Genomic comparison between a virulent type A1 strain of Francisella tularensis and its attenuated O-antigen mutant.
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ABSTRACT:
SUBMITTER: Modise T
PROVIDER: S-EPMC3347185 | biostudies-literature | 2012 May
REPOSITORIES: biostudies-literature
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Genomic comparison between a virulent type A1 strain of Francisella tularensis and its attenuated O-antigen mutant.
Modise Thero T Ryder Cheryl C Mane Shrinivasrao P SP Bandara Aloka B AB Jensen Roderick V RV Inzana Thomas J TJ
Journal of bacteriology 20120501 10
We report the complete genome sequences of TI0902, a highly virulent type A1 strain, and TIGB03, a related, attenuated chemical mutant strain. Compared to the wild type, the mutant strain had 45 point mutations and a 75.9-kb duplicated region that had not been previously observed in Francisella species. ...[more]
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Project description:Francisella tularensis, the causative agent of tularemia, has been designated a CDC category A select agent because of its low infective dose (<10 CFU), its ready transmission by aerosol, and its ability to produce severe morbidity and high mortality. The identification and characterization of this organism's virulence determinants will facilitate the development of a safe and effective vaccine. We report that inactivation of the wbtA-encoded dehydratase of the O-antigen polysaccharide (O-PS) locus of the still-unlicensed live vaccine strain of F. tularensis (LVS) results in a mutant (the LVS wbtA mutant) with remarkably attenuated virulence. Western blot analysis and immune electron microscopy studies associate this loss of virulence with a complete lack of surface O-PS expression. A likely mechanism for attenuation is shown to be the transformation from serum resistance in the wild-type strain to serum sensitivity in the mutant. Despite this significant attenuation in virulence, the LVS wbtA mutant remains immunogenic and confers protective immunity on mice against challenge with an otherwise lethal dose of either F. tularensis LVS or a fully virulent clinical isolate of F. tularensis type B. Recognition and characterization of the pivotal role of O-PS in the virulence of this intracellular bacterial pathogen may have broad implications for the creation of a safe and efficacious vaccine.
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Project description:Tularemia is a severe infectious zoonotic disease caused by Francisella tularensis. Although F. tularensis is considered to be a potential biological weapon due to its high infectivity and mortality rate, no vaccine has been currently licensed. Recently, we reported that F. tularensis SCHU P9 derived ΔpdpC strain lacking the pathogenicity determinant protein C gene conferred stable and good protection in a mouse lethal model. In this study, the protective effect of ΔpdpC was evaluated using a monkey lethal model. Two cynomolgus macaques (Macaca fascicularis) intratracheally challenged with the virulent strain SCHU P9 were euthanized on 7 and 11 days post-challenge after the development of severe clinical signs. The bacterial replication in alveolar macrophages and type II epithelial cells in the lungs would cause severe pneumonia accompanied by necrosis. Conversely, two animals subcutaneously immunized with ΔpdpC survived 3 weeks after SCHU P9 challenge. Though one of the two animals developed mild symptoms of tularemia, bacterial replication was limited in the respiratory organs, which may be due to a high level of humoral and cellular immune responses against F. tularensis. These results suggest that the ΔpdpC mutant would be a safe and promising candidate as a live attenuated tularemia vaccine.
| S-EPMC6591246 | biostudies-literature
Project description:Francisella tularensis, the causative agent of tularemia, is a category A bioterrorism agent. A vaccine that is safer and more effective than the currently available unlicensed F. tularensis live vaccine strain (LVS) is needed to protect against intentional release of aerosolized F. tularensis, the most dangerous type of exposure. In this study, we employed a heterologous prime-boost vaccination strategy comprising intradermally administered LVS ?capB (highly attenuated capB-deficient LVS mutant) as the primer vaccine and rLm/iglC (recombinant attenuated Listeria monocytogenes expressing the F. tularensis immunoprotective antigen IglC) as the booster vaccine. Boosting LVS ?capB-primed mice with rLm/iglC significantly enhanced T cell immunity; their splenic T cells secreted significantly more gamma interferon (IFN-?) and had significantly more cytokine (IFN-? and/or tumor necrosis factor [TNF] and/or interleukin-2 [IL-2])-producing CD4(+) and CD8(+) T cells upon in vitro IglC stimulation. Importantly, mice primed with LVS ?capB or rLVS ?capB/IglC, boosted with rLm/iglC, and subsequently challenged with 10 50% lethal doses (LD50) of aerosolized highly virulent F. tularensis Schu S4 had a significantly higher survival rate and mean survival time than mice immunized with only LVS ?capB (P < 0.0001); moreover, compared with mice immunized once with LVS, primed-boosted mice had a higher survival rate (75% versus 62.5%) and mean survival time during the first 21 days postchallenge (19 and 20 days for mice boosted after being primed with LVS ?capB and rLVS ?capB/IglC, respectively, versus 17 days for mice immunized with LVS) and maintained their weight significantly better (P < 0.01). Thus, the LVS ?capB-rLm/iglC prime-boost vaccination strategy holds substantial promise for a vaccine that is safer and at least as potent as LVS.
