine clades of HIV-1 are responsible for the majority of infections worldwide. Recent studies demonstrate that different HIV-1 clades have biological differences in relation to transmission, replication, and disease progression. In this study, we show that the cysteine to serine mutation at position 31, found in >90% of HIV-1 clade C Tat proteins, results in a marked decrease in IL-10 production in monocytes compared with clade B Tat. Additionally, the C31S mutation found in C Tat is responsible for the inability of these Tat proteins to produce high IL-10 levels in monocytes due to its inability to induce intracellular calcium flux through L-type calcium channels. Moreover, we show that p38α/p38β and phosphoinositide 3-kinase are crucial to Tat-induced IL-10 production. These findings provide further evidence that HIV-1 clades differ in their biological properties that may impact HIV-1 pathogenesis and disease progression. PMID:20378550

  19. Control of Intracellular Francisella tularensis by Different Cell Types and the Role of Nitric Oxide

    PubMed Central

    Newstead, Sarah L.; Gates, Amanda J.; Hartley, M. Gillian; Rowland, Caroline A.; Williamson, E. Diane; Lukaszewski, Roman A.

    2014-01-01

    Reactive nitrogen is critical for the clearance of Francisella tularensis infections. Here we assess the role of nitric oxide in control of intracellular infections in two murine macrophage cell lines of different provenance: the alveolar macrophage cell line, MH-S, and the widely used peritoneal macrophage cell line, J774A.1. Cells were infected with the highly virulent Schu S4 strain or with the avirulent live vaccine strain (LVS) with and without stimuli. Compared to MH-S cells, J774A.1 cells were unresponsive to stimulation and were able to control the intracellular replication of LVS bacteria, but not of Schu S4. In MH-S cells, Schu S4 demonstrated control over cellular NO production. Despite this, MH-S cells stimulated with LPS or LPS and IFN-γ were able to control intracellular Schu S4 numbers. However, only stimulation with LPS induced significant cellular NO production. Combined stimulation with LPS and IFN-γ produced a significant reduction in intracellular bacteria that occurred whether high levels of NO were produced or not, indicating that NO secretion is not the only defensive cellular mechanism operating in virulent Francisella infections. Understanding how F. tularensis interacts with host macrophages will help in the rational design of new and effective therapies. PMID:25170518

  20. Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication

    PubMed Central

    Checroun, Claire; Wehrly, Tara D.; Fischer, Elizabeth R.; Hayes, Stanley F.; Celli, Jean

    2006-01-01

    Intracellular bacterial pathogens evade the bactericidal functions of mammalian cells by physical escape from their phagosome and replication into the cytoplasm or through the modulation of phagosome maturation and biogenesis of a membrane-bound replicative organelle. Here, we detail in murine primary macrophages the intracellular life cycle of Francisella tularensis, a highly infectious bacterium that survives and replicates within mammalian cells. After transient interactions with the endocytic pathway, bacteria escaped from their phagosome by 1 h after infection and underwent replication in the cytoplasm from 4 to 20 h after infection. Unexpectedly, the majority of bacteria were subsequently found to be enclosed within large, juxtanuclear, LAMP-1-positive vacuoles called Francisella-containing vacuoles (FCVs). FCV formation required intracytoplasmic replication of bacteria. Using electron and fluorescence microscopy, we observed that the FCVs contained morphologically intact bacteria, despite fusing with lysosomes. FCVs are multimembranous structures that accumulate monodansylcadaverine and display the autophagy-specific protein LC3 on their membrane. Formation of FCVs was significantly inhibited by 3-methyladenine, confirming a role for the autophagic pathway in the biogenesis of these organelles. Taken together, our results demonstrate that, via autophagy, F. tularensis reenters the endocytic pathway after cytoplasmic replication, a process thus far undescribed for intracellular pathogens. PMID:16983090

  1. Humoral immune response of cottontail rabbits naturally infected with Francisella tularensis in southern Illinois.

    PubMed

    Shoemaker, D; Woolf, A; Kirkpatrick, R; Cooper, M

    1997-10-01

    Cottontail rabbits (Sylvilagus floridanus) usually are thought to succumb to infection with Francisella tularensis. Reports of a rabbit population from southern Illinois (USA) with a high prevalence of F. tularensis antibodies suggested that some cottontails survived infection with this typically fatal bacterium. Our goal was to examine the humoral response of cottontails from a study area in southern Illinois for which multiple serum samples existed. Multiple sera were collected from 79 cottontails from 1986 to 1990 and 63% gained, lost, or maintained ELISA titers of IgM and IgG isotype antibodies. The typical pattern of antibody response appeared to be IgM isotype antibodies first, followed by IgG isotype antibodies, with both generally increasing to high titers. Negative culture attempts of liver tissue from 51 cottontails with varying antibody responses suggested that chronic infection did not occur in rabbits that developed antibody. The significance of the cottontail antibody response in resolution or prevention of tularemia infection remains unclear. PMID:9391956

  2. Immunodetection of inactivated Francisella tularensis bacteria by using a quartz crystal microbalance with dissipation monitoring.

    PubMed

    Kleo, K; Schäfer, D; Klar, S; Jacob, D; Grunow, R; Lisdat, F

    2012-08-01

    Francisella tularensis are very small, gram-negative bacteria which are capable of infecting a number of mammals. As a highly pathogenic species, it is a potential bioterrorism agent. In this work we demonstrate a fast immunological detection system for whole F. tularensis bacteria. The technique is based on a quartz crystal microbalance with dissipation monitoring (QCMD), which uses sensor chips modified by a specific antibody. This antibody is useful as a capture molecule to capture the lipopolysaccharide structure on the surface of the bacterial cell wall. The QCMD technique is combined with a microfluidic system and allows the label-free online detection of the binding of whole bacteria to the sensor surface in a wide dynamic concentration range. A detection limit of about 4 × 10(3) colony-forming units per milliliter can be obtained. Furthermore, a rather short analysis time and a clear discrimination against other bacteria can be achieved. Additionally, we demonstrate two possibilities for specific and significant signal enhancement by using antibody-functionalized gold nanoparticles or an enzymatic precipitation reaction. These additional steps can be seen as further proof of the specificity and validity. PMID:22722745

  3. Typical Preparation of Francisella tularensis O-antigen Yields a Mixture of Three Types of Saccharides†

    PubMed Central

    Wang, Qi; Shi, Xiaofeng; Leymarie, Nancy; Madico, Guillermo; Sharon, Jacqueline; Costello, Catherine E.; Zaia, Joseph

    2011-01-01

    Tularemia is a severe infectious disease in humans caused by the gram-negative bacterium Francisella tularensis (Ft). Due to its low infectious dose, high mortality rate, and the threat of its large-scale dissemination in weaponized form, development of vaccines and immunotherapeutics against Ft is essential. Ft lipopolysaccharide (LPS), which contains the linear graded-length saccharide component O-antigen (OAg) attached to a core oligosaccharide, has been reported as a protective antigen. Purification of LPS saccharides of defined length and composition is necessary to reveal the epitopes targeted by protective antibodies. In this study, we purified saccharides from LPS preparations from both the Ft subspecies holarctica live vaccine strain (LVS) and the virulent Ft subspecies tularensis SchuS4 strain using liquid chromatography. We then characterized the fractions using high resolution mass spectrometry and tandem mass spectrometry. Three types of saccharides were observed in both the LVS and SchuS4 preparations: two consisting of OAg tetrasaccharide repeats attached to one of two core-oligosaccharide variants, and one consisting of tetrasaccharide repeats only (coreless). The coreless OAg oligosaccharides were shown to contain Qui4NFm (4,6-dideoxy-4-formamido-D-glucose) at the non-reducing end and QuiNAc (2-acetamido-2,6-dideoxy-O-D-glucose) at the reducing end. Purified homogeneous preparations of saccharides of each type will allow mapping of protective epitopes in Ft LPS. PMID:22091710

  4. A Full-Genomic Sequence-Verified Protein-Coding Gene Collection for Francisella tularensis

    PubMed Central

    Murthy, Tal; Rolfs, Andreas; Hu, Yanhui; Shi, Zhenwei; Raphael, Jacob; Moreira, Donna; Kelley, Fontina; McCarron, Seamus; Jepson, Daniel; Taycher, Elena; Zuo, Dongmei; Mohr, Stephanie E.; Fernandez, Mauricio; Brizuela, Leonardo; LaBaer, Joshua

    2007-01-01

    The rapid development of new technologies for the high throughput (HT) study of proteins has increased the demand for comprehensive plasmid clone resources that support protein expression. These clones must be full-length, sequence-verified and in a flexible format. The generation of these resources requires automated pipelines supported by software management systems. Although the availability of clone resources is growing, current collections are either not complete or not fully sequence-verified. We report an automated pipeline, supported by several software applications that enabled the construction of the first comprehensive sequence-verified plasmid clone resource for more than 96% of protein coding sequences of the genome of F. tularensis, a highly virulent human pathogen and the causative agent of tularemia. This clone resource was applied to a HT protein purification pipeline successfully producing recombinant proteins for 72% of the genes. These methods and resources represent significant technological steps towards exploiting the genomic information of F. tularensis in discovery applications. PMID:17593976

  5. Clades reach highest morphological disparity early in their evolution

    NASA Astrophysics Data System (ADS)

    Hughes, Martin; Gerber, Sylvain; Albion Wills, Matthew

    2013-08-01

    There are few putative macroevolutionary trends or rules that withstand scrutiny. Here, we test and verify the purported tendency for animal clades to reach their maximum morphological variety relatively early in their evolutionary histories (early high disparity). We present a meta-analysis of 98 metazoan clades radiating throughout the Phanerozoic. The disparity profiles of groups through time are summarized in terms of their center of gravity (CG), with values above and below 0.50 indicating top- and bottom-heaviness, respectively. Clades that terminate at one of the "big five" mass extinction events tend to have truncated trajectories, with a significantly top-heavy CG distribution overall. The remaining 63 clades show the opposite tendency, with a significantly bottom-heavy mean CG (relatively early high disparity). Resampling tests are used to identify groups with a CG significantly above or below 0.50; clades not terminating at a mass extinction are three times more likely to be significantly bottom-heavy than top-heavy. Overall, there is no clear temporal trend in disparity profile shapes from the Cambrian to the Recent, and early high disparity is the predominant pattern throughout the Phanerozoic. Our results do not allow us to distinguish between ecological and developmental explanations for this phenomenon. To the extent that ecology has a role, however, the paucity of bottom-heavy clades radiating in the immediate wake of mass extinctions suggests that early high disparity more probably results from the evolution of key apomorphies at the base of clades rather than from physical drivers or catastrophic ecospace clearing.

  6. Clades reach highest morphological disparity early in their evolution

    PubMed Central

    Hughes, Martin; Gerber, Sylvain; Wills, Matthew Albion

    2013-01-01

    There are few putative macroevolutionary trends or rules that withstand scrutiny. Here, we test and verify the purported tendency for animal clades to reach their maximum morphological variety relatively early in their evolutionary histories (early high disparity). We present a meta-analysis of 98 metazoan clades radiating throughout the Phanerozoic. The disparity profiles of groups through time are summarized in terms of their center of gravity (CG), with values above and below 0.50 indicating top- and bottom-heaviness, respectively. Clades that terminate at one of the “big five” mass extinction events tend to have truncated trajectories, with a significantly top-heavy CG distribution overall. The remaining 63 clades show the opposite tendency, with a significantly bottom-heavy mean CG (relatively early high disparity). Resampling tests are used to identify groups with a CG significantly above or below 0.50; clades not terminating at a mass extinction are three times more likely to be significantly bottom-heavy than top-heavy. Overall, there is no clear temporal trend in disparity profile shapes from the Cambrian to the Recent, and early high disparity is the predominant pattern throughout the Phanerozoic. Our results do not allow us to distinguish between ecological and developmental explanations for this phenomenon. To the extent that ecology has a role, however, the paucity of bottom-heavy clades radiating in the immediate wake of mass extinctions suggests that early high disparity more probably results from the evolution of key apomorphies at the base of clades rather than from physical drivers or catastrophic ecospace clearing. PMID:23884651

  7. Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita, Osamu; Mizoguchi, Toshio; Shindo, Junji; Park, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamada, Akio; Morikawa, Shigeru; Tanabayashi, Kiyoshi

    2014-04-01

    Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild. PMID:24689989

  8. Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

    PubMed Central

    Jawaid, Safdar; Seidle, Heather; Zhou, Weidong; Abdirahman, Hafsa; Abadeer, Maher; Hix, Joseph H.; van Hoek, Monique L.; Couch, Robin D.

    2009-01-01

    Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (KMDXP = 104 µM, KMNADPH = 13 µM, kcatDXP = 2 s−1, kcatNADPH = 1.3 s−1), an IC50 for fosmidomycin of 247 nM, and a Ki for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg+2 as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis. PMID:20011597

  9. Identification of trkH, Encoding a Potassium Uptake Protein Required for Francisella tularensis Systemic Dissemination in Mice

    PubMed Central

    Alkhuder, Khaled; Meibom, Karin L.; Dubail, Iharilalao; Dupuis, Marion; Charbit, Alain

    2010-01-01

    Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. During its infectious cycle, F. tularensis is not only exposed to the intracellular environment of macrophages but also resides transiently in extracellular compartments, in particular during its systemic dissemination. The screening of a bank of F. tularensis LVS transposon insertion mutants on chemically defined medium (CDM) led us to identify a gene, designated trkH, encoding a homolog of the potassium uptake permease TrkH. Inactivation of trkH impaired bacterial growth in CDM. Normal growth of the mutant was only restored when CDM was supplemented with potassium at high concentration. Strikingly, although not required for intracellular survival in cell culture models, TrkH appeared to be essential for bacterial virulence in the mouse. In vivo kinetics of bacterial dissemination revealed a severe defect of multiplication of the trkH mutant in the blood of infected animals. The trkH mutant also showed impaired growth in blood ex vivo. Genome sequence analyses suggest that the Trk system constitutes the unique functional active potassium transporter in both tularensis and holarctica subspecies. Hence, the impaired survival of the trkH mutant in vivo is likely to be due to its inability to survive in the low potassium environment (1–5 mM range) of the blood. This work unravels thus the importance of potassium acquisition in the extracellular phase of the F. tularensis infectious cycle. More generally, potassium could constitute an important mineral nutrient involved in other diseases linked to systemic dissemination of bacterial pathogens. PMID:20126460

  10. Cell-mediated and humoral immune responses induced by scarification vaccination of human volunteers with a new lot of the live vaccine strain of Francisella tularensis.

    PubMed Central

    Waag, D M; Galloway, A; Sandstrom, G; Bolt, C R; England, M J; Nelson, G O; Williams, J C

    1992-01-01

    Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis. We evaluated a new lot of live F. tularensis vaccine for its immunogenicity in human volunteers. Scarification vaccination induced humoral and cell-mediated immune responses. Indications of a positive immune response after vaccination included an increase in specific antibody levels, which were measured by enzyme-linked immunosorbent and immunoblot assays, and the ability of peripheral blood lymphocytes to respond to whole F. tularensis bacteria as recall antigens. Vaccination caused a significant rise (P less than 0.05) in immunoglobulin A (IgA), IgG, and IgM titers. Lymphocyte stimulation indices were significantly increased (P less than 0.01) in vaccinees 14 days after vaccination. These data verify that this new lot of live F. tularensis vaccine is immunogenic. Images PMID:1400988

  11. Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation.

    PubMed

    Genersch, Elke; Forsgren, Eva; Pentikäinen, Jaana; Ashiralieva, Ainura; Rauch, Sandra; Kilwinski, Jochen; Fries, Ingemar

    2006-03-01

    A polyphasic taxonomic study of the two subspecies of Paenibacillus larvae, Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens, supported the reclassification of the subspecies into one species, Paenibacillus larvae, without subspecies separation. Our conclusions are based on the analysis of six reference strains of P. larvae subsp. pulvifaciens and three reference strains and 44 field isolates of P. larvae. subsp. larvae. The latter originated from brood or honey of clinically diseased honey bee colonies or from honey of both clinically diseased and asymptomatic colonies from Sweden, Finland and Germany. Colony and spore morphology, as well as the metabolism of mannitol and salicin, did not allow a clear identification of the two subspecies and SDS-PAGE of whole-cell proteins did not support the subspecies differentiation. For genomic fingerprinting, repetitive element-PCR fingerprinting using ERIC primers and PFGE of bacterial DNA were performed. The latter method is a high-resolution DNA fingerprinting method proven to be superior to most other methods for biochemical and molecular typing and has not previously been used to characterize P. larvae. ERIC-PCR identified four different genotypes, while PFGE revealed two main clusters. One cluster included most of the P. larvae subsp. larvae field isolates, as well as all P. larvae subsp. pulvifaciens reference strains. The other cluster comprised the pigmented variants of P. larvae subsp. larvae. 16S rRNA gene sequences were determined for some strains. Finally, exposure bioassays demonstrated that reference strains of P. larvae subsp. pulvifaciens were pathogenic for honey bee larvae, producing symptoms similar to reference strains of P. larvae subsp. larvae. In comparison with the type strain for P. larvae subsp. larvae, ATCC 9545T, the P. larvae subsp. pulvifaciens strains tested were even more virulent, since they showed a shorter LT100. An emended description of the species is given

  12. Clades and clans: a comparison study of two evolutionary models.

    PubMed

    Zhu, Sha; Than, Cuong; Wu, Taoyang

    2015-07-01

    The Yule-Harding-Kingman (YHK) model and the proportional to distinguishable arrangements (PDA) model are two binary tree generating models that are widely used in evolutionary biology. Understanding the distributions of clade sizes under these two models provides valuable insights into macro-evolutionary processes, and is important in hypothesis testing and Bayesian analyses in phylogenetics. Here we show that these distributions are log-convex, which implies that very large clades or very small clades are more likely to occur under these two models. Moreover, we prove that there exists a critical value κ(n) for each n ≥ 4 such that for a given clade with size k, the probability that this clade is contained in a random tree with n leaves generated under the YHK model is higher than that under the PDA model if 1 < k < κ(n),and lower if κ(n) < k < n. Finally, we extend our results to binary unrooted trees, and obtain similar results for the distributions of clan sizes. PMID:25047805

  13. Complete Genome Sequence of Mycobacterium abscessus subsp. bolletii

    PubMed Central

    Spilker, Theodore; LiPuma, John J.

    2016-01-01

    We report the complete genome sequence of a Mycobacterium abscessus subsp. bolletii isolate recovered from a sputum culture from an individual with cystic fibrosis. This sequence is the first completed whole-genome sequence of M. abscessus subsp. bolletii and adds value to studies of M. abscessus complex genomics. PMID:27284156

  14. Treponema pallidum subsp. pertenue displays pathogenic properties different from those of T. pallidum subsp. pallidum.

    PubMed

    Wicher, K; Wicher, V; Abbruscato, F; Baughn, R E

    2000-06-01

    The present study described the susceptibility of C4D guinea pigs to cutaneous infection with Treponema pallidum subsp. pertenue Haiti B strain. The general manifestations of the disease in adults and neonates differ, to a certain degree, from those induced by T. pallidum subsp. pallidum Nichols strain. Noticeable differences between the infections were reflected in the character of the skin lesions, their onset and persistence, and the kinetics of the humoral response. The incidence and dissemination of cutaneous yaws lesions in very young guinea pigs were remarkably different from the low frequency observed in a similar age group of syphilis infection, 100 versus 17%, respectively. Moreover, as opposed to T. pallidum subsp. pallidum, T. pallidum subsp. pertenue does not cross the placenta. Offspring born to yaws-infected mothers did not produce immunoglobulin M antibodies and their organs, examined by PCR and rabbit infectivity test (RIT), were all negative. Examination of a large number of tissues and organs in adult, neonate, and maternal yaws by PCR and RIT clearly demonstrated that, unlike syphilis, there was a low incidence and short persistence of the yaws pathogen in internal organs. These findings stress the dermotropic rather than the organotropic character of yaws and provide further evidence of distinctive biological and pathological differences between yaws and venereal syphilis. PMID:10816466

  15. Primate phylogeny: molecular evidence for a pongid clade excluding humans and a prosimian clade containing tarsiers.

    PubMed

    Huang, Shi

    2012-08-01

    Unbiased readings of fossils are well known to contradict some of the popular molecular groupings among primates, particularly with regard to great apes and tarsiers. The molecular methodologies today are however flawed as they are based on a mistaken theoretical interpretation of the genetic equidistance phenomenon that originally started the field. An improved molecular method the 'slow clock' was here developed based on the Maximum Genetic Diversity hypothesis, a more complete account of the unified changes in genotypes and phenotypes. The method makes use of only slow evolving sequences and requires no uncertain assumptions or mathematical corrections and hence is able to give definitive results. The findings indicate that humans are genetically more distant to orangutans than African apes are and separated from the pongid clade ∼17.6 million years ago. Also, tarsiers are genetically closer to lorises than simian primates are. Finally, the fossil times for the radiation of mammals at the K/T boundary and for the Eutheria-Metatheria split in the Early Cretaceous were independently confirmed from molecular dating calibrated using the fossil split times of gorilla-orangutan, mouse-rat, and opossum-kangaroo. Therefore, the re-established primate phylogeny indicates a remarkable unity between molecules and fossils. PMID:22932887

  16. EmrA1 Membrane Fusion Protein of Francisella tularensis LVS is required for Resistance to Oxidative Stress, Intramacrophage Survival and Virulence in Mice

    PubMed Central

    Ma, Zhuo; Banik, Sukalyani; Rane, Harshita; Mora, Vanessa T.; Rabadi, Seham M.; Doyle, Christopher R.; Thanassi, David G.; Bakshi, Chandra Shekhar; Malik, Meenakshi

    2014-01-01

    Francisella tularensis is a Category A Biodefense agent that causes a fatal human disease known as tularemia. The pathogenicity of F. tularensis depends on its ability to persist inside host immune cells primarily by resisting an attack from host-generated reactive oxygen and nitrogen species (ROS/RNS). Based on the ability of F. tularensis to resist high ROS/RNS levels, we have hypothesized that additional unknown factors act in conjunction with known antioxidant defenses to render ROS resistance. By screening a transposon insertion library of F. tularensis LVS in the presence of hydrogen peroxide, we have identified an oxidant sensitive mutant in putative EmrA1 (FTL_0687) secretion protein. The results demonstrate that the emrA1 mutant is highly sensitive to oxidants and several antimicrobial agents, and exhibits diminished intramacrophage growth that can be restored to wild type F. tularensis LVS levels either by transcomplementation, inhibition of ROS generation, or infection in NADPH oxidase deficient (gp91Phox−/−) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox−/− mice. Further, EmrA1 contributes to oxidative stress resistance by affecting secretion of Francisella antioxidant enzymes SodB and KatG. This study exposes unique links between transporter activity and the antioxidant defense mechanisms of F. tularensis. PMID:24397487

  17. Worldwide emergence of multiple clades of enterovirus 68

    PubMed Central

    Firth, Cadhla; Madhi, Shabir A.; Howie, Stephen R. C.; Wu, Winfred; Sall, Amadou Alpha; Haq, Saddef; Briese, Thomas; Lipkin, W. Ian

    2012-01-01

    Human enterovirus 68 (EV-D68) is a historically rarely reported virus linked with respiratory disease. In the past 3 years, a large increase in respiratory disease associated with EV-D68 has been reported, with documented outbreaks in North America, Europe and Asia. In several outbreaks, genetic differences were identified among the circulating strains, indicating the presence of multiple clades. In this report, we analyse archived and novel EV-D68 strains from Africa and the USA, obtained from patients with respiratory illness. Phylogenetic analysis of all EV-D68 sequences indicates that, over the past two decades, multiple clades of the virus have emerged and spread rapidly worldwide. All clades appear to be currently circulating and contributing to respiratory disease. PMID:22694903

  18. Data supporting phylogenetic reconstructions of the Neotropical clade Gymnotiformes

    PubMed Central

    Tagliacollo, Victor A.; Bernt, Maxwell J.; Craig, Jack M.; Oliveira, Claudio; Albert, James S.

    2016-01-01

    Data is presented in support of model-based total evidence (MBTE) phylogenetic reconstructions of the Neotropical clade of Gymnotiformes “Model-based total evidence phylogeny of Neotropical electric knifefishes (Teleostei, Gymnotiformes)” (Tagliacollo et al., 2016) [1]). The MBTE phylogenies were inferred using a comprehensive dataset comprised of six genes (5277 bp) and 223 morphological characters for an ingroup taxon sample of 120 of 218 valid species and 33 of the 34 extant genera. The data in this article include primer sequences for gene amplification and sequencing, voucher information and GenBank accession numbers, descriptions of morphological characters, morphological synapomorphies for the recognized clades of Gymnotiformes, a supermatrix comprised of concatenated molecular and morphological data, and computer scripts to replicate MBTE inferences. We also included here Maximum-likelihood and Bayesian topologies, which support two main gymnotiform clades: Gymnotidae and Sternopygoidei, the latter comprised of Rhamphichthyoidea (Rhamphichthyidae+Hypopomidae) and Sinusoidea (Sternopygidae+Apteronotidae). PMID:26955648

  19. Complete genome sequences of Campylobacter hyointestinalis subsp. hyointestinalis strain LMG9260 and Campylobacter hyointestinalis subsp. lawsonii strain LMG15993

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter hyointestinalis is isolated primarily from ruminants and swine, but is also occasionally isolated from humans. C. hyointestinalis is currently divided into two subspecies: subsps. hyointestinalis and lawsonii. This study describes the first closed whole-genome sequences of the subsp. h...

  20. FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence.

    PubMed

    Wu, Xiaojun; Ren, Guoping; Gunning, William T; Weaver, David A; Kalinoski, Andrea L; Khuder, Sadik A; Huntley, Jason F

    2016-01-01

    Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in

  1. FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence

    PubMed Central

    Wu, Xiaojun; Ren, Guoping; Gunning, William T.; Weaver, David A.; Kalinoski, Andrea L.; Khuder, Sadik A.; Huntley, Jason F.

    2016-01-01

    Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in

  2. Expanding the World of Marine Bacterial and Archaeal Clades.

    PubMed

    Yilmaz, Pelin; Yarza, Pablo; Rapp, Josephine Z; Glöckner, Frank O

    2015-01-01

    Determining which microbial taxa are out there, where they live, and what they are doing is a driving approach in marine microbial ecology. The importance of these questions is underlined by concerted, large-scale, and global ocean sampling initiatives, for example the International Census of Marine Microbes, Ocean Sampling Day, or Tara Oceans. Given decades of effort, we know that the large majority of marine Bacteria and Archaea belong to about a dozen phyla. In addition to the classically culturable Bacteria and Archaea, at least 50 "clades," at different taxonomic depths, exist. These account for the majority of marine microbial diversity, but there is still an underexplored and less abundant portion remaining. We refer to these hitherto unrecognized clades as unknown, as their boundaries, names, and classifications are not available. In this work, we were able to characterize up to 92 of these unknown clades found within the bacterial and archaeal phylogenetic diversity currently reported for marine water column environments. We mined the SILVA 16S rRNA gene datasets for sequences originating from the marine water column. Instead of the usual subjective taxa delineation and nomenclature methods, we applied the candidate taxonomic unit (CTU) circumscription system, along with a standardized nomenclature to the sequences in newly constructed phylogenetic trees. With this new phylogenetic and taxonomic framework, we performed an analysis of ICoMM rRNA gene amplicon datasets to gain insights into the global distribution of the new marine clades, their ecology, biogeography, and interaction with oceanographic variables. Most of the new clades we identified were interspersed by known taxa with cultivated members, whose genome sequences are available. This result encouraged us to perform metabolic predictions for the novel marine clades using the PICRUSt approach. Our work also provides an update on the taxonomy of several phyla and widely known marine clades as our

  3. Expanding the World of Marine Bacterial and Archaeal Clades

    PubMed Central

    Yilmaz, Pelin; Yarza, Pablo; Rapp, Josephine Z.; Glöckner, Frank O.

    2016-01-01

    Determining which microbial taxa are out there, where they live, and what they are doing is a driving approach in marine microbial ecology. The importance of these questions is underlined by concerted, large-scale, and global ocean sampling initiatives, for example the International Census of Marine Microbes, Ocean Sampling Day, or Tara Oceans. Given decades of effort, we know that the large majority of marine Bacteria and Archaea belong to about a dozen phyla. In addition to the classically culturable Bacteria and Archaea, at least 50 “clades,” at different taxonomic depths, exist. These account for the majority of marine microbial diversity, but there is still an underexplored and less abundant portion remaining. We refer to these hitherto unrecognized clades as unknown, as their boundaries, names, and classifications are not available. In this work, we were able to characterize up to 92 of these unknown clades found within the bacterial and archaeal phylogenetic diversity currently reported for marine water column environments. We mined the SILVA 16S rRNA gene datasets for sequences originating from the marine water column. Instead of the usual subjective taxa delineation and nomenclature methods, we applied the candidate taxonomic unit (CTU) circumscription system, along with a standardized nomenclature to the sequences in newly constructed phylogenetic trees. With this new phylogenetic and taxonomic framework, we performed an analysis of ICoMM rRNA gene amplicon datasets to gain insights into the global distribution of the new marine clades, their ecology, biogeography, and interaction with oceanographic variables. Most of the new clades we identified were interspersed by known taxa with cultivated members, whose genome sequences are available. This result encouraged us to perform metabolic predictions for the novel marine clades using the PICRUSt approach. Our work also provides an update on the taxonomy of several phyla and widely known marine clades as

  4. The Class A β-Lactamase FTU-1 Is Native to Francisella tularensis

    PubMed Central

    Antunes, Nuno T.; Frase, Hilary; Toth, Marta

    2012-01-01

    The class A β-lactamase FTU-1 produces resistance to penicillins and ceftazidime but not to any other β-lactam antibiotics tested. FTU-1 hydrolyzes penicillin antibiotics with catalytic efficiencies of 105 to 106 M−1 s−1 and cephalosporins and carbapenems with catalytic efficiencies of 102 to 103 M−1 s−1, but the monobactam aztreonam and the cephamycin cefoxitin are not substrates for the enzyme. FTU-1 shares 21 to 34% amino acid sequence identity with other class A β-lactamases and harbors two cysteine residues conserved in all class A carbapenemases. FTU-1 is the first weak class A carbapenemase that is native to Francisella tularensis. PMID:22083489

  5. Genome sequencing identifies Listeria fleischmannii subsp. coloradonensis subsp. nov., isolated from a ranch.

    PubMed

    den Bakker, Henk C; Manuel, Clyde S; Fortes, Esther D; Wiedmann, Martin; Nightingale, Kendra K

    2013-09-01

    Twenty Listeria-like isolates were obtained from environmental samples collected on a cattle ranch in northern Colorado; all of these isolates were found to share an identical partial sigB sequence, suggesting close relatedness. The isolates were similar to members of the genus Listeria in that they were Gram-stain-positive, short rods, oxidase-negative and catalase-positive; the isolates were similar to Listeria fleischmannii because they were non-motile at 25 °C. 16S rRNA gene sequencing for representative isolates and whole genome sequencing for one isolate was performed. The genome of the type strain of Listeria fleischmannii (strain LU2006-1(T)) was also sequenced. The draft genomes were very similar in size and the average MUMmer nucleotide identity across 91% of the genomes was 95.16%. Genome sequence data were used to design primers for a six-gene multi-locus sequence analysis (MLSA) scheme. Phylogenies based on (i) the near-complete 16S rRNA gene, (ii) 31 core genes and (iii) six housekeeping genes illustrated the close relationship of these Listeria-like isolates to Listeria fleischmannii LU2006-1(T). Sufficient genetic divergence of the Listeria-like isolates from the type strain of Listeria fleischmannii and differing phenotypic characteristics warrant these isolates to be classified as members of a distinct infraspecific taxon, for which the name Listeria fleischmannii subsp. coloradonensis subsp. nov. is proposed. The type strain is TTU M1-001(T) ( =BAA-2414(T) =DSM 25391(T)). The isolates of Listeria fleischmannii subsp. coloradonensis subsp. nov. differ from the nominate subspecies by the inability to utilize melezitose, turanose and sucrose, and the ability to utilize inositol. The results also demonstrate the utility of whole genome sequencing to facilitate identification of novel taxa within a well-described genus. The genomes of both subspecies of Listeria fleischmannii contained putative enhancin genes; the Listeria fleischmannii subsp

  6. Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis

    PubMed Central

    Batten, Laura E.; Parnell, Alice E.; Wells, Neil J.; Murch, Amber L.; Oyston, Petra C. F.; Roach, Peter L.

    2015-01-01

    The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using 31P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors. PMID:26582818

  7. Growth conditions and environmental factors impact aerosolization but not virulence of Francisella tularensis infection in mice

    PubMed Central

    Faith, Seth A.; Smith, Le'Kneitah P.; Swatland, Angela S.; Reed, Douglas S.

    2012-01-01

    In refining methodology to develop a mouse model for inhalation of Francisella tularensis, it was noted that both relative humidity and growth media impacted the aerosol concentration of the live vaccine strain (LVS) of F. tularensis. A relative humidity of less than 55% had a negative impact on the spray factor, the ratio between the concentration of LVS in the aerosol and the nebulizer. The spray factor was significantly higher for LVS grown in brain heart infusion (BHI) broth than LVS grown in Mueller–Hinton broth (MHb) or Chamberlain's chemically defined medium (CCDM). The variability between aerosol exposures was also considerably less with BHI. LVS grown in BHI survived desiccation far longer than MHb-grown or CCDM-grown LVS (~70% at 20 min for BHI compared to <50% for MHb and CCDM). Removal of the capsule by hypertonic treatment impacted the spray factor for CCDM-grown LVS or MHb-grown LVS but not BHI-grown LVS, suggesting the choice of culture media altered the adherence of the capsule to the cell membrane. The choice of growth media did not impact the LD50 of LVS but the LD99 of BHI-grown LVS was 1 log lower than that for MHb-grown LVS or CCDM-grown LVS. Splenomegaly was prominent in mice that succumbed to MHb- and BHI-grown LVS but not CCDM-grown LVS. Environmental factors and growth conditions should be evaluated when developing new animal models for aerosol infection, particularly for vegetative bacterial pathogens. PMID:23087911

  8. Detection of Francisella tularensis within Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler

    PubMed Central

    Emanuel, Peter A.; Bell, Ryan; Dang, Jessica L.; McClanahan, Rebecca; David, John C.; Burgess, Robert J.; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

    2003-01-01

    The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5′ hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field. PMID:12574268

  9. Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis.

    PubMed

    Batten, Laura E; Parnell, Alice E; Wells, Neil J; Murch, Amber L; Oyston, Petra C F; Roach, Peter L

    2016-01-01

    The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using (31)P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors. PMID:26582818

  10. Characterization of the PT clade of oligopeptide transporters in rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oligopeptide transporters (OPTs) are a group of membrane-localized proteins with a broad range of substrate transport capabilities, and which are thought to contribute to many biological processes. Nine OPTs belonging to the peptide transport (PT) clade were identified in the rice (Oryza sativa L.) ...

