Towards proteome database of Francisella tularensis

Hubálek, Martin; Hernychová, Lenka; Havlasová, J; Kasalová, Irena; Neubauerová, Vĕra; Stulı́k, Jiřı́; Macela, Aleš; Lundqvist, Margaretha; Larsson, Pär

doi:10.1016/s1570-0232(02)00730-4

Cited by 31 publications

(29 citation statements)

References 88 publications

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“…2 and 3). While our optimized method was rigorous and subject to some experimental variability (see Results), our results appear to have much greater utility than those garnered from the use of detergents, lithium chloride, or sodium carbonate (2,20,33,41,46,51). We identified 15 bona fide F. tularensis OMPs, which colocalized at densities between 1.17 and 1.20 g/ml.…”

Section: Discussionmentioning

confidence: 82%

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Characterization ofFrancisella tularensisOuter Membrane Proteins

et al. 2007

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Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.

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Section: Discussionmentioning

confidence: 82%

“…More than 1,500 proteins were visualized when total cellular proteins were solubilized from F. tularensis and separated by 2DE (20). However, only three of those identified by mass spectrometry were proposed to be membrane proteins, including TUL4.…”

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confidence: 99%

Characterization ofFrancisella tularensisOuter Membrane Proteins

et al. 2007

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“…Similarly, a Bfr protein spot unique to F. tularensis subsp. holarctica was recently reported in a comparative proteome analysis of the bacterial cell lysates from F. tularensis subspecies holarctica and tularensis (17). Analysis of the cytosol of F. tularensis-infected macrophages demonstrated that KatG and GroEL were released into the host cell cytosol.…”

Section: Discussionmentioning

confidence: 83%

Identification, Recombinant Expression, Immunolocalization in Macrophages, and T-Cell Responsiveness of the Major Extracellular Proteins of Francisella tularensis

2006

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A safer and more effective vaccine than the previously developed live attenuated vaccine is needed for combating Francisella tularensis, a highly infectious bacterial pathogen. To search for potential candidates for inclusion in a new vaccine, we characterized the proteins present in the culture filtrates of a virulent recent clinical isolate and the attenuated live vaccine strain of F. tularensis using a proteomic approach. We identified a total of 12 proteins; among these, catalase-peroxidase was much more abundant in the culture filtrate of the virulent clinical isolate, whereas bacterioferritin was more abundant in the culture filtrate of the live vaccine