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Project description:Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in 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.
| S-EPMC3031720 | biostudies-literature
Project description:Francisella tularensis is a highly infectious intracellular bacterium that causes the zoonotic infection tularemia. While much literature exists on the host response to F. tularensis infection, the vast majority of work has been conducted using attenuated strains of Francisella that do not cause disease in humans. However, emerging data indicate that the protective immune response against attenuated F. tularensis versus F. tularensis type A differs. Several groups have recently reported that interleukin-17 (IL-17) confers protection against the live vaccine strain (LVS) of Francisella. While we too have found that IL-17R?(-/-) mice are more susceptible to F. tularensis LVS infection, our studies, using a virulent type A strain of F. tularensis (SchuS4), indicate that IL-17R?(-/-) mice display organ burdens and pulmonary gamma interferon (IFN-?) responses similar to those of wild-type mice following infection. In addition, oral LVS vaccination conferred equivalent protection against pulmonary challenge with SchuS4 in both IL-17R?(-/-) and wild-type mice. While IFN-? was found to be critically important for survival in a convalescent model of SchuS4 infection, IL-17 neutralization from either wild-type or IFN-?(-/-) mice had no effect on morbidity or mortality in this model. IL-17 protein levels were also higher in the lungs of mice infected with the LVS rather than F. tularensis type A, while IL-23p19 mRNA expression was found to be caspase-1 dependent in macrophages infected with LVS but not SchuS4. Collectively, these results demonstrate that IL-17 is dispensable for host immunity to type A F. tularensis infection, and that induced and protective immunity differs between attenuated and virulent strains of F. tularensis.
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Project description:Francisella tularensis is the causative agent of tularemia, which is a highly lethal disease from nature and potentially from a biological weapon. This species contains four recognized subspecies including the North American endemic F. tularensis subsp. tularensis (type A), whose genetic diversity is correlated with its geographic distribution including a major population subdivision referred to as A.I and A.II. The biological significance of the A.I - A.II genetic differentiation is unknown, though there are suggestive ecological and epidemiological correlations. In order to understand the differentiation at the genomic level, we have determined the complete sequence of an A.II strain (WY96-3418) and compared it to the genome of Schu S4 from the A.I population. We find that this A.II genome is 1,898,476 bp in size with 1,820 genes, 1,303 of which code for proteins. While extensive genomic variation exists between "WY96" and Schu S4, there is only one whole gene difference. This one gene difference is a hypothetical protein of unknown function. In contrast, there are numerous SNPs (3,367), small indels (1,015), IS element differences (7) and large chromosomal rearrangements (31), including both inversions and translocations. The rearrangement borders are frequently associated with IS elements, which would facilitate intragenomic recombination events. The pathogenicity island duplicated regions (DR1 and DR2) are essentially identical in WY96 but vary relative to Schu S4 at 60 nucleotide positions. Other potential virulence-associated genes (231) varied at 559 nucleotide positions, including 357 non-synonymous changes. Molecular clock estimates for the divergence time between A.I and A.II genomes for different chromosomal regions ranged from 866 to 2131 years before present. This paper is the first complete genomic characterization of a member of the A.II clade of Francisella tularensis subsp. tularensis.
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Project description: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.
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Project description: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.
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Project description:Francisella tularensis secretes tubular outer membrane vesicles (OMVs) that contain a number of immunoreactive proteins as well as virulence factors. We have reported previously that isolated Francisella OMVs enter macrophages, cumulate inside, and induce a strong pro-inflammatory response. In the current article, we present that OMVs treatment of macrophages also enhances phagocytosis of the bacteria and suppresses their intracellular replication. On the other hand, the subsequent infection with Francisella is able to revert to some extent the strong pro-inflammatory effect induced by OMVs in macrophages. Being derived from the bacterial surface, isolated OMVs may be considered a "non-viable mixture of Francisella antigens" and as such, they present a promising protective material. Immunization of mice with OMVs isolated from a virulent F. tularensis subsp. holarctica strain FSC200 prolonged the survival time but did not fully protect against the infection with a lethal dose of the parent strain. However, the sera of the immunized animals revealed unambiguous cytokine and antibody responses and proved to recognize a set of well-known Francisella immunoreactive proteins. For these reasons, Francisella OMVs present an interesting material for future protective studies.
| S-EPMC10963506 | biostudies-literature
Project description:Francisella tularensis is a pathogenic, aerobic gram-negative coccobacillus bacterium. It is the causative agent of tularemia, a rare infectious disease that can attack skin, lungs, eyes, and lymph nodes. The genome of F. tularensis has been sequenced, and ~16% of the proteome is still uncharacterized. Characterizations of these proteins are essential to find new drug targets for better therapeutics. In silico characterization of proteins has become an extremely important approach to determine the functionality of proteins as experimental functional elucidation is unable to keep pace with the current growth of the sequence database. Initially, we have annotated 577 Hypothetical Proteins (HPs) of F. tularensis strain SCHU4 with seven bioinformatics tools which characterized them based on the family, domain and motif. Out of 577 HPs, 119 HPs were annotated by five or more tools and are further screened to predict their virulence properties, subcellular localization, transmembrane helices as well as physicochemical parameters. VirulentPred predicted 66 HPs out of 119 as virulent. These virulent proteins were annotated to find the interacting partner using STRING, and proteins with high confidence interaction scores were used to predict their 3D structures using Phyre2. The three virulent proteins Q5NH99 (phosphoserine phosphatase), Q5NG42 (Cystathionine beta-synthase) and Q5NG83 (Rrf2-type helix turn helix domain) were predicted to involve in modulation of cytoskeletal and innate immunity of host, H2S (hydrogen sulfide) based antibiotic tolerance and nitrite and iron metabolism of bacteria. The above predicted virulent proteins can serve as novel drug targets in the era of antibiotic resistance.
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