  11. Collecting reliable clades using the Greedy Strict Consensus Merger

    PubMed Central

    Böcker, Sebastian

    2016-01-01

    Supertree methods combine a set of phylogenetic trees into a single supertree. Similar to supermatrix methods, these methods provide a way to reconstruct larger parts of the Tree of Life, potentially evading the computational complexity of phylogenetic inference methods such as maximum likelihood. The supertree problem can be formalized in different ways, to cope with contradictory information in the input. Many supertree methods have been developed. Some of them solve NP-hard optimization problems like the well-known Matrix Representation with Parsimony, while others have polynomial worst-case running time but work in a greedy fashion (FlipCut). Both can profit from a set of clades that are already known to be part of the supertree. The Superfine approach shows how the Greedy Strict Consensus Merger (GSCM) can be used as preprocessing to find these clades. We introduce different scoring functions for the GSCM, a randomization, as well as a combination thereof to improve the GSCM to find more clades. This helps, in turn, to improve the resolution of the GSCM supertree. We find this modifications to increase the number of true positive clades by 18% compared to the currently used Overlap scoring. PMID:27375971

  12. Identification of potent maturation inhibitors against HIV-1 clade C.

    PubMed

    Timilsina, Uddhav; Ghimire, Dibya; Timalsina, Bivek; Nitz, Theodore J; Wild, Carl T; Freed, Eric O; Gaur, Ritu

    2016-01-01

    Antiretroviral therapy has led to a profound improvement in the clinical care of HIV-infected patients. However, drug tolerability and the evolution of drug resistance have limited treatment options for many patients. Maturation inhibitors are a new class of antiretroviral agents for treatment of HIV-1. They act by interfering with the maturation of the virus by blocking the last step in Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA by the viral protease (PR). The first-in-class maturation inhibitor bevirimat (BVM) failed against a subset of HIV-1 isolates in clinical trials due to polymorphisms present in the CA-SP1 region of the Gag protein. Sequence analysis indicated that these polymorphisms are more common in non-clade B strains of HIV-1 such as HIV-1 clade C. Indeed, BVM was found to be ineffective against HIV-1 clade C molecular clones tested in this study. A number of BVM analogs were synthesized by chemical modifications at the C-28 position to improve its activity. The new BVM analogs displayed potent activity against HIV-1 clade B and C and also reduced infectivity of the virus. This study identifies novel and broadly active BVM analogs that may ultimately demonstrate efficacy in the clinic. PMID:27264714

  13. Photosynthetic pigments of oceanic Chlorophyta belonging to prasinophytes clade VII.

    PubMed

    Lopes Dos Santos, Adriana; Gourvil, Priscillia; Rodríguez, Francisco; Garrido, José Luis; Vaulot, Daniel

    2016-02-01

    The ecological importance and diversity of pico/nanoplanktonic algae remains poorly studied in marine waters, in part because many are tiny and without distinctive morphological features. Amongst green algae, Mamiellophyceae such as Micromonas or Bathycoccus are dominant in coastal waters while prasinophytes clade VII, yet not formerly described, appear to be major players in open oceanic waters. The pigment composition of 14 strains representative of different subclades of clade VII was analyzed using a method that improves the separation of loroxanthin and neoxanthin. All the prasinophytes clade VII analyzed here showed a pigment composition similar to that previously reported for RCC287 corresponding to pigment group prasino-2A. However, we detected in addition astaxanthin for which it is the first report in prasinophytes. Among the strains analyzed, the pigment signature is qualitatively similar within subclades A and B. By contrast, RCC3402 from subclade C (Picocystis) lacks loroxanthin, astaxanthin, and antheraxanthin but contains alloxanthin, diatoxanthin, and monadoxanthin that are usually found in diatoms or cryptophytes. For subclades A and B, loroxanthin was lowest at highest light irradiance suggesting a light-harvesting role of this pigment in clade VII as in Tetraselmis. PMID:26987097

  14. Identification of potent maturation inhibitors against HIV-1 clade C

    PubMed Central

    Timilsina, Uddhav; Ghimire, Dibya; Timalsina, Bivek; Nitz, Theodore J.; Wild, Carl T.; Freed, Eric O.; Gaur, Ritu

    2016-01-01

    Antiretroviral therapy has led to a profound improvement in the clinical care of HIV-infected patients. However, drug tolerability and the evolution of drug resistance have limited treatment options for many patients. Maturation inhibitors are a new class of antiretroviral agents for treatment of HIV-1. They act by interfering with the maturation of the virus by blocking the last step in Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA by the viral protease (PR). The first-in-class maturation inhibitor bevirimat (BVM) failed against a subset of HIV-1 isolates in clinical trials due to polymorphisms present in the CA-SP1 region of the Gag protein. Sequence analysis indicated that these polymorphisms are more common in non-clade B strains of HIV-1 such as HIV-1 clade C. Indeed, BVM was found to be ineffective against HIV-1 clade C molecular clones tested in this study. A number of BVM analogs were synthesized by chemical modifications at the C-28 position to improve its activity. The new BVM analogs displayed potent activity against HIV-1 clade B and C and also reduced infectivity of the virus. This study identifies novel and broadly active BVM analogs that may ultimately demonstrate efficacy in the clinic. PMID:27264714

  15. Alahopcin, a new dipeptide antibiotic produced by Streptomyces albulus subsp. ochragerus subsp. nov.

    PubMed

    Higashide, E; Horii, S; Ono, H; Mizokami, N; Yamazaki, T; Shibata, M; Yoneda, M

    1985-03-01

    An actinomycete strain No. B-52653 was found to produce an antibiotic selectively active against the in vitro antibiotic resistant mutant of Staphylococcus aureus. Based on taxonomic studies, the name Streptomyces albulus subsp. ochragerus subsp. nov. is proposed for the strain. The microorganism produced two kinds of antibiotics; one identical with gougerotin, the other an amphoteric water soluble dipeptide containing L-alanine. The latter has the molecular formula C9H15N3O6 and is named alahopcin. It has a broad antibacterial spectrum and a synergistic effect with some other antibiotics against some antibiotic resistant staphylococci. Alahopcin has a low toxicity and was effective against experimental infections in mice caused by Staphylococcus aureus. PMID:3839222

  16. Serosurveillance for Francisella tularensis Among Wild Animals in Japan Using a Newly Developed Competitive Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita, Osamu; Mizoguchi, Toshio; Shindo, Junji; Park, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamada, Akio; Morikawa, Shigeru

    2014-01-01

    Abstract Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis–seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild. PMID:24689989

  17. TLR4-mediated activation of dendritic cells by the heat shock protein DnaK from Francisella tularensis

    PubMed Central

    Ashtekar, Amit R.; Zhang, Ping; Katz, Jannet; Deivanayagam, Champion C. S.; Rallabhandi, Prasad; Vogel, Stefanie N.; Michalek, Suzanne M.

    2008-01-01

    Francisella tularensis is the causative agent of tularemia, a severe, debilitating disease of humans and other mammals. As this microorganism is also classified as a “category-A pathogen” and a potential biowarfare agent, there is a need for an effective vaccine. Several antigens of F. tularensis, including the heat shock protein DnaK, have been proposed for use in a potential subunit vaccine. In this study, we characterized the innate immune response of murine bone marrow-derived dendritic cells (DC) to F. tularensis DnaK. Recombinant DnaK was produced using a bacterial expression system and purified using affinity, ion-exchange, and size-exclusion chromatography. DnaK induced the activation of MAPKs and NF-κB in DC and the production of the proinflammatory cytokines IL-6, TNF-α, and IL-12 p40, as well as low levels of IL-10. DnaK induced phenotypic maturation of DC, as demonstrated by an up-regulation of costimulatory molecules CD40, CD80, and CD86. DnaK stimulated DC through TLR4 and the adapters MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF) that mediated differential responses. DnaK induced activation of MAPKs and NF-κB in a MyD88- or TRIF-dependent manner. However, the presence of MyD88- and TRIF-dependent signaling pathways was essential for an optimal, DnaK-induced cytokine response in DC. In contrast, DnaK induced DC maturation in a TRIF-dependent, MyD88-independent manner. These results provide insight about the molecular interactions between an immunodominant antigen of F. tularensis and host immune cells, which is crucial for the rational design and development of a safe and efficacious vaccine against tularemia. PMID:18708593

  18. Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains.

    PubMed Central

    Pelletier, C; Bouley, C; Cayuela, C; Bouttier, S; Bourlioux, P; Bellon-Fontaine, M N

    1997-01-01

    Hydrophilic and electrostatic cell surface properties of eight Lactobacillus strains were characterized by using the microbial adhesion to solvents method and microelectrophoresis, respectively. All strains appeared relatively hydrophilic. The strong microbial adhesion to chloroform, an acidic solvent, in comparison with microbial adhesion to hexadecane, an apolar n-alkane, demonstrated the particularity of lactobacilli to have an important electron donor and basic character and consequently their potential ability to generate Lewis acid-base interactions with a support. Regardless of their electrophoretic mobility (EM), strains were in general slightly negatively charged at alkaline pH. A pH-dependent behavior concerning cell surface charges was observed. The EM decreased progressively with more acidic pHs for the L. casei subsp. casei and L. paracasei subsp. paracasei strains until the isoelectric point (IEP), i.e., the pH value for which the EM is zero. On the other hand, the EM for the L. rhamnosus strains was stable from pH 8 to pH 3 to 4, at which point there was a shift near the IEP. Both L. casei subsp. casei and L. paracasei subsp. paracasei strains were characterized by an IEP of around 4, whereas L. rhamnosus strains possessed a markedly lower IEP of 2. The present study showed that the cell surface physicochemical properties of lactobacilli seem to be, at least in part and under certain experimental conditions, particular to the bacterial species. Such differences detected between species are likely to be accompanied by some particular changes in cell wall chemical composition. PMID:9143109

  19. Lipidation of the FPI protein IglE contributes to Francisella tularensis ssp. novicida intramacrophage replication and virulence.

    PubMed

    Nguyen, Jesse Q; Gilley, Ryan P; Zogaj, Xhavit; Rodriguez, Stephen A; Klose, Karl E

    2014-10-01

    Francisella tularensis is a Gram-negative bacterium responsible for the human disease tularemia. The Francisella pathogenicity island (FPI) encodes a secretion system related to type VI secretion systems (T6SS) which allows F. tularensis to escape the phagosome and replicate within the cytosol of infected macrophages and ultimately cause disease. A lipoprotein is typically found encoded within T6SS gene clusters and is believed to anchor portions of the secretion apparatus to the outer membrane. We show that the FPI protein IglE is a lipoprotein that incorporates (3)H-palmitate and localizes to the outer membrane. A C22G IglE mutant failed to be lipidated and failed to localize to the outer membrane, consistent with C22 being the site of lipidation. Francisella tularensis ssp. novicida expressing IglE C22G is defective for replication in macrophages and unable to cause disease in mice. Bacterial two-hybrid analysis demonstrated that IglE interacts with the C-terminal portion of the FPI inner membrane protein PdpB, and PhoA fusion analysis indicated the PdpB C-terminus is located within the periplasm. We predict this interaction facilitates channel formation to allow secretion through this system. PMID:24616435

  20. Lipidation of the FPI protein IglE contributes to Francisella tularensis ssp. novicida intramacrophage replication and virulence

    PubMed Central

    Nguyen, Jesse Q.; Gilley, Ryan P.; Zogaj, Xhavit; Rodriguez, Stephen A.; Klose, Karl E.

    2014-01-01

    Francisella tularensis is a Gram-negative bacterium responsible for the human disease tularemia. The Francisella pathogenicity island (FPI) encodes a secretion system related to type VI secretion systems (T6SS) which allows F. tularensis to escape the phagosome and replicate within the cytosol of infected macrophages and ultimately cause disease. A lipoprotein is typically found encoded within T6SS gene clusters and is believed to anchor portions of the secretion apparatus to the outer membrane. We show that the FPI protein IglE is a lipoprotein that incorporates 3H-palmitate and localizes to the outer membrane. A C22G IglE mutant failed to be lipidated and failed to localize to the outer membrane, consistent with C22 being the site of lipidation. Francisella tularensis ssp. novicida expressing IglE C22G is defective for replication in macrophages and unable to cause disease in mice. Bacterial two-hybrid analysis demonstrated that IglE interacts with the C-terminal portion of the FPI inner membrane protein PdpB, and PhoA fusion analysis indicated the PdpB C-terminus is located within the periplasm. We predict this interaction facilitates channel formation to allow secretion through this system. PMID:24616435

  1. Generation and Characterization of an Attenuated Mutant in a Response Regulator Gene of Francisella tularensis Live Vaccine Strain (LVS)

    PubMed Central

    Sammons-Jackson, Wendy L.; McClelland, Karen; Manch-Citron, Jean N.; Metzger, Dennis W.; Bakshi, Chandra Shekhar; Garcia, Emilio; Rasley, Amy

    2008-01-01

    Francisella tularensis is a zoonotic bacterium that must exist in diverse environments ranging from arthropod vectors to mammalian hosts. To better understand how virulence genes are regulated in these different environments, a transcriptional response regulator gene (genome locus FTL0552) was deleted in F. tularensis live vaccine strain (LVS). The FTL0552 deletion mutant exhibited slightly reduced rates of extracellular growth but was unable to replicate or survive in mouse macrophages and was avirulent in the mouse model using either BALB/c or C57BL/6 mice. Mice infected with the FTL0552 mutant produced reduced levels of inflammatory cytokines, exhibited reduced histopathology, and cleared the bacteria quicker than mice infected with LVS. Mice that survived infection with the FTL0552 mutant were afforded partial protection when challenged with a lethal dose of the virulent SchuS4 strain (4 of 10 survivors, day 21 postinfection) when compared to naive mice (0 of 10 survivors by day 7 postinfection). Microarray experiments indicate that 148 genes are regulated by FTL0552. Most of the genes are downregulated, indicating that FTL0552 controls transcription of genes in a positive manner. Genes regulated by FTL0552 include genes located within the Francisella pathogenicity island that are essential for intracellular survival and virulence of F. tularensis. Further, a mutant in FTL0552 or the comparable locus in SchuS4 (FTT1557c) may be an alternative candidate vaccine for tularemia. PMID:18613792

  2. Prior Inoculation with Type B Strains of Francisella tularensis Provides Partial Protection against Virulent Type A Strains in Cottontail Rabbits

    PubMed Central

    Brown, Vienna R.; Adney, Danielle R.; Olea-Popelka, Francisco; Bowen, Richard A.

    2015-01-01

    Francisella tularensis is a highly virulent bacterium that is capable of causing severe disease (tularemia) in a wide range of species. This organism is characterized into two distinct subspecies: tularensis (type A) and holarctica (type B) which vary in several crucial ways, with some type A strains having been found to be considerably more virulent in humans and laboratory animals. Cottontail rabbits have been widely implicated as a reservoir species for this subspecies; however, experimental inoculation in our laboratory revealed type A organisms to be highly virulent, resulting in 100% mortality following challenge with 50–100 organisms. Inoculation of cottontail rabbits with the same number of organisms from type B strains of bacteria was found to be rarely lethal and to result in a robust humoral immune response. The objective of this study was to characterize the protection afforded by a prior challenge with type B strains against a later inoculation with a type A strain in North American cottontail rabbits (Sylvilagus spp). Previous infection with a type B strain of organism was found to lengthen survival time and in some cases prevent death following inoculation with a type A2 strain of F. tularensis. In contrast, inoculation of a type A1b strain was uniformly lethal in cottontail rabbits irrespective of a prior type B inoculation. These findings provide important insight about the role cottontail rabbits may play in environmental maintenance and transmission of this organism. PMID:26474413

  3. Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument.

    PubMed

    Mölsä, Markos; Hemmilä, Heidi; Katz, Anna; Niemimaa, Jukka; Forbes, Kristian M; Huitu, Otso; Stuart, Peter; Henttonen, Heikki; Nikkari, Simo

    2015-08-01

    In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis, Bacillus anthracis and Yersinia pestis. In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100 fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100 min, respectively. In field testing for F. tularensis, results were obtained with the PikoReal system in 95 min, as the pre-PCR preparation, including DNA extraction, required an additional 45 min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods. PMID:26043838

  4. Fatal pneumonia due to Serratia proteamaculans subsp. quinovora.

    PubMed Central

    Bollet, C; Grimont, P; Gainnier, M; Geissler, A; Sainty, J M; De Micco, P

    1993-01-01

    Serratia proteamaculans subsp. quinovora was isolated from several samples (blood cultures, tracheal aspirates, pleural effusion) from a patient with pneumonia. This is the first clinical isolate and the first documented human infection caused by this organism. PMID:8432835

  5. Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine

    PubMed Central

    Harris, N. Beth; Barletta, Raúl G.

    2001-01-01

    Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures. PMID:11432810

  6. Description of Klebsiella quasipneumoniae sp. nov., isolated from human infections, with two subspecies, Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a junior heterotypic synonym of Klebsiella variicola.

    PubMed

    Brisse, Sylvain; Passet, Virginie; Grimont, Patrick A D

    2014-09-01

    Strains previously classified as members of Klebsiella pneumoniae phylogroups KpI, KpII-A, KpII-B and KpIII were characterized by 16S rRNA (rrs) gene sequencing, multilocus sequence analysis based on rpoB, fusA, gapA, gyrA and leuS genes, average nucleotide identity and biochemical characteristics. Phylogenetic analysis demonstrated that KpI and KpIII corresponded to K. pneumoniae and Klebsiella variicola, respectively, whereas KpII-A and KpII-B formed two well-demarcated sequence clusters distinct from other members of the genus Klebsiella. Average nucleotide identity between KpII-A and KpII-B was 96.4 %, whereas values lower than 94 % were obtained for both groups when compared with K. pneumoniae and K. variicola. Biochemical properties differentiated KpII-A, KpII-B, K. pneumoniae and K. variicola, with acid production from adonitol and l-sorbose and ability to use 3-phenylproprionate, 5-keto-d-gluconate and tricarballylic acid as sole carbon sources being particularly useful. Based on their genetic and phenotypic characteristics, we propose the names Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and K. quasipneumoniae subsp. similipneumoniae subsp. nov. for strains of KpII-A and KpII-B, respectively. The type strain of K. quasipneumoniae sp. nov. and of K. quasipneumoniae subsp. quasipneumoniae subsp. nov. is 01A030(T) ( = SB11(T) = CIP 110771(T) = DSM 28211(T)). The type strain of K. quasipneumoniae subsp. similipneumoniae subsp. nov. is 07A044(T) ( = SB30(T) = CIP 110770(T) = DSM 28212(T)). Both strains were isolated from human blood cultures. This work also showed that Klebsiella singaporensis is a junior heterotypic synonym of K. variicola. PMID:24958762

  7. A phylogenetic overview of the antrodia clade (Basidiomycota, Polyporales).

    PubMed

    Ortiz-Santana, Beatriz; Lindner, Daniel L; Miettinen, Otto; Justo, Alfredo; Hibbett, David S

    2013-01-01

    Phylogenetic relationships among members of the antrodia clade were investigated with molecular data from two nuclear ribosomal DNA regions, LSU and ITS. A total of 123 species representing 26 genera producing a brown rot were included in the present study. Three DNA datasets (combined LSU-ITS dataset, LSU dataset, ITS dataset) comprising sequences of 449 isolates were evaluated with three different phylogenetic analyses (maximum likelihood, maximum parsimony, Bayesian inference). We present a phylogenetic overview of the five main groups recovered: the fibroporia, laetiporus, postia, laricifomes and core antrodia groups. Not all of the main groups received strong support in the analyses, requiring further research. We were able to identify a number of well supported clades within the main groups. PMID:23935025

  8. Distinct Processes Drive Diversification in Different Clades of Gesneriaceae.

    PubMed

    Roalson, Eric H; Roberts, Wade R

    2016-07-01

    Using a time-calibrated phylogenetic hypothesis including 768 Gesneriaceae species (out of [Formula: see text]3300 species) and more than 29,000 aligned bases from 26 gene regions, we test Gesneriaceae for diversification rate shifts and the possible proximal drivers of these shifts: geographic distributions, growth forms, and pollination syndromes. Bayesian Analysis of Macroevolutionary Mixtures analyses found five significant rate shifts in Beslerieae, core Nematanthus, core Columneinae, core Streptocarpus, and Pacific Cyrtandra These rate shifts correspond with shifts in diversification rates, as inferred by Binary State Speciation and Extinction Model and Geographic State Speciation and Extinction model, associated with hummingbird pollination, epiphytism, unifoliate growth, and geographic area. Our results suggest that diversification processes are extremely variable across Gesneriaceae clades with different combinations of characters influencing diversification rates in different clades. Diversification patterns between New and Old World lineages show dramatic differences, suggesting that the processes of diversification in Gesneriaceae are very different in these two geographic regions. PMID:26880147

  9. The historical biogeography of groupers: Clade diversification patterns and processes.

    PubMed

    Ma, Ka Yan; Craig, Matthew Thomas; Choat, John Howard; van Herwerden, Lynne

    2016-07-01

    Groupers (family Epinephelidae) are a clade of species-rich, biologically diverse reef fishes. Given their ecological variability and widespread distribution across ocean basins, it is important to scrutinize their evolutionary history that underlies present day distributions. This study investigated the patterns and processes by which grouper biodiversity has been generated and what factors have influenced their present day distributions. We reconstructed a robust, time-calibrated molecular phylogeny of Epinephelidae with comprehensive (∼87%) species sampling, whereby diversification rates were estimated and ancestral ranges were reconstructed. Our results indicate that groupers originated in what is now the East Atlantic during the mid-Eocene and diverged successively to form six strongly supported main clades. These clades differ in age (late Oligocene to mid-Miocene), geographic origin (West Atlantic to West Indo-Pacific) and temporal-spatial diversification pattern, ranging from constant rates of diversification to episodes of rapid radiation. Overall, divergence within certain biogeographic regions was most prevalent in groupers, while vicariant divergences were more common in Tropical Atlantic and East Pacific groupers. Our findings reveal that both biological and geographical factors have driven grouper diversification. They also underscore the importance of scrutinizing group-specific patterns to better understand reef fish evolution. PMID:26908372

  10. Anuran trypanosomes: phylogenetic evidence for new clades in Brazil.

    PubMed

    da S Ferreira, Juliana I G; da Costa, Andrea P; Ramirez, Diego; Roldan, Jairo A M; Saraiva, Danilo; da S Founier, Gislene F R; Sue, Ana; Zambelli, Erick R; Minervino, Antonio H H; Verdade, Vanessa K; Gennari, Solange M; Marcili, Arlei

    2015-05-01

    Trypanosomes of anurans and fish are grouped into the Aquatic Clade which includes species isolated from fish, amphibians, turtles and platypus, usually transmitted by leeches and phlebotomine sand flies. Trypanosomes from Brazilian frogs are grouped within the Aquatic Clade with other anuran trypanosome species, where there seems to be coevolutionary patterns with vertebrate hosts and association to Brazilian biomes (Atlantic Forest, Pantanal and Amazonia Rainforest). We characterised the anuran trypanosomes from two different areas of the Cerrado biome and examined their phylogenetic relationships based on the SSU rRNA gene. A total of 112 anurans of six species was analysed and trypanosome prevalence evaluated through haemoculture was found to be 7% (8 positive frogs). However, only three isolates (2.7%) from two anuran species were recovered and cryopreserved. Analysis including SSU rDNA sequences from previous studies segregated the anuran trypanosomes into six groups, the previously reported An01 to An04, and An05 and An06 reported herein. Clade An05 comprises the isolates from Leptodactylus latrans (Steffen) and Pristimantis sp. captured in the Cerrado biome and Trypanosoma chattoni Mathis & Leger, 1911. The inclusion of new isolates in the phylogenetic analyses provided evidence for a new group (An06) of parasites from phlebotomine hosts. Our results indicate that the diversity of trypanosome species is underestimated since studies conducted in Brazil and other regions of the world are still few. PMID:25862033

  11. A broadly protective vaccine against globally dispersed clade 1 and clade 2 H5N1 influenza viruses.

    PubMed

    Hoelscher, Mary A; Singh, Neetu; Garg, Sanjay; Jayashankar, Lakshmi; Veguilla, Vic; Pandey, Aseem; Matsuoka, Yumi; Katz, Jacqueline M; Donis, Ruben; Mittal, Suresh K; Sambhara, Suryaprakash

    2008-04-15

    Development of effective and immunogenic vaccines against highly pathogenic avian influenza H5N1 viruses with the potential to cause a pandemic is a public health priority. The global demand for a vaccine cannot be met in the event of an influenza pandemic because of the limited capacity to manufacture egg-derived vaccines as well as potential problems with the availability of embryonated eggs. Thus, there is an urgent need to develop alternative, egg-independent vaccines. We developed an adenoviral vector-based vaccine that contains hemagglutinin protein from clade 1 and clade 2 viruses, as well as conserved nucleoprotein, to broaden the vaccine coverage against H5N1 viruses. PMID:18462165

  12. The Trichoderma brevicompactum clade: a new lineage with new species, new peptaibiotics and mycotoxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new lineage is recognized in Trichoderma/Hypocrea, the Brevicompactum clade. This clade includes T. brevicompactum and the new species T. arundinaceum, T. turrialbense, T. protrudens and Hypocrea rodmanii. With the exception of H. rodmanii, all members of this clade produce trichothecenes harzian...

  13. Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle.

    PubMed

    Ahlstrom, Christina; Barkema, Herman W; Stevenson, Karen; Zadoks, Ruth N; Biek, Roman; Kao, Rowland; Trewby, Hannah; Haupstein, Deb; Kelton, David F; Fecteau, Gilles; Labrecque, Olivia; Keefe, Greg P; McKenna, Shawn L B; Tahlan, Kapil; De Buck, Jeroen

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne's disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six "Bison type" isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale. PMID:26871723

  14. Molecular fingerprinting of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica derby isolated from tropical seafood in South India.

    PubMed

    Kumar, Rakesh; Surendran, P K; Thampuran, Nirmala

    2008-09-01

    Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby strains isolated from different seafood were genotyped by PCR-ribotyping and ERIC-PCR assays. This study has ascertained the genetic relatedness among serovars prevalent in tropical seafood. PCR-ribotyping exhibited genetic variation in both Salmonella serovars, and ribotype profile (II) was most predominant, which was observed in 10/18 of Salmonella enterica subsp. enterica Typhimurium and 7/17 Salmonella enterica subsp. enterica Derby isolates. Cluster analysis of ERIC-PCR for Salmonella enterica subsp. enterica Typhimurium strains exhibited nine different banding patterns and four strains showed >95% genetic homology within the cluster pairs. ERIC-PCR produced more genetic variations in Salmonella enterica subsp. enterica Typhimurium; nevertheless, both methods were found to be comparable for Salmonella enterica subsp. enterica Derby isolates. Discrimination index of PCR-ribotyping for Salmonella enterica subsp. enterica Typhimurium isolates was obtained at 0.674 and index value 0.714 was observed for Salmonella enterica subsp. enterica Derby strains. Molecular fingerprinting investigation highlighted the hypothesis of diverse routes of Salmonella contamination in seafood as multiple clones of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby were detected in same or different seafood throughout the study period. PMID:18480975

  15. Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms

    PubMed Central

    Lehtola, Markku J.; Torvinen, Eila; Miettinen, Ilkka T.; Keevil, C. William

    2006-01-01

    Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply. PMID:16391126

  16. Detoxified endotoxin vaccine (J5dLPS/OMP) protects mice against lethal respiratory challenge with Francisella tularensis SchuS4.

    PubMed

    Gregory, Stephen H; Chen, Wilbur H; Mott, Stephanie; Palardy, John E; Parejo, Nicholas A; Heninger, Sara; Anderson, Christine A; Artenstein, Andrew W; Opal, Steven M; Cross, Alan S

    2010-04-01

    Francisella tularensis is a category A select agent. J5dLPS/OMP is a novel vaccine construct consisting of detoxified, O-polysaccharide side chain-deficient, lipopolysaccharide non-covalently complexed with the outer membrane protein of N. meningitidis group B. Immunization elicits high-titer polyclonal antibodies specific for the highly-conserved epitopes expressed within the glycolipid core that constitutes gram-negative bacteria (e.g., F. tularensis). Mice immunized intranasally with J5dLPS/OMP exhibited protective immunity to intratracheal challenge with the live vaccine strain, as well as the highly-virulent SchuS4 strain, of F. tularensis. The efficacy of J5dLPS/OMP vaccine suggests its potential utility in immunizing the general population against several different gram-negative select agents concurrently. PMID:20170768

  17. Denitrification capabilities of two biological phosphorus removal sludges dominated by different "Candidatus Accumulibacter" clades.

    PubMed

    Flowers, Jason J; He, Shaomei; Yilmaz, Safak; Noguera, Daniel R; McMahon, Katherine D

    2009-12-01

    The capability of "Candidatus Accumulibacter" to use nitrate as an electron acceptor for phosphorus uptake was investigated using two activated sludge communities. The two communities were enriched in Accumulibacter clade IA and clade IIA, respectively. By performing a series of batch experiments, we found that clade IA was able to couple nitrate reduction with phosphorus uptake, but clade IIA could not. These results agree with a previously proposed hypothesis that different populations of Accumulibacter have different nitrate reduction capabilities, and they will help to understand the ecological roles that these two clades provide. PMID:20808723

  18. Neonatal mortality in puppies due to bacteremia by Streptococcus dysgalactiae subsp. dysgalactiae.

    PubMed

    Vela, Ana I; Falsen, Enevold; Simarro, Isabel; Rollan, Eduardo; Collins, Matthew D; Domínguez, Lucas; Fernandez-Garayzabal, Jose F

    2006-02-01

    We report a case of bacteremia in puppies caused by Streptococcus dysgalactiae subsp. dysgalactiae. Identification was achieved by phenotypic and molecular genetic methods. This is the first report of the recovery of S. dysgalactiae subsp. dysgalactiae from dogs. PMID:16455943

  19. Bioavailability and Efficacy of Levofloxacin against Francisella tularensis in the Common Marmoset (Callithrix jacchus)▿

    PubMed Central

    Nelson, Michelle; Lever, Mark S.; Dean, Rachel E.; Pearce, Peter C.; Stevens, Daniel J.; Simpson, Andrew J. H.

    2010-01-01

    Pharmacokinetic and efficacy studies with levofloxacin were performed in the common marmoset (Callithrix jacchus) model of inhalational tularemia. Plasma levofloxacin pharmacokinetics were determined in six animals in separate single-dose and multidose studies. Plasma drug concentrations were analyzed using liquid chromatography-tandem mass spectrometry-electrospray ionization. On day 7 of a twice-daily dosing regimen of 40 mg/kg, the levofloxacin half-life, maximum concentration, and area under the curve in marmoset plasma were 2.3 h, 20.9 μg/ml, and 81.4 μg/liter/h, respectively. An efficacy study was undertaken using eight treated and two untreated control animals. Marmosets were challenged with a mean of 1.5 × 102 CFU of Francisella tularensis by the airborne route. Treated animals were administered 16.5 mg/kg levofloxacin by mouth twice daily, based on the pharmacokinetic parameters, beginning 24 h after challenge. Control animals had a raised core body temperature by 57 h postchallenge and died from infection by day 5. All of the other animals survived, remained afebrile, and lacked overt clinical signs. No bacteria were recovered from the organs of these animals at postmortem after culling at day 24 postchallenge. In conclusion, postexposure prophylaxis with orally administered levofloxacin was efficacious against acute inhalational tularemia in the common marmoset. The marmoset appears to be an appropriate animal model for the evaluation of postexposure therapies. PMID:20625157

  20. Establishment of lethal inhalational infection with Francisella tularensis (tularaemia) in the common marmoset (Callithrix jacchus)

    PubMed Central

    Nelson, Michelle; Lever, Mark S; Savage, Victoria L; Salguero, Francisco Javier; Pearce, Peter C; Stevens, Daniel J; Simpson, Andrew J H

    2009-01-01

    Susceptibility and lethality studies of inhalational tularaemia were undertaken using the common marmoset (Callithrix jacchus) to determine its suitability as a non-human primate model. Pairs of marmosets were exposed to varying challenge doses of Francisella tularensis by the airborne route and monitored for up to 14 days postchallenge (p.c.). Lethal infection was achieved following a retained dose of less than 10 bacterial colony-forming units (CFU). However, precise LD50 determination was not possible. The model was characterized using a target challenge dose of approximately 100 CFU. Increased core body temperature was the first indicator of disease, at approximately 2.5 days p.c. Overt clinical signs were first observed 12–18 h after the temperature increase. Significantly decreased activity was observed after approximately 3 days. All animals succumbed to infection between 4.5 and 7 days p.c. At postmortem examination, gross pathology was evident in the liver, spleen and lungs of all animals and high bacterial numbers were detected in all the organs assessed. Bacteraemia was demonstrated in all animals postmortem. Histopathological observations included severe suppurative bronchopneumonia, severe multifocal pyogranulomatous hepatitis, splenitis and lymphadenitis. Tularaemia disease progression in the common marmoset therefore appears to be consistent with the disease seen in humans and other animal models. The common marmoset may therefore be considered a suitable model for further studies of inhalational tularaemia. PMID:19335549

  1. Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.

    PubMed

    del Río, Jonathan Sabaté; Yehia Adly, Nouran; Acero-Sánchez, Josep Lluis; Henry, Olivier Y F; O'Sullivan, Ciara K

    2014-04-15

    Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers. PMID:24334283

  2. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of macrophage growth locus A (MglA) protein from Francisella tularensis

    SciTech Connect

    Subburaman, P.; Austin, B.P.; Shaw, G.X.; Waugh, D.S.; Ji, X.

    2010-11-03

    Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 {angstrom} resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 125, c = 54 {angstrom}.

  3. Candidatus Accumulibacter phosphatis clades enriched under cyclic anaerobic and microaerobic conditions simultaneously use different electron acceptors.

    PubMed

    Camejo, Pamela Y; Owen, Brian R; Martirano, Joseph; Ma, Juan; Kapoor, Vikram; Santo Domingo, Jorge; McMahon, Katherine D; Noguera, Daniel R

    2016-10-01

    Lab- and pilot-scale simultaneous nitrification, denitrification and phosphorus removal-sequencing batch reactors were operated under cyclic anaerobic and micro-aerobic conditions. The use of oxygen, nitrite, and nitrate as electron acceptors by Candidatus Accumulibacter phosphatis during the micro-aerobic stage was investigated. A complete clade-level characterization of Accumulibacter in both reactors was performed using newly designed qPCR primers targeting the polyphosphate kinase gene (ppk1). In the lab-scale reactor, limited-oxygen conditions led to an alternated dominance of Clade IID and IC over the other clades. Results from batch tests when Clade IC was dominant (i.e., >92% of Accumulibacter) showed that this clade was capable of using oxygen, nitrite and nitrate as electron acceptors for P uptake. A more heterogeneous distribution of clades was found in the pilot-scale system (Clades IIA, IIB, IIC, IID, IA, and IC), and in this reactor, oxygen, nitrite and nitrate were also used as electron acceptors coupled to phosphorus uptake. However, nitrite was not an efficient electron acceptor in either reactor, and nitrate allowed only partial P removal. The results from the Clade IC dominated reactor indicated that either organisms in this clade can simultaneously use multiple electron acceptors under micro-aerobic conditions, or that the use of multiple electron acceptors by Clade IC is due to significant microdiversity within the Accumulibacter clades defined using the ppk1 gene. PMID:27340814

  4. Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

    PubMed

    Monroe, Nicole; Hill, Christopher P

    2016-05-01

    Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. PMID:26555750

  5. Dengue virus type 1 clade replacement in recurring homotypic outbreaks

    PubMed Central

    2013-01-01

    Background Recurring dengue outbreaks occur in cyclical pattern in most endemic countries. The recurrences of dengue virus (DENV) infection predispose the population to increased risk of contracting the severe forms of dengue. Understanding the DENV evolutionary mechanism underlying the recurring dengue outbreaks has important implications for epidemic prediction and disease control. Results We used a set of viral envelope (E) gene to reconstruct the phylogeny of DENV-1 isolated between the periods of 1987–2011 in Malaysia. Phylogenetic analysis of DENV-1 E gene revealed that genotype I virus clade replacements were associated with the cyclical pattern of major DENV-1 outbreaks in Malaysia. A total of 9 non-conservative amino acid substitutions in the DENV-1 E gene consensus were identified; 4 in domain I, 3 in domain II and 2 in domain III. Selection pressure analyses did not reveal any positively selected codon site within the full length E gene sequences (1485 nt, 495 codons). A total of 183 (mean dN/dS = 0.0413) negatively selected sites were found within the Malaysian isolates; neither positive nor negative selection was noted for the remaining 312 codons. All the viruses were cross-neutralized by the respective patient sera suggesting no strong support for immunological advantage of any of the amino acid substitutions. Conclusion DENV-1 clade replacement is associated with recurrences of major DENV-1 outbreaks in Malaysia. Our findings are consistent with those of other studies that the DENV-1 clade replacement is a stochastic event independent of positive selection. PMID:24073945

  6. Biofilm formation of Francisella noatunensis subsp. orientalis

    USGS Publications Warehouse

    Soto, Esteban; Halliday-Wimmonds, Iona; Kearney, Michael T; Hansen, John D.

    2015-01-01

    Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC)and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon®, bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in theiglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.

  7. Biofilm formation of Francisella noatunensis subsp. orientalis.

    PubMed

    Soto, Esteban; Halliday-Simmonds, Iona; Francis, Stewart; Kearney, Michael T; Hansen, John D

    2015-12-31

    Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC) and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon(®), bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in the iglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums. PMID:26507830

  8. Disparate host immunity to Mycobacterium avium subsp. paratuberculosis antigens in calves inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and criticism of current tests for the detection of paratuberculosis. In the present study, host immune responses to antigen preparations of Mycobacterium avium subsp. paratuberculosis (MAP), consis...

  9. Potential Transmission Pathways of Streptococcus gallolyticus subsp. gallolyticus

    PubMed Central

    Dumke, Jessika; Hinse, Dennis; Vollmer, Tanja; Schulz, Jochen; Knabbe, Cornelius; Dreier, Jens

    2015-01-01

    Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus subsp. gallolyticus), a member of group D streptococci, is an inhabitant of the animal and human gastrointestinal tract. Furthermore, it is a facultative pathogen which causes e.g. endocarditis, septicemia and mastitis. S. gallolyticus subsp. gallolyticus may be transmitted either directly or indirectly between animals and humans. However, the transmission routes are an unsolved issue. In this study, we present systematic analyses of an S. gallolyticus subsp. gallolyticus isolate of an infective endocarditis patient in relation to isolates of his laying hen flock. Isolates from pooled droppings of laying hens, pooled dust samples and human blood culture were characterized by using multilocus sequence typing (MLST) and DNA fingerprinting. MLST revealed the same allelic profile of isolates from the human blood culture and from the droppings of laying hens. In addition, these isolates showed clonal identity regarding a similar DNA fingerprinting pattern. For the first time, we received a hint that transmission of S. gallolyticus subsp. gallolyticus between poultry and humans may occur. This raises the question about the zoonotic potential of isolates from poultry and should be considered in future studies. PMID:25978355

  10. Juvenile skeletogenesis in anciently diverged sea urchin clades.

    PubMed

    Gao, Feng; Thompson, Jeffrey R; Petsios, Elizabeth; Erkenbrack, Eric; Moats, Rex A; Bottjer, David J; Davidson, Eric H

    2015-04-01

    Mechanistic understanding of evolutionary divergence in animal body plans devolves from analysis of those developmental processes that, in forms descendant from a common ancestor, are responsible for their morphological differences. The last common ancestor of the two extant subclasses of sea urchins, i.e., euechinoids and cidaroids, existed well before the Permian/Triassic extinction (252 mya). Subsequent evolutionary divergence of these clades offers in principle a rare opportunity to solve the developmental regulatory events underlying a defined evolutionary divergence process. Thus (i) there is an excellent and fairly dense (if yet incompletely analyzed) fossil record; (ii) cladistically confined features of the skeletal structures of modern euechinoid and cidaroid sea urchins are preserved in fossils of ancestral forms; (iii) euechinoids and cidaroids are among current laboratory model systems in molecular developmental biology (here Strongylocentrotus purpuratus [Sp] and Eucidaris tribuloides [Et]); (iv) skeletogenic specification in sea urchins is uncommonly well understood at the causal level of interactions of regulatory genes with one another, and with known skeletogenic effector genes, providing a ready arsenal of available molecular tools. Here we focus on differences in test and perignathic girdle skeletal morphology that distinguish all modern euechinoid from all modern cidaroid sea urchins. We demonstrate distinct canonical test and girdle morphologies in juveniles of both species by use of SEM and X-ray microtomography. Among the sharply distinct morphological features of these clades are the internal skeletal structures of the perignathic girdle to which attach homologous muscles utilized for retraction and protraction of Aristotles׳ lantern and its teeth. We demonstrate that these structures develop de novo between one and four weeks after metamorphosis. In order to study the underlying developmental processes, a method of section whole mount in

  11. The automation and evaluation of nested clade phylogeographic analysis.

    PubMed

    Panchal, Mahesh; Beaumont, Mark A

    2007-06-01

    Nested clade phylogeographic analysis (NCPA) is a popular method for reconstructing the demographic history of spatially distributed populations from genetic data. Although some parts of the analysis are automated, there is no unique and widely followed algorithm for doing this in its entirety, beginning with the data, and ending with the inferences drawn from the data. This article describes a method that automates NCPA, thereby providing a framework for replicating analyses in an objective way. To do so, a number of decisions need to be made so that the automated implementation is representative of previous analyses. We review how the NCPA procedure has evolved since its inception and conclude that there is scope for some variability in the manual application of NCPA. We apply the automated software to three published datasets previously analyzed manually and replicate many details of the manual analyses, suggesting that the current algorithm is representative of how a typical user will perform NCPA. We simulate a large number of replicate datasets for geographically distributed, but entirely random-mating, populations. These are then analyzed using the automated NCPA algorithm. Results indicate that NCPA tends to give a high frequency of false positives. In our simulations we observe that 14% of the clades give a conclusive inference that a demographic event has occurred, and that 75% of the datasets have at least one clade that gives such an inference. This is mainly due to the generation of multiple statistics per clade, of which only one is required to be significant to apply the inference key. We survey the inferences that have been made in recent publications and show that the most commonly inferred processes (restricted gene flow with isolation by distance and contiguous range expansion) are those that are commonly inferred in our simulations. However, published datasets typically yield a richer set of inferences with NCPA than obtained in our random

  12. Simultaneous detection of Clavibacter michiganensis subsp. nebraskensis and Pantoea stewartii subsp. stewartii based on microsphere immunoreaction.

    PubMed

    Zhang, Fan; Li, Jinfeng; Zou, Mingqiang; Chen, Yan; Wang, Yanfei; Qi, Xiaohua

    2013-04-01

    Clavibacter michiganensis subsp. nebraskensis (Cmn) and Pantoea stewartii subsp. stewartii (Pss) are two plant pathogens that can cause tremendous agricultural economic losses. This novel method based on microsphere immunoreaction was developed for the simultaneous detection of Cmn and Pss in maize. This multiplex method was constructed based on microsphere immunodetection with fluorescent labels such as quantum dots (QDs) and R-phycoerythrin (R-PE) for the detection of Cmn and Pss. Captured QDs and R-PE serve as signal reporters for fluorescent readout. The principle of this method is based on a sandwich immunoreaction. Cmn and Pss captured by the microspheres were detected using flow cytometry. The limit of detection of this method was 10 times lower than the enzyme-linked immunosorbent assay (ELISA), and its analysis time (1 h) was much shorter compared with ELISA (6-8 h). The method, which has been proven to be an effective approach to multiplex detection of plant bacteria (Cmn and Pss as models), not only increased the varieties but also improved the sensitivity. The microsphere immunoreaction provides a universal method for the multiplex determination of microbes because of its high sensitivity, specificity, and speed. In the future, the method will be more fully validated in vivo to detect diversiform bacteria. PMID:23169888

  13. Sugar Utilization and Acid Production by Free and Entrapped Cells of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactococcus lactis subsp. lactis in a Whey Permeate Medium

    PubMed Central

    Audet, Pascal; Paquin, Celine; Lacroix, Christophe

    1989-01-01

    Cells of Streptococcus salivarius subsp. thermophilus and Lactococcus lactis subsp. lactis entrapped in k-carrageenan-locust bean gum gel performed similarly to free cells in the conversion of lactose to lactic acid. Bead diameter influenced the fermentation rate. Cells entrapped in smaller beads (0.5 to 1.0 mm) showed higher release rates, higher lactose, glucose, and formic acid utilization, higher galactose accumulation, and higher lactic acid production than did cells entrapped in larger beads (1.0 to 2.0 mm). Values for smaller beads were comparable with those for free cells. Immobilization affected the fermentation rate of lactic acid bacteria, especially Lactobacillus delbrueckii subsp. bulgaricus. Entrapped cells of L. delbrueckii subsp. bulgaricus demonstrated a lower lactic acid production than did free cells in batch fermentation. The kinetics of the production of formic and pyruvic acids by L. lactis subsp. lactis and S. salivarius subsp. thermophilus are presented. PMID:16347822

  14. Symbiodinium clade C dominates zooxanthellate corals (Scleractinia) in the temperate region of Japan.

    PubMed

    Lien, Yi-Ting; Fukami, Hironobu; Yamashita, Yoh

    2012-03-01

    Endosymbiotic algae of the genus Symbiodinium have been divided into nine clades (A-I) following genetic classification; some clades are known to have physiological properties that enable the coral hosts to adapt to different environmental conditions. To understand the relationships of coral-alga symbioses, we focused on Symbiodinium diversity in zooxanthellate corals living under the severe environmental conditions of the temperate region (30°-35°N) of Japan. We investigated Symbiodinium clades in 346 colonies belonging to 58 coral species from six locations. We then selected three coral species-Acropora hyacinthus, Acropora japonica, and Cyphastrea chalcidicum-to investigate whether Symbiodinium clades changed during winter or summer over the course of year (May 2009-Apr 2010) in Tanabe Bay, Japan. Three Symbiodinium clades (C, D, and F) were detected in corals in the temperate region. Notably, 56 coral species contained Symbiodinium clade C. Oulastrea crispata predominantly contained clade D, but traces of clade C were also detected in all samples. The temperate-specific species Alveopora japonica contained clades C and F simultaneously. Seasonal change of symbiont clades did not occur in the three coral species during the investigation period where SSTs range on 12.5-29.2°C. However, we found Acropora (2 spp.) and Cyphastrea (1 sp.) contained different subcladal types of clade C. These results reveal that most coral species harbored Symbiodinium clade C stably throughout the year, suggesting that Symbiodinium clade C shows low-temperature tolerance, and that two hypothetical possibilities; genetic differences of subcladal types generating physiological differences or wide physiological flexibility in the clade C. PMID:22379984

  15. Description of Teunomyces gen. nov. for the Candida kruisii clade, Suhomyces gen. nov. for the Candida tanzawaensis clade and Suhomyces kilbournensis sp. nov.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA sequence analysis has shown that species of the Candida kruisii clade and species of the Candida tanzawaensis clade represent phylogenetically circumscribed genera, which are described as Teunomyces gen. nov., type species T. kruisii, and Suhomyces gen. nov., type species S. tanzawaensis. Many o...

  16. Live Attenuated Mutants of Francisella tularensis Protect Rabbits against Aerosol Challenge with a Virulent Type A Strain

    PubMed Central

    Smith, Le'Kneitah P.; Cole, Kelly Stefano; Santiago, Araceli E.; Mann, Barbara J.; Barry, Eileen M.

    2014-01-01

    Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD50). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection. PMID:24614653

  17. [Investigation of the presence of Francisella tularensis by culture, serology and molecular methods in mice of Thrace Region, Turkey].

    PubMed

    Unal Yilmaz, Gülizar; Gurcan, Saban; Ozkan, Beytullah; Karadenizli, Aynur

    2014-04-01

    Tularemia is a disease that has been reported in Turkey since 1936. Although mice are considered to have a role in the transmission of Francisella tularensis to man, this has not been exactly confirmed yet. The aim of this study was to investigate the presence of F. tularensis in mice by using culture, serology and molecular methods. For this purpose, four villages (Edirne-Demirkoy, Kirklareli-Kaynarca, Tekirdag-Muzruplu, Tekirdag-Sinanli) were selected in Thrace Region of Turkey where tularemia cases had been reported previously. A total of 126 live-catch mouse traps were established in warehouses, barns, areas near wells, water tanks and creeks in the villages in December 2012. Traps were kept overnight and the next day the animals collected were identified at species-level. The live-captured mice were anesthetized and their heart blood samples were obtained. Subsequently, liver and spleen tissues were removed from every mouse under aseptic conditions in the class-2 safety cabinet. These tissues were cultivated in Francis medium containing 5% sheep blood, 0.1% cystein, 1% glucose and incubated for seven days in both normal atmosphere and 5% carbondioxide incubator at 37°C. Tularemia microagglutination test was performed by using the sera which were obtained from live-captured mice. Finally, DNAs were isolated from both liver and spleen tissues of mice, and real-time polymerase chain reaction (Tularemia RT-PCR; Public Health Agency of Turkey, Ankara) were performed. In our study, a total of 19 mice were captured and of these 11 were alive. Ten mice were identified as Apodemus flavicollis, seven were Mus macedonicus and two were Mus musculus. There were no Francisella tularensis isolation in the cultures of mice liver and spleen tissues. Serological tests yielded negative results for 10 mice whose serum samples could be obtained. In RT-PCR, positivity were detected in spleen tissues of two mice which were captured from Kaynarca where first tularemia cases in

  18. Decoupled form and function in disparate herbivorous dinosaur clades

    PubMed Central

    Lautenschlager, Stephan; Brassey, Charlotte A.; Button, David J.; Barrett, Paul M.

    2016-01-01

    Convergent evolution, the acquisition of morphologically similar traits in unrelated taxa due to similar functional demands or environmental factors, is a common phenomenon in the animal kingdom. Consequently, the occurrence of similar form is used routinely to address fundamental questions in morphofunctional research and to infer function in fossils. However, such qualitative assessments can be misleading and it is essential to test form/function relationships quantitatively. The parallel occurrence of a suite of morphologically convergent craniodental characteristics in three herbivorous, phylogenetically disparate dinosaur clades (Sauropodomorpha, Ornithischia, Theropoda) provides an ideal test case. A combination of computational biomechanical models (Finite Element Analysis, Multibody Dynamics Analysis) demonstrate that despite a high degree of morphological similarity between representative taxa (Plateosaurus engelhardti, Stegosaurus stenops, Erlikosaurus andrewsi) from these clades, their biomechanical behaviours are notably different and difficult to predict on the basis of form alone. These functional differences likely reflect dietary specialisations, demonstrating the value of quantitative biomechanical approaches when evaluating form/function relationships in extinct taxa. PMID:27199098

  19. Decoupled form and function in disparate herbivorous dinosaur clades.

    PubMed

    Lautenschlager, Stephan; Brassey, Charlotte A; Button, David J; Barrett, Paul M

    2016-01-01

    Convergent evolution, the acquisition of morphologically similar traits in unrelated taxa due to similar functional demands or environmental factors, is a common phenomenon in the animal kingdom. Consequently, the occurrence of similar form is used routinely to address fundamental questions in morphofunctional research and to infer function in fossils. However, such qualitative assessments can be misleading and it is essential to test form/function relationships quantitatively. The parallel occurrence of a suite of morphologically convergent craniodental characteristics in three herbivorous, phylogenetically disparate dinosaur clades (Sauropodomorpha, Ornithischia, Theropoda) provides an ideal test case. A combination of computational biomechanical models (Finite Element Analysis, Multibody Dynamics Analysis) demonstrate that despite a high degree of morphological similarity between representative taxa (Plateosaurus engelhardti, Stegosaurus stenops, Erlikosaurus andrewsi) from these clades, their biomechanical behaviours are notably different and difficult to predict on the basis of form alone. These functional differences likely reflect dietary specialisations, demonstrating the value of quantitative biomechanical approaches when evaluating form/function relationships in extinct taxa. PMID:27199098

  20. Decoupled form and function in disparate herbivorous dinosaur clades

    NASA Astrophysics Data System (ADS)

    Lautenschlager, Stephan; Brassey, Charlotte A.; Button, David J.; Barrett, Paul M.

    2016-05-01

    Convergent evolution, the acquisition of morphologically similar traits in unrelated taxa due to similar functional demands or environmental factors, is a common phenomenon in the animal kingdom. Consequently, the occurrence of similar form is used routinely to address fundamental questions in morphofunctional research and to infer function in fossils. However, such qualitative assessments can be misleading and it is essential to test form/function relationships quantitatively. The parallel occurrence of a suite of morphologically convergent craniodental characteristics in three herbivorous, phylogenetically disparate dinosaur clades (Sauropodomorpha, Ornithischia, Theropoda) provides an ideal test case. A combination of computational biomechanical models (Finite Element Analysis, Multibody Dynamics Analysis) demonstrate that despite a high degree of morphological similarity between representative taxa (Plateosaurus engelhardti, Stegosaurus stenops, Erlikosaurus andrewsi) from these clades, their biomechanical behaviours are notably different and difficult to predict on the basis of form alone. These functional differences likely reflect dietary specialisations, demonstrating the value of quantitative biomechanical approaches when evaluating form/function relationships in extinct taxa.

  1. Amphitremida (Poche, 1913) Is a New Major, Ubiquitous Labyrinthulomycete Clade

    PubMed Central

    Gomaa, Fatma; Mitchell, Edward A. D.; Lara, Enrique

    2013-01-01

    Micro-eukaryotic diversity is poorly documented at all taxonomic levels and the phylogenetic affiliation of many taxa – including many well-known and common organisms - remains unknown. Among these incertae sedis taxa are Archerella flavum (Loeblich and Tappan, 1961) and Amphitrema wrightianum (Archer, 1869) (Amphitremidae), two filose testate amoebae commonly found in Sphagnum peatlands. To clarify their phylogenetic position, we amplified and sequenced the SSU rRNA gene obtained from four independent DNA extractions of A. flavum and three independent DNA extractions of A. wrightianum. Our molecular data demonstrate that genera Archerella and Amphitrema form a fully supported deep-branching clade within the Labyrinthulomycetes (Stramenopiles), together with Diplophrys sp. (ATCC50360) and several environmental clones obtained from a wide range of environments. This newly described clade we named Amphitremida is diverse genetically, ecologically and physiologically. Our phylogenetic analysis suggests that osmotrophic species evolved most likely from phagotrophic ancestors and that the bothrosome, an organelle that produces cytoplasmic networks used for attachment to the substratum and to absorb nutrients from the environments, appeared lately in labyrithulomycete evolution. PMID:23341921

  2. Mechanisms involved in quinolone resistance in Mycoplasma mycoides subsp. capri.

    PubMed

    Antunes, Nuno T; Assunção, Patrícia; Poveda, José B; Tavío, María M

    2015-06-01

    Mycoplasma mycoides subsp. capri is a causative agent of contagious agalactia in goats. In this study, M. mycoides subsp. capri mutants were selected for resistance to fluoroquinolones (norfloxacin, enrofloxacin and ciprofloxacin) by serial passes in broth with increasing concentrations of antibiotic. Mutations conferring cross-resistance to the three fluoroquinolones were found in the quinolone resistance determining regions of the four genes encoding DNA gyrase and topoisomerase IV. Different mutations in the DNA gyrase GyrA subunit suggest a different mechanism of inhibition between norfloxacin and the other tested fluoroquinolones. The presence of an adenosine triphosphate-dependent efflux system was suggested through the use of the inhibitor orthovanadate. PMID:25951987

  3. Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis*

    PubMed Central

    Kim, Tae-Hyun; Sebastian, Shite; Pinkham, Jessica T.; Ross, Robin A.; Blalock, LeeAnn T.; Kasper, Dennis L.

    2010-01-01

    The O-antigen polymerase of Gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly176, Asp177, Gly323, and Tyr324) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface. PMID:20605777

  4. Roles of reactive oxygen species-degrading enzymes of Francisella tularensis SCHU S4.

    PubMed

    Binesse, Johan; Lindgren, Helena; Lindgren, Lena; Conlan, Wayne; Sjöstedt, Anders

    2015-06-01

    Francisella tularensis is a facultative intracellular bacterium utilizing macrophages as its primary intracellular habitat and is therefore highly capable of resisting the effects of reactive oxygen species (ROS), potent mediators of the bactericidal activity of macrophages. We investigated the roles of enzymes presumed to be important for protection against ROS. Four mutants of the highly virulent SCHU S4 strain with deletions of the genes encoding catalase (katG), glutathione peroxidase (gpx), a DyP-type peroxidase (FTT0086), or double deletion of FTT0086 and katG showed much increased susceptibility to hydrogen peroxide (H2O2) and slightly increased susceptibility to paraquat but not to peroxynitrite (ONOO(-)) and displayed intact intramacrophage replication. Nevertheless, mice infected with the double deletion mutant showed significantly longer survival than SCHU S4-infected mice. Unlike the aforementioned mutants, deletion of the gene coding for alkyl-hydroperoxide reductase subunit C (ahpC) generated a mutant much more susceptible to paraquat and ONOO(-) but not to H2O2. It showed intact replication in J774 cells but impaired replication in bone marrow-derived macrophages and in internal organs of mice. The live vaccine strain, LVS, is more susceptible than virulent strains to ROS-mediated killing and possesses a truncated form of FTT0086. Expression of the SCHU S4 FTT0086 gene rendered LVS more resistant to H2O2, which demonstrates that the SCHU S4 strain possesses additional detoxifying mechanisms. Collectively, the results demonstrate that SCHU S4 ROS-detoxifying enzymes have overlapping functions, and therefore, deletion of one or the other does not critically impair the intracellular replication or virulence, although AhpC appears to have a unique function. PMID:25802058

  5. B-Cell Epitopes in GroEL of Francisella tularensis

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J.; Madico, Guillermo; Li, Sheng; Yang, Chiou-Ying; Perkins, Hillary M.; Sompuram, Seshi R.; Kodela, Vani; Liu, Tong; Morris, Timothy; Wang, Daphne; Roche, Marly I.; Seaton, Barbara A.; Sharon, Jacqueline

    2014-01-01

    The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other’s binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange–mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL. PMID:24968190

  6. T-bet regulates immunity to Francisella tularensis live vaccine strain infection, particularly in lungs.

    PubMed

    Melillo, Amanda A; Foreman, Oded; Bosio, Catharine M; Elkins, Karen L

    2014-04-01

    Upregulation of the transcription factor T-bet is correlated with the strength of protection against secondary challenge with the live vaccine strain (LVS) of Francisella tularensis. Thus, to determine if this mediator had direct consequences in immunity to LVS, we examined its role in infection. Despite substantial in vivo gamma interferon (IFN-γ) levels, T-bet-knockout (KO) mice infected intradermally (i.d.) or intranasally (i.n.) with LVS succumbed to infection with doses 2 log units less than those required for their wild-type (WT) counterparts, and exhibited significantly increased bacterial burdens in the lung and spleen. Lungs of LVS-infected T-bet-KO mice contained fewer lymphocytes and more neutrophils and interleukin-17 than WT mice. LVS-vaccinated T-bet-KO mice survived lethal LVS intraperitoneal secondary challenge but not high doses of LVS i.n. challenge, independently of the route of vaccination. Immune T lymphocytes from the spleens of i.d. LVS-vaccinated WT or KO mice controlled intracellular bacterial replication in an in vitro coculture system, but cultures with T-bet-KO splenocyte supernatants contained less IFN-γ and increased amounts of tumor necrosis factor alpha. In contrast, immune T-bet-KO lung lymphocytes were greatly impaired in controlling intramacrophage growth of LVS; this functional defect is the likely mechanism underpinning the lack of respiratory protection. Taken together, T-bet is important in host resistance to primary LVS infection and i.n. secondary challenge. Thus, T-bet represents a true, useful correlate for immunity to LVS. PMID:24421047

  7. Francisella tularensis novicida proteomic and transcriptomic data integration and annotation based on semantic web technologies

    PubMed Central

    Anwar, Nadia; Hunt, Ela

    2009-01-01

    Background This paper summarises the lessons and experiences gained from a case study of the application of semantic web technologies to the integration of data from the bacterial species Francisella tularensis novicida (Fn). Fn data sources are disparate and heterogeneous, as multiple laboratories across the world, using multiple technologies, perform experiments to understand the mechanism of virulence. It is hard to integrate these data sources in a flexible manner that allows new experimental data to be added and compared when required. Results Public domain data sources were combined in RDF. Using this connected graph of database cross references, we extended the annotations of an experimental data set by superimposing onto it the annotation graph. Identifiers used in the experimental data automatically resolved and the data acquired annotations in the rest of the RDF graph. This happened without the expensive manual annotation that would normally be required to produce these links. This graph of resolved identifiers was then used to combine two experimental data sets, a proteomics experiment and a transcriptomic experiment studying the mechanism of virulence through the comparison of wildtype Fn with an avirulent mutant strain. Conclusion We produced a graph of Fn cross references which enabled the combination of two experimental datasets. Through combination of these data we are able to perform queries that compare the results of the two experiments. We found that data are easily combined in RDF and that experimental results are easily compared when the data are integrated. We conclude that semantic data integration offers a convenient, simple and flexible solution to the integration of published and unpublished experimental data. PMID:19796400

  8. Roles of Reactive Oxygen Species-Degrading Enzymes of Francisella tularensis SCHU S4

    PubMed Central

    Binesse, Johan; Lindgren, Helena; Lindgren, Lena; Conlan, Wayne

    2015-01-01

    Francisella tularensis is a facultative intracellular bacterium utilizing macrophages as its primary intracellular habitat and is therefore highly capable of resisting the effects of reactive oxygen species (ROS), potent mediators of the bactericidal activity of macrophages. We investigated the roles of enzymes presumed to be important for protection against ROS. Four mutants of the highly virulent SCHU S4 strain with deletions of the genes encoding catalase (katG), glutathione peroxidase (gpx), a DyP-type peroxidase (FTT0086), or double deletion of FTT0086 and katG showed much increased susceptibility to hydrogen peroxide (H2O2) and slightly increased susceptibility to paraquat but not to peroxynitrite (ONOO−) and displayed intact intramacrophage replication. Nevertheless, mice infected with the double deletion mutant showed significantly longer survival than SCHU S4-infected mice. Unlike the aforementioned mutants, deletion of the gene coding for alkyl-hydroperoxide reductase subunit C (ahpC) generated a mutant much more susceptible to paraquat and ONOO− but not to H2O2. It showed intact replication in J774 cells but impaired replication in bone marrow-derived macrophages and in internal organs of mice. The live vaccine strain, LVS, is more susceptible than virulent strains to ROS-mediated killing and possesses a truncated form of FTT0086. Expression of the SCHU S4 FTT0086 gene rendered LVS more resistant to H2O2, which demonstrates that the SCHU S4 strain possesses additional detoxifying mechanisms. Collectively, the results demonstrate that SCHU S4 ROS-detoxifying enzymes have overlapping functions, and therefore, deletion of one or the other does not critically impair the intracellular replication or virulence, although AhpC appears to have a unique function. PMID:25802058

  9. T-bet Regulates Immunity to Francisella tularensis Live Vaccine Strain Infection, Particularly in Lungs

    PubMed Central

    Melillo, Amanda A.; Foreman, Oded; Bosio, Catharine M.

    2014-01-01

    Upregulation of the transcription factor T-bet is correlated with the strength of protection against secondary challenge with the live vaccine strain (LVS) of Francisella tularensis. Thus, to determine if this mediator had direct consequences in immunity to LVS, we examined its role in infection. Despite substantial in vivo gamma interferon (IFN-γ) levels, T-bet-knockout (KO) mice infected intradermally (i.d.) or intranasally (i.n.) with LVS succumbed to infection with doses 2 log units less than those required for their wild-type (WT) counterparts, and exhibited significantly increased bacterial burdens in the lung and spleen. Lungs of LVS-infected T-bet-KO mice contained fewer lymphocytes and more neutrophils and interleukin-17 than WT mice. LVS-vaccinated T-bet-KO mice survived lethal LVS intraperitoneal secondary challenge but not high doses of LVS i.n. challenge, independently of the route of vaccination. Immune T lymphocytes from the spleens of i.d. LVS-vaccinated WT or KO mice controlled intracellular bacterial replication in an in vitro coculture system, but cultures with T-bet-KO splenocyte supernatants contained less IFN-γ and increased amounts of tumor necrosis factor alpha. In contrast, immune T-bet-KO lung lymphocytes were greatly impaired in controlling intramacrophage growth of LVS; this functional defect is the likely mechanism underpinning the lack of respiratory protection. Taken together, T-bet is important in host resistance to primary LVS infection and i.n. secondary challenge. Thus, T-bet represents a true, useful correlate for immunity to LVS. PMID:24421047

  10. Structural Analysis of a Protective Epitope of the Francisella tularensis O-Polysaccharide†

    PubMed Central

    Rynkiewicz, Michael J.; Lu, Zhaohua; Hui, Julia H.; Sharon, Jacqueline; Seaton, Barbara A.

    2012-01-01

    Francisella tularensis (Ft), the Gram negative facultative intracellular bacterium that causes tularemia, is considered a biothreat due to its high infectivity and the high mortality rate of respiratory disease. The Ft lipopolysaccharide (Ft LPS) is thought to be a main protective antigen in mice and humans, and we have previously demonstrated the protective effect of the Ft LPS-specific monoclonal antibody Ab52 in a mouse model of respiratory tularemia. Immunochemical characterization has shown that the epitope recognized by Ab52 is contained within two internal repeat units of the O-polysaccharide [O-antigen (OAg)] of Ft LPS. To further localize the Ab52 epitope and understand the molecular interactions between the antibody and the saccharide, we now solved the X-ray crystal structure of the Fab fragment of Ab52 and derived an antibody-antigen complex using molecular docking. The docked complex, refined through energy minimization, reveals an antigen binding site in the shape of a large canyon with a central pocket that accommodates a V-shaped epitope consisting of six sugar residues, α-D-GalpNAcAN(1→4)-α-D-GalpNAcAN(1→3)-β-D-QuipNAc(1→2)-β-D-Quip4NFm(1→4)-α-D-GalpNAcAN(1→4)-α-D-GalpNAcAN. These results inform the development of vaccines and immunotherapeutic/immunoprophylactic antibodies against Ft by suggesting a desired topology for antibody binding to internal epitopes of Ft LPS. This is the first report of an X-ray crystal structure of a monoclonal antibody that targets a protective Ft B cell epitope. PMID:22747335

  11. A rapid real-time quantitative PCR assay to determine the minimal inhibitory extracellular concentration of antibiotics against an intracellular Francisella tularensis Live Vaccine Strain

    PubMed Central

    Aloni-Grinstein, Ronit; Shifman, Ohad; Lazar, Shlomi; Steinberger-Levy, Ida; Maoz, Sharon; Ber, Raphael

    2015-01-01

    Francisella tularensis is a highly virulent facultative intracellular bacterium. The lack of a safe and efficient vaccine makes antibiotics the preferred treatment. F. tularensis antibiotic susceptibility tests are based on the in vitro standard CLSI-approved microdilution method for determining the MIC. However, limited data are available regarding the minimal inhibitory extracellular concentration (MIEC) needed to eradicate intracellular bacteria. Here, we evaluated the MIEC values of various WHO-recommended antibiotics and compared the MIEC values to the established MICs. We describe a rapid 3-h quantitative PCR (qPCR) intracellular antibiogram assay, which yields comparable MIEC values to those obtained by the classical 72-h cfu assay. This rapid qPCR assay is highly advantageous in light of the slow growth rates of F. tularensis. Our results showed that the MIECs obtained for doxycycline, chloramphenicol and ciprofloxacin were indicative of intracellular activity. Gentamicin was not effective against intracellular bacteria for at least 32 h post treatment, raising the question of whether slow-penetrating gentamicin should be used for certain stages of the disease. We suggest that the qPCR intracellular antibiogram assay may be used to screen for potentially active antibiotics against intracellular F. tularensis as well as to detect strains with acquired resistance to recommended antibiotics. PMID:26579112

  12. Characterization of Francisella tularensis Schu S4 mutants identified from a transposon library screened for O-antigen and capsule deficiencies

    PubMed Central

    Rasmussen, Jed A.; Fletcher, Joshua R.; Long, Matthew E.; Allen, Lee-Ann H.; Jones, Bradley D.

    2015-01-01

    The lipopolysaccharide (LPS) and O-antigen polysaccharide capsule structures of Francisella tularensis play significant roles in helping these highly virulent bacteria avoid detection within a host. We previously created pools of F. tularensis mutants that we screened to identify strains that were not reactive to a monoclonal antibody to the O-antigen capsule. To follow up previously published work, we characterize further seven of the F. tularensis Schu S4 mutant strains identified by our screen. These F. tularensis strains carry the following transposon mutations: FTT0846::Tn5, hemH::Tn5, wbtA::Tn5, wzy::Tn5, FTT0673p/prsA::Tn5, manB::Tn5, or dnaJ::Tn5. Each of these strains displayed sensitivity to human serum, to varying degrees, when compared to wild-type F. tularensis Schu S4. By Western blot, only FTT0846::Tn5, wbtA::Tn5, wzy::Tn5, and manB::Tn5 strains did not react to the capsule and LPS O-antigen antibody 11B7, although the wzy::Tn5 strain did have a single O-antigen reactive band that was detected by the FB11 monoclonal antibody. Of these strains, manB::Tn5 and FTT0846 appear to have LPS core truncations, whereas wbtA::Tn5 and wzy::Tn5 had LPS core structures that are similar to the parent F. tularensis Schu S4. These strains were also shown to have poor growth within human monocyte derived macrophages (MDMs) and bone marrow derived macrophages (BMDMs). We examined the virulence of these strains in mice, following intranasal challenge, and found that each was attenuated compared to wild type Schu S4. Our results provide additional strong evidence that LPS and/or capsule are F. tularensis virulence factors that most likely function by providing a stealth shield that prevents the host immune system from detecting this potent pathogen. PMID:25999917

  13. Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

    PubMed

    Peterson, Johnny W; Moen, Scott T; Healy, Daniel; Pawlik, Jennifer E; Taormina, Joanna; Hardcastle, Jason; Thomas, John M; Lawrence, William S; Ponce, Cindy; Chatuev, Bagram M; Gnade, Bryan T; Foltz, Sheri M; Agar, Stacy L; Sha, Jian; Klimpel, Gary R; Kirtley, Michelle L; Eaves-Pyles, Tonyia; Chopra, Ashok K

    2010-01-01

    Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 μg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities. PMID:21127743

  14. Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis

    PubMed Central

    Peterson, Johnny W; Moen, Scott T; Healy, Daniel; Pawlik, Jennifer E; Taormina, Joanna; Hardcastle, Jason; Thomas, John M; Lawrence, William S; Ponce, Cindy; Chatuev, Bagram M; Gnade, Bryan T; Foltz, Sheri M; Agar, Stacy L; Sha, Jian; Klimpel, Gary R; Kirtley, Michelle L; Eaves-Pyles, Tonyia; Chopra, Ashok K

    2010-01-01

    Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 μg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities. PMID:21127743

  15. Characterization of the Receptor-Ligand Pathways Important for Entry and Survival of Francisella tularensis in Human Macrophages

    PubMed Central

    Balagopal, Ashwin; MacFarlane, Amanda Shearer; Mohapatra, Nrusingh; Soni, Shilpa; Gunn, John S.; Schlesinger, Larry S.

    2006-01-01

    Inhalational pneumonic tularemia, caused by Francisella tularensis, is lethal in humans. F. tularensis is phagocytosed by macrophages followed by escape from phagosomes into the cytoplasm. Little is known of the phagocytic mechanisms for Francisella, particularly as they relate to the lung and alveolar macrophages. Here we examined receptors on primary human monocytes and macrophages which mediate the phagocytosis and intracellular survival of F. novicida. F. novicida association with monocyte-derived macrophages (MDM) was greater than with monocytes. Bacteria were readily ingested, as shown by electron microscopy. Bacterial association was significantly increased in fresh serum and only partially decreased in heat-inactivated serum. A role for both complement receptor 3 (CR3) and Fcγ receptors in uptake was supported by studies using a CR3-expressing cell line and by down-modulation of Fcγ receptors on MDM, respectively. Consistent with Fcγ receptor involvement, antibody in nonimmune human serum was detected on the surface of Francisella. In the absence of serum opsonins, competitive inhibition of mannose receptor (MR) activity on MDM with mannan decreased the association of F. novicida and opsonization of F. novicida with lung collectin surfactant protein A (SP-A) increased bacterial association and intracellular survival. This study demonstrates that human macrophages phagocytose more Francisella than monocytes with contributions from CR3, Fcγ receptors, the MR, and SP-A present in lung alveoli. PMID:16926403

  16. Differential niche dynamics among major marine invertebrate clades

    PubMed Central

    Hopkins, Melanie J; Simpson, Carl; Kiessling, Wolfgang

    2014-01-01

    The degree to which organisms retain their environmental preferences is of utmost importance in predicting their fate in a world of rapid climate change. Notably, marine invertebrates frequently show strong affinities for either carbonate or terrigenous clastic environments. This affinity is due to characteristics of the sediments as well as correlated environmental factors. We assessed the conservatism of substrate affinities of marine invertebrates over geological timescales, and found that niche conservatism is prevalent in the oceans, and largely determined by the strength of initial habitat preference. There is substantial variation in niche conservatism among major clades with corals and sponges being among the most conservative. Time-series analysis suggests that niche conservatism is enhanced during times of elevated nutrient flux, whereas niche evolution tends to occur after mass extinctions. Niche evolution is not necessarily elevated in genera exhibiting higher turnover in species composition. PMID:24313951

  17. Acyl-Homoserine Lactone Quorum Sensing in the Roseobacter Clade

    PubMed Central

    Zan, Jindong; Liu, Yue; Fuqua, Clay; Hill, Russell T.

    2014-01-01

    Members of the Roseobacter clade are ecologically important and numerically abundant in coastal environments and can associate with marine invertebrates and nutrient-rich marine snow or organic particles, on which quorum sensing (QS) may play an important role. In this review, we summarize current research progress on roseobacterial acyl-homoserine lactone-based QS, particularly focusing on three relatively well-studied representatives, Phaeobacter inhibens DSM17395, the marine sponge symbiont Ruegeria sp. KLH11 and the dinoflagellate symbiont Dinoroseobacter shibae. Bioinformatic survey of luxI homologues revealed that over 80% of available roseobacterial genomes encode at least one luxI homologue, reflecting the significance of QS controlled regulatory pathways in adapting to the relevant marine environments. We also discuss several areas that warrant further investigation, including studies on the ecological role of these diverse QS pathways in natural environments. PMID:24402124

  18. Hidden Gems in the Mycobacterium avium subsp. paratuberculosis Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    If 4,350 genes annotated in the M. avium subsp paratuberculosis strain K-10 genome wasn’t already enough to study, more genes have recently been uncovered, hidden deep within this genome sequence. Genomic and proteomic studies, both published and unpublished, have revealed a handful of new genes mi...

  19. Cellular Interactions in Mycobacterium avium subsp. paratuberculosis Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study of host immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) is complicated by a number of factors, including the protracted nature of the disease and the stealthy nature of the pathogen. Noted as one of the more fastidious mycobacteria, infection with MAP is often chara...

  20. Toxic shock syndrome related to Streptococcus equi subsp zooepidemicus

    PubMed Central

    Saleh, Mohamed; Vialette, Véronique

    2013-01-01

    We describe a documented streptococcal toxic shock syndrome linked to horse-to-man transmission of Streptococcus equi subsp zooepidemicus. The patient was treated successfully with respiratory and haemodynamic support in conjunction with antibiotic treatment associated with polyvalent human immunoglobulin and high-volume venovenous haemofiltration. PMID:24014562

  1. Staphylococcus aureus subsp. anaerobius strain ST1464 genome sequence

    PubMed Central

    Elbir, Haitham; Robert, Catherine; Nguyen, Ti Thien; Gimenez, Grégory; El Sanousi, Sulieman M.; Flock, Jan-Ingmar; Raoult, Didier

    2013-01-01

    Staphylococcus aureus subsp. anaerobius is responsible for Morel's disease in animals and a cause of abscess in humans. It is characterized by a microaerophilic growth, contrary to the other strains of S. aureus. The 2,604,446-bp genome (32.7% GC content) of S. anaerobius ST1464 comprises one chromosome and no plasmids. The chromosome contains 2,660 open reading frames (ORFs), 49 tRNAs and three complete rRNAs, forming one complete operon. The size of ORFs ranges between 100 to 4,600 bp except for two ORFs of 6,417 and 7,173 bp encoding segregation ATPase and non-ribosomal peptide synthase, respectively. The chromosome harbors Staphylococcus phage 2638A genome and incomplete Staphylococcus phage genome PT1028, but no detectable CRISPRS. The antibiotic resistance gene for tetracycline was found although Staphylococcus aureus subsp. anaerobius is susceptible to tetracycline in-vitro. Intact oxygen detoxification genes encode superoxide dismutase and cytochrome quinol oxidase whereas the catalase gene is impaired by a stop codon. Based on the genome, in-silico multilocus sequence typing indicates that S. aureus subsp. anaerobius emerged as a clone separated from all other S. aureus strains, illustrating host-adaptation linked to missing functions. Availability of S. aureus subsp. anaerobius genome could prompt the development of post-genomic tools for its rapid discrimination from S. aureus. PMID:24501641

  2. Recurrent Streptococcus equi subsp. zooepidemicus Bacteremia in an Infant

    PubMed Central

    Watson, Joshua R.; Leber, Amy; Velineni, Sridhar; Timoney, John F.

    2015-01-01

    We describe a case of an infant with recurrent bacteremia caused by Streptococcus equi subsp. zooepidemicus, likely transmitted from mother to infant. Our case highlights the importance of an epidemiological history and molecular diagnostics in ascertaining insights into transmission, pathogenesis, and optimal management. PMID:26179301

  3. Laminaria japonica Extract, an Inhibitor of Clavibater michiganense Subsp. Sepedonicum

    PubMed Central

    Cai, Jin; Feng, Jia; Xie, Shulian; Wang, Feipeng; Xu, Qiufeng

    2014-01-01

    Bacterial ring rot of potato is one of the most serious potato plant and tuber diseases. Laminaria japonica extract was investigated for its antimicrobial activity against Clavibater michiganense subsp. sepedonicum (Spieckermann & Kotthoff) Davis et al., the causative agent of bacterial ring rot of potato. The results showed that the optimum extraction conditions of antimicrobial substances from L. japonica were an extraction temperature of 80°C, an extraction time of 12 h, and a solid to liquid ratio of 1∶25. Active compounds of L. japonica were isolated by solvent partition, thin layer chromatography (TLC) and column chromatography. All nineteen fractionations had antimicrobial activities against C. michiganense subsp. sepedonicum, while Fractionation three (Fr.3) had the highest (P<0.05) antimicrobial activity. Chemical composition analysis identified a total of 26 components in Fr.3. The main constituents of Fr.3 were alkanes (80.97%), esters (5.24%), acids (4.87%) and alcohols (2.21%). Antimicrobial activity of Fr.3 against C. michiganense subsp. sepedonicum could be attributed to its ability to damage the cell wall and cell membrane, induce the production of reactive oxygen species (ROS), increase cytosolic Ca2+ concentration, inhibit the glycolytic pathway (EMP) and tricarboxylic acid (TCA) cycle, inhibit protein and nucleic acid synthesis, and disrupt the normal cycle of DNA replication. These findings indicate that L. japonica extracts have potential for inhibiting C. michiganense subsp. sepedonicum. PMID:24714388

  4. Complete Genome Sequence of Anaplasma marginale subsp. centrale

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anaplasma marginale subsp. centrale is a naturally attenuated subtype that has been used as a vaccine for a century. We sequenced the genome of this organism and compared it to those of virulent senso stricto A. marginale strains. The comparison markedly narrows the number of outer membrane protein ...

  5. Draft Genome Sequence of Streptomyces hygroscopicus subsp. hygroscopicus NBRC 16556.

    PubMed

    Komaki, Hisayuki; Ichikawa, Natsuko; Oguchi, Akio; Hamada, Moriyuki; Tamura, Tomohiko; Suzuki, Ken-Ichiro; Fujita, Nobuyuki

    2016-01-01

    Here, we report the draft genome sequence of strain NBRC 16556, deposited as Streptomyces hygroscopicus subsp. hygroscopicus into the NBRC culture collection. An average nucleotide identity analysis confirmed that the taxonomic identification is correct. The genome sequence will serve as a valuable reference for genome mining to search new secondary metabolites. PMID:27198007

  6. Description and history of Syringa oblata subsp. oblata 'Frank Meyer'

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An accession of Syringa oblata subsp. oblata (PI 23031) collected in China by Frank Meyer in 1908was given the name ‘Frank Meyer’ by Father Fiala in 1988. To be established according to the International Code of Nomenclature for Cultivated Plants, a new cultivar name must be accompanied by a descrip...

  7. Mycobacterium avium subsp. paratuberculosis infection, immunology and pathology of livestock

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) infection in ruminants leads to a chronic and progressive enteric disease (Johne’s disease) that results in loss of intestinal function, poor body condition, and eventual death. Transmission is primarily through a fecal-oral route in neonates but con...

  8. Lactococcus lactis subsp. tructae subsp. nov. isolated from the intestinal mucus of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss).

    PubMed

    Pérez, Tania; Balcázar, José Luis; Peix, Alvaro; Valverde, Angel; Velázquez, Encarna; de Blas, Ignacio; Ruiz-Zarzuela, Imanol

    2011-08-01

    The species Lactococcus lactis currently includes three subspecies; L. lactis subsp. lactis and L. lactis subsp. cremoris, isolated from milk sources, and L. lactis subsp. hordniae, isolated from the leafhopper Hordnia circellata. In this study, three strains, designated L105(T), I3 and L101, were isolated from the intestinal mucus of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss). These strains were closely related to members of the species Lactococcus lactis. Strain L105(T) showed 99.4 % 16S rRNA gene sequence similarity to that of the type strains L. lactis subsp. lactis NCDO 604(T) and L. lactis subsp. hordniae NCDO 2181(T) and showed 99.9 % similarity to the type strain Lactococcus lactis subsp. cremoris NCDO 607(T). Analysis of two housekeeping genes, rpoB and recA, confirmed the close relationship between the novel strains and L. lactis subsp. cremoris with similarities of 99.3 and 99.7 %, respectively. The three strains could, however, be differentiated from their closest relatives on the basis of several phenotypic characteristics, as was the case for L. lactis subsp. lactis and L. lactis subsp. hordniae, which were also closely related on the basis of 16S rRNA, rpoB and recA gene sequence similarities. The strains isolated in this study represent a new subspecies, for which the name Lactococcus lactis subsp. tructae subsp. nov. is proposed. The type strain is L105(T) ( = LMG 24662(T)  = DSM 21502(T)). PMID:20833888

  9. Analysis of labdane-type diterpenes from Cistus creticus (subsp. creticus and subsp. eriocephalus), by GC and GC-MS.

    PubMed

    Anastasaki, T; Demetzos, C; Perdetzoglou, D; Gazouli, M; Loukis, A; Harvala, C

    1999-12-01

    The qualitative and quantitative analysis of labdane-type diterpenes of the hexane extracts and of the essential oils of the leaves, fruits and resin "Ladano", of Cistus creticus subsp. creticus and Cistus creticus subsp. eriocephalus, have been carried out by GC and GC-MS analysis using two capillary chromatographic columns, i.e., HP-5MS and CP-Wax. The methanolic extract of the fruits of C. creticus subsp. creticus was examined and seven labdane diterpenes were isolated and identified by spectroscopic methods. Data on the investigation of labdane diterpenes by GC and GC-MS is limited and most of them have never been analysed by this method. The results obtained by this analysis could be useful for identifying them in crude plant extracts. Manoyl oxides were studied further for the percentage content of their isomers. The hexane extracts of the two subspecies as well as the manoyl oxide isomers isolated from the methanolic extract of the fruits of C. creticus subsp. creticus, were tested for their antimicrobial activity against Gram-positive and Gram-negative bacteria. Global numerical differences of these C. creticus subspecies, based on labdane diterpenes content in the hexane extracts as well as in the essential oils, were established by statistical methods. Phenotypic differences are discussed. PMID:10630116

  10. Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle

    PubMed Central

    Ahlstrom, Christina; Barkema, Herman W.; Stevenson, Karen; Zadoks, Ruth N.; Biek, Roman; Kao, Rowland; Trewby, Hannah; Haupstein, Deb; Kelton, David F.; Fecteau, Gilles; Labrecque, Olivia; Keefe, Greg P.; McKenna, Shawn L. B.; Tahlan, Kapil; De Buck, Jeroen

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale. PMID:26871723

  11. Comparison of whole mitochondrial genome sequences from two clades of the invasive ascidian, Didemnum vexillum.

    PubMed

    Smith, Kirsty F; Abbott, Cathryn L; Saito, Yasunori; Fidler, Andrew E

    2015-02-01

    The mitochondria are the main source of cellular energy production and have an important role in development, fertility, and thermal limitations. Adaptive mitochondrial DNA mutations have the potential to be of great importance in determining aspects of the life history of an organism. Phylogenetic analyses of the globally invasive marine ascidian Didemnum vexillum using the mitochondrial cytochrome c oxidase 1 (COX1) coding region, revealed two distinct clades. Representatives of one clade (denoted by 'B') are geographically restricted to D. vexillum's native region (north-west Pacific Ocean, including Japan), whereas members of the other clade (denoted by 'A') have been introduced and become invasive in temperate coastal areas around the world. Persistence of clade B's restricted distribution may reflect it being inherently less invasive than clade A. To investigate this we sought to determine if the two clades differ significantly in other mitochondrial genes of functional significance, specifically, alterations in amino acids encoded in mitochondrial enzyme subunits. Differences in functional mitochondrial genes could indicate an increased ability for clade A colonies to tolerate a wider range of environmental temperature. Full mitochondrial genomic sequences from D. vexillum clades A and B were obtained and they predict significant sequence differences in genes encoding for enzymes involved in oxidative phosphorylation. Diversity levels were relatively high and showed divergence across almost all genes, with p-distance values between the two clades indicating recent divergence. Both clades showed an excess of rare variants, which is consistent with balancing selection or a recent population expansion. Results presented here will inform future research focusing on examining the functional properties of the corresponding mitochondrial respiration enzymes, of A and B clade enzymes. By comparing closely related taxa that have differing distributions it is possible

  12. Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

    SciTech Connect

    Singh, Harkewal; Felts, Richard L.; Schuermann, Jonathan P.; Reilly, Thomas J.; Tanner, John J.

    2009-12-01

    Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-{angstrom}-resolution structure of FtHAP complexed with the competitive inhibitor L(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 {angstrom}, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 {angstrom} resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an {alpha}/{beta} core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3'-AMP binds in one end of a 15-{angstrom}-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2'-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K{sub m} by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small

  13. Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins.

    PubMed Central

    Von Tersch, M A; Robbins, H L; Jany, C S; Johnson, T B

    1991-01-01

    Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids. Images PMID:2014985

  14. [Evaluation of a newly-developed ready-to-use commercial PCR kit for the molecular diagnosis of Francisella tularensis].

    PubMed

    Celebi, Bekir; Kılıç, Selçuk; Yeşilyurt, Murat; Acar, Bülent

    2014-01-01

    Tularemia is a rare zoonotic infection, however, considerations of tularemia as a biological weapon and several recent major epidemics have caused renewed interest in this disease. Laboratory diagnosis of tularemia is done in the presence of appropriate epidemiological data, by the demonstration of specific antibodies in the serum samples obtained with 1-2 week intervals following the development of symptoms. It is an a posteriori analysis with limited use for prompt diagnosis of the patient during the early symptomatic phase and deliberate release of biological agents. Limitations in both culture and serology have led to substantial research in the development of new diagnostic techniques. Several PCR methods for tularemia have been developed, both for conventional and real-time polymerase chain reaction (rtPCR). However, PCR methods are hard to be deployed in remote endemic areas that lack sufficient infrastructure. Recently a "Toolbox" which includes all instruments, equipments and solutions [DNA4U® Bacteria Genomic DNA Isolation Kit, CubeCycler® (Personal Thermal Cycler), PCR4U® Bioterrorism Agents Detection Kit, electrophoresis tank, power supply, ready-agarose gel and electrophoresis buffer] necessary for conventional PCR, was developed for the identification of bioterrorism agents in the field. In this study we aimed to evaluate the efficacy of a ready-to-use commercial PCR kit (Nanobiz, Ankara, Turkey) targeting the tul4 gene, for the diagnosis of tularemia and to compare the results with an in-house conventional PCR and a rtPCR test. We applied the assay to a collection of four F.tularensis standard strains, 15 field isolates (from humans, animals, water), 13 non-Francisella strains which are phylogenetically related to F.tularensis and a total of 60 lymph node aspirates obtained from suspected tularemia cases. Compared to the in-house PCR method used in our laboratory, the sensitivity, specificity, positive and negative predictive values of Nanobiz PCR

  15. Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles.

    PubMed

    Nandi, Tannistha; Holden, Matthew T G; Holden, Mathew T G; Didelot, Xavier; Mehershahi, Kurosh; Boddey, Justin A; Beacham, Ifor; Peak, Ian; Harting, John; Baybayan, Primo; Guo, Yan; Wang, Susana; How, Lee Chee; Sim, Bernice; Essex-Lopresti, Angela; Sarkar-Tyson, Mitali; Nelson, Michelle; Smither, Sophie; Ong, Catherine; Aw, Lay Tin; Hoon, Chua Hui; Michell, Stephen; Studholme, David J; Titball, Richard; Chen, Swaine L; Parkhill, Julian; Tan, Patrick

    2015-01-01

    Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. PMID:25236617

  16. Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles

    PubMed Central

    Nandi, Tannistha; Holden, Matthew T.G.; Didelot, Xavier; Mehershahi, Kurosh; Boddey, Justin A.; Beacham, Ifor; Peak, Ian; Harting, John; Baybayan, Primo; Guo, Yan; Wang, Susana; How, Lee Chee; Sim, Bernice; Essex-Lopresti, Angela; Sarkar-Tyson, Mitali; Nelson, Michelle; Smither, Sophie; Ong, Catherine; Aw, Lay Tin; Hoon, Chua Hui; Michell, Stephen; Studholme, David J.; Titball, Richard; Chen, Swaine L.; Parkhill, Julian

    2015-01-01

    Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. PMID:25236617

  17. A functional genomic analysis of Arabidopsis thaliana PP2C clade D

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the reference dicot plant Arabidopsis thaliana, the PP2C family of P-protein phosphatases includes the products of 80 genes that have been separated into 10 multi-protein clades plus six singletons. Clade D includes the products of nine genes distributed among 3 chromosomes (PPD1, At3g12620; PPD2...

  18. Clade age and species richness are decoupled across the eukaryotic tree of life.

    PubMed

    Rabosky, Daniel L; Slater, Graham J; Alfaro, Michael E

    2012-08-01

    Explaining the dramatic variation in species richness across the tree of life remains a key challenge in evolutionary biology. At the largest phylogenetic scales, the extreme heterogeneity in species richness observed among different groups of organisms is almost certainly a function of many complex and interdependent factors. However, the most fundamental expectation in macroevolutionary studies is simply that species richness in extant clades should be correlated with clade age: all things being equal, older clades will have had more time for diversity to accumulate than younger clades. Here, we test the relationship between stem clade age and species richness across 1,397 major clades of multicellular eukaryotes that collectively account for more than 1.2 million described species. We find no evidence that clade age predicts species richness at this scale. We demonstrate that this decoupling of age and richness is unlikely to result from variation in net diversification rates among clades. At the largest phylogenetic scales, contemporary patterns of species richness are inconsistent with unbounded diversity increase through time. These results imply that a fundamentally different interpretative paradigm may be needed in the study of phylogenetic diversity patterns in many groups of organisms. PMID:22969411

  19. Interplay between the genetic clades of Micromonas and their viruses in the Western English Channel.

    PubMed

    Baudoux, A-C; Lebredonchel, H; Dehmer, H; Latimier, M; Edern, R; Rigaut-Jalabert, F; Ge, P; Guillou, L; Foulon, E; Bozec, Y; Cariou, T; Desdevises, Y; Derelle, E; Grimsley, N; Moreau, H; Simon, N

    2015-10-01

    The genus Micromonas comprises distinct genetic clades that commonly dominate eukaryotic phytoplankton community from polar to tropical waters. This phytoplankter is also recurrently infected by abundant and genetically diverse prasinoviruses. Here we report on the interplay between prasinoviruses and Micromonas with regard to the genetic diversity of this host. For 1 year, we monitored the abundance of three clades of Micromonas and their viruses in the Western English Channel, both in the environment using clade-specific probes and flow cytometry, and in the laboratory using clonal strains of Micromonas clades to assay for their viruses by plaque-forming units. We showed that the seasonal fluctuations of Micromonas clades were closely mirrored by the abundance of their corresponding viruses, indicating that the members of Micromonas genus are susceptible to viral infection, regardless of their genetic affiliation. The characterization of 45 viral isolates revealed that Micromonas clades are attacked by specific virus populations, which exhibit distinctive clade specificity, life strategies and genetic diversity. However, some viruses can also cross-infect different host clades, suggesting a mechanism of horizontal gene transfer within the Micromonas genus. This study provides novel insights into the impact of viral infection for the ecology and evolution of the prominent phytoplankter Micromonas. PMID:26081716

  20. Clade Age and Species Richness Are Decoupled Across the Eukaryotic Tree of Life

    PubMed Central

    2012-01-01

    Explaining the dramatic variation in species richness across the tree of life remains a key challenge in evolutionary biology. At the largest phylogenetic scales, the extreme heterogeneity in species richness observed among different groups of organisms is almost certainly a function of many complex and interdependent factors. However, the most fundamental expectation in macroevolutionary studies is simply that species richness in extant clades should be correlated with clade age: all things being equal, older clades will have had more time for diversity to accumulate than younger clades. Here, we test the relationship between stem clade age and species richness across 1,397 major clades of multicellular eukaryotes that collectively account for more than 1.2 million described species. We find no evidence that clade age predicts species richness at this scale. We demonstrate that this decoupling of age and richness is unlikely to result from variation in net diversification rates among clades. At the largest phylogenetic scales, contemporary patterns of species richness are inconsistent with unbounded diversity increase through time. These results imply that a fundamentally different interpretative paradigm may be needed in the study of phylogenetic diversity patterns in many groups of organisms. PMID:22969411

  1. Draft Genome Sequence of Environmental Bacterium Vibrio vulnificus CladeA-yb158

    PubMed Central

    Danin-Poleg, Yael; Raz, Nili; Roig, Francisco J.; Amaro, Carmen

    2015-01-01

    We report the genome sequence of the environmental Vibrio vulnificus biotype 1_cladeA. This draft genome of the CladeA-yb158 strain, isolated in Israel, represents this newly emerged clonal group that contains both clinical and environmental strains. PMID:26205875

  2. Bioinformatics of varicella-zoster virus: Single nucleotide polymorphisms define clades and attenuated vaccine genotypes

    PubMed Central

    Chow, Vincent T.; Tipples, Graham A.; Grose, Charles

    2012-01-01

    Varicella zoster virus (VZV) is one of the human herpesviruses. To date, over 40 complete VZV genomes have been sequenced and analyzed. The VZV genome contains around 125,000 base pairs including 70 open reading frames (ORFs). Enumeration of single nucleotide polymorphisms (SNPs) has determined that the following ORFs are the most variable (in descending order): 62, 22, 29, 28, 37, 21, 54, 31, 1 and 55. ORF 62 is the major immediate early regulatory VZV gene. Further SNP analysis across the entire genome has led to the observation that VZV strains can be broadly grouped into clades within a phylogenetic tree. VZV strains collected in Singapore provided important sequence data for construction of the phylogenetic tree. Currently 5 VZV clades are recognized; they have been designated clades 1 through 5. Clades 1 and 3 include European/North American strains; clade 2 includes Asian strains, especially from Japan; and clade 5 includes strains from India. Clade 4 includes some strains from Europe, but its geographic origins need further documentation.. Within clade 1, five variant viruses have been isolated with a missense mutation in the gE (ORF 68) glycoprotein; these strains have an altered increased cell spread phenotype. Bioinformatics analyses of the attenuated vaccine strains have also been performed, with a subsequent discovery of a stop-codon SNP in ORFO as a likely attenuation determinant. Taken together, these VZV bioinformatics analyses have provided enormous insights into VZV phylogenetics as well as VZV SNPs associated with attenuation. PMID:23183312

  3. Progress to extinction: increased specialisation causes the demise of animal clades.

    PubMed

    Raia, P; Carotenuto, F; Mondanaro, A; Castiglione, S; Passaro, F; Saggese, F; Melchionna, M; Serio, C; Alessio, L; Silvestro, D; Fortelius, M

    2016-01-01

    Animal clades tend to follow a predictable path of waxing and waning during their existence, regardless of their total species richness or geographic coverage. Clades begin small and undifferentiated, then expand to a peak in diversity and range, only to shift into a rarely broken decline towards extinction. While this trajectory is now well documented and broadly recognised, the reasons underlying it remain obscure. In particular, it is unknown why clade extinction is universal and occurs with such surprising regularity. Current explanations for paleontological extinctions call on the growing costs of biological interactions, geological accidents, evolutionary traps, and mass extinctions. While these are effective causes of extinction, they mainly apply to species, not clades. Although mass extinctions is the undeniable cause for the demise of a sizeable number of major taxa, we show here that clades escaping them go extinct because of the widespread tendency of evolution to produce increasingly specialised, sympatric, and geographically restricted species over time. PMID:27507121

  4. Progress to extinction: increased specialisation causes the demise of animal clades

    NASA Astrophysics Data System (ADS)

    Raia, P.; Carotenuto, F.; Mondanaro, A.; Castiglione, S.; Passaro, F.; Saggese, F.; Melchionna, M.; Serio, C.; Alessio, L.; Silvestro, D.; Fortelius, M.

    2016-08-01

    Animal clades tend to follow a predictable path of waxing and waning during their existence, regardless of their total species richness or geographic coverage. Clades begin small and undifferentiated, then expand to a peak in diversity and range, only to shift into a rarely broken decline towards extinction. While this trajectory is now well documented and broadly recognised, the reasons underlying it remain obscure. In particular, it is unknown why clade extinction is universal and occurs with such surprising regularity. Current explanations for paleontological extinctions call on the growing costs of biological interactions, geological accidents, evolutionary traps, and mass extinctions. While these are effective causes of extinction, they mainly apply to species, not clades. Although mass extinctions is the undeniable cause for the demise of a sizeable number of major taxa, we show here that clades escaping them go extinct because of the widespread tendency of evolution to produce increasingly specialised, sympatric, and geographically restricted species over time.

  5. Progress to extinction: increased specialisation causes the demise of animal clades

    PubMed Central

    Raia, P.; Carotenuto, F.; Mondanaro, A.; Castiglione, S.; Passaro, F.; Saggese, F.; Melchionna, M.; Serio, C.; Alessio, L.; Silvestro, D.; Fortelius, M.

    2016-01-01

    Animal clades tend to follow a predictable path of waxing and waning during their existence, regardless of their total species richness or geographic coverage. Clades begin small and undifferentiated, then expand to a peak in diversity and range, only to shift into a rarely broken decline towards extinction. While this trajectory is now well documented and broadly recognised, the reasons underlying it remain obscure. In particular, it is unknown why clade extinction is universal and occurs with such surprising regularity. Current explanations for paleontological extinctions call on the growing costs of biological interactions, geological accidents, evolutionary traps, and mass extinctions. While these are effective causes of extinction, they mainly apply to species, not clades. Although mass extinctions is the undeniable cause for the demise of a sizeable number of major taxa, we show here that clades escaping them go extinct because of the widespread tendency of evolution to produce increasingly specialised, sympatric, and geographically restricted species over time. PMID:27507121

  6. Genome size increases in recently diverged hornwort clades.

    PubMed

    Bainard, Jillian D; Villarreal, Juan Carlos

    2013-08-01

    As our knowledge of plant genome size estimates continues to grow, one group has continually been neglected: the hornworts. Hornworts (Anthocerotophyta) have been traditionally grouped with liverworts and mosses because they share a haploid dominant life cycle; however, recent molecular studies place hornworts as the sister lineage to extant tracheophytes. Given the scarcity of information regarding the DNA content of hornworts, our objective was to estimate the 1C-value for a range of hornwort species within a phylogenetic context. Using flow cytometry, we estimated genome size for 36 samples representing 24 species. This accounts for roughly 10% of known hornwort species. Haploid genome sizes (1C-value) ranged from 160 Mbp or 0.16 pg (Leiosporoceros dussii) to 719 Mbp or 0.73 pg (Nothoceros endiviifolius). The average 1C-value was 261 ± 104 Mbp (0.27 ± 0.11 pg). Ancestral reconstruction of genome size on a hornwort phylogeny suggests a small ancestral genome size and revealed increases in genome size in the most recently divergent clades. Much more work is needed to understand DNA content variation in this phylogenetically important group, but this work has significantly increased our knowledge of genome size variation in hornworts. PMID:24168626

  7. Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

    SciTech Connect

    Shams-Eldin, Hosam Santos de Macedo, Cristiana; Niehus, Sebastian; Dorn, Caroline; Kimmel, Juergen; Azzouz, Nahid; Schwarz, Ralph T.

    2008-06-06

    Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker's yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.

  8. Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    SciTech Connect

    Seiner, Derrick R.; Colburn, Heather A.; Baird, Cheryl L.; Bartholomew, Rachel A.; Straub, Tim M.; Victry, Kristin D.; Hutchison, Janine R.; Valentine, Nancy B.; Bruckner-Lea, Cindy J.

    2013-04-29

    To evaluate the sensitivity and specificity of the Idaho Technologies FilmArray® Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft), and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. Methods and Results: DNA samples from Ba, Ft and Yp strains and near-neighbors, and live Ba spores were analyzed using the Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA suggest a limit of detection of 250 genome equivalents (GEs) per sample. Furthermore, the correct call of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 Ba Sterne spores, at least one of the two possible Ba markers were identified in all samples tested. We observed no cross-reactivity with near-neighbor DNAs.

  9. Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes

    PubMed Central

    Amigo, Natalia; Mercado, Elsa; Bentancor, Adriana; Singh, Pallavi; Vilte, Daniel; Gerhardt, Elisabeth; Zotta, Elsa; Ibarra, Cristina; Manning, Shannon D.; Larzábal, Mariano; Cataldi, Angel

    2015-01-01

    The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina. PMID:26030198

  10. Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes.

    PubMed

    Amigo, Natalia; Mercado, Elsa; Bentancor, Adriana; Singh, Pallavi; Vilte, Daniel; Gerhardt, Elisabeth; Zotta, Elsa; Ibarra, Cristina; Manning, Shannon D; Larzábal, Mariano; Cataldi, Angel

    2015-01-01

    The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina. PMID:26030198

  11. Draft Genome Sequences of Salmonella enterica subsp. enterica Serovars Typhimurium and Nottingham Isolated from Food Products

    PubMed Central

    Zheng, Jie; Ayers, Sherry; Melka, David C.; Curry, Phillip E.; Payne, Justin S.; Laasri, Anna; Wang, Charles; Hammack, Thomas S.; Brown, Eric W.

    2016-01-01

    A quantitative real-time PCR (qPCR) designed to detect Salmonella enterica subsp. enterica serovar Enteritidis, targeting the sdf gene, generated positive results for S. enterica subsp. enterica serovar Typhimurium (CFSAN033950) and S. enterica subsp. enterica serovar Nottingham (CFSAN006803) isolated from food samples. Both strains show pulsed-field gel electrophoresis (PFGE) patterns distinct from those of S. Enteritidis. Here, we report the genome sequences of these two strains. PMID:27445384

  12. Draft Genome Sequence of Fusobacterium necrophorum subsp. funduliforme Bovine Liver Abscess Isolate B35

    PubMed Central

    Calcutt, Michael J.; Foecking, Mark F.; Nagaraja, Tiruvoor G.

    2014-01-01

    Fusobacterium necrophorum is a Gram-negative anaerobic bacterium that causes foot rot and liver abscesses in cattle. F. necrophorum subsp. necrophorum and the less virulent organism F. necrophorum subsp. funduliforme are recognized. We present here a draft genome sequence of the bovine liver abscess isolate F. necrophorum subsp. funduliforme strain B35, which affords a genomic perspective of virulence and bovine adaptation. PMID:24786958

  13. Francisella tularensis Vaccines Elicit Concurrent Protective T- and B-Cell Immune Responses in BALB/cByJ Mice

    PubMed Central

    De Pascalis, Roberto; Mittereder, Lara; Chou, Alicia Y.; Kennett, Nikki J.; Elkins, Karen L.

    2015-01-01

    In the last decade several new vaccines against Francisella tularensis, which causes tularemia, have been characterized in animal models. Whereas many of these vaccine candidates showed promise, it remains critical to bridge the preclinical studies to human subjects, ideally by taking advantage of correlates of protection. By combining in vitro intramacrophage LVS replication with gene expression data through multivariate analysis, we previously identified and quantified correlative T cell immune responses that discriminate vaccines of different efficacy. Further, using C57BL/6J mice, we demonstrated that the relative levels of gene expression vary according to vaccination route and between cell types from different organs. Here, we extended our studies to the analysis of T cell functions of BALB/cByJ mice to evaluate whether our approach to identify correlates of protection also applies to a Th2 dominant mouse strain. BALB/cByJ mice had higher survival rates than C57BL/6J mice when they were immunized with suboptimal vaccines and challenged. However, splenocytes derived from differentially vaccinated BALB/cByJ mice controlled LVS intramacrophage replication in vitro in a pattern that reflected the hierarchy of protection observed in C57BL/6J mice. In addition, gene expression of selected potential correlates revealed similar patterns in splenocytes of BALB/cByJ and C57BL/6J mice. The different survival patterns were related to B cell functions, not necessarily to specific antibody production, which played an important protective role in BALB/cByJ mice when vaccinated with suboptimal vaccines. Our studies therefore demonstrate the range of mechanisms that operate in the most common mouse strains used for characterization of vaccines against F. tularensis, and illustrate the complexity necessary to define a comprehensive set of correlates. PMID:25973794

  14. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis.

    PubMed

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E; Nikolaev, Evgeni V; Magni, Giulio; Zhang, Hong; Osterman, Andrei L

    2009-03-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-A resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN --> NMN --> NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN --> NaAD --> NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis. PMID:19204287

  15. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

    PubMed Central

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A.; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E.; Nikolaev, Evgeni V.; Magni, Giulio; Zhang, Hong; Osterman, Andrei L.

    2009-01-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-Å resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN → NMN → NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN → NaAD → NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis. PMID:19204287

  16. Rapid Focused Sequencing: A Multiplexed Assay for Simultaneous Detection and Strain Typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    PubMed Central

    Zolotova, Anna; Tan, Eugene; Selden, Richard F.

    2013-01-01

    Background The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. Methodology/Principal Findings We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed “Rapid Focused Sequencing,” allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. Conclusions/Significance The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental background strains. The

  17. Microinjection of Francisella tularensis and Listeria monocytogenes reveals the importance of bacterial and host factors for successful replication.

    PubMed

    Meyer, Lena; Bröms, Jeanette E; Liu, Xijia; Rottenberg, Martin E; Sjöstedt, Anders

    2015-08-01

    Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., ΔiglA, ΔiglÇ ΔiglG, ΔiglI, and ΔpdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC(-/-) BMDM, MyD88(-/-) BMDM, J774 cells, or HeLa cells, LVS, ΔpdpE and ΔiglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the ΔiglA and ΔiglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC(-/-) BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88(-/-) BMDM and LVS showed no replication in either BMDM or MyD88(-/-) BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection. PMID:26034213

  18. Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

    PubMed Central

    Meyer, Lena; Bröms, Jeanette E.; Liu, Xijia; Rottenberg, Martin E.

    2015-01-01

    Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., ΔiglA, ΔiglÇ ΔiglG, ΔiglI, and ΔpdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC−/− BMDM, MyD88−/− BMDM, J774 cells, or HeLa cells, LVS, ΔpdpE and ΔiglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the ΔiglA and ΔiglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC−/− BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88−/− BMDM and LVS showed no replication in either BMDM or MyD88−/− BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection. PMID:26034213

  19. Reproducible and Quantitative Model of Infection of Dermacentor variabilis with the Live Vaccine Strain of Francisella tularensis

    PubMed Central

    Coburn, Jenifer; Maier, Tamara; Casey, Monika; Padmore, Lavinia; Sato, Hiromi

    2014-01-01

    Pathogen life cycles in mammalian hosts have been studied extensively, but studies with arthropod vectors represent considerable challenges. In part this is due to the difficulty of delivering a reproducible dose of bacteria to follow arthropod-associated replication. We have established reproducible techniques to introduce known numbers of Francisella tularensis strain LVS from mice into Dermacentor variabilis nymphs. Using this model infection system, we performed dose-response infection experiments and followed bacterial replication through the molt to adults and at later time points. During development to adults, bacteria replicate to high numbers and can be found associated with the gut tissues, salivary glands, and hemolymph of adult ticks. Further, we can transmit a mutant of LVS (LVS ΔpurMCD) that cannot replicate in macrophages in vitro or in mice to nymphs. Our data show that the LVS ΔpurMCD mutant cannot be transstadially transmitted from nymphs to adult ticks. We then show that a plasmid-complemented strain of this mutant is recoverable in adult ticks and necessary for bacterial replication during the molt. In a mixed-infection assay (ΔpurMCD mutant versus ΔpurMCD complement), 98% of the recovered bacteria retained the plasmid marker. These data suggest that the ΔpurMCD mutation cannot be rescued by the presence a complemented strain in a mixed infection. Importantly, our infection model provides a platform to test specific mutants for their replication in ticks, perform competition studies, and use other genetic techniques to identify F. tularensis genes that are expressed or required in this unique environment. PMID:25362054

  20. Comparing the Effects of Symbiotic Algae (Symbiodinium) Clades C1 and D on Early Growth Stages of Acropora tenuis

    PubMed Central

    Yuyama, Ikuko; Higuchi, Tomihiko

    2014-01-01

    Reef-building corals switch endosymbiotic algae of the genus Symbiodinium during their early growth stages and during bleaching events. Clade C Symbiodinium algae are dominant in corals, although other clades — including A and D — have also been commonly detected in juvenile Acroporid corals. Previous studies have been reported that only molecular data of Symbiodinium clade were identified within field corals. In this study, we inoculated aposymbiotic juvenile polyps with cultures of clades C1 and D Symbiodinium algae, and investigated the different effect of these two clades of Symbiodinium on juvenile polyps. Our results showed that clade C1 algae did not grow, while clade D algae grew rapidly during the first 2 months after inoculation. Polyps associated with clade C1 algae exhibited bright green fluorescence across the body and tentacles after inoculation. The growth rate of polyp skeletons was lower in polyps associated with clade C1 algae than those associated with clade D algae. On the other hand, antioxidant activity (catalase) of corals was not significantly different between corals with clade C1 and clade D algae. Our results suggested that clade D Symbiodinium algae easily form symbiotic relationships with corals and that these algae could contribute to coral growth in early symbiosis stages. PMID:24914677

  1. Comparing the effects of symbiotic algae (Symbiodinium) clades C1 and D on early growth stages of Acropora tenuis.

    PubMed

    Yuyama, Ikuko; Higuchi, Tomihiko

    2014-01-01

    Reef-building corals switch endosymbiotic algae of the genus Symbiodinium during their early growth stages and during bleaching events. Clade C Symbiodinium algae are dominant in corals, although other clades - including A and D - have also been commonly detected in juvenile Acroporid corals. Previous studies have been reported that only molecular data of Symbiodinium clade were identified within field corals. In this study, we inoculated aposymbiotic juvenile polyps with cultures of clades C1 and D Symbiodinium algae, and investigated the different effect of these two clades of Symbiodinium on juvenile polyps. Our results showed that clade C1 algae did not grow, while clade D algae grew rapidly during the first 2 months after inoculation. Polyps associated with clade C1 algae exhibited bright green fluorescence across the body and tentacles after inoculation. The growth rate of polyp skeletons was lower in polyps associated with clade C1 algae than those associated with clade D algae. On the other hand, antioxidant activity (catalase) of corals was not significantly different between corals with clade C1 and clade D algae. Our results suggested that clade D Symbiodinium algae easily form symbiotic relationships with corals and that these algae could contribute to coral growth in early symbiosis stages. PMID:24914677

  2. Colonization patterns of an mCherry-tagged Pectobacterium carotovorum subsp. brasiliense strain in potato plants.

    PubMed

    Kubheka, Gugulethu C; Coutinho, Teresa A; Moleleki, Ntsane; Moleleki, Lucy N

    2013-12-01

    Pectobacterium carotovorum subsp. brasiliense is a newly identified member of the potato soft rot enterobacteriaceae. The pathogenesis of this pathogen is still poorly understood. In this study, an mCherry-P. carotovorum subsp. brasiliense-tagged strain was generated to study P. carotovorum subsp. brasiliense-potato plant interactions. Prior to use, the tagged strain was evaluated for in vitro growth, plasmid stability, and virulence on potato tubers and shown to be similar to the wild type. Four potato cultivars were evaluated for stem-based resistance against P. carotovorum subsp. brasiliense. Confocal laser-scanning microscopy and in vitro viable cell counts showed that P. carotovorum subsp. brasiliense is able to penetrate roots of a susceptible potato cultivar as early as 12 h postinoculation and migrate upward into aerial stem parts. Due to the phenotypic differences observed between tolerant and susceptible cultivars, a comparison of P. carotovorum subsp. brasiliense colonization patterns in these cultivars was undertaken. In the susceptible cultivar, P. carotovorum subsp. brasiliense cells colonized the xylem tissue, forming "biofilm-like" aggregates that led to occlusion of some of the vessels. In contrast, in the tolerant cultivar, P. carotovorum subsp. brasiliense appeared as free-swimming planktonic cells with no specific tissue localization. This suggests that there are resistance mechanisms in the tolerant cultivar that limit aggregation of P. carotovorum subsp. brasiliense in planta and, hence, the lack of symptom development in this cultivar. PMID:23758294

  3. Characterization of Tetragenococcus strains from sugar thick juice reveals a novel species, Tetragenococcus osmophilus sp. nov., and divides Tetragenococcus halophilus into two subspecies, T. halophilus subsp. halophilus subsp. nov. and T. halophilus subsp. flandriensis subsp. nov.

    PubMed

    Justé, A; Van Trappen, S; Verreth, C; Cleenwerck, I; De Vos, P; Lievens, B; Willems, K A

    2012-01-01

    Most bacteria recovered so far from sugar thick juice during storage represent strains of the species Tetragenococcus halophilus. Recently, several Gram-positive, non-motile, non-spore-forming cocci with other physiological and genetic traits were isolated from sugar thick juice samples from different origins. In this study, representative isolates were investigated using a polyphasic taxonomic approach. The 16S rRNA gene sequence similarity between these isolates and their closest relative, Tetragenococcus muriaticus, was 97.4%. The level of DNA-DNA relatedness between isolate T1(T), representing the newly found Tetragenococcus isolates, and T. muriaticus was 57%. Isolate T1(T) had a DNA G+C content of 36.7 mol%. Phylogenetic data and genomic and phenotypic features demonstrated that the isolates represent a novel species, for which the name Tetragenococcus osmophilus sp. nov. is proposed with T1(T) as the type strain (=LMG 26041(T) =DSM 23765(T)). Additionally, T. halophilus isolates from high-salt and high-sugar environments showed clear differences in several physiological and genetic characteristics like RAPD fingerprints and 16S rRNA gene sequences. DNA-DNA hybridizations, however, showed 79 to 80% relatedness between osmophilic and halophilic T. halophilus isolates, demonstrating that the different strains belong to the same species. Based on the phenotypic and genotypic differences observed, as well as the different origins of the strains and the industrial relevance of thick juice degradation, two subspecies of T. halophilus are described in this manuscript: T. halophilus subsp. halophilus subsp. nov. for the strains isolated from salt media and T. halophilus subsp. flandriensis subsp. nov. for the strains isolated from sugar-rich environments, which were first isolated in Flanders, Belgium. The type strains for the subspecies are IAM 1676(T) (=LMG 11490(T) =DSM 20339(T)) and T5(T) (=LMG 26042(T) =DSM 23766(T)), respectively. PMID:21357458

  4. Hylid frog phylogeny and sampling strategies for speciose clades.

    PubMed

    Wiens, John J; Fetzner, James W; Parkinson, Christopher L; Reeder, Tod W

    2005-10-01

    How should characters and taxa be sampled to resolve efficiently the phylogeny of ancient and highly speciose groups? We addressed this question empirically in the treefrog family Hylidae, which contains > 800 species and may be nonmonophyletic with respect to other anuran families. We sampled 81 species (54 hylids and 27 outgroups) for two mitochondrial genes (12S, ND1), two nuclear genes (POMC, c-myc), and morphology (144 characters) in an attempt to resolve higher-level relationships. We then added 117 taxa to the combined data set, many of which were sampled for only one gene (12S). Despite the relative incompleteness of the majority of taxa, the resulting trees placed all taxa in the expected higher-level clades with strong support, despite some taxa being > 90% incomplete. Furthermore, we found no relationship between the completeness of a taxon and the support (parsimony bootstrap or Bayesian posterior probabilities) for its localized placement on the tree. Separate analysis of the data set with the most taxa (12S) gives a somewhat problematic estimate of higher-level relationships, suggesting that data sets scored only for some taxa (ND1, nuclear genes, morphology) are important in determining the outcome of the combined analysis. The results show that hemiphractine hylids are not closely related to other hylids and should be recognized as a distinct family. They also show that the speciose genus Hyla is polyphyletic, but that its species can be arranged into three monophyletic genera. A new classification of hylid frogs is proposed. Several potentially misleading signals in the morphological data are discussed. PMID:16243760

  5. Serological Investigation of Wild Boars (Sus scrofa) and Red Foxes (Vulpes vulpes) As Indicator Animals for Circulation of Francisella tularensis in Germany

    PubMed Central

    Chaignat, Valerie; Klimpel, Diana; Diller, Roland; Melzer, Falk; Müller, Wolfgang; Tomaso, Herbert

    2014-01-01

    Abstract Tularemia outbreaks in humans have recently been reported in many European countries, but data on the occurrence in the animal population are scarce. In North America, seroconversion of omnivores and carnivores was used as indicator for the presence of tularemia, for the European fauna, however, data are barely available. Therefore, the suitability of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicators for the circulation of F. tularensis in Germany was evaluated. Serum samples from 566 wild boars and 457 red foxes were collected between 1995 and 2012 in three federal states in Central Germany (Hesse, Saxony-Anhalt, and Thuringia). The overall rate of seropositive animals was 1.1% in wild boars and 7.4% in red foxes. In conclusion, serological examination of red foxes is recommended, because they can be reliably used as indicator animals for the presence of F. tularensis in the environment. PMID:24359418

  6. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

    SciTech Connect

    Fischer, N. O.

    2015-01-13

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the third quarter of the third year, F344 rats vaccinated with adjuvanted NLP formulations were challenged with F. tularensis SCHU S4 at Battelle. Preliminary data indicate that up to 65% of females vaccinated intranasally with an NLP-based formulation survived this challenge, compared to only 20% survival of naïve animals. In addition, NLPs were successfully formulated with Burkholderia protein antigens. IACUC approval for immunological assessments in BALB/c mice was received and we anticipate that these assessments will begin by March 2015, pending ACURO approval.

  7. Structural and Enzymatic Analyses Reveal the Binding Mode of a Novel Series of Francisella tularensis Enoyl Reductase (FabI) Inhibitors

    SciTech Connect

    Mehboob, Shahila; Hevener, Kirk E.; Truong, Kent; Boci, Teuta; Santarsiero, Bernard D.; Johnson, Michael E.

    2012-10-10

    Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.

  8. A Francisella tularensis Schu S4 Purine Auxotroph Is Highly Attenuated in Mice but Offers Limited Protection against Homologous Intranasal Challenge

    PubMed Central

    Pechous, Roger D.; McCarthy, Travis R.; Mohapatra, Nrusingh P.; Soni, Shilpa; Penoske, Renee M.; Salzman, Nita H.; Frank, Dara W.; Gunn, John S.; Zahrt, Thomas C.

    2008-01-01

    Background Francisella tularensis is a Gram-negative coccobacillus that causes the febrile illness tularemia. Subspecies that are pathogenic for humans include those comprising the type A (subspecies tularensis) or type B (subspecies holarctica) biovars. An attenuated live vaccine strain (LVS) developed from a type B isolate has previously been used to vaccinate at-risk individuals, but offers limited protection against high dose (>1000 CFUs) challenge with type A strains delivered by the respiratory route. Due to differences between type A and type B F. tularensis strains at the genetic level, it has been speculated that utilization of an attenuated type A strain as a live vaccine might offer better protection against homologous respiratory challenge compared with LVS. Here, we report the construction and characterization of an unmarked ΔpurMCD mutant in the highly virulent type A strain Schu S4. Methodology/Principal Findings Growth of Schu S4 ΔpurMCD was severely attenuated in primary human peripheral blood monocyte-derived macrophages and in the A549 human lung epithelial cell line. The Schu S4 ΔpurMCD mutant was also highly attenuated in mice when delivered via either the intranasal or intradermal infection route. Mice vaccinated intranasally with Schu S4 ΔpurMCD were well protected against high dose intradermal challenge with virulent type A or type B strains of F. tularensis. However, intranasal vaccination with Schu S4 ΔpurMCD induced tissue damage in the lungs, and conferred only limited protection against high dose Schu S4 challenge delivered by the same route. The level of protection observed was similar to that conferred following vaccination with wild-type LVS or the analogous LVS ΔpurMCD mutant. Conclusions/Significance Collectively, these results argue that development of the next generation live attenuated vaccine for Francisella should be based on use of the less pathogenic type B biovar rather than the more reactogenic type A biovar. PMID

  9. Francisella tularensis-induced in vitro gamma interferon, tumor necrosis factor alpha, and interleukin 2 responses appear within 2 weeks of tularemia vaccination in human beings.

    PubMed Central

    Karttunen, R; Surcel, H M; Andersson, G; Ekre, H P; Herva, E

    1991-01-01

    Cell-mediated immunity is essential for protection against the intracellular bacterium Francisella tularensis, which causes tularemia. Positive in vitro T-cell responses in the form of lymphocyte proliferation and lymphokine interleukin 2 (IL-2) and gamma interferon (IFN-gamma) secretion are found in memory immunity. Studies on the secretion of lymphokines with regard to the developing immunity to F. tularensis have not been published. Therefore, 14 subjects with no clinical history of tularemia were vaccinated with a live F. tularensis vaccine strain. The in vitro responses of five subjects (antigen-induced mononuclear cell and whole blood culture DNA synthesis and cytokine secretion) were measured twice a week throughout the period from 0 to 35 days after vaccination, and the peripheral blood lymphocyte subpopulations of nine subjects were determined between days 0 and 14. Positive reactions, i.e., responses exceeding those on day 0, were reached on day 10 with regard to the whole blood culture DNA synthesis response and IL-2 and IFN-gamma secretion and on day 14 with regard to the mononuclear cell DNA synthesis response and tumor necrosis factor alpha (TNF-alpha) secretion. No measurable IL-4 was found in either the immune or nonimmune supernatants. Since the secretion of TNF-alpha was related to immunization, this points to the specificity of the phenomenon, even though the type of secreting cell is not yet known. If it is shown later that specific T cells produce it, the TNF-alpha response and the negative IL-4 finding may speak for the importance of the Th1-like pattern in immunity to F. tularensis. PMID:1909711

  10. Phylogeny, evolutionary trends and classification of the Spathelia–Ptaeroxylon clade: morphological and molecular insights

    PubMed Central

    Appelhans, M. S.; Smets, E.; Razafimandimbison, S. G.; Haevermans, T.; van Marle, E. J.; Couloux, A.; Rabarison, H.; Randrianarivelojosia, M.; Keßler, P. J. A.

    2011-01-01

    Background and Aims The Spathelia–Ptaeroxylon clade is a group of morphologically diverse plants that have been classified together as a result of molecular phylogenetic studies. The clade is currently included in Rutaceae and recognized at a subfamilial level (Spathelioideae) despite the fact that most of its genera have traditionally been associated with other families and that there are no obvious morphological synapomorphies for the clade. The aim of the present study is to construct phylogenetic trees for the Spathelia–Ptaeroxylon clade and to investigate anatomical characters in order to decide whether it should be kept in Rutaceae or recognized at the familial level. Anatomical characters were plotted on a cladogram to help explain character evolution within the group. Moreover, phylogenetic relationships and generic limits within the clade are also addressed. Methods A species-level phylogenetic analysis of the Spathelia–Ptaeroxylon clade based on five plastid DNA regions (rbcL, atpB, trnL–trnF, rps16 and psbA–trnH) was conducted using Bayesian, maximum parsimony and maximum likelihood methods. Leaf and seed anatomical characters of all genera were (re)investigated by light and scanning electron microscopy. Key Results With the exception of Spathelia, all genera of the Spathelila–Ptaeroxylon clade are monophyletic. The typical leaf and seed anatomical characters of Rutaceae were found. Further, the presence of oil cells in the leaves provides a possible synapomorphy for the clade. Conclusions The Spathelia–Ptaeroxylon clade is well placed in Rutaceae and it is reasonable to unite the genera into one subfamily (Spathelioideae). We propose a new tribal classification of Spathelioideae. A narrow circumscription of Spathelia is established to make the genus monophyletic, and Sohnreyia is resurrected to accommodate the South American species of Spathelia. The most recent common ancestor of Spathelioideae probably had leaves with secretory cavities

  11. Reticulate evolution, cryptic species, and character convergence in the core East Asian clade of Gaultheria (Ericaceae).

    PubMed

    Lu, Lu; Fritsch, Peter W; Cruz, Boni C; Wang, Hong; Li, De-Zhu

    2010-10-01

    Phylogenetic relationships of 84 samples representing 30 species in the core East Asian clade of the wintergreen group of Gaultheria (Angiospermae: Ericaceae: Gaultherieae) were estimated from separate and combined DNA sequence data from five genic regions (ITS, matK, rpl16, trnL-trnF, and trnS-trnG) with parsimony, likelihood and Bayesian analyses. Two major clades were recovered, one comprising several sections and series with leaves generally more than 1 cm long [the ser. Leucothoides sensu lato (s.l.) clade] and another comprising the species of ser. Trichophyllae, with leaves generally less than 1 cm long. The ITS region yielded little phylogenetic resolution, whereas in the combined chloroplast analysis the samples from individual morphospecies in both clades were often nonmonophyletic. This was postulated to result from reticulate evolution in the ser. Leucothoides s.l. clade, particularly in two specific cases of hybridization and a crown clade with likely chloroplast capture following localized introgression. In the ser. Trichophyllae clade, such nonmonophyly was largely attributed to cryptic species and character convergence resulting at least partly from extreme morphological reduction. The relatively low-elevation habitats in which the species of the ser. Leucothoides s.l. clade generally grow are thought to have promoted opportunities for sympatry and reticulation, whereas the high-alpine habitats of ser. Trichophyllae are more likely to have spawned isolated populations and narrow endemism. As in other Sino-Himalayan plant groups, overall low sequence divergence and reticulate evolution suggest rapid radiation in the core East Asian clade of Gaultheria. PMID:20558306

  12. Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection Running Title: Airway Barrier Functions during Bacterial Infections.

    PubMed

    Blume, Cornelia; David, Jonathan; Bell, Rachel E; Laver, Jay R; Read, Robert C; Clark, Graeme C; Davies, Donna E; Swindle, Emily J

    2016-01-01

    The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human bronchial epithelium were analysed. Polarised 16HBE14o- or differentiated primary human bronchial epithelial cells (BECs) were exposed to increasing multiplicities of infection (MOI) of B. thailandensis or F. tularensis Live Vaccine Strain and barrier responses monitored over 24-72 h. Challenge of polarized BECs with either bacterial species caused an MOI- and time-dependent increase in ionic permeability, disruption of tight junctions, and bacterial passage from the apical to the basolateral compartment. B. thailandensis was found to be more invasive than F. tularensis. Both bacterial species induced an MOI-dependent increase in TNF-α release. An increase in ionic permeability and TNF-α release was induced by B. thailandensis in differentiated BECs. Pretreatment of polarised BECs with the corticosteroid fluticasone propionate reduced bacterial-dependent increases in ionic permeability, bacterial passage, and TNF-α release. TNF blocking antibody Enbrel(®) reduced bacterial passage only. BEC barrier properties are disrupted during respiratory bacterial infections and targeting with corticosteroids or anti-TNF compounds may represent a therapeutic option. PMID:27527221

  13. Two parallel pathways for ferric and ferrous iron acquisition support growth and virulence of the intracellular pathogen Francisella tularensis Schu S4.

    PubMed

    Pérez, Natalie; Johnson, Richard; Sen, Bhaswati; Ramakrishnan, Girija

    2016-06-01

    Iron acquisition mechanisms in Francisella tularensis, the causative agent of tularemia, include the Francisella siderophore locus (fsl) siderophore operon and a ferrous iron-transport system comprising outer-membrane protein FupA and inner-membrane transporter FeoB. To characterize these mechanisms and to identify any additional iron uptake systems in the virulent subspecies tularensis, single and double deletions were generated in the fsl and feo iron acquisition systems of the strain Schu S4. Deletion of the entire fsl operon caused loss of siderophore production that could be restored by complementation with the biosynthetic genes fslA and fslC and Major Facilitator Superfamily (MFS) transporter gene fslB. (55) Fe-transport assays demonstrated that siderophore-iron uptake required the receptor FslE and MFS transporter FslD. A ΔfeoB' mutation resulted in loss of ability to transport ferrous iron ((55) Fe(2+) ). A ΔfeoB' ΔfslA mutant that required added exogenous siderophore for growth in vitro was unable to grow within tissue culture cells and was avirulent in mice, indicating that no compensatory cryptic iron uptake systems were induced in vivo. These studies demonstrate that the fsl and feo pathways function independently and operate in parallel to effectively support virulence of F. tularensis. PMID:26918301

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

    PubMed Central

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

    2008-01-01

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

  15. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of Francisella tularensis

    PubMed Central

    Hua, Fei; Zhang, Pingping; Zhang, Fuli; Zhao, Yong; Li, Chunfeng; Sun, Chongyun; Wang, Xiaochen; Yang, Ruifu; Wang, Chengbin; Yu, Ailian; Zhou, Lei

    2015-01-01

    Francisella tularensis is a potential biowarfare/bioterrorism agent and zoonotic pathogen that causes tularemia; thus, surveillance of F. tularensis and first-level emergency response using point-of-care testing (POCT) are essential. The UPT-LF POCT assay was established to quantitatively detect F. tularensis within 15 min, and the sensitivity of the assay was 104 CFU · mL−1 (100 CFU/test). The linear quantitative range covered five orders of magnitude, and the coefficients of variation were less than 10%. Except Shigella dysenteriae, UPT-LF showed excellent specificity to four strains that are also potential biowarfare/bioterrorism agents and 13 food-borne pathogenic strains. Samples with pH 2–13, high ion strengths (≥2 mol · L−1 solution of KCl and NaCl), high viscosities (≤50 mg · mL−1 PEG20000 or ≥20% glycerol), and high concentrations of biomacromolecules (≥400 mg · mL−1 bovine serum albumin or ≥80 mg · mL−1 casein) showed little influence on the assay. For practical utilization, the tolerance limits for seven powders and eight viscera were determined, and operation errors of liquid measurement demonstrated a minor influence on the strip. Ftu-UPT-LF is a candidate POCT method because of its excellent sensitivity, specificity, and stability in complex samples, as well as low operation error. PMID:26608358

  16. Levofloxacin Rescues Mice from Lethal Intra-nasal Infections with Virulent Francisella tularensis and Induces Immunity and Production of Protective Antibody

    PubMed Central

    Klimpel, Gary R.; Eaves-Pyles, Tonyia; Moen, Scott T.; Taormina, Joanna; Peterson, Johnny W.; Chopra, Ashok K.; Niesel, David W.; Carness, Paige; Haithcoat, Judith L.; Kirtley, Michelle; Ben Nasr, Abdelhakim

    2009-01-01

    The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with Francisella tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72 hours post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to rechallenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4 specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis. PMID:18930100

  17. Levofloxacin rescues mice from lethal intra-nasal infections with virulent Francisella tularensis and induces immunity and production of protective antibody.

    PubMed

    Klimpel, Gary R; Eaves-Pyles, Tonyia; Moen, Scott T; Taormina, Joanna; Peterson, Johnny W; Chopra, Ashok K; Niesel, David W; Carness, Paige; Haithcoat, Judith L; Kirtley, Michelle; Nasr, Abdelhakim Ben

    2008-12-01

    The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with F. tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72h post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to re-challenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4-specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis. PMID:18930100

  18. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of Francisella tularensis.

    PubMed

    Hua, Fei; Zhang, Pingping; Zhang, Fuli; Zhao, Yong; Li, Chunfeng; Sun, Chongyun; Wang, Xiaochen; Yang, Ruifu; Wang, Chengbin; Yu, Ailian; Zhou, Lei

    2015-01-01

    Francisella tularensis is a potential biowarfare/bioterrorism agent and zoonotic pathogen that causes tularemia; thus, surveillance of F. tularensis and first-level emergency response using point-of-care testing (POCT) are essential. The UPT-LF POCT assay was established to quantitatively detect F. tularensis within 15 min, and the sensitivity of the assay was 10(4) CFU · mL(-1) (100 CFU/test). The linear quantitative range covered five orders of magnitude, and the coefficients of variation were less than 10%. Except Shigella dysenteriae, UPT-LF showed excellent specificity to four strains that are also potential biowarfare/bioterrorism agents and 13 food-borne pathogenic strains. Samples with pH 2-13, high ion strengths (≥ 2 mol · L(-1) solution of KCl and NaCl), high viscosities (≤ 50 mg · mL(-1) PEG20000 or ≥ 20% glycerol), and high concentrations of biomacromolecules (≥ 400 mg · mL(-1) bovine serum albumin or ≥ 80 mg · mL(-1) casein) showed little influence on the assay. For practical utilization, the tolerance limits for seven powders and eight viscera were determined, and operation errors of liquid measurement demonstrated a minor influence on the strip. Ftu-UPT-LF is a candidate POCT method because of its excellent sensitivity, specificity, and stability in complex samples, as well as low operation error. PMID:26608358

  19. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Annual Technical Report

    SciTech Connect

    Fischer, N. O.

    2015-04-16

    The goal of this proposal is to demonstrate that co-localization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of recombinant subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. NLPs are are biocompatible, high-density lipoprotein mimetics that are amenable to the incorporation of multiple, chemically-disparate adjuvant and antigen molecules. We hypothesize that the ability to co-localize optimized adjuvant formulations with subunit antigens within a single particle will enhance the stimulation and activation of key immune effector cells, increasing the protective efficacy of subunit antigen-based vaccines. While Burkholderia spp. and F. tularensis subunit antigens are the focus of this proposal, we anticipate that this approach is applicable to a wide range of DOD-relevant biothreat agents. The F344 rat aerosol challenge model for F. tularensis has been successfully established at Battelle under this contract, and Year 3 efficacy studies performed at Battelle demonstrated that an NLP vaccine formulation was able to enhance survival of female F344 rats relative to naïve animals. In addition, Year 3 focused on the incorporation of multiple Burkholderia antigens (both polysaccharides and proteins) onto adjuvanted NLPs, with immunological analysis poised to begin in the next quarter.

  20. PEDIOCIN PRODUCTION IN MILK BY PEDIOCOCCUS ACIDILACTICI IN CO-CULTURE WITH STREPTOCOCCUS THERMOPHILUS AND LACTOBACILLUS DELBRUECKII SUBSP. BULGARICUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The production of pediocin in milk by Pediococcus acidilactici was evaluated in co-culture with the dairy fermentation cultures Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactococcus lactis subsp. lactis. The cultures were tested singly or in different combinations...

  1. Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov.

    PubMed Central

    Sawabe, Tomoo; Ogura, Yoshitoshi; Matsumura, Yuta; Feng, Gao; Amin, AKM Rohul; Mino, Sayaka; Nakagawa, Satoshi; Sawabe, Toko; Kumar, Ramesh; Fukui, Yohei; Satomi, Masataka; Matsushima, Ryoji; Thompson, Fabiano L.; Gomez-Gil, Bruno; Christen, Richard; Maruyama, Fumito; Kurokawa, Ken; Hayashi, Tetsuya

    2013-01-01

    To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); (2) clades amended since the 2007 proposal with recently described new species; (3) orphan clades of genomospecies F6 and F10; (4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and (5) description of V. tritonius sp. nov., which is a member of the “Porteresiae” clade. PMID:24409173

  2. Description of Teunomyces gen. nov. for the Candida kruisii clade, Suhomyces gen. nov. for the Candida tanzawaensis clade and Suhomyces kilbournensis sp. nov.

    PubMed

    Kurtzman, Cletus P; Robnett, Christie J; Blackwell, Meredith

    2016-08-01

    DNA sequence analysis has shown that species of the Candida kruisii clade and species of the C. tanzawaensis clade represent phylogenetically circumscribed genera, which are described as Teunomyces gen. nov., type species T kruisii, and Suhomyces gen. nov., type species S tanzawaensis Many of the species are distributed worldwide and they are often isolated from fungus-feeding insects and their habitats. Included is the description of S. kilbournensis (type strain NRRL Y-17864, CBS 14276), a species found almost exclusively on maize kernels (Zea mays) in IL, USA. PMID:27188882

  3. Transmission of a heterologous clade C Symbiodinium in a model anemone infection system via asexual reproduction

    PubMed Central

    Chen, Wan-Nan U.; Hsiao, Ya-Ju; Mayfield, Anderson B.; Young, Ryan; Hsu, Ling-Lan

    2016-01-01

    Anemones of genus Exaiptasia are used as model organisms for the study of cnidarian-dinoflagellate (genus Symbiodinium) endosymbiosis. However, while most reef-building corals harbor Symbiodinium of clade C, Exaiptasia spp. anemones mainly harbor clade B Symbiodinium (ITS2 type B1) populations. In this study, we reveal for the first time that bleached Exaiptasia pallida anemones can establish a symbiotic relationship with a clade C Symbiodinium (ITS2 type C1). We further found that anemones can transmit the exogenously supplied clade C Symbiodinium cells to their offspring by asexual reproduction (pedal laceration). In order to corroborate the establishment of stable symbiosis, we used microscopic techniques and genetic analyses to examine several generations of anemones, and the results of these endeavors confirmed the sustainability of the system. These findings provide a framework for understanding the differences in infection dynamics between homologous and heterologous dinoflagellate types using a model anemone infection system.

  4. Dispersal and niche evolution jointly shape the geographic turnover of phylogenetic clades across continents

    PubMed Central

    Eiserhardt, Wolf L.; Svenning, Jens-Christian; Baker, William J.; Couvreur, Thomas L. P.; Balslev, Henrik

    2013-01-01

    The turnover of phylogenetic clades across space is a fundamental biodiversity pattern that may depend on long-term evolutionary processes, and that has downstream effects on other aspects of diversity including species richness and community structure. Limited niche evolution and limited dispersal are two major processes causing spatial restriction, and thus turnover, of clades. We studied the determinants of clade turnover within the World's richest floristic kingdom, the Neotropics, using the palm family (Arecaceae) as a model. We show that continental-scale clade turnover is driven by a combination of limited niche evolution — with respect to temperature and soil tolerances — and limited dispersal. These findings are consistent with strong dispersal barriers within the Neotropics, and the observation that some palm lineages are most diverse in certain biomes or climates. The importance of such deep-time effects suggest that palms might be slow to adapt or disperse in response to anthropogenic climate change. PMID:23383367

  5. Mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

    DOEpatents

    Korber, Bette T.; Fischer, William; Liao, Hua-Xin; Haynes, Barton F.; Letvin, Norman; Hahn; Beatrice H.

    2011-05-31

    The present invention relates to mosaic clade M HIV-1 Env polypeptides and to compositions comprising same. The polypeptides of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  6. Sequential and Simultaneous Immunization of Rabbits with HIV-1 Envelope Glycoprotein SOSIP.664 Trimers from Clades A, B and C.

    PubMed

    Klasse, P J; LaBranche, Celia C; Ketas, Thomas J; Ozorowski, Gabriel; Cupo, Albert; Pugach, Pavel; Ringe, Rajesh P; Golabek, Michael; van Gils, Marit J; Guttman, Miklos; Lee, Kelly K; Wilson, Ian A; Butera, Salvatore T; Ward, Andrew B; Montefiori, David C; Sanders, Rogier W; Moore, John P

    2016-09-01

    We have investigated the immunogenicity in rabbits of native-like, soluble, recombinant SOSIP.664 trimers based on the env genes of four isolates of human immunodeficiency virus type 1 (HIV-1); specifically BG505 (clade A), B41 (clade B), CZA97 (clade C) and DU422 (clade C). The various trimers were delivered either simultaneously (as a mixture of clade A + B trimers) or sequentially over a 73-week period. Autologous, Tier-2 neutralizing antibody (NAb) responses were generated to the clade A and clade B trimers in the bivalent mixture. When delivered as boosting immunogens to rabbits immunized with the clade A and/or clade B trimers, the clade C trimers also generated autologous Tier-2 NAb responses, the CZA97 trimers doing so more strongly and consistently than the DU422 trimers. The clade C trimers also cross-boosted the pre-existing NAb responses to clade A and B trimers. We observed heterologous Tier-2 NAb responses albeit inconsistently, and with limited overall breath. However, cross-neutralization of the clade A BG505.T332N virus was consistently observed in rabbits immunized only with clade B trimers and then boosted with clade C trimers. The autologous NAbs induced by the BG505, B41 and CZA97 trimers predominantly recognized specific holes in the glycan shields of the cognate virus. The shared location of some of these holes may account for the observed cross-boosting effects and the heterologous neutralization of the BG505.T332N virus. These findings will guide the design of further experiments to determine whether and how multiple Env trimers can together induce more broadly neutralizing antibody responses. PMID:27627672

  7. Immunogenicity of a novel engineered HIV-1 clade C synthetic consensus-based envelope DNA vaccine.

    PubMed

    Yan, Jian; Corbitt, Natasha; Pankhong, Panyupa; Shin, Thomas; Khan, Amir; Sardesai, Niranjan Y; Weiner, David B

    2011-09-22

    DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence. We also shortened the V1 and V2 loops to approximate early transmitter isolate sequences and the cytoplasmic tail was truncated to prevent envelope recycling. When studied as a DNA vaccine in Balb/c mice, compared to a primary codon-optimized clade C envelope DNA vaccine (p96ZM651gp140-CD5), this novel construct is up to three times more potent in driving CTL responses. Importantly this construct not only induces stronger cross-reactive cellular responses within clade C, it also induces stronger immune responses against clade B and group M envelope peptide pools than p96ZM651gp140-CD5. Epitope mapping demonstrated that EY3E1-C was able to induce clade C envelope-specific immune responses against 15 peptide pools, clade B envelope-specific immune responses against 19 peptide pools and group M envelope-specific immune responses against 16 peptide pools out of 29, respectively, indicating that a significant increase in the breadth of induced immune responses. The analysis of antibody responses suggested that vaccination of pEY3E1-C could induce a clade C envelope-specific antibody response. The cellular immune responses of pEY3E1-C could be further enhanced when the DNA was delivered by using electroporation (EP). Thus, the synthetic engineered consensus EY3E1-C gene is capable of eliciting stronger and broader CTL responses than primary clade C envelopes. This finding

  8. Draft Genome Sequence of Photorhabdus luminescens subsp. laumondii HP88, an Entomopathogenic Bacterium Isolated from Nematodes.

    PubMed

    Ghazal, Shimaa; Oshone, Rediet; Simpson, Stephen; Morris, Krystalynne; Abebe-Akele, Feseha; Thomas, W Kelley; Khalil, Kamal M; Tisa, Louis S

    2016-01-01

    Photorhabdus luminescens subsp. laumondii HP88 is an entomopathogenic bacterium that forms a symbiotic association with Heterorhabditis nematodes. We report here a 5.27-Mbp draft genome sequence for P. luminescens subsp. laumondii HP88, with a G+C content of 42.4% and containing 4,243 candidate protein-coding genes. PMID:26988056

  9. Draft Genome Sequence of Photorhabdus luminescens subsp. laumondii HP88, an Entomopathogenic Bacterium Isolated from Nematodes

    PubMed Central

    Ghazal, Shimaa; Oshone, Rediet; Simpson, Stephen; Morris, Krystalynne; Abebe-Akele, Feseha; Thomas, W. Kelley; Khalil, Kamal M.

    2016-01-01

    Photorhabdus luminescens subsp. laumondii HP88 is an entomopathogenic bacterium that forms a symbiotic association with Heterorhabditis nematodes. We report here a 5.27-Mbp draft genome sequence for P. luminescens subsp. laumondii HP88, with a G+C content of 42.4% and containing 4,243 candidate protein-coding genes. PMID:26988056

  10. Development and Characterization of Monoclonal Antibodies and Aptamers Against Major Antigens of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne’s disease (JD). To fill this gap in JD research, monoclonal antibodies (mAbs) against M. avium subsp. paratuberculosis were produced fr...

  11. Complete genome sequence of Mycobacterium avium subsp. paratuberculosis, isolated from human breast milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp paratuberculosis is the etiologic agent of Johne’s disease. We report the draft genome sequences of six M. avium subsp paratuberculosis isolates obtained from diverse hosts including bison, cattle and sheep. These sequences will deepen our understanding of host association ...

  12. Draft Genome Sequence of Lactococcus lactis subsp. lactis Strain YF11

    PubMed Central

    Du, Yuhui; Song, Lifu; Feng, Wenjing; Pei, Guangsheng; Zheng, Ping; Yu, Zhichao; Sun, Jibin

    2013-01-01

    Lactococcus lactis subsp. lactis strain YF11 is a food preservative bacterium with a high capacity to produce nisin. Here, we announce the draft genome sequence of Lactococcus lactis subsp. lactis YF11 (2,527,433 bp with a G+C content of 34.81%). PMID:23929487

  13. Complete genome sequence of Streptococcus equi subsp. zooepidemicus strain ATCC 35246.

    PubMed

    Ma, Zhe; Geng, Jianing; Zhang, Hui; Yu, Haiying; Yi, Li; Lei, Meng; Lu, Cheng-ping; Fan, Hong-jie; Hu, Songnian

    2011-10-01

    Streptococcus equi subsp. zooepidemicus is an opportunistic pathogen. It has caused a very large economic loss in the swine industry of China and has become a threat to human health. We announce the complete genome sequence of S. equi subsp. zooepidemicus strain ATCC 35246, which provides opportunities to understand its pathogenesis mechanism and genetic basis. PMID:21914890

  14. First isolation of Mycobacterium avium subsp Paratuberculosis from commercial pasteurized milk in Argentina

    PubMed Central

    Paolicchi, Fernando; Cirone, Karina; Morsella, Claudia; Gioffré, Andrea

    2012-01-01

    Mycobacterium avium subsp paratuberculosis was isolated from two out of seventy samples (2.86 %) of pasteurized and ultra-pasteurized milk. The isolates were positives to IS900 PCR and showed a C17 RFLP pattern, the most prevalent in Argentina. The present study is the first report of Mycobacterium avium subsp paratuberculosis culture from pasteurized milk in Argentina. PMID:24031925

  15. Draft Genome Sequence of a Virulent Pectobacterium carotovorum subsp. brasiliense Isolate Causing Soft Rot of Cucumber.

    PubMed

    Onkendi, Edward M; Ramesh, Aadi Moolam; Kwenda, Stanford; Naidoo, Sanushka; Moleleki, Lucy

    2016-01-01

    Pectobacterium carotovorum subsp. brasiliense causes soft rot and blackleg diseases on potatoes, ornamentals, and other crops of economic importance. Here, we report a draft genome sequence of a highly virulent P. carotovorum subsp. brasiliense strain, PcbHPI01, isolated from a cucumber in South Africa. PMID:26744374

  16. Proposal for designation of F38-type caprine mycoplasmas as Mycoplasma capricolum subsp. capripneumoniae subsp. nov. and consequent obligatory relegation of strains currently classified as M. capricolum (Tully, Barile, Edward, Theodore, and Ernø 1974) to an additional new subspecies, M. capricolum subsp. capricolum subsp. nov.

    PubMed

    Leach, R H; Ernø, H; MacOwan, K J

    1993-07-01

    A subspecies relationship with the existing species Mycoplasma capricolum is appropriate for the F38 group of mycoplasmas, the causative agent of classical contagious caprine pleuropneumonia. We believe that this classification is justified on the basis of the close DNA-DNA relationship recently reported for isolates belonging to the two groups and the other known serological and biological similarities and differences of these organisms. Strain F38T (T = type strain) and taxonomically indistinguishable strains are therefore proposed as members of a new subspecies of M. capricolum, M. capricolum subsp. capripneumoniae. Strain F38 (= NCTC 10192) is the type strain of M. capricolum subsp. capripneumoniae subsp. nov. As a consequence of this subdivision of the species M. capricolum, strains previously classified as M. capricolum are now necessarily relegated to subspecies status, as M. capricolum subsp. capricolum subsp. nov. Strain California kid (= ATCC 27343 = NCTC 10154) is the type strain of M. capricolum, as well as of M. capricolum subsp. capricolum. A taxonomic description of M. capricolum subsp. capripneumoniae and a brief amended description of M. capricolum subsp. capricolum are presented. PMID:8347517

  17. Complete Genome Sequence of Leuconostoc mesenteroides subsp. mesenteroides Strain J18, Isolated from Kimchi

    PubMed Central

    Jung, Ji Young; Lee, Seung Hyeon; Lee, Se Hee

    2012-01-01

    Leuconostoc mesenteroides subsp. mesenteroides is one of the most predominant lactic acid bacterial groups during kimchi fermentation. Here, we report the complete genome sequence of L. mesenteroides subsp. mesenteroides J18, which was isolated from kimchi. The genome of the strain consists of a 1,896,561-bp chromosome and five plasmids. PMID:22247530

  18. Genome Sequence of an Epidemic Isolate of Mycobacterium abscessus subsp. bolletii from Rio de Janeiro, Brazil.

    PubMed

    Davidson, Rebecca M; Reynolds, Paul R; Farias-Hesson, Eveline; Duarte, Rafael Silva; Jackson, Mary; Strong, Michael

    2013-01-01

    Multiple isolates of Mycobacterium abscessus subsp. bolletii, collectively called BRA100, were associated with outbreaks of postsurgical skin infections across various regions of Brazil from 2003 to 2009. We announce the draft genome sequence of a newly sequenced BRA100 strain, M. abscessus subsp. bolletii CRM-0020, isolated from a patient in Rio de Janeiro, Brazil. PMID:23950125

  19. Global distribution patterns of distinct clades of the photosynthetic picoeukaryote Ostreococcus.

    PubMed

    Demir-Hilton, Elif; Sudek, Sebastian; Cuvelier, Marie L; Gentemann, Chelle L; Zehr, Jonathan P; Worden, Alexandra Z

    2011-07-01

    Ostreococcus is a marine picophytoeukaryote for which culture studies indicate there are 'high-light' and 'low-light' adapted ecotypes. Representatives of these ecotypes fall within two to three 18S ribosomal DNA (rDNA) clades for the former and one for the latter. However, clade distributions and relationships to this form of niche partitioning are unknown in nature. We developed two quantitative PCR primer-probe sets and enumerated the proposed ecotypes in the Pacific Ocean as well as the subtropical and tropical North Atlantic. Statistical differences in factors such as salinity, temperature and NO(3) indicated the ecophysiological parameters behind clade distributions are more complex than irradiance alone. Clade OII, containing the putatively low-light adapted strains, was detected at warm oligotrophic sites. In contrast, Clade OI, containing high-light adapted strains, was present in cooler mesotrophic and coastal waters. Maximal OI abundance (19 555±37 18S rDNA copies per ml) was detected in mesotrophic waters at 40 m depth, approaching the nutricline. OII was often more abundant at the deep chlorophyll maximum, when nutrient concentrations were significantly higher than at the surface (stratified euphotic zone waters). However, in mixed euphotic-zone water columns, relatively high numbers (for example, 891±107 18S rDNA copies per ml, Sargasso Sea, springtime) were detected at the surface. Both Clades OI and OII were found at multiple euphotic zone depths, but co-occurrence at the same geographical location appeared rare and was detected only in continental slope waters. In situ growth rate estimates using these primer-probes and better comprehension of physiology will enhance ecological understanding of Ostreococcus Clades OII and OI which appear to be oceanic and coastal clades, respectively. PMID:21289652

  20. Neurocognitive Impairment in HIV-1 Clade C versus B Infected Individuals in Southern Brazil

    PubMed Central

    de Almeida, Sergio Monteiro; Ribeiro, Clea Elisa; de Pereira, Ana Paula; Badiee, Jayraan; Cherner, Mariana; Smith, Davey; Maich, Ingrid; Raboni, Sonia Mara; Rotta, Indianara; Barbosa, Francisco Jaime; Heaton, Robert K.; Umlauf, Anya; Ellis, Ronald J.

    2014-01-01

    HIV-1 clade C isolates show reduced Tat protein chemoattractant activity compared with clade B. This might influence neuropathogenesis by altering trafficking of monocytes into the CNS. A previous study suggested low rates of HIV-associated dementia in clade C infected individuals. The present study evaluated neurocognitive impairment rates in clade B- and C-infected individuals from the same local population. HIV+ and HIV- participants were recruited from the same geographic region in southern Brazil. We evaluated neuropsychological (NP) impairment using a screening instrument (the International HIV Dementia Scale; IHDS), as well as a Brazilian Portuguese adaptation of a comprehensive battery that has demonstrated sensitivity to HIV associated neurocognitive disorders (HAND) internationally. NP performance in controls was used to generate T-scores and impairment ratings by the global deficit score (GDS) method. Clade assignments were ascertained by sequencing pol and env. Blood and cerebrospinal fluid (CSF) were collected from all HIV+ participants. HIV+ and HIV- participants were comparable on demographic characteristics. HIV+ participants overall were more likely to be impaired than HIV- by the IHDS and the GDS. Clade B and C infected individuals were demographically similar and did not differ significantly in rates of impairment. The prevalence of pleocytosis, a marker of intrathecal cellular chemotaxis, also did not differ between clade B and C infections. Clade B and C HIV-infected individuals from the same geographic region, when ascertained using comparable methods, did not differ in their rates of neurocognitive impairment, and there was no evidence of differences in CNS chemotaxis. PMID:24277437

  1. Comparative genomics of two ‘Candidatus Accumulibacter' clades performing biological phosphorus removal

    PubMed Central

    Flowers, Jason J; He, Shaomei; Malfatti, Stephanie; del Rio, Tijana Glavina; Tringe, Susannah G; Hugenholtz, Philip; McMahon, Katherine D

    2013-01-01

    Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80–90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience. PMID:23887171

  2. Comparative genomics of two 'Candidatus Accumulibacter' clades performing biological phosphorus removal.

    PubMed

    Flowers, Jason J; He, Shaomei; Malfatti, Stephanie; del Rio, Tijana Glavina; Tringe, Susannah G; Hugenholtz, Philip; McMahon, Katherine D

    2013-12-01

    Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80-90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience. PMID:23887171

  3. Onshore-offshore trends in benthic faunal change: drive by clade origination

    SciTech Connect

    Bottjer, D.J.; Jablonski, D.

    1985-01-01

    Higher taxa in Phanerozoic benthic communities appear to arise preferentially in nearshore habitats and expand outwards across the continental shelf. This pattern is demonstrably independent of sea-level fluctuations, and could be driven by a variety of evolutionary processes, including: 1) differential origination of novelties nearshore; and 2) equal occurrence of innovations across the shelf, but with offshore novelties more extinction-prone than onshore novelties in the early phases of diversification. To test these mechanism, it is necessary to analyze the pattern on a clade-by-clade basis, rather than leaving the clades embedded within their communities. Review and synthesis of the literature indicates that a number of clades have their first species originating onshore with a subsequent history that shows either (A) migration offshore while losing onshore components or (B) expansion offshore while persisting onshore. In a detailed study of pattern A the authors have compiled presence/absence data for the crinoid order Isocrinida in 70 post-Paleozoic benthic communities, which shows migration across the shelf in the mid-Mesozoic. These studies show that different, contemporaneous clades proceeded across the shelf at different times and rates; no evidence is found for cohesive community migration. The supposed pattern of community migration may be an epiphenomenon, underlain by individualistic clade histories that appear to act in concert when viewed on a coarse time scale.

  4. Development of Quantitative Real-time PCR Assays for Different Clades of "Candidatus Accumulibacter".

    PubMed

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-01-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual "Candidatus Accumulibacter" (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85-112% (R(2) = 0.962-0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants. PMID:27142574

  5. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    NASA Astrophysics Data System (ADS)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-05-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.

  6. Clade age and not diversification rate explains species richness among animal taxa.

    PubMed

    McPeek, Mark A; Brown, Jonathan M

    2007-04-01

    Animal taxa show remarkable variability in species richness across phylogenetic groups. Most explanations for this disparity postulate that taxa with more species have phenotypes or ecologies that cause higher diversification rates (i.e., higher speciation rates or lower extinction rates). Here we show that clade longevity, and not diversification rate, has primarily shaped patterns of species richness across major animal clades: more diverse taxa are older and thus have had more time to accumulate species. Diversification rates calculated from 163 species-level molecular phylogenies were highly consistent within and among three major animal phyla (Arthropoda, Chordata, Mollusca) and did not correlate with species richness. Clades with higher estimated diversification rates were younger, but species numbers increased with increasing clade age. A fossil-based data set also revealed a strong, positive relationship between total extant species richness and crown group age across the orders of insects and vertebrates. These findings do not negate the importance of ecology or phenotype in influencing diversification rates, but they do show that clade longevity is the dominant signal in major animal biodiversity patterns. Thus, some key innovations may have acted through fostering clade longevity and not by heightening diversification rate. PMID:17427118

  7. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    PubMed Central

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-01-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants. PMID:27142574

  8. Biosystematic studies on Enicostema axillare (Lam.) A. Raynal subsp. Axillare (Gentianaceae) in peninsular India.

    PubMed

    Shahina, P M; Nampy, Santhosh

    2014-05-01

    The pantropical genus Enicostema (Gentianaceae) has three species and two sub species world over, namely, E. verticillatum (L.) Engl. (America), E. elizabethae Veldkamp (Madagascar) and E. axillare having 3 subsp. viz., subsp. axillare (Lam.) A. Raynal (India), subsp. latilobum (N.E. Br.) A. Raynal (East Africa) and subsp. littorale (Blume) A. Raynal (Indonesia). The present study aims to delimit the Indian taxa based on field and herbarium studies. Comparative morphology is studied using live as well as consulting wide range of specimens housed at various herbaria. The anatomy of leaf, stem, and root is studied using free hand sections and from epidermal peelings. The seed and pollen morphology are studied under SEM. Information on anatomy, palynology and seed micromorphology of E. axillare subsp. axillare is provided for the first time. PMID:26031003

  9. Taxonomic evaluation of species in the Streptomyces hirsutus clade using multi-locus sequence analysis and proposals to reclassify several species in this clade

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous phylogenetic analyses of species of Streptomyces based on 16S rRNA gene sequences resulted in a statistically well-supported clade (100% bootstrap value) containing 8 species that exhibited very similar gross morphology in producing open looped (Retinaculum-Apertum) to spiral (Spira) chains...

  10. Trading Capsule for Increased Cytotoxin Production: Contribution to Virulence of a Newly Emerged Clade of emm89 Streptococcus pyogenes

    PubMed Central

    Zhu, Luchang; Olsen, Randall J.; Nasser, Waleed; de la Riva Morales, Ivan

    2015-01-01

    ABSTRACT Strains of emm89 Streptococcus pyogenes have become one of the major causes of invasive infections worldwide in the last 10 years. We recently sequenced the genome of 1,125 emm89 strains and identified three major phylogenetic groups, designated clade 1, clade 2, and the epidemic clade 3. Epidemic clade 3 strains, which now cause the great majority of infections, have two distinct genetic features compared to clade 1 and clade 2 strains. First, all clade 3 organisms have a variant 3 nga promoter region pattern, which is associated with increased production of secreted cytolytic toxins SPN (S. pyogenes NADase) and SLO (streptolysin O). Second, all clade 3 strains lack the hasABC locus mediating hyaluronic acid capsule synthesis, whereas this locus is intact in clade 1 and clade 2 strains. We constructed isogenic mutant strains that produce different levels of SPN and SLO toxins and capsule (none, low, or high). Here we report that emm89 strains with elevated toxin production are significantly more virulent than low-toxin producers. Importantly, we also show that capsule production is dispensable for virulence in strains that already produce high levels of SPN and SLO. Our results provide new understanding about the molecular mechanisms contributing to the rapid emergence and molecular pathogenesis of epidemic clade 3 emm89 S. pyogenes. PMID:26443457

  11. Szidat's rule re-tested: relationships between flea and host phylogenetic clade ranks in four biogeographic realms.

    PubMed

    Krasnov, Boris R; Kiefer, Daniel; Warburton, Elizabeth M; Khokhlova, Irina S

    2016-05-01

    We tested Szidat's rule (the more primitive the host, the more primitive the parasites it harbours) by analysing the relationships between phylogenetic clade ranks of fleas and their small mammalian hosts in four biogeographic realms (Afrotropics, Neotropics, Nearctic and Palearctic). From the host perspective, we tested the association between host clade rank and the mean clade rank of all fleas collected from this host. From the flea perspective, we tested the relationships between flea clade rank and the mean clade rank of hosts on which this flea was recorded. First, we tested whether the analysis of the relationships between host and flea clade ranks should be controlled for phylogenetic dependence among either host or flea species. Then, we tested for the associations between host and flea clade ranks separately for each realm using either a phylogenetic general least-squares analysis or an ordinary least-squares analysis. In all realms, the mean clade rank of fleas parasitic on a given host increased with an increase of this host's clade rank, and the mean clade rank of hosts recorded on a given flea increased with an increase of this flea's clade rank, suggesting that Szidat's rule, at least to some extent, holds for fleas. PMID:26887914

  12. Complete Genome and Methylome Sequences of Salmonella enterica subsp. enterica Serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica Serovar Sloterdijk (ATCC 15791).

    PubMed

    Yao, Kuan; Muruvanda, Tim; Roberts, Richard J; Payne, Justin; Allard, Marc W; Hoffmann, Maria

    2016-01-01

    Salmonella enterica spp. are pathogenic bacteria commonly associated with food-borne outbreaks in human and animals. Salmonella enterica spp. are characterized into more than 2,500 different serotypes, which makes epidemiological surveillance and outbreak control more difficult. In this report, we announce the first complete genome and methylome sequences from two Salmonella type strains associated with food-borne outbreaks, Salmonella enterica subsp. enterica serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica serovar Sloterdijk (ATCC 15791). PMID:26988049

  13. Complete Genome and Methylome Sequences of Salmonella enterica subsp. enterica Serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica Serovar Sloterdijk (ATCC 15791)

    PubMed Central

    Yao, Kuan; Muruvanda, Tim; Roberts, Richard J.; Payne, Justin; Allard, Marc W.

    2016-01-01

    Salmonella enterica spp. are pathogenic bacteria commonly associated with food-borne outbreaks in human and animals. Salmonella enterica spp. are characterized into more than 2,500 different serotypes, which makes epidemiological surveillance and outbreak control more difficult. In this report, we announce the first complete genome and methylome sequences from two Salmonella type strains associated with food-borne outbreaks, Salmonella enterica subsp. enterica serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica serovar Sloterdijk (ATCC 15791). PMID:26988049

  14. Systematics within Gyps vultures: a clade at risk

    PubMed Central

    Johnson, Jeff A; Lerner, Heather RL; Rasmussen, Pamela C; Mindell, David P

    2006-01-01

    Background Populations of the Oriental White-backed Vulture (Gyps bengalensis) have declined by over 95% within the past decade. This decline is largely due to incidental consumption of the non-steroidal anti-inflammatory veterinary pharmaceutical diclofenac, commonly used to treat domestic livestock. The conservation status of other Gyps vultures in southern Asia is also of immediate concern, given the lack of knowledge regarding status of their populations and the continuing existence of taxonomic uncertainties. In this study, we assess phylogenetic relationships for all recognized species and the majority of subspecies within the genus Gyps. The continuing veterinary use of diclofenac is an unknown but potential risk to related species with similar feeding habits to Gyps bengalensis. Therefore, an accurate assessment of the phylogenetic relationships among Gyps vultures should aid in their conservation by clarifying taxonomic uncertainties, and enabling inference of their respective relatedness to susceptible G. bengalensis. Results Phylogenetic results using mitochondrial cytB, ND2 and control region sequence data indicate a recent and rapid diversification within the genus Gyps. All recognized species formed monophyletic groups with high statistical support, with the exception of the Eurasian Vulture, for which specimens identified as subspecies G. fulvus fulvescens appear closely related to the Himalayan Vulture (G. himalayensis). In all analyses, the earliest divergence separated the Oriental White-backed Vulture from other Gyps taxa, with the next diverging taxon being either the African White-backed Vulture (G. africanus), or the Himalayan Vulture. All analyses supported a sister relationship between the Eurasian Vulture (G. f. fulvus), and Rüppell's Vulture (G. rueppellii), with this clade being sister to another consisting of the two taxa of "Long-billed" Vulture (G. indicus indicus and G. i. tenuirostris), and the Cape Vulture (G. coprotheres). These

  15. Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

    PubMed Central

    Tkachuk, Victoria L.; Krause, Denis O.; McAllister, Tim A.; Buckley, Katherine E.; Reuter, Tim; Hendrick, Steve

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne's disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle

  16. Assessing the inactivation of Mycobacterium avium subsp. paratuberculosis during composting of livestock carcasses.

    PubMed

    Tkachuk, Victoria L; Krause, Denis O; McAllister, Tim A; Buckley, Katherine E; Reuter, Tim; Hendrick, Steve; Ominski, Kim H

    2013-05-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne's disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle

  17. Subspeciation of Bifidobacterium longum by multilocus approaches and amplified fragment length polymorphism: Description of B. longum subsp. suillum subsp. nov., isolated from the faeces of piglets.

    PubMed

    Yanokura, Emiko; Oki, Kaihei; Makino, Hiroshi; Modesto, Monica; Pot, Bruno; Mattarelli, Paola; Biavati, Bruno; Watanabe, Koichi

    2015-07-01

    The species Bifidobacterium longum is currently divided into three subspecies, B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis. This classification was based on an assessment of accumulated information on the species' phenotypic and genotypic features. The three subspecies of B. longum were investigated using genotypic identification [amplified-fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and multilocus sequence typing (MLST)]. By using the AFLP and the MLSA methods, we allocated 25 strains of B. longum into three major clusters corresponding to the three subspecies; the cluster comprising the strains of B. longum subsp. suis was further divided into two subclusters differentiable by the ability to produce urease. By using the MLST method, the 25 strains of B. longum were divided into eight groups: four major groups corresponding to the results obtained by AFLP and MLSA, plus four minor disparate groups. The results of AFLP, MLSA and MLST analyses were consistent and revealed a novel subspeciation of B. longum, which comprised three known subspecies and a novel subspecies of urease-negative B. longum, for which the name B. longum subsp. suillum subsp. nov. is proposed, with type strain Su 851(T)=DSM 28597(T)=JCM 19995(T). PMID:26007614

  18. Intraspecific diversity and distribution of the cosmopolitan species Pseudo-nitzschia pungens (Bacillariophyceae): morphology, genetics, and ecophysiology of the three clades.

    PubMed

    Kim, Jin Ho; Park, Bum Soo; Kim, Joo-Hwan; Wang, Pengbin; Han, Myung-Soo

    2015-02-01

    Three clades of Pseudo-nitzschia pungens, determined by the internal transcribed space (ITS) region, are distributed throughout the world. We studied 15 P. pungens clones from various geographical locations and confirmed the existence of the three clades within P. pungens, based on ITS sequencing and described the three subgroups (IIIaa, IIIab, and IIIb) of clade III. Clade III (clade IIIaa) populations were reported for the first time in Korean coastal waters and the East China Sea. In morphometric analysis, we found the ultrastructural differences in the number of fibulae, striae, and poroids that separate the three clades. We carried out physiological tests on nine clones belonging to the three clades growing under various culture conditions. In temperature tests, only clade III clones could not grow at lower temperatures (10°C and 15°C), although clade I and II clones grew well. The estimated optimal growth range of clade I clones was wider than that of clades II and III. Clade II clones were considered to be adapted to lower temperatures and clade III to higher temperatures. In salinity tests, clade II and III clones did not grow well at a salinity of 40. Clade I clones were regarded as euryhaline and clade II and III clones were stenohaline. This supports the hypothesis that P. pungens clades have different ecophysiological characteristics based on their habitats. Our data show that physiological and morphological features are correlated with genetic intraspecific differentiation in P. pungens. PMID:26986266

  19. Using a grass substrate to compare decay among two clades of brown rot fungi.

    PubMed

    Kaffenberger, Justin T; Schilling, Jonathan S

    2013-10-01

    Interest in the mechanisms of wood-degrading fungi has grown in tandem with lignocellulose bioconversion efforts, yet many potential biomass feedstocks are non-woody. Using corn stover (Zea mays) as a substrate, we tracked degradative capacities among brown rot fungi from the Antrodia clade, including Postia placenta, the first brown rot fungus to have its genome sequenced. Decay dynamics were compared against Gloeophyllum trabeum from the Gloeophyllum clade. Weight loss induced by P. placenta (6.2 %) and five other Antrodia clade isolates (average 7.4 %) on corn stalk after 12 weeks demonstrated inefficiency among these fungi, relative to decay induced by G. trabeum (44.4 %). Using aspen (Populus sp.) as a woody substrate resulted in, on average, a fourfold increase in weight loss induced by Antrodia clade fungi, while G. trabeum results matched those on stover. The sequence and trajectories of chemical constituent losses differed as a function of substrate but not fungal clade. Instead, chemical data suggest that characters unique to stover limit decay by the Antrodia clade, rather than disparities in growth rate or extractives toxicity. High p-coumaryl lignin content, lacking the methoxy groups characteristically cleaved during brown rot, is among potential rate-distinguishing characters in grasses. This ineptitude among Antrodia clade fungi on grasses was supported by meta-analysis of other unrelated studies using grass substrates. Concerning application, results expose a problem if adopting the strategy of the model decay fungus P. placenta to treat corn stover, a widely available plant feedstock. Overall, the results insinuate phylogenetically distinct modes of brown rot and demonstrate the benefit of using non-woody substrates to probe wood degradation mechanisms. PMID:23917637

  20. Generation and Evaluation of Clade C Simian-Human Immunodeficiency Virus Challenge Stocks

    PubMed Central

    Chang, Hui-Wen; Tartaglia, Lawrence J.; Whitney, James B.; Lim, So-Yon; Sanisetty, Srisowmya; Lavine, Christy L.; Seaman, Michael S.; Rademeyer, Cecelia; Williamson, Carolyn; Ellingson-Strouss, Katharine; Stamatatos, Leonidas; Kublin, James

    2014-01-01

    ABSTRACT The development of a panel of mucosally transmissible simian-human immunodeficiency virus (SHIV) challenge stocks from multiple virus clades would facilitate preclinical evaluation of candidate HIV-1 vaccines and therapeutics. The majority of SHIV stocks that have been generated to date have been derived from clade B HIV-1 env sequences from viruses isolated during chronic infection and typically required serial animal-to-animal adaptation for establishing mucosal transmissibility and pathogenicity. To capture essential features of mucosal transmission of clade C viruses, we produced a series of SHIVs with early clade C HIV-1 env sequences from acutely HIV-1-infected individuals from South Africa. SHIV-327c and SHIV-327cRM expressed env sequences that were 99.7 to 100% identical to the original HIV-1 isolate and did not require in vivo passaging for mucosal infectivity. These challenge stocks infected rhesus monkeys efficiently by both intrarectal and intravaginal routes, replicated to high levels during acute infection, and established chronic setpoint viremia in 13 of 17 (76%) infected animals. The SHIV-327cRM challenge stock was also titrated for both single, high-dose intrarectal challenges and repetitive, low-dose intrarectal challenges in rhesus monkeys. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing clade C HIV-1 infection. IMPORTANCE We describe the development of two related clade C SHIV challenge stocks. These challenge stocks should prove useful for preclinical testing of vaccines and other interventions aimed at preventing clade C HIV-1 infection. PMID:25473043

  1. Biological activity of harpin produced by Pantoea stewartii subsp. stewartii.

    PubMed

    Ahmad, M; Majerczak, D R; Pike, S; Hoyos, M E; Novacky, A; Coplin, D L

    2001-10-01

    Pantoea stewartii subsp. stewartii causes Stewart's wilt of sweet corn. A hypersensitive response and pathogenicity (Hrp) secretion system is needed to produce water-soaking and wilting symptoms in corn and to cause a hypersensitive response (HR) in tobacco. Sequencing of the hrp cluster revealed a putative harpin gene, hrpN. The product of this gene was overexpressed in Escherichia coli and shown to elicit the HR in tobacco and systemic resistance in radishes. The protein was designated HrpN(Pnss). Like other harpins, it was heat stable and protease sensitive, although it was three- to fourfold less active biologically than Erwinia amylovora harpin. We used antibodies to purified HrpN(Pnss) to verify that hrpN mutants could not produce harpin. This protein was secreted into the culture supernatant and was produced by strains of P. stewartii subsp. indologenes. In order to determine the importance of HrpN(Pnss) in pathogenesis on sweet corn, three hrpN::Tn5 mutants were compared with the wild-type strain with 50% effective dose, disease severity, response time, and growth rate in planta as parameters. In all tests, HrpN(Pnss) was not required for infection, growth, or virulence in corn or endophytic growth in related grasses. PMID:11605962

  2. Intestinal colonization of neonatal animals by Campylobacter fetus subsp. jejuni.

    PubMed Central

    Field, L H; Underwood, J L; Pope, L M; Berry, L J

    1981-01-01

    Neonatal mice (2.3 to 2.8 g) were inoculated intragastrically with different human isolates of Campylobacter fetus subsp. jejuni. At weekly intervals thereafter, mice were sacrificed and dilution plate counts were performed on segments of the gastrointestinal tract. Mice were uniformly colonized by some strains for 2 weeks, whereas other strains were being cleared at that time. One strain (BO216) persisted in some mice for 3 weeks. The greatest number of organisms (10(7)) was recovered from the cecum and large intestine. The small intestine had from 10(2) to 10(5) colony-forming units. Colonization of the stomach was not found consistently. One strain killed 13% of the infected mice. Deaths occurred between 1 and 5 days postinfection. Two other strains killed a smaller percentage of challenged animals, and two additional strains killed none. Retarded weight gain was noticed in some, but not all, of the infected mice. The intestines of neonatal rats and rabbits were colonized much the same as those of mice, whereas hamsters were resistant to colonization. Preweanling mice, up to about 6.5 to 7.0 g, could be colonized with C. fetus subsp. jejuni after intragastric challenge, but weanling mice of larger weight (9.8 g) and young adult mice (18.3 g) could not. Scanning electron photomicrographs of the lower ileum showed campylobacters in and below the dried mucous gel that lines the intestines. The use of this model for additional studies is discussed. Images PMID:7287188

  3. Cell-mediated and humoral immune responses after vaccination of human volunteers with the live vaccine strain of Francisella tularensis.

    PubMed

    Waag, D M; McKee, K T; Sandstrom, G; Pratt, L L; Bolt, C R; England, M J; Nelson, G O; Williams, J C

    1995-03-01

    The specific humoral and cell-mediated immune responses of human volunteers vaccinated with the Francisella tularensis live vaccine strain (LVS) were evaluated. In the search for an optimal antigen to measure the immunogenicity of the vaccine in an enzyme-linked immunosorbent assay, we tested irradiation-killed LVS, an aqueous ether extract of the LVS (EEx), lipopolysaccharide (LPS) from LVS, and a virulent strain (SCHU4). Volunteers were immunized with LVS by scarification. Immunoglobulin G (IgG) responses to LVS and LPS gave the highest background titers when tested with sera from unimmunized volunteers, whereas IgA, IgG, and IgM background titers to EEx and SCHU4 were low. Vaccination caused a significant rise (P < 0.01) in IgA, IgG, and IgM titers to all antigens tested, except for the IgG response to LPS. Eighty percent of vaccinated volunteers developed a positive IgG response to EEx 14 days postvaccination, while 50% were positive to LVS. By day 14 after vaccination, 70% of immunized volunteers exhibited a positive response to EEx in an in vitro peripheral blood lymphocyte proliferation assay. EEx, a specific and sensitive antigen for evaluating immune responses of vaccinated volunteers, may be a superior antigen for the diagnosis of tularemia. PMID:7697521

  4. From microfluidic modules to an integrated Lab-on-a-chip system for the detection of Francisella tularensis

    NASA Astrophysics Data System (ADS)

    Hlawatsch, Nadine; Krumbholz, Marco; Prüfer, Anna; Moche, Christian; Becker, Holger; Gärtner, Claudia

    2013-05-01

    Lab-on-a-chip (LoC) systems translating the whole process of pathogen analysis to an integrated, miniaturized, and automatically functioning microfluidic platform are generally expected to be very promising future diagnostic approaches. The development of such a LoC system for the detection of bacterial pathogens applied to the example pathogen Francisella tularensis is described in this report. To allow functional testing of the whole process cascade before final device integration, various bio-analytical steps such as cell lysis, DNA extraction and purification, continuous-flow PCR and analyte detection have been adapted to unique functional microfluidic modules. As a successive step, positively tested modules for pathogen detection have been successfully assembled to an integrated chip. Moreover, technical solutions for a smooth interaction between sample input from the outer world as well as microfluidic chip and chip driving instrument have been developed. In conclusion, a full repertoire of analytical tools have been developed and successfully tested in the concerted manner of a functionally integrated microfluidic device representing a tool for future diagnostic approaches.

  5. Aerosol delivery of liposome-encapsulated ciprofloxacin: aerosol characterization and efficacy against Francisella tularensis infection in mice.

    PubMed

    Conley, J; Yang, H; Wilson, T; Blasetti, K; Di Ninno, V; Schnell, G; Wong, J P

    1997-06-01

    The aerosol delivery of liposome-encapsulated ciprofloxacin by using 12 commercially available jet nebulizers was evaluated in this study. Aerosol particles containing liposome-encapsulated ciprofloxacin generated by the nebulizers were analyzed with a laser aerodynamic particle sizer. Mean mass aerodynamic diameters (MMADs) and geometric standard deviations (GSDs) were determined, and the drug contents of the sampling filters from each run onto which aerosolized liposome-encapsulated ciprofloxacin had been deposited were analyzed spectrophotometrically. The aerosol particles of liposome-encapsulated ciprofloxacin generated by these nebulizers ranged from 1.94 to 3.5 microm, with GSDs ranging from 1.51 to 1.84 microm. The drug contents of the sampling filters exposed for 1 min to aerosolized liposome-encapsulated ciprofloxacin range from 12.7 to 40.5 microg/ml (0.06 to 0.2 mg/filter). By using the nebulizer selected on the basis of most desirable MMADs, particle counts, and drug deposition, aerosolized liposome-encapsulated ciprofloxacin was used for the treatment of mice infected with 10 times the 50% lethal dose of Francisella tularensis. All mice treated with aerosolized liposome-encapsulated ciprofloxacin survived the infection, while all ciprofloxacin-treated or untreated control mice succumbed to the infection (P < 0.001). These results suggest that aerosol delivery of liposome-encapsulated ciprofloxacin to the lower respiratory tract is feasible and that it may provide an effective therapy for the treatment of respiratory tract infections. PMID:9174185

  6. Structural Heterogeneity and Environmentally Regulated Remodeling of Francisella tularensis subspecies novicida Lipid A Characterized by Tandem Mass Spectrometry

    PubMed Central

    Shaffer, Scott A.; Harvey, Megan D.; Goodlett, David R.; Ernst, Robert K.

    2009-01-01

    The structural characterization of environmentally-regulated lipid A derived from Francisella tularensis subspecies novicida (Fn) U112 is described using negative electrospray ionization with a linear ion trap Fourier transform ion cyclotron resonance (IT-FT-ICR) hybrid mass spectrometer. The results indicate that a unique profile of lipid A molecular structures are synthesized in response to Fn growth at 25 °C versus 37 °C. Molecular species were found to be tetra-acylated, sharing a conserved glucosamine disaccharide backbone, a galactosamine-1-phosphate linked to the reducing glucosamine, and multiple O- and N-linked fatty acyl groups. Deprotonated molecules were interrogated by MSn scanning techniques at both high and nominal mass resolution and were found to be complex heterogeneous mixtures where structures differed based on the positions and identities of the O- and N-linked fatty acyl substituents. For the dominant ion series, which consisted of five peaks, 30 unique lipid A structures were identified. Estimates for the relative abundance of each structure were derived from MS relative abundance ratios and fragment ion ratios from comparable dissociation pathways from MS2 through MS4 experiments. The results suggest a remodeling pathway in which the amide linked fatty acid of the reducing glucosamine favors a 3-hydroxyhexadecanoic acid substituent for growth conditions at 25 °C versus a 3-hydroxyoctadecanoic acid substituent for growth conditions at 37 °C. PMID:17446084

  7. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    SciTech Connect

    Lu, H.; England, K; Ende, C; Truglio, J; Luckner, S; Reddy, B; Marlenee, N; Knudson, S; Knudson, D; et. al.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

  8. Genomic and SNP Analyses Demonstrate a Distant Separation of the Hospital and Community-Associated Clades of Enterococcus faecium

    PubMed Central

    Latorre, Mauricio; Qin, Xiang; Murray, Barbara E.

    2012-01-01

    Recent studies have pointed to the existence of two subpopulations of Enterococcus faecium, one containing primarily commensal/community-associated (CA) strains and one that contains most clinical or hospital-associated (HA) strains, including those classified by multi-locus sequence typing (MLST) as belonging to the CC17 group. The HA subpopulation more frequently has IS16, pathogenicity island(s), and plasmids or genes associated with antibiotic resistance, colonization, and/or virulence. Supporting the two clades concept, we previously found a 3–10% difference between four genes from HA-clade strains vs. CA-clade strains, including 5% difference between pbp5-R of ampicillin-resistant, HA strains and pbp5-S of ampicillin-sensitive, CA strains. To further investigate the core genome of these subpopulations, we studied 100 genes from 21 E. faecium genome sequences; our analyses of concatenated sequences, SNPs, and individual genes all identified two distinct groups. With the concatenated sequence, HA-clade strains differed by 0–1% from one another while CA clade strains differed from each other by 0–1.1%, with 3.5–4.2% difference between the two clades. While many strains had a few genes that grouped in one clade with most of their genes in the other clade, one strain had 28% of its genes in the CA clade and 72% in the HA clade, consistent with the predicted role of recombination in the evolution of E. faecium. Using estimates for Escherichia coli, molecular clock calculations using sSNP analysis indicate that these two clades may have diverged ≥1 million years ago or, using the higher mutation rate for Bacillus anthracis, ∼300,000 years ago. These data confirm the existence of two clades of E. faecium and show that the differences between the HA and CA clades occur at the core genomic level and long preceded the modern antibiotic era. PMID:22291916

  9. Comprehensive Cross-Clade Neutralization Analysis of a Panel of Anti-Human Immunodeficiency Virus Type 1 Monoclonal Antibodies

    PubMed Central

    Binley, James M.; Wrin, Terri; Korber, Bette; Zwick, Michael B.; Wang, Meng; Chappey, Colombe; Stiegler, Gabriela; Kunert, Renate; Zolla-Pazner, Susan; Katinger, Hermann; Petropoulos, Christos J.; Burton, Dennis R.

    2004-01-01

    Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV+ plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (≤7%). MAbs b6 (directed against the CD4 binding site) and X5 (directed against a CD4-induced epitope of gp120) neutralized only sensitive primary clade B viruses. The HIV+ plasma neutralized 70% of the viruses, including some from all major clades. Further analysis revealed five neutralizing immunotypes that were somewhat associated with clades. As well as the significance for vaccine design, our data have implications for passive-immunization studies in countries where clade C viruses are common, given that only MAbs b12 and 4E10 were effective against viruses from this clade. PMID:15542675

  10. Strong genetic differentiation between North American and European populations of Phytophthora alni subsp. uniformis.

    PubMed

    Aguayo, Jaime; Adams, Gerard C; Halkett, Fabien; Catal, Mursel; Husson, Claude; Nagy, Zoltán Á; Hansen, Everett M; Marçais, Benoît; Frey, Pascal

    2013-02-01

    Alder decline caused by Phytophthora alni has been one of the most important diseases of natural ecosystems in Europe during the last 20 years. The emergence of P. alni subsp. alni -the pathogen responsible for the epidemic-is linked to an interspecific hybridization event between two parental species: P. alni subsp. multiformis and P. alni subsp. uniformis. One of the parental species, P. alni subsp. uniformis, has been isolated in several European countries and, recently, in North America. The objective of this work was to assess the level of genetic diversity, the population genetic structure, and the putative reproduction mode and mating system of P. alni subsp. uniformis. Five new polymorphic microsatellite markers were used to contrast both geographical populations. The study comprised 71 isolates of P. alni subsp. uniformis collected from eight European countries and 10 locations in North America. Our results revealed strong differences between continental populations (Fst = 0.88; Rst = 0.74), with no evidence for gene flow. European isolates showed extremely low genetic diversity compared with the North American collection. Selfing appears to be the predominant mating system in both continental collections. The results suggest that the European P. alni subsp. uniformis population is most likely alien and derives from the introduction of a few individuals, whereas the North American population probably is an indigenous population. PMID:23095465

  11. Nested PCR for ultrasensitive detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus.

    PubMed Central

    Lee, I M; Bartoszyk, I M; Gundersen, D E; Mogen, B; Davis, R E

    1997-01-01

    Oligonucleotide primers derived from sequences of the 16S rRNA gene (CMR16F1, CMR16R1, CMR16F2, and CMR16R2) and insertion element IS1121 of Clavibacter michiganensis subsp. sepedonicus (CMSIF1, CMSIR1, CMSIF2, and CMISR2) were used in nested PCR to detect the potato ring rot bacterium C. michiganensis subsp. sepedonicus. Nested PCR with primer pair CMSIF1-CMSIR1 followed by primer pair CMSIF2-CMSIR2 specifically detected C. michiganensis subsp. sepedonicus, while nested PCR with CMR16F1-CMR16R1 followed by CMR16F2-CMR16R2 detected C. michiganensis subsp. sepedonicus and the other C. michiganensis subspecies. In the latter case, C. michiganensis subsp. sepedonicus can be differentiated from the other subspecies by restriction fragment length polymorphism (RFLP) analyses of the nested PCR products (16S rDNA sequences). The nested PCR assays developed in this work allow ultrasensitive detection of very low titers of C. michiganensis subsp. sepedonicus which may be present in symptomiess potato plants or tubers and which cannot be readily detected by direct PCR (single PCR amplification). RFLP analysis of PCR products provides for an unambiguous confirmation of the identify of C. michiganensis subsp. sepedonicus. PMID:9212412

  12. Role of Blossoms in Watermelon Seed Infestation by Acidovorax avenae subsp. citrulli.

    PubMed

    Walcott, R R; Gitaitis, R D; Castro, A C

    2003-05-01

    ABSTRACT The role of watermelon blossom inoculation in seed infestation by Acidovorax avenae subsp. citrulli was investigated. Approximately 98% (84/87) of fruit developed from blossoms inoculated with 1 x 10(7) or 1 x 10(9) CFU of A. avenae subsp. citrulli per blossom were asymptomatic. Using immunomagnetic separation and the polymerase chain reaction, A. avenae subsp. citrulli was detected in 44% of the seed lots assayed, despite the lack of fruit symptoms. Furthermore, viable colonies were recovered from 31% of the seed lots. Of these lots, 27% also yielded seedlings expressing bacterial fruit blotch symptoms when planted under conditions of 30 degrees C and 90% relative humidity. A. avenae subsp. citrulli was detected and recovered from the pulp of 33 and 19%, respectively, of symptomless fruit whose blossoms were inoculated with A. avenae subsp. citrulli. The ability to penetrate watermelon flowers was not unique to A. avenae subsp. citrulli, because blossoms inoculated with Pantoea ananatis also resulted in infested seed and pulp. The data indicate that watermelon blossoms are a potential site of ingress for fruit and seed infestation by A. avenae subsp. citrulli. PMID:18942974

  13. Clade perseverance from Mesozoic to present: a multidisciplinary approach to interpretation of pattern and process.

    PubMed

    Barnes, David K A

    2002-10-01

    Two clades of marine bryozoans, cyclostomes and cheilostomes, exemplify the benefits of applying a multidisciplinary approach to the interpretation of long-term evolutionary patterns. The cyclostome bryozoans were dominant in the Mesozoic; since that era, they have decreased in absolute terms and the cheilostomes have come to exceed them in both abundance and diversity. Many studies of living assemblages of the encrusting members of these two clades indicate that cheilostomes are superior space competitors, but paleontological studies suggest that competition between the two taxa has not been escalating over geological time. Both clades occur throughout the world's oceans and seas, and recent work in the geographical extremes has shown that the relative success of the clades varies markedly from place to place. In this study, the importance of differential patterns of recruitment and cumulative space occupation in the two clades was evaluated over four years and in two environments, one temperate and one polar. In both of these environments, peaks of recruitment and space occupation by the two clades were out of phase. The different strategies and outcomes of spatial competition are examined, largely using data from the literature. Only recently has it been realized that tied outcomes of competition are stable alternative results and not simply transitory phases. Many competitive encounters involving cyclostomes result in ties, implying that their strategy is based on persistence rather than dominance. When different indices and models are used to analyze competition data from the two clades, the interpretation varies markedly with methodology. The differences in patterns of recruitment, space occupation, and spatial competition have influenced both our understanding of how the two clades have persisted alongside each other and our perception of cheilostome superiority. Analysis of fluid dynamics has shown that small differences in the mechanical structure of

  14. [Comparative foliar anatomy and systematics of the Trichocentrum-clade with emphasis in Cohniella (Asparagales: Orchidaceae)].

    PubMed

    Cetzal-Ix, William; Noguera-Savelli, Eliana; Jáuregui, Damelis; Carnevali, Germáin

    2013-12-01

    The genera Cohniella, Lophiarella, Lophiaris, and Trichocentrum are included in the Trichocentrum-clade. These genera are distributed from Florida and Northern Mexico to Southern Brazil and Northern Argentina, growing in tropical deciduous forests or tropical rain forests and thorn scrub forests to pine-oak forest, from sea level to 1700 m. The leaf anatomical structure of 23 members of the Trichocentrum-clade was explored as a source of taxonomic and phylogenetic characters. A total of 11 species of Cohniella, three species of Lophiarella, seven species of Lophiaris, two species of Trichocentrum, and other four species were included as outgroup. Anatomical characters were studied by cross sections and paradermic observations of the middle portion of fresh leaves. Although anatomical characters were fairly homogeneous throughout the clade, twelve vegetative anatomical, phylogenetically informative characters were selected and coded for an analysis that was performed using an exhaustive search (implicit enumeration) implemented through TNT. The strict consensus of 2692 most parsimonious trees resulted in a poorly resolved polytomy, which however recovers the Trichocentrum-clade with a monophyletic, strongly supported Cohniella nested within it with unifacial leaves and the presence of cellular inclusions in the epidermis as synapomorphies. We concluded that the anatomy characters alone are insufficient to assess the relationships amongst the genera of the Trichocentrum-clade. However, the two synapomorphies recovered for Cohniella strongly support its monophyly when these are analyzed in conjunction with other data sources (e.g., molecular and morphological characters). PMID:24432538

  15. Four New Vining Species of Solanum (Dulcamaroid Clade) from Montane Habitats in Tropical America

    PubMed Central

    Knapp, Sandra

    2010-01-01

    Background Solanum (Solanaceae), with approximately 1500 species, is one of the largest genera of flowering plants, and has a centre of diversity in the New World tropics. The genus is divided into 13 major clades, of which two, the Dulcamaroid clade and the “African Non-Spiny” clade, exhibit vine morphology with twining petioles. I am currently preparing a worldwide monograph of these two groups, comprising some 70 species. Methods I formally describe here four new species of Solanum from montane Mexico and South America all belonging to the Dulcamaroid clade (including the traditionally recognised section Jasminosolanum Bitter). Descriptions, discussions of closely related species and preliminary conservation assessments are provided for all species; all species are illustrated. This paper is also a test case for the electronic publication of new names in flowering plants. Conclusions These new species are all relatively rare, but not currently of conservation concern. Solanum aspersum sp. nov. is distributed in Colombia and Ecuador, S. luculentum sp. nov. in Colombia and Venezuela, S. sanchez-vegae sp. nov. is endemic to northern Peru and S. sousae sp. nov. to southern Mexico. Solanum luculentum has the morphology of a dioecious species; this is the first report of this breeding system in the Dulcamaroid clade. PMID:20463921

  16. The role of clade competition in the diversification of North American canids

    PubMed Central

    Silvestro, Daniele; Antonelli, Alexandre; Salamin, Nicolas; Quental, Tiago B.

    2015-01-01

    The history of biodiversity is characterized by a continual replacement of branches in the tree of life. The rise and demise of these branches (clades) are ultimately determined by changes in speciation and extinction rates, often interpreted as a response to varying abiotic and biotic factors. However, understanding the relative importance of these factors remains a major challenge in evolutionary biology. Here we analyze the rich North American fossil record of the dog family Canidae and of other carnivores to tease apart the roles of competition, body size evolution, and climate change on the sequential replacement of three canid subfamilies (two of which have gone extinct). We develop a novel Bayesian analytic framework to show that competition from multiple carnivore clades successively drove the demise and replacement of the two extinct canid subfamilies by increasing their extinction rates and suppressing their speciation. Competitive effects have likely come from ecologically similar species from both canid and felid clades. These results imply that competition among entire clades, generally considered a rare process, can play a more substantial role than climate change and body size evolution in determining the sequential rise and decline of clades. PMID:26124128

  17. Mitochondrial DNA variation reveals recent evolutionary history of main Boa constrictor clades.

    PubMed

    Hynková, Ivana; Starostová, Zuzana; Frynta, Daniel

    2009-09-01

    We sequenced a 1114-bp fragment of cytochrome b gene in six subspecies (115 samples) of Boa constrictor and detected 67 haplotypes. Our analyses revealed the presence of two distinct clades, one from Central America (CA) including the neighboring part of South America west of the Andes, and the other covering the rest of South America (SA). Sequence divergence between CA and SA clades is about 5-7%, which roughly corresponds to a separation at the time of uplift of the Colombian Andes following formation of the Panama Isthmus before 3.5 Myr Sequence divergence within the SA and CA clades is only 2-3%, suggesting a fairly recent spread of these clades Into their current geographic ranges. Thus, we may not be dealing with taxa with a markedly old evolutionary history. Because juveniles of B. constrictor feed mostly on small rodents, we hypothesized that spread of this species was allowed by a new food source represented by murold rodents that appeared after closure of the Panama portal. With respect to the taxonomy, B. c. imperator may be elevated to full species rank. Within the SA clade, a haplotype of Argentinian B. c. occidentalis is markedly distinct, while the remaining haplotype groups analyzed are distributed throughout large ranges and may all belong to a single nominotypic subspecies. PMID:19799513

  18. Safety and Immunogenicity of a Single Low Dose or High Dose of Clade 2 Influenza A(H5N1) Inactivated Vaccine in Adults Previously Primed With Clade 1 Influenza A(H5N1) Vaccine.

    PubMed

    Winokur, Patricia L; Patel, Shital M; Brady, Rebecca; Chen, Wilbur H; El-Kamary, Samer S; Edwards, Kathryn; Creech, C Buddy; Frey, Sharon; Keitel, Wendy A; Belshe, Robert; Walter, Emmanuel; Bellamy, Abbie; Hill, Heather

    2015-08-15

    Influenza A(H5N1) vaccination strategies that improve the speed of the immunological response and cross-clade protection are desired. We compared the immunogenicity of a single 15-μg or 90-μg dose of A/H5N1/Indonesia/05/05 (clade 2) vaccine in adults who were previously primed with A/H5N1/Vietnam/1203/2004 (clade 1) vaccine. High-dose vaccine resulted in significantly higher titers to both clade 1 and 2 antigens. Clade 2 titers were unaffected by the previous dose of clade 1 vaccine. Low-dose priming with a mismatched pandemic influenza A(H5N1) vaccine would improve the rapidity, magnitude, and cross-reactivity of the immunological response following a single high-dose, unadjuvanted, pandemic vaccine. PMID:25712967

  19. Profiling Bovine Antibody Responses to Mycobacterium avium subsp. paratuberculosis Infection by Using Protein Arrays▿ †

    PubMed Central

    Bannantine, John P.; Paustian, Michael L.; Waters, W. Ray; Stabel, Judith R.; Palmer, Mitchell V.; Li, Lingling; Kapur, Vivek

    2008-01-01

    With the genome sequence of Mycobacterium avium subsp. paratuberculosis determined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against M. avium subsp. paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp. paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. avium subsp. paratuberculosis. Also included were previously reported or known M. avium subsp. paratuberculosis antigens to serve as a frame of reference. Sera from healthy control cattle (n = 3) and cattle infected with either M. avium subsp. avium and Mycobacterium bovis were exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. avium subsp. avium proteins that was higher than the degree of cross-reactivity with the more distantly related M. bovis proteins. Finally, sera from naturally infected cattle (n = 3) as well as cattle experimentally infected with M. avium subsp. paratuberculosis (n = 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp. paratuberculosis. PMID:18039835

  20. Alcaligenes faecalis subsp. parafaecalis subsp. nov., a bacterium accumulating poly-beta-hydroxybutyrate from acetone-butanol bioprocess residues.

    PubMed

    Schroll, G; Busse, H J; Parrer, G; Rölleke, S; Lubitz, W; Denner, E B

    2001-04-01

    The authors have previously isolated a solvent tolerant bacterium, strain G(T), (T = type strain) capable to convert acetone-butanol bioprocess residues into poly-beta-hydroxybutyrate. Strain G(T) was initially identified as Alcaligenes spp by standard bacteriological tests. In this study the taxonomic position of the bacterium was investigated in detail. The 165 rDNA sequence analysis, the G + C content of DNA (56 mol%) and the presence of ubiquinone Q-8 confirmed strain G(T) as a representative of the genus Alcaligenes. In the polyamine pattern of the bacterium putrescine and cadaverine were detected, but only trace amounts of 2-hydroxyputrescine. The extremely low content of 2-hydroxyputrescine is remarkable, since this unique diamine is a common marker for beta-proteobacteria. Phylogenetic analyses of 16S rDNA demonstrated that Alcaligenes sp. G(T) is most closely related to the species Alcaligenes faecalis (99.6% sequence similarity to A. faecalis HR4 and 98.7% sequence similarity to A. faecalis [ATCC 8750T = DSM 30030T]. On the basis of DNA-DNA relatedness (56% similarity), the unique polyamine pattern, the physiological and biochemical differences strain G(T) could be distinguished from the species A. faecalis. Therefore, a new subspecies for the species Alcaligenes faecalis is proposed; Alcaligenes faecalis subsp. parafaecalis subsp. nov. PMID:11403397

  1. Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor.

    PubMed

    Rehfuss, Marc; Urban, James

    2005-07-01

    A Gram (-) coccobacillary bacterium, J(T), was isolated from a graywater bioprocessor. 16S rRNA and biochemical analysis has revealed strain J(T) closely resembles Alcaligenes faecalis ATCC 8750T and A. faecalis subsp. parafaecalis DSM 13975T, but is a distinct, previously uncharacterized isolate. Strain J(T), along with the type strain of A. faecalis and its previously described subspecies share the ability to aerobically degrade phenol. The degradation rates of phenol for strain J(T) and reference phenol degrading bacteria were determined by photometrically measuring the change in optical density when grown on 0.1% phenol as the sole carbon source, followed by addition of Gibb's reagent to measure depletion of substrate. The phenol degradation rates of strain J(T) was found to exceed that of the phenol hydroxylase group III bacterium Pseudomonas pseudoalcaligenes, with isolate J(T) exhibiting a doubling time of 4.5 h. The presence of the large subunit of the multicomponent phenol hydroxylase gene in strain J(T) was confirmed by PCR. The presence of the nirK nitrite reductase gene as demonstrated by PCR as well as results obtained from nitrite media indicated denitrification at least to N2O. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA DNA hybridization, we propose assigning a novel subspecies of Alcaligenes faecalis, to be named Alcaligenes faecalis subsp. phenolicus with the type strain J(T) (= DSM 16503) (= NRRL B-41076). PMID:16094869

  2. A proposal to unify two subspecies of Staphylococcus equorum: Staphylococcus equorum subsp. equorum and Staphylococcus equorum subsp. linens.

    PubMed

    Jeong, Do-Won; Kim, Hye-Rim; Han, Seulhwa; Jeon, Che Ok; Lee, Jong-Hoon

    2013-12-01

    Twelve isolates from jeotgal, a Korean high-salt-fermented seafood, identified as Staphylococcus equorum were compared by phenotypic and genotypic methods to determine their precise taxonomic identities at the subspecies level. Four strains and three strains had complete 16S rRNA gene sequence matches with S. equorum subsp. equorum DSM 20674(T) and S. equorum subsp. linens DSM 15097(T), respectively. Five strains showed 99.9 % identity with the sequences of both type strains. In our DNA-DNA hybridization analyses among two type strains and two isolates, the similarities were over 72 % and were higher than the similarities presented at the subspecies proposal. Physiological characteristics such as sugar utilization, β-galactosidase activity, novobiocin resistance and salt tolerance, which were adopted for subspecies separation, could not be applied to assign the isolates to a taxonomic unit. Antibiotic susceptibility, hemolytic activity, biofilm formation and protein profiles did not present markers to divide the isolates into either of the subspecies. Multilocus sequence typing of the sequences of the 16S rRNA gene and five housekeeping genes did not produce any coherent relationship among the isolates and type strains. Repetitive element-PCR fingerprinting using ERIC (enterobacterial repetitive intergenic consensus) primers classified 12 isolates to three genotypes, and the genotypes of both type strains coincided with two isolates expressing different characteristics. Based on these phenotypic and genotypic analyses results, we propose to unify the present two subspecies of S. equorum into one species, S. equorum. PMID:24057981

  3. Seroprevalence of Bartonella vinsonii subsp. berkhoffii in urban and rural dogs in Turkey.

    PubMed

    Celebi, Bekir; Taylan Ozkan, Aysegul; Kilic, Selcuk; Akca, Atilla; Koenhemsi, Lora; Pasa, Serdar; Yildiz, Kader; Mamak, Nuri; Guzel, Murat

    2010-11-01

    The seroprevalence of Bartonella vinsonii subsp. berkhoffii was investigated in stray urban dogs and shepherd and farm guard dogs from rural areas sampled from 10 provinces of Turkey. Sera from 855 dogs were examined for the presence of anti-B. vinsonii subsp. berkhoffii antibodies by indirect fluorescent antibody test. Overall, 56 (6.6%) of the 855 dogs examined, including 16 (3%) of the 522 stray dogs and 40 (12%) of the 333 rural dogs, were seropositive. This is the first report on prevalence of antibodies to B. vinsonii subsp. berkhoffii in dogs in Turkey. PMID:20574140

  4. High quality draft genome sequence of Staphylococcus cohnii subsp. cohnii strain hu-01

    PubMed Central

    Hu, XinJun; Li, Ang; Lv, LongXian; Yuan, Chunhui; Guo, Lihua; Jiang, Xiawei; Jiang, Haiyin; Qian, GuiRong; Zheng, BeiWen; Guo, Jing; Li, LanJuan

    2014-01-01

    Staphylococcus cohnii subsp. cohnii belongs to the family Staphylococcaceae in the order Bacillales, class Bacilli and phylum Firmicutes. The increasing relevance of S. cohnii to human health prompted us to determine the genomic sequence of Staphylococcus cohnii subsp. cohnii strain hu-01, a multidrug-resistant isolate from a hospital in China. Here we describe the features of S. cohnii subsp. cohnii strain hu-01, together with the genome sequence and its annotation. This is the first genome sequence of the species Staphylococcus cohnii. PMID:25197460

  5. Vibrio panuliri sp. nov., a marine bacterium isolated from spiny lobster, Panulirus penicillatus and transfer of Vibrio ponticus from Scophthalmi clade to the newly proposed Ponticus clade.

    PubMed

    Kumari, Prabla; Poddar, Abhijit; Schumann, Peter; Das, Subrata K

    2014-12-01

    A novel marine bacterium, strain LBS2(T) was isolated from eggs carried on pleopods of the spiny lobster collected from Andaman Sea. Heterotrophic growth occurred at 1-7% NaCl. 16S rRNA gene sequence similarity revealed the strain LBS2(T) belonged to the genus Vibrio and showed above 97% similarity with eight type strains of the genus Vibrio. Multilocus analysis based on ftsZ, gapA, gyrB, mreB, pyrH recA, rpoA, and topA revealed LBS2(T) formed a separate cluster with Vibrio ponticus DSM 16217(T) with 89.8% multilocus gene sequence similarity. However, strain LBS2(T) is distantly related with other members of the Scophthalmi clade in terms of 16S rRNA signatures, phenotypic variations and multilocus gene sequence similarity, for which we propose LBS2(T) belongs to a new clade i.e. Ponticus clade with V. ponticus DSM 16217(T) as the representative type strain of the clade. DNA-DNA homologies between strain LBS2(T) and closely related strains were well below 70%. DNA G + C content was 45.3 mol%. On the basis of our polyphasic study, strain LBS2(T) represents a novel species of the genus Vibrio, for which the name Vibrio panuliri sp. nov. is proposed. The type strain is LBS2(T) (= JCM 19500(T) = DSM 27724(T) = LMG 27902(T)). PMID:25445014

  6. MiR-155 Induction by F. novicida but Not the Virulent F. tularensis Results in SHIP Down-Regulation and Enhanced Pro-Inflammatory Cytokine Response

    PubMed Central

    Cremer, Thomas J.; Ravneberg, David H.; Clay, Corey D.; Piper-Hunter, Melissa G.; Marsh, Clay B.; Elton, Terry S.; Gunn, John S.; Amer, Amal; Kanneganti, Thirumala-Devi; Schlesinger, Larry S.; Butchar, Jonathan P.; Tridandapani, Susheela

    2009-01-01

    The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3′UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella. PMID:20041145

  7. The binding sites of monoclonal antibodies to the non-reducing end of Francisella tularensis O-antigen accommodate mainly the terminal saccharide

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J; Yang, Chiou-Ying; Madico, Guillermo; Perkins, Hillary M; Wang, Qi; Costello, Catherine E; Zaia, Joseph; Seaton, Barbara A; Sharon, Jacqueline

    2013-01-01

    We have previously described two types of protective B-cell epitopes in the O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti-OAg monoclonal antibodies (mAbs), and a non-overlapping epitope at the non-reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non-reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope. Like FB11, the new mAbs, Ab63 (IgG3), N213 (IgG3) and N62 (IgG2b), had higher antigen-binding bivalent avidity than internally binding anti-OAg mAbs, and an oligosaccharide containing a single OAg repeat was sufficient for optimal inhibition of their antigen-binding. The X-ray crystal structure of N62 Fab showed that the antigen-binding site is lined mainly by aromatic amino acids that form a small cavity, which can accommodate no more than one and a third sugar residues, indicating that N62 binds mainly to the terminal Qui4NFm residue at the nonreducing end of OAg. In efficacy studies with mice infected intranasally with the highly virulent F. tularensis strain SchuS4, N62, N213 and Ab63 prolonged survival and reduced blood bacterial burden. These results yield insights into how antibodies to non-reducing ends of microbial polysaccharides can contribute to immune protection despite the smaller size of their target epitopes compared with antibodies to internal polysaccharide regions. PMID:23844703

  8. Importance of PdpC, IglC, IglI, and IglG for modulation of a host cell death pathway induced by Francisella tularensis.

    PubMed

    Lindgren, Marie; Eneslätt, Kjell; Bröms, Jeanette E; Sjöstedt, Anders

    2013-06-01

    Modulation of host cell death pathways appears to be a prerequisite for the successful lifestyles of many intracellular pathogens. The facultative intracellular bacterium Francisella tularensis is highly pathogenic, and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand the prerequisites of this cell death, macrophages were infected with the F. tularensis live vaccine strain (LVS), and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of the pdpC, iglC, iglG, or iglI genes, which encode components of the Francisella pathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and, after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the ΔiglI or ΔiglC mutants, although the former strain replicated. The ΔiglG mutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the ΔpdpC mutant showed no replication but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to those obtained with LVS. The unique phenotypes of the mutants provide insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from the F. tularensis infection. PMID:23529623

  9. The binding sites of monoclonal antibodies to the non-reducing end of Francisella tularensis O-antigen accommodate mainly the terminal saccharide.

    PubMed

    Lu, Zhaohua; Rynkiewicz, Michael J; Yang, Chiou-Ying; Madico, Guillermo; Perkins, Hillary M; Wang, Qi; Costello, Catherine E; Zaia, Joseph; Seaton, Barbara A; Sharon, Jacqueline

    2013-11-01

    We have previously described two types of protective B-cell epitopes in the O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti-OAg monoclonal antibodies (mAbs), and a non-overlapping epitope at the non-reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non-reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope. Like FB11, the new mAbs, Ab63 (IgG3), N213 (IgG3) and N62 (IgG2b), had higher antigen-binding bivalent avidity than internally binding anti-OAg mAbs, and an oligosaccharide containing a single OAg repeat was sufficient for optimal inhibition of their antigen-binding. The X-ray crystal structure of N62 Fab showed that the antigen-binding site is lined mainly by aromatic amino acids that form a small cavity, which can accommodate no more than one and a third sugar residues, indicating that N62 binds mainly to the terminal Qui4NFm residue at the nonreducing end of OAg. In efficacy studies with mice infected intranasally with the highly virulent F. tularensis strain SchuS4, N62, N213 and Ab63 prolonged survival and reduced blood bacterial burden. These results yield insights into how antibodies to non-reducing ends of microbial polysaccharides can contribute to immune protection despite the smaller size of their target epitopes compared with antibodies to internal polysaccharide regions. PMID:23844703

  10. The Estimation of Tree Posterior Probabilities Using Conditional Clade Probability Distributions

    PubMed Central

    Larget, Bret

    2013-01-01

    In this article I introduce the idea of conditional independence of separated subtrees as a principle by which to estimate the posterior probability of trees using conditional clade probability distributions rather than simple sample relative frequencies. I describe an algorithm for these calculations and software which implements these ideas. I show that these alternative calculations are very similar to simple sample relative frequencies for high probability trees but are substantially more accurate for relatively low probability trees. The method allows the posterior probability of unsampled trees to be calculated when these trees contain only clades that are in other sampled trees. Furthermore, the method can be used to estimate the total probability of the set of sampled trees which provides a measure of the thoroughness of a posterior sample. [Bayesian phylogenetics; conditional clade distributions; improved accuracy; posterior probabilities of trees.] PMID:23479066

  11. Phylogeny of the sea hares in the aplysia clade based on mitochondrial DNA sequence data

    SciTech Connect

    Medina, Monica; Collins, Timothy; Walsh, Patrick J.

    2004-02-20

    Sea hare species within the Aplysia clade are distributed worldwide. Their phylogenetic and biogeographic relationships are, however, still poorly known. New molecular evidence is presented from a portion of the mitochondrial cytochrome oxidase c subunit 1 gene (cox1) that improves our understanding of the phylogeny of the group. Based on these data a preliminary discussion of the present distribution of sea hares in a biogeographic context is put forward. Our findings are consistent with only some aspects of the current taxonomy and nomenclatural changes are proposed. The first, is the use of a rank free classification for the different Aplysia clades and subclades as opposed to previously used genus and subgenus affiliations. The second, is the suggestion that Aplysia brasiliana (Rang, 1828) is a junior synonym of Aplysia fasciata (Poiret, 1789). The third, is the elimination of Neaplysia since its only member is confirmed to be part of the large Varria clade.

  12. Six genetically distinct clades of Palola (Eunicidae, Annelida) from Lizard Island, Great Barrier Reef, Australia.

    PubMed

    Schulze, Anja

    2015-01-01

    A total of 36 lots of Palola spp. (Eunicidae, Annelida) were collected during the Lizard Island Polychaete Workshop on Lizard Island, Great Barrier Reef, Queensland, Australia. Of these, 21 specimens were sequenced for a portion of the mitochondrial cytochrome c oxidase I gene. These sequences were analysed in conjunction with existing sequences of Palola spp. from other geographic regions. The samples from Lizard Island form six distinct clades, although none of them can clearly be assigned to any of the nominal species. Four of the six Lizard Island clades fall into species group A and the remaining two into species group B (which also includes the type species, Palola viridis). All sequenced specimens were characterized morphologically as far as possible and a dichotomous key was assembled. Based on this key, the remaining samples were identified as belonging to one of the clades. PMID:26624083

  13. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

    SciTech Connect

    Fischer, N. O.

    2015-01-06

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the second quarter of the third year, LLNL finalized all immunological assessments of NLP vaccine formulations in the F344 model. Battelle has immunized rats with three unique NLP formulations by either intramuscular or intranasal administration. All inoculations have been completed, and protective efficacy against an aerosolized challenge will begin at the end of October, 2014.

  14. Sarcoptes scabiei mites in humans are distributed into three genetically distinct clades.

    PubMed

    Andriantsoanirina, V; Ariey, F; Izri, A; Bernigaud, C; Fang, F; Charrel, R; Foulet, F; Botterel, F; Guillot, J; Chosidow, O; Durand, R

    2015-12-01

    Scabies is an ectoparasitic infestation caused by the mite Sarcoptes scabiei. Currently, S. scabiei is taxonomically divided into different varieties on the basis of host origin. Genetics-based research on scabies has been conducted, but the data on genetic diversity of populations of this mite in humans in Europe are lacking. We evaluated the genetic diversity of populations of S. scabiei. A large series of mites obtained from humans in France and the data of mites from various hosts and geographical areas retrieved from GenBank were included to investigate whether mites are divided into distinct populations. The study of cytochrome c oxidase subunit 1 gene polymorphisms were found to be best suited for phylogenetic analysis. S. scabiei mites were distributed into three genetically distinct clades, with most mites clustering in clades B and C. The Fst value and the Nm value calculated for mites included in clades B and C indicated a strong population structure and a very low gene flow between mites of those clades. The results of the present study not only support the rejection of the hypothesis of panmixia for S. scabiei in humans but also suggest that mites belonging to different clades are genetically isolated. Moreover, the results suggest that the subdivision of S. scabies in varieties according to animal or human hosts is not warranted. In conclusion, S. scabiei mites in humans do not constitute a homogeneous population. Further investigations are now required to assess whether different clinical forms of scabies are associated with particular haplotypes or clades. PMID:26278670

  15. A molecular assessment of the iron stress response in the two phylogenetic clades of Trichodesmium.

    PubMed

    Chappell, P Dreux; Webb, Eric A

    2010-01-01

    Trichodesmium spp. play key roles in global carbon and nitrogen budgets and thus defining what controls their productivity is important for understanding climate change. While iron availability has been shown to be an important chemical factor for controlling both growth and nitrogen fixation rates in Trichodesmium, all culture experiments to date have focused solely on representatives from one clade of Trichodesmium. Genomic sequence analysis determined that the Trichodesmium erythraeum (IMS101) genome contains many of the archetypical genes involved in the prokaryotic iron stress response. Focusing on three of these genes, isiB, idiA and feoB, we found that all three showed an iron stress response in axenic T. erythraeum (IMS101), and their sequences were well conserved across four species in our Trichodesmium culture collection [consisting of two T. erythraeum strains (IMS101 and GBRTRLI101), two Trichodesmium tenue strains (Z-1 and H9-4), Trichodesmium thiebautii and Trichodesmium spiralis]. With clade-specific quantitative PCR (qPCR) primers for one of these genes, isiB, we found that high isiB expression at low Fe levels corresponded to specific reductions in N(2) fixation rates in both major phylogenetic clades of Trichodesmium (the T. erythraeum clade and T. tenue clade). With regard to the two clades, the most significant difference determined was temperature optima, while more subtle differences in growth, N(2) fixation rate and gene expression responses to Fe stress were also observed. However the apparent conservation of the Fe stress response in the Trichodesmium genus suggests that it is an important adaptation for their niche in the oligotrophic ocean. PMID:19708870

  16. FISH-Flow: a quantitative molecular approach for describing mixed clade communities of Symbiodinium

    NASA Astrophysics Data System (ADS)

    McIlroy, S. E.; Smith, G. J.; Geller, J. B.

    2014-03-01

    Our understanding of reef corals and their fate in a changing climate is limited by our ability to monitor the diversity and abundance of the dinoflagellate endosymbionts that sustain them. This study combined two well-known methods in tandem: fluorescent in situ hybridization (FISH) for genotype-specific labeling of Symbiodinium and flow cytometry to quantify the abundance of each symbiont clade in a sample. This technique (FISH-Flow) was developed with cultured Symbiodinium representing four distinct clades (based on large subunit rDNA) and was used to distinguish and quantify these types with high efficiency and few false positives. This technique was also applied to freshly isolated symbionts of Orbicella faveolata and Orbicella annularis. Isolates from acutely bleached coral tissues had significantly lower labeling efficiency; however, isolates from healthy tissue had efficiencies comparable to cultured Symbiodinium trials. RNA degradation in bleaching samples may have interfered with labeling of cells. Nevertheless, we were able to determine that, with and without thermal stress, experimental columns of the coral O. annularis hosted a majority of clade B and B/C symbionts on the top and side of the coral column, respectively. We demonstrated that, for cultured Symbiodinium and Symbiodinium freshly isolated from healthy host tissues, the relative ratio of clades could be accurately determined for clades present at as low as 7 % relative abundance. While this method does not improve upon PCR-based techniques in identifying clades at background levels, FISH-Flow provides a high precision, flexible system for targeting, quantifying and isolating Symbiodinium genotypes of interest.

  17. Microsatellite typing identifies the major clades of the human pathogen Candida albicans.

    PubMed

    Chávez-Galarza, Julio; Pais, Célia; Sampaio, Paula

    2010-07-01

    Candida albicans population studies showed that this species could be divided into sub-groups of closely related strains, designated by clades. Since the emergence of microsatellite analysis as a PCR based method, this technique has been successfully used as a tool to differentiate C. albicans isolates but has never been tested regarding clustering of the five major clades. In this study we tested microsatellite length polymorphism (MLP) ability to group 29 C. albicans strains previously defined as belonging to clades I, II, III, E and SA, nine atypical strains from Angola and Madagascar, and 78 Portuguese clinical isolates. MLP typing of the total 116 strains analyzed yielded 87 different multilocus allelic combinations which resulted in a high discriminatory power index, of 0.987, with only two markers, CA1 and CEF3. Cluster analysis of the 29 previously defined strains grouped them according to their clade designation with a matrix cophenetic correlation of r=0.963 after a normalized Mantel statistic. Clustering analysis of the 116 strains maintained the same groupings, clearly defining the five major C. albicans clades. The cophenetic value obtained was of r=0.839, and the one-tail probability of the normalized Mantel statistic out of 1000 random permutations was P=0.0020. The proportion of Portuguese isolates in the groups I, II, III and SA was of 2.7%, 15.4%, 3.8% and 0%, respectively. None of the isolates co-clustered with the atypical strains. These results confirm MLP typing as a good method both to type and differentiate C. albicans isolates and to group isolates, identifying the major C. albicans clades, similarly to Ca3 fingerprinting and multilocus sequence typing (MLST). PMID:20348035

  18. Comparative Genomic Hybridizations Reveal Genetic Regions within the Mycobacterium avium Complex That Are Divergent from Mycobacterium avium subsp. paratuberculosis Isolates†

    PubMed Central

    Paustian, Michael L.; Kapur, Vivek; Bannantine, John P.

    2005-01-01

    Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs. PMID:15774884

  19. Integrative Cloning, Expression, and Stability of the cryIA(c) Gene from Bacillus thuringiensis subsp. kurstaki in a Recombinant Strain of Clavibacter xyli subsp. cynodontis

    PubMed Central

    Lampel, Jay S.; Canter, Gayle L.; Dimock, Michael B.; Kelly, Jeffrey L.; Anderson, James J.; Uratani, Brenda B.; Foulke, James S.; Turner, John T.

    1994-01-01

    A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The cryIA(c) gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp. cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the cryIA(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The cryIA(c) gene and the tetM gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control. Images PMID:16349179

  20. The in vitro effect of six antimicrobials against Mycoplasma putrefaciens, Mycoplasma mycoides subsp. mycoides LC and Mycoplasma capricolum subsp. capricolum isolated from sheep and goats in Jordan.

    PubMed

    Al-Momani, W; Nicholas, R A J; Janakat, S; Abu-Basha, E; Ayling, R D

    2006-01-01

    Respiratory disease in sheep and goats is a major problem in Jordan and is often associated with Mycoplasma species. Without effective vaccines, control is mainly by chemotherapy, but the uncontrolled use of antimicrobials has led to concerns about the potential development of antimicrobial resistance. The in vitro effect of chloramphenicol, florfenicol, enrofloxacin, tylosin, erythromycin and oxytetracycline was determined against 32 isolates of Mycoplasma species-M. mycoides subsp. mycoides LC (6), M. capricolum subsp. capricolum (8) and M. putrefaciens (18), all isolated from either nasal swabs or milk, from sheep and goats in different regions of Jordan. The antimicrobial susceptibility showed some Mycoplasma species-specific differences, with M. capricolum subsp. capricolum being more susceptible to tylosin and erythromycin. Chloramphenicol and florfenicol were the least effective for all three Mycoplasma species. No trends or significant differences in antimicrobial susceptibilities were observed between sheep and goat isolates, between milk or nasal swab isolates, or between isolates from different regions of Jordan. Some isolates of M. capricolum subsp. capricolum and M. putrefaciens showed higher MIC levels with oxytetracycline, as did two isolates of M. mycoides subsp. mycoides LC with tylosin, possibly indicating signs of development of antimicrobial resistance. PMID:17405622

  1. Clade age and diversification rate variation explain disparity in species richness among water scavenger beetle (Hydrophilidae) lineages.

    PubMed

    Bloom, Devin D; Fikáček, Martin; Short, Andrew E Z

    2014-01-01

    Explaining the disparity of species richness across the tree of life is one of the great challenges in evolutionary biology. Some lineages are exceptionally species rich, while others are relatively species poor. One explanation for heterogeneity among clade richness is that older clades are more species rich because they have had more time to accrue diversity than younger clades. Alternatively, disparity in species richness may be due to among-lineage diversification rate variation. Here we investigate diversification in water scavenger beetles (Hydrophilidae), which vary in species richness among major lineages by as much as 20 fold. Using a time-calibrated phylogeny and comparative methods, we test for a relationship between clade age and species richness and for shifts in diversification rate in hydrophilids. We detected a single diversification rate increase in Megasternini, a relatively young and species rich clade whose diversity might be explained by the stunning diversity of ecological niches occupied by this clade. We find that Amphiopini, an old clade, is significantly more species poor than expected, possibly due to its restricted geographic range. The remaining lineages show a correlation between species richness and clade age, suggesting that both clade age and variation in diversification rates explain the disparity in species richness in hydrophilids. We find little evidence that transitions between aquatic, semiaquatic, and terrestrial habitats are linked to shifts in diversification rates. PMID:24887453

  2. Clade Age and Diversification Rate Variation Explain Disparity in Species Richness among Water Scavenger Beetle (Hydrophilidae) Lineages

    PubMed Central

    Bloom, Devin D.; Fikáček, Martin; Short, Andrew E. Z.

    2014-01-01

    Explaining the disparity of species richness across the tree of life is one of the great challenges in evolutionary biology. Some lineages are exceptionally species rich, while others are relatively species poor. One explanation for heterogeneity among clade richness is that older clades are more species rich because they have had more time to accrue diversity than younger clades. Alternatively, disparity in species richness may be due to among-lineage diversification rate variation. Here we investigate diversification in water scavenger beetles (Hydrophilidae), which vary in species richness among major lineages by as much as 20 fold. Using a time-calibrated phylogeny and comparative methods, we test for a relationship between clade age and species richness and for shifts in diversification rate in hydrophilids. We detected a single diversification rate increase in Megasternini, a relatively young and species rich clade whose diversity might be explained by the stunning diversity of ecological niches occupied by this clade. We find that Amphiopini, an old clade, is significantly more species poor than expected, possibly due to its restricted geographic range. The remaining lineages show a correlation between species richness and clade age, suggesting that both clade age and variation in diversification rates explain the disparity in species richness in hydrophilids. We find little evidence that transitions between aquatic, semiaquatic, and terrestrial habitats are linked to shifts in diversification rates. PMID:24887453

  3. Composition and potency characterization of Mycobacterium avium subsp. paratuberculosis purified protein derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain ATCC 19698. Traditional production consists of floating culture incubation at 37oC, organism inactivation by autoclaving, coarse filtrat...

  4. Complete genome sequence of Campylobacter fetus subsp. testudinum type strain 03-427T

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter fetus subsp. testudinum has been isolated from reptiles and humans. This Campylobacter subspecies is genetically distinct from other C. fetus subspecies. Here we present the first whole genome sequence for this C. fetus subspecies....

  5. Pantoea stewartii subsp. stewartii produces an endoglucanase that is required for full virulence in sweet corn.

    PubMed

    Mohammadi, Mojtaba; Burbank, Lindsey; Roper, M Caroline

    2012-04-01

    Pantoea stewartii subsp. stewartii, a xylem-dwelling bacterium, is the causal agent of Stewart's wilt and blight of sweet corn. The goal of this study was to characterize the only gene in the P. stewartii subsp. stewartii genome predicted to encode an endoglucanase (EGase); this gene was designated engY. Culture supernatants from P. stewartii subsp. stewartii and Escherichia coli expressing recombinant EngY protein possessed both EGase and xylanase activities. Deletion of engY abolished EGase and xylanase activity, demonstrating that EngY appears to be the major EGase or xylanase produced by P. stewartii subsp. stewartii. Most importantly, our results show that EngY contributes to movement in the xylem and disease severity during the wilting phase of Stewart's wilt but is not required for water-soaked lesion formation. PMID:22122328

  6. Comparative genomics of Bifidobacterium animalis subsp. lactis reveals a strict monophyletic bifidobacterial taxon.

    PubMed

    Milani, Christian; Duranti, Sabrina; Lugli, Gabriele Andrea; Bottacini, Francesca; Strati, Francesco; Arioli, Stefania; Foroni, Elena; Turroni, Francesca; van Sinderen, Douwe; Ventura, Marco

    2013-07-01

    Strains of Bifidobacterium animalis subsp. lactis are extensively exploited by the food industry as health-promoting bacteria, although the genetic variability of members belonging to this taxon has so far not received much scientific attention. In this article, we describe the complete genetic makeup of the B. animalis subsp. lactis Bl12 genome and discuss the genetic relatedness of this strain with other sequenced strains belonging to this taxon. Moreover, a detailed comparative genomic analysis of B. animalis subsp. lactis genomes was performed, which revealed a closely related and isogenic nature of all currently available B. animalis subsp. lactis strains, thus strongly suggesting a closed pan-genome structure of this bacterial group. PMID:23645200

  7. Exploring the Genome of Cheese Starter Lactic Acid Bacterium Lactococcus lactis subsp. lactis CECT 4433

    PubMed Central

    Tschoeke, Diogo Antonio; Moreira, Ana Paula B.; Chimetto Tonon, Luciane A.; de Mesquita, Milene Miranda A.; Gregoracci, Gustavo B.; Gomez-Gil, Bruno; Valle, Rogério; Thompson, Cristiane C.

    2014-01-01

    Here, we present the draft genome sequences of Lactococcus lactis subsp. lactis CECT 4433, a cheese fermentation starter strain. The genome provides further insight into the genomic plasticity, biocomplexity (including gene strain specifics), and evolution of these genera. PMID:25395632

  8. Exploring the Genome of Cheese Starter Lactic Acid Bacterium Lactococcus lactis subsp. lactis CECT 4433.

    PubMed

    Tschoeke, Diogo Antonio; Moreira, Ana Paula B; Chimetto Tonon, Luciane A; de Mesquita, Milene Miranda A; Gregoracci, Gustavo B; Gomez-Gil, Bruno; Valle, Rogério; Thompson, Cristiane C; Thompson, Fabiano L

    2014-01-01

    Here, we present the draft genome sequences of Lactococcus lactis subsp. lactis CECT 4433, a cheese fermentation starter strain. The genome provides further insight into the genomic plasticity, biocomplexity (including gene strain specifics), and evolution of these genera. PMID:25395632

  9. Experimental Paratuberculosis in Calves following Inoculation with a Rabbit Isolate of Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Beard, P. M.; Stevenson, K.; Pirie, A.; Rudge, K.; Buxton, D.; Rhind, S. M.; Sinclair, M. C.; Wildblood, L. A.; Jones, D. G.; Sharp, J. M.

    2001-01-01

    The role of wildlife species in the epidemiology of paratuberculosis has been the subject of increased research efforts following the discovery of natural paratuberculosis in free-living rabbits from farms in east Scotland. This paper describes the experimental inoculation of young calves with an isolate of Mycobacterium avium subsp. paratuberculosis recovered from a free-living rabbit. After a 6-month incubation period, all eight calves inoculated with the rabbit isolate had developed histopathological and/or microbiological evidence of M. avium subsp. paratuberculosis infection. Similar results were obtained from a group of calves infected with a bovine isolate of M. avium subsp. paratuberculosis. The virulence of the rabbit isolate for calves demonstrated in this study suggests that rabbits are capable of passing paratuberculosis to domestic ruminants and that wildlife reservoirs of M. avium subsp. paratuberculosis should therefore be considered when formulating control plans for the disease. PMID:11526132

  10. Neisseria elongata subsp elongata infective endocarditis following endurance exercise.

    PubMed

    Jenkins, Joanne May; Fife, Amanda; Baghai, Max; Dworakowski, Rafal

    2015-01-01

    A 31-year-old Argentinian woman presented with a 3-week history of fever, night sweats, myalgia and lethargy following a work trip to Uganda where she ran a marathon. Malarial screens were negative but C reactive protein, erythrocyte sedimentation rate and neutrophil count were raised and she was anaemic. A new pansystolic murmur was heard over the mitral valve and the transthoracic echocardiogram showed a large vegetation (>1 cm) with at least moderate mitral regurgitation. Blood cultures grew Neisseria elongata, subsp elongata treated initially with ceftriaxone then oral ciprofloxacin to complete 4 weeks of treatment. CT scan revealed a wedge-shaped area of low attenuation in the spleen in keeping with a splenic infarct. Seven days postadmission, the patient underwent a successful mitral valve repair. Recovery was complicated by a likely embolic infarct in the right frontal lobe, but the patient was discharged 12 days postoperative with no neurological sequelae. PMID:26655669

  11. Efficient production of nonactin by Streptomyces griseus subsp. griseus.

    PubMed

    Zhan, Yulian; Zheng, Shaolun

    2016-08-01

    Here we report the production of the cyclic macrotetrolide nonactin from the fermentation culture of Streptomyces griseus subsp. griseus. Nonactin is a member of a family of naturally occurring cyclic ionophores known as the macrotetrolide antibiotics. Our fermentation procedure of Streptomyces griseus was performed at 30 °C and 200 rev·min(-1) for 5 days on a rotary shaker. Diaion HP-20 and Amberlite XAD-16 were added to the fermentation medium. Isolated yield of nonactin was up to 80 mg·L(-1) using our methodology. Nonactin is commonly known as an ammonium ionophore and also exhibits antibacterial, antiviral, and antitumor activities. It is also widely used for the preparation of ion-selective electrodes and sensors. Chemical synthesis of nonactin has been achieved by some groups; however, overall yields are very low, making efficient biosynthesis an attractive means of production. PMID:27405846

  12. Volatile Components Emitted from the Liverwort Marchantia paleacea subsp. diptera.

    PubMed

    Sakurai, Kazutoshi; Tomiyama, Kenichi; Kawakami, Yukihiko; Ochiai, Nozomi; Yabe, Shigeki; Nakagawa, Tomomi; Asakawa, Yoshinori

    2016-02-01

    The volatile components from the thalloid liverwort, Marchantia paleacea subsp. diptera were investigated by HS-SPME-GC-MS analysis. The monocyclic monoterpene aldehyde, perillaldehyde was identified for the first time as the major component and its content was about 50% of the volatiles, along with β-pinene, limonene, β-caryophyllene, α-selinene and β-selinene as minor volatiles. Using MD (Multi-dimensional) GC-MS analysis equipped with a chiral column as the second column, the chirality was determined of both perillaldehyde and limonene, which was considered as the precursor of perillaldehyde. Both compounds were (S)-(-)-enantiomers (over 99.0 %) and (R)-enantiomers (less than 0.5 %). This is the first report of the existence of perillaldehyde in liverworts. PMID:27032216

  13. Bibenzyls and dihydroisocoumarins from white salsify (Tragopogon porrifolius subsp. porrifolius).

    PubMed

    Zidorn, Christian; Lohwasser, Ulrike; Pschorr, Susanne; Salvenmoser, Daniela; Ongania, Karl-Hans; Ellmerer, Ernst P; Börner, Andreas; Stuppner, Hermann

    2005-07-01

    A phytochemical investigation of three accessions of Tragopogon porrifolius L. subsp. porrifolius (Asteraceae, Lactuceae) yielded three new bibenzyl derivatives, 5,4'-dihydroxy-3-alpha-l-rhamnopyranosyl-(1-->3)-beta-d-xylopyranosyloxybibenzyl, 2-carboxyl-3,4'-dihydroxy-5-beta-d-xylopyranosyloxybibenzyl, tragopogonic acid (2'carboxyl-3',5',4''-trihydroxyphenylethanone) and three dihydroisocoumarin derivatives, including the new natural product 6-O-methylscorzocreticoside I. One of the isolated bibenzyl derivatives is considered to be a precursor to the biosynthesis of dihydroisocoumarins. Structures of new compounds were established by HR mass spectrometry, extensive 1D and 2D NMR spectroscopy, and CD spectroscopy. Moreover, radical scavenging activities of the polyphenolic compounds were measured using the 2,2-diphenyl-1-picrylhydrazyl assay; two of the bibenzyls showed moderate and two of the dihydroisocoumarins showed weak radical scavenging activities. The chemosystematic impact of bibenzyls and dihydroisocoumarins is discussed briefly. PMID:15964041

  14. Carbon sources of natural cyanamide in Vicia villosa subsp. varia.

    PubMed

    Kamo, Tsunashi; Kasahara, Ryohei; Abe, Shun; Hirota, Mitsuru; Sugano, Mami; Yamaya, Hiroko; Hiradate, Syuntaro; Fujii, Yoshiharu

    2010-10-01

    The ¹³C labels of [¹³C]carbon dioxide and D-[¹³C₆]glucose were incorporated into cyanamide (NH₂CN) when they were administered to Vicia villosa subsp. varia shoots. In contrast, the administration of sodium [2,3-¹³C₂]pyruvate did not affect the relative area of the [M + 1]+ ion of cyanamide in the gas chromatography-mass spectrometry analysis. [2,3-¹³C₂]pyruvate was incorporated into organic acids that are part of the citric acid cycle, such as succinate and fumarate, confirming that the shoots absorbed and metabolised it. These observations demonstrated that the carbon atom of cyanamide is derived from any of the carbohydrates that are present upstream of pyruvate in the metabolic pathway. PMID:20954091

  15. Novel fermentation media for production of Bacillus thuringiensis subsp. israelensis.

    PubMed

    Poopathi, Subbiah; Kumar, K Anup

    2003-08-01

    The production of Bacillus thuringiensis subsp. israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology. In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti. Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani). Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium. These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs. PMID:14503573

  16. Molecular Characterization of Three Lactobacillus delbrueckii subsp. bulgaricus Phages

    PubMed Central

    Casey, Eoghan; Mahony, Jennifer; O'Connell-Motherway, Mary; Bottacini, Francesca; Cornelissen, Anneleen; Neve, Horst; Heller, Knut J.; Noben, Jean-Paul; Dal Bello, Fabio

    2014-01-01

    In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations. PMID:25002431

  17. Seed-associated subspecies of the genus Clavibacter are clearly distinguishable from Clavibacter michiganensis subsp. michiganensis.

    PubMed

    Yasuhara-Bell, Jarred; Alvarez, Anne M

    2015-03-01

    The genus Clavibacter contains one recognized species, Clavibacter michiganensis. Clavibacter michiganensis is subdivided into subspecies based on host specificity and bacteriological characteristics, with Clavibacter michiganensis subsp. michiganensis causing bacterial canker of tomato. Clavibacter michiganensis subsp. michiganensis is often spread through contaminated seed leading to outbreaks of bacterial canker in tomato production areas worldwide. The frequent occurrence of non-pathogenic Clavibacter michiganensis subsp. michiganensis-like bacteria (CMB) is a concern for seed producers because Clavibacter michiganensis subsp. michiganensis is a quarantine organism and detection of a non-pathogenic variant may result in destruction of an otherwise healthy seed lot. A thorough biological and genetic characterization of these seed-associated CMB strains was performed using standard biochemical tests, cell wall analyses, metabolic profiling using Biolog, and single-gene and multilocus sequence analyses. Combined, these tests revealed two distinct populations of seed-associated members of the genus Clavibacter that differed from each other, as well as from all other described subspecies of Clavibacter michiganensis. DNA-DNA hybridization values are 70 % or higher, justifying placement into the single recognized species, C. michiganensis, but other analyses justify separate subspecies designations. Additionally, strains belonging to the genus Clavibacter isolated from pepper also represent a distinct population and warrant separate subspecies designation. On the basis of these data we propose subspecies designations for separate non-pathogenic subpopulations of Clavibacter michiganensis: Clavibacter michiganensis subsp. californiensis subsp. nov. and Clavibacter michiganensis subsp. chilensis subsp. nov. for seed-associated strains represented by C55(T) ( = ATCC BAA-2691(T) = CFBP 8216(T)) and ZUM3936(T) ( = ATCC BAA-2690(T) = CFBP 8217(T

  18. Interacting Factors That Influence Long-term Storage of Live Pasteurella tularensis Vaccine and Rift Valley Fever Virus

    PubMed Central

    Klein, Frederick; Walker, Jerry S.; Mahlandt, Bill G.; Carter, Richard C.; Orlando, Michael D.; Weirether, Francis J.; Lincoln, Ralph E.

    1969-01-01

    Studies were conducted on the interaction of various parameters which affect the storage stability and growth potential of liquid cultures of Pasteurella tularensis live vaccine strain (LVS) and Rift Valley fever virus Van Wyk strain (RVFV). Storage variables studied with LVS included four storage temperatures (4, -20, -65, -175 C), single and multiple freeze-thaw cycles, two freezing and two thawing rates (slow and fast), various inoculum levels (1, 3, 5, and 10%) for the determination of growth potential, and the retention of immunizing potential (mice and guinea pig) after storage. Neither the freezing rate nor the number of freeze-thaw cycles seriously affected the growth of LVS after storage at -175C; however, the slow rate of thaw proved deleterious as were all temperatures of storage except -175 C after 1 year of storage, as shown by both criteria of evaluation. RVFV produced in two combinations of cell lines and media (LM cell line-199 peptone medium and LDR cell line-Eagle's minimum essential medium) was stored at three serum levels (10, 20, 40%), three pH values (6.2., 7.0, 7.8), and three temperatures (-20, -65, -175 C). These studies indicated: (i) virus produced in the LDR cell line and Eagle's medium was more stable than that produced in the LM cell line and 199 peptone medium for either short- or long-term storage; (ii) serum levels did not affect stability; and (iii) low pH resulted in losses during long-term storage under all conditions tested. Thus, cryogenic storage is advantageous for stock culture maintenance of bacteria and viruses and for other similar applications. PMID:5780399

  19. Functional and Structural Characterization of Francisella tularensis O-Antigen Antibodies at the Low End of Antigen Reactivity

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J.; Yang, Chiou-Ying; Madico, Guillermo; Perkins, Hillary M.; Roche, Marly I.; Seaton, Barbara A.

    2014-01-01

    The O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis (Ft), which is both a capsular polysaccharide and a component of lipopolysaccharide, is comprised of tetrasaccharide repeats and induces antibodies mainly against repeating internal epitopes. We previously reported on several BALB/c mouse monoclonal antibodies (MAbs) that bind to internal Ft OAg epitopes and are protective in mouse models of respiratory tularemia. We now characterize three new internal Ft OAg IgG2a MAbs, N203, N77, and N24, with 10- to 100-fold lower binding potency than previously characterized internal-OAg IgG2a MAbs, despite sharing one or more variable region germline genes with some of them. In a mouse model of respiratory tularemia with the highly virulent Ft type A strain SchuS4, the three new MAbs reduced blood bacterial burden with potencies that mirror their antigen-binding strength; the best binder of the new MAbs, N203, prolonged survival in a dose-dependent manner, but was at least 10-fold less potent than the best previously characterized IgG2a MAb, Ab52. X-ray crystallographic studies of N203 Fab showed a flexible binding site in the form of a partitioned groove, which cannot provide as many contacts to OAg as does the Ab52 binding site. These results reveal structural features of antibodies at the low end of reactivity with multi-repeat microbial carbohydrates and demonstrate that such antibodies still have substantial protective effects against infection. PMID:25171003

  20. FTT0831c/FTL_0325 Contributes to Francisella tularensis Cell Division, Maintenance of Cell Shape, and Structural Integrity

    PubMed Central

    Robertson, Gregory T.; Case, Elizabeth Di Russo; Dobbs, Nicole; Ingle, Christine; Balaban, Murat; Celli, Jean

    2014-01-01

    The Francisella FTT0831c/FTL_0325 gene encodes amino acid motifs to suggest it is a lipoprotein and that it may interact with the bacterial cell wall as a member of the OmpA-like protein family. Previous studies have suggested that FTT0831c is surface exposed and required for virulence of Francisella tularensis by subverting the host innate immune response (M. Mahawar et al., J. Biol. Chem. 287:25216–25229, 2012). We also found that FTT0831c is required for murine pathogenesis and intramacrophage growth of Schu S4, but we propose a different model to account for the proinflammatory nature of the resultant mutants. First, inactivation of FTL_0325 from live vaccine strain (LVS) or FTT0831c from Schu S4 resulted in temperature-dependent defects in cell viability and morphology. Loss of FTT0831c was also associated with an unusual defect in lipopolysaccharide O-antigen synthesis, but loss of FTL_0325 was not. Full restoration of these properties was observed in complemented strains expressing FTT0831c in trans, but not in strains lacking the OmpA motif, suggesting that cell wall contact is required. Finally, growth of the LVS FTL_0325 mutant in Mueller-Hinton broth at 37°C resulted in the appearance of membrane blebs at the poles and midpoint, prior to the formation of enlarged round cells that showed evidence of compromised cellular membranes. Taken together, these data are more consistent with the known structural role of OmpA-like proteins in linking the OM to the cell wall and, as such, maintenance of structural integrity preventing altered surface exposure or release of Toll-like receptor 2 agonists during rapid growth of Francisella in vitro and in vivo. PMID:24778115

  1. Pork Meat as a Potential Source of Salmonella enterica subsp. arizonae Infection in Humans

    PubMed Central

    Kritas, Spyridon; Govaris, Alexander; Burriel, Angeliki R.

    2014-01-01

    Salmonella enterica subsp. arizonae was isolated from 13 of 123 slaughtered pigs in central Greece. The samples cultured were feces, ileum tissue, mesenteric lymph nodes, and gallbladder swabs. A total of 74 isolates from 492 samples were identified as Salmonella spp. by use of standard laboratory culture media and two commercial micromethods and by use of a polyvalent slide agglutination test for the detection of O and H antigens. Among them were 19 (25.68%) suspected to be S. enterica subsp. arizonae according to analysis with standard laboratory culture media. Of those, 14 were identified as S. enterica subsp. arizonae by the API 20E (bioMérieux, France) and the Microgen GnA+B-ID (Microgen Bioproducts, Ltd., United Kingdom) identification systems. All the isolates were tested for resistance to 23 antimicrobials. Strains identified as S. enterica subsp. arizonae were resistant to 17 (70.8%) antibiotics. The highest proportions of resistance were observed for sulfamethoxazole-trimethoprim (71.4%), tetracycline (71.4%), ampicillin (64.3%), and amoxicillin (57.1%). Two isolates were resistant to aztreonam (7.1%) and tigecycline (7.1%), used only for the treatment of humans. Thus, pork meat may play a role in the transmission of antibiotic-resistant S. enterica subsp. arizonae to human consumers. This is the first report of S. enterica subsp. arizonae isolation from pigs. PMID:24335956

  2. Detection of Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis in Maize by Loop-Mediated Amplification.

    PubMed

    Yasuhara-Bell, Jarred; de Silva, Asoka; Heuchelin, Scott A; Chaky, Jennifer L; Alvarez, Anne M

    2016-03-01

    The Goss's wilt pathogen, Clavibacter michiganensis subsp. nebraskensis, can cause considerable losses in maize (Zea mays) production. Diagnosis of Goss's wilt currently is based on symptomology and identification of C. michiganensis subsp. nebraskensis, following isolation on a semiselective medium and/or serological testing. In an effort to provide a more efficient identification method, a loop-mediated amplification (LAMP) assay was developed to detect the tripartite ATP-independent periplasmic (TRAP)-type C4-dicarboxylate transport system large permease component and tested using strains of C. michiganensis subsp. nebraskensis, all other C. michiganensis subspecies and several genera of nontarget bacteria. Only strains of C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay. The LAMP assay was then used to identify bacterial isolates from diseased maize. 16S rDNA and dnaA sequence analyses were used to confirm the identity of the maize isolates and validate assay specificity. The Cmm ImmunoStrip assay was included as a presumptive identification test of C. michiganensis subsp. nebraskensis at the species level. The Cmn-LAMP assay was further tested using symptomatic leaf tissue. The Cmn-LAMP assay was run in a hand-held real-time monitoring device (SMART-DART) and performed equally to in-lab quantitative polymerase chain reaction equipment. The Cmn-LAMP assay accurately identified C. michiganensis subsp. nebraskensis and has potential as a field test. The targeted sequence also has potential application in other molecular detection platforms. PMID:26595113

  3. Tomato fruit and seed colonization by Clavibacter michiganensis subsp. michiganensis through external and internal routes.

    PubMed

    Tancos, Matthew A; Chalupowicz, Laura; Barash, Isaac; Manulis-Sasson, Shulamit; Smart, Christine D

    2013-11-01

    The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial wilt and canker of tomato, is an economically devastating pathogen that inflicts considerable damage throughout all major tomato-producing regions. Annual outbreaks continue to occur in New York, where C. michiganensis subsp. michiganensis spreads via infected transplants, trellising stakes, tools, and/or soil. Globally, new outbreaks can be accompanied by the introduction of contaminated seed stock; however, the route of seed infection, especially the role of fruit lesions, remains undefined. In order to investigate the modes of seed infection, New York C. michiganensis subsp. michiganensis field strains were stably transformed with a gene encoding enhanced green fluorescent protein (eGFP). A constitutively eGFP-expressing virulent C. michiganensis subsp. michiganensis isolate, GCMM-22, was used to demonstrate that C. michiganensis subsp. michiganensis could not only access seeds systemically through the xylem but also externally through tomato fruit lesions, which harbored high intra- and intercellular populations. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed, once the fruits began to ripen. These results highlight the ability of C. michiganensis subsp. michiganensis to invade tomato fruits and seeds through multiple entry routes. PMID:24014525

  4. Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection.

    PubMed

    Huntley, J F; Stabel, J R; Paustian, M L; Reinhardt, T A; Bannantine, J P

    2005-10-01

    Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization. PMID:16177367

  5. Decreased Toxicity of Bacillus thuringiensis subsp. israelensis to Mosquito Larvae after Contact with Leaf Litter

    PubMed Central

    Stalinski, Renaud; Kersusan, Dylann; Veyrenc, Sylvie; David, Jean-Philippe; Reynaud, Stéphane; Després, Laurence

    2012-01-01

    Bacillus thuringiensis subsp. israelensis is a bacterium producing crystals containing Cry and Cyt proteins, which are toxic for mosquito larvae. Nothing is known about the interaction between crystal toxins and decaying leaf litter, which is a major component of several mosquito breeding sites and represents an important food source. In the present work, we investigated the behavior of B. thuringiensis subsp. israelensis toxic crystals sprayed on leaf litter. In the presence of leaf litter, a 60% decrease in the amount of Cyt toxin detectable by immunology (enzyme-linked immunosorbent assays [ELISAs]) was observed, while the respective proportions of Cry toxins were not affected. The toxicity of Cry toxins toward Aedes aegypti larvae was not affected by leaf litter, while the synergistic effect of Cyt toxins on all B. thuringiensis subsp. israelensis Cry toxins was decreased by about 20% when mixed with leaf litter. The toxicity of two commercial B. thuringiensis subsp. israelensis strains (VectoBac WG and VectoBac 12AS) and a laboratory-produced B. thuringiensis subsp. israelensis strain decreased by about 70% when mixed with leaf litter. Taken together, these results suggest that Cyt toxins interact with leaf litter, resulting in a decreased toxicity of B. thuringiensis subsp. israelensis in litter-rich environments and thereby dramatically reducing the efficiency of mosquitocidal treatments. PMID:22610426

  6. Genetic analysis reveals candidate species in the Scinax catharinae clade (Amphibia: Anura) from Central Brazil

    PubMed Central

    Nogueira, Lídia; Solé, Mirco; Siqueira, Sérgio; Affonso, Paulo Roberto Antunes de Mello; Strüssmann, Christine; Sampaio, Iracilda

    2016-01-01

    Abstract Scinax (Anura: Hylidae) is a species-rich genus of amphibians (113 spp.), divided into five species groups by morphological features. Cladistic analyses however revealed only two monophyletic clades in these groups: Scinax catharinae and Scinax ruber. Most species from the S. catharinae clade are found in Atlantic rainforest, except for Scinax canastrensis,S. centralis, S. luizotavioi, S. machadoi,S. pombali and S. skaios. In the present work, specimens of Scinax collected in Chapada dos Guimarães, central Brazil, were morphologically compatible with species from theS. catharinae group. On the other hand, genetic analysis based on mitochondrial (16S and 12S) and nuclear (rhodopsin) sequences revealed a nucleotide divergence of 6 to 20% between Scinax sp. and other congeners from the Brazilian savannah (Cerrado). Accordingly, Bayesian inference placed Scinax sp. in the S. catharinae clade with high support values. Hence, these findings strongly indicate the presence of a new species in the S. catharinae clade from the southwestern portion of the Brazilian savannah. To be properly validated as a novel species, detailed comparative morphological and bioacustic studies with other taxa from Brazil such asS. canastrensis, S. centralis, S. luizotavioi, S. machadoi, S. pombali and S. skaios are required. PMID:27007898

  7. Novel Clade of Alphaproteobacterial Endosymbionts Associated with Stinkbugs and Other Arthropods

    PubMed Central

    Kikuchi, Yoshitomo; Meng, Xian Ying; Koga, Ryuichi; Fukatsu, Takema

    2012-01-01

    Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus Nysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont Schneideria nysicola within a pair of bacteriomes in the abdomen. Our survey of Nysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of Wolbachia. Diagnostic PCR survey of 343 individuals representing 24 populations of four Nysius species revealed overall detection rates of the alphaproteobacteria at 77.6% in Nysius plebeius, 87.7% in Nysius sp. 1, 81.0% in Nysius sp. 2, and 100% in Nysius expressus. Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the order Rickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, and Midichloria spp., the mitochondrion-associated endosymbionts of ticks. In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of N. plebeius, including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation “Candidatus Lariskella arthropodarum” is proposed for the endosymbiont clade. PMID:22504806

  8. A novel sister clade to the enterobacteria microviruses (family Microviridae) identified in methane seep sediments.

    PubMed

    Bryson, Samuel Joseph; Thurber, Andrew R; Correa, Adrienne M S; Orphan, Victoria J; Vega Thurber, Rebecca

    2015-10-01

    Methane seep microbial communities perform a key ecosystem service by consuming the greenhouse gas methane prior to its release into the hydrosphere, minimizing the impact of marine methane sources on our climate. Although previous studies have examined the ecology and biochemistry of these communities, none has examined viral assemblages associated with these habitats. We employed virus particle purification, genome amplification, pyrosequencing and gene/genome reconstruction and annotation on two metagenomic libraries, one prepared for ssDNA and the other for all DNA, to identify the viral community in a methane seep. Similarity analysis of these libraries (raw and assembled) revealed a community dominated by phages, with a significant proportion of similarities to the Microviridae family of ssDNA phages. We define these viruses as the Eel River Basin Microviridae (ERBM). Assembly and comparison of 21 ERBM closed circular genomes identified five as members of a novel sister clade to the Microvirus genus of Enterobacteria phages. Comparisons among other metagenomes and these Microviridae major-capsid sequences indicated that this clade of phages is currently unique to the Eel River Basin sediments. Given this ERBM clade's relationship to the Microviridae genus Microvirus, we define this sister clade as the candidate genus Pequeñovirus. PMID:25640518

  9. Expanding our view of genomic diversity in Candidatus Accumulibacter clades.

    PubMed

    Skennerton, Connor T; Barr, Jeremy J; Slater, Frances R; Bond, Philip L; Tyson, Gene W

    2015-05-01

    Enhanced biological phosphorus removal (EBPR) is an important industrial wastewater treatment process mediated by polyphosphate-accumulating organisms (PAOs). Members of the genus Candidatus Accumulibacter are one of the most extensively studied PAO as they are commonly enriched in lab-scale EBPR reactors. Members of different Accumulibacter clades are often enriched through changes in reactor process conditions; however, the two currently sequenced Accumulibacter genomes show extensive metabolic similarity. Here, we expand our understanding of Accumulibacter genomic diversity through recovery of eight population genomes using deep metagenomics, including seven from phylogenetic clades with no previously sequenced representative. Comparative genomic analysis revealed a core of shared genes involved primarily in carbon and phosphorus metabolism; however, each Accumulibacter genome also encoded a substantial number of unique genes (> 700 genes). A major difference between the Accumulibacter clades was the type of nitrate reductase encoded and the capacity to perform subsequent steps in denitrification. The Accumulibacter clade IIF genomes also contained acetaldehyde dehydrogenase that may allow ethanol to be used as carbon source. These differences in metabolism between Accumulibacter genomes provide a molecular basis for niche differentiation observed in lab-scale reactors and may offer new opportunities for process optimization. PMID:25088527

  10. Contrarian clade confirms the ubiquity of spatial origination patterns in the production of latitudinal diversity gradients

    PubMed Central

    Krug, Andrew Z.; Jablonski, David; Valentine, James W.

    2007-01-01

    The latitudinal diversity gradient (LDG), wherein the number of species and higher taxa peaks in the tropics and decreases toward the poles, is the best-documented large-scale diversity pattern on Earth, but hypotheses explaining the standard LDG must also account for rare “contrarian” taxa that show diversity maxima outside of the tropics. For marine bivalves, one of the few groups that provide spatially explicit temporal data on a global scale, we show that a major contrarian group, the Anomalodesmata, unexpectedly exhibits the same large-scale dynamics as related clades having normal LDGs in two key respects. First, maxima in standing genus diversity and genus origination rates coincide spatially. Second, the strength of a clade's present-day LDG is significantly related to the proportion of its living genera that originated in the tropics during the late Cenozoic, with the contrarian gradient strength at both species and genus level predicted quantitatively by the values for the other clades. Geologic age distributions indicate that the anomalous LDG results from origination that is damped in the tropics rather than heightened in the temperate zones. The pervasive role of spatial origination patterns in shaping LDGs, regardless of the position of their diversity maxima, corroborates hypotheses based on clades showing standard gradients and underscores the insights that contrarian groups can provide into general principles of diversity dynamics. PMID:17989214

  11. Testing Evolutionary Hypotheses in the Classroom with MacClade Software.

    ERIC Educational Resources Information Center

    Codella, Sylvio G.

    2002-01-01

    Introduces MacClade which is a Macintosh-based software package that uses the techniques of cladistic analysis to explore evolutionary patterns. Describes a novel and effective exercise that allows undergraduate biology majors to test a hypothesis about behavioral evolution in insects. (Contains 13 references.) (Author/YDS)

  12. Full-Genome Sequence of a Novel Varicella-Zoster Virus Clade Isolated in Mexico

    PubMed Central

    Rodríguez-Castillo, Araceli; Ortiz-Alcántara, Joanna María; Gonzalez-Durán, Elizabeth; Segura-Candelas, José Miguel; Pérez-Agüeros, Sandra Ivette; Escobar-Escamilla, Noé; Méndez-Tenorio, Alfonso; Diaz-Quiñonez, José Alberto

    2015-01-01

    Varicella-zoster virus (VZV) is a member of the Herpesviridae family, which causes varicella (chicken pox) and herpes zoster (shingles) in humans. Here, we report the complete genome sequence of varicella-zoster virus, isolated from a vesicular fluid sample, revealing the circulation of VZV clade VIII in Mexico. PMID:26159533

  13. Genetic analysis reveals candidate species in the Scinax catharinae clade (Amphibia: Anura) from Central Brazil.

    PubMed

    Nogueira, Lídia; Solé, Mirco; Siqueira, Sérgio; Affonso, Paulo Roberto Antunes de Mello; Strüssmann, Christine; Sampaio, Iracilda

    2016-03-01

    Scinax (Anura: Hylidae) is a species-rich genus of amphibians (113 spp.), divided into five species groups by morphological features. Cladistic analyses however revealed only two monophyletic clades in these groups: Scinax catharinae and Scinax ruber. Most species from the S. catharinae clade are found in Atlantic rainforest, except for Scinax canastrensis,S. centralis, S. luizotavioi, S. machadoi,S. pombali and S. skaios. In the present work, specimens of Scinax collected in Chapada dos Guimarães, central Brazil, were morphologically compatible with species from theS. catharinae group. On the other hand, genetic analysis based on mitochondrial (16S and 12S) and nuclear (rhodopsin) sequences revealed a nucleotide divergence of 6 to 20% between Scinax sp. and other congeners from the Brazilian savannah (Cerrado). Accordingly, Bayesian inference placed Scinax sp. in the S. catharinae clade with high support values. Hence, these findings strongly indicate the presence of a new species in the S. catharinae clade from the southwestern portion of the Brazilian savannah. To be properly validated as a novel species, detailed comparative morphological and bioacustic studies with other taxa from Brazil such asS. canastrensis, S. centralis, S. luizotavioi, S. machadoi, S. pombali and S. skaios are required. PMID:27007898

  14. Characterization of Virulence-Related Phenotypes in Candida Species of the CUG Clade.

    PubMed

    Priest, Shelby J; Lorenz, Michael C

    2015-09-01

    Candida species cause a variety of mucosal and invasive infections and are, collectively, the most important human fungal pathogens in the developed world. The majority of these infections result from a few related species within the "CUG clade," so named because they use a nonstandard translation for that codon. Some members of the CUG clade, such as Candida albicans, present significant clinical problems, whereas others, such as Candida (Meyerozyma) guilliermondii, are uncommon in patients. The differences in incidence rates are imperfectly correlated with virulence in animal models of infection, but comparative analyses that might provide an explanation for why some species are effective pathogens and others are not have been rare or incomplete. To better understand the phenotypic basis for these differences, we characterized eight CUG clade species--C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, Clavispora lusitaniae, M. guilliermondii, Debaryomyces hansenii, and Lodderomyces elongisporus--for host-relevant phenotypes, including nutrient utilization, stress tolerance, morphogenesis, interactions with phagocytes, and biofilm formation. Two species deviated from expectations based on animal studies and human incidence. C. dubliniensis was quite robust, grouping in nearly all assays with the most virulent species, C. albicans and C. tropicalis, whereas C. parapsilosis was substantially less fit than might be expected from its clinical importance. These findings confirm the utility of in vitro measures of virulence and provide insight into the evolution of virulence in the CUG clade. PMID:26150417

  15. Multilocus Sequence Analysis of the redefined clade Scophthalmi in the genus Vibrio.

    PubMed

    Tarazona, Eva; Pérez-Cataluña, Alba; Lucena, Teresa; Arahal, David R; Macián, M Carmen; Pujalte, María J

    2015-05-01

    A Multilocus Sequence Analysis (MLSA) was performed on members of the Scophthalmi clade in the genus Vibrio, including type and reference strains of the species V. scophthalmi, V. ichthyoenteri, and 39 strains phenotypically identified as Vibrio ichthyoenteri-like, with the aim of better defining boundaries between these two closely related, fish-associated species. The type strain of V. ponticus, recently added to the clade Scophthalmi, was also included. The study was based on partial sequences of the protein-coding housekeeping genes rpoD, mreB, recA, ftsZ, and gyrB, and the 16S rRNA. While the 16S rRNA gene-based trees were unable to pull apart members of V. scophthalmi or V. ichthyoenteri, both the other individual gene trees and the trees obtained from the five-genes concatenated sequences were able to consistently differentiate four subclades within the main clade, corresponding to the bona fide V. scophthalmi, V. ichthyoenteri, and two small ones that may represent a new species each. The best genes to differentiate V. scophthalmi from V. ichthyoenteri were rpoD, recA, and mreB. Vibrio ponticus failed to associate to the clade in the MLSA and in most single gene trees for which it should not be considered part of it. In this study we also confirm using genomic indexes that V. ichthyoenteri and V. scophthalmi are two separate species. PMID:25861826

  16. Full-Genome Sequence of a Novel Varicella-Zoster Virus Clade Isolated in Mexico.

    PubMed

    Garcés-Ayala, Fabiola; Rodríguez-Castillo, Araceli; Ortiz-Alcántara, Joanna María; Gonzalez-Durán, Elizabeth; Segura-Candelas, José Miguel; Pérez-Agüeros, Sandra Ivette; Escobar-Escamilla, Noé; Méndez-Tenorio, Alfonso; Diaz-Quiñonez, José Alberto; Ramirez-González, José Ernesto

    2015-01-01

    Varicella-zoster virus (VZV) is a member of the Herpesviridae family, which causes varicella (chicken pox) and herpes zoster (shingles) in humans. Here, we report the complete genome sequence of varicella-zoster virus, isolated from a vesicular fluid sample, revealing the circulation of VZV clade VIII in Mexico. PMID:26159533

  17. Characterization of Virulence-Related Phenotypes in Candida Species of the CUG Clade

    PubMed Central

    Priest, Shelby J.

    2015-01-01

    Candida species cause a variety of mucosal and invasive infections and are, collectively, the most important human fungal pathogens in the developed world. The majority of these infections result from a few related species within the “CUG clade,” so named because they use a nonstandard translation for that codon. Some members of the CUG clade, such as Candida albicans, present significant clinical problems, whereas others, such as Candida (Meyerozyma) guilliermondii, are uncommon in patients. The differences in incidence rates are imperfectly correlated with virulence in animal models of infection, but comparative analyses that might provide an explanation for why some species are effective pathogens and others are not have been rare or incomplete. To better understand the phenotypic basis for these differences, we characterized eight CUG clade species—C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, Clavispora lusitaniae, M. guilliermondii, Debaryomyces hansenii, and Lodderomyces elongisporus—for host-relevant phenotypes, including nutrient utilization, stress tolerance, morphogenesis, interactions with phagocytes, and biofilm formation. Two species deviated from expectations based on animal studies and human incidence. C. dubliniensis was quite robust, grouping in nearly all assays with the most virulent species, C. albicans and C. tropicalis, whereas C. parapsilosis was substantially less fit than might be expected from its clinical importance. These findings confirm the utility of in vitro measures of virulence and provide insight into the evolution of virulence in the CUG clade. PMID:26150417

  18. Production of sophorolipids biosurfactants by multiple species of the Starmerella (Candida) bombicola yeast clade

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sophorolipid production was tested for 26 strains representing 19 species of the Starmerella yeast clade, including S. bombicola and Candida apicola, which were previously reported to produce sophorolipids. Five of the 19 species tested showed significant production of sophorolipids: S. bombicola, ...

  19. Developing Clade-Specific Microsatellite Markers: A Case Study in the Filamentous Fungal Genus Aspergillus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite markers are highly variable and very commonly used in population genetics studies. However, microsatellite loci are typically poorly conserved and cannot be used in distant related species. Thus, development of clade-specific microsatellite markers would increase efficiency and allow ...

  20. Poly (beta-L-malic acid) production by diverse phylogenetic clades of Aureobasidium pullulans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Poly (beta-L-malic acid) (PMA) is a natural biopolyester that has pharmaceutical applications and other potential uses. Here we examine PMA production by genetically diverse phylogenetic clades of the fungus A. pullulans. Thirty-six strains of A. pullulans were isolated for this study from various...

  1. Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

    DOEpatents

    Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

    2015-04-21

    The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  2. Glucosylation and other biotransformations of T-2 toxin by yeasts of the Trichomonascus clade

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty-five yeasts assigned to the Trichomonascus clade (Saccharomycotina, Ascomycota), including three Trichomonascus species and 22 anamorphic species presently classified in Blastobotrys, were tested for their ability to convert T-2 toxin, a Fusarium trichothecene mycotoxin, to less toxic product...

  3. A divergent clade of circular single-stranded DNA viruses from pig feces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using metagenomics and molecular cloning methods, we characterized five novel small circular viral genomes from pig feces distantly related to chimpanzee and porcine stool-associated circular viruses, (ChiSCV and PoSCV1). Phylogenetic analysis placed these viruses into a new, highly divergent, clade...

  4. Early Tertiary mammals from North Africa reinforce the molecular Afrotheria clade

    PubMed Central

    Tabuce, Rodolphe; Marivaux, Laurent; Adaci, Mohammed; Bensalah, Mustapha; Hartenberger, Jean-Louis; Mahboubi, Mohammed; Mebrouk, Fateh; Tafforeau, Paul; Jaeger, Jean-Jacques

    2007-01-01

    The phylogenetic pattern and timing of the radiation of mammals, especially the geographical origins of major crown clades, are areas of controversy among molecular biologists, morphologists and palaeontologists. Molecular phylogeneticists have identified an Afrotheria clade, which includes several taxa as different as tenrecs (Tenrecidae), golden moles (Chrysochloridae), elephant-shrews (Macroscelididae), aardvarks (Tubulidentata) and paenungulates (elephants, sea cows and hyracoids). Molecular data also suggest a Cretaceous African origin for Afrotheria within Placentalia followed by a long period of endemic evolution on the Afro-Arabian continent after the mid-Cretaceous Gondwanan breakup (approx. 105–25 Myr ago). However, there was no morphological support for such a natural grouping so far. Here, we report new dental and postcranial evidence of Eocene stem hyrax and macroscelidid from North Africa that, for the first time, provides a congruent phylogenetic view with the molecular Afrotheria clade. These new fossils imply, however, substantial changes regarding the historical biogeography of afrotheres. Their long period of isolation in Africa, as assumed by molecular inferences, is now to be reconsidered inasmuch as Eocene paenungulates and elephant-shrews are here found to be related to some Early Tertiary Euramerican ‘hyopsodontid condylarths’ (archaic hoofed mammals). As a result, stem members of afrotherian clades are not strictly African but also include some Early Paleogene Holarctic mammals. PMID:17329227

  5. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD[superscript +] and triclosan

    SciTech Connect

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2010-11-19

    Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD{sup +} has been solved to a resolution of 2.1 {angstrom}. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD{sup +} reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD{sup +} cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors.

  6. Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensis Live Vaccine Strain (LVS) Supports a General Host Suppression and Bacterial Uptake by Macropinocytosis*

    PubMed Central

    Bradburne, Christopher E.; Verhoeven, Anne B.; Manyam, Ganiraju C.; Chaudhry, Saira A.; Chang, Eddie L.; Thach, Dzung C.; Bailey, Charles L.; van Hoek, Monique L.

    2013-01-01

    Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2–6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell. PMID:23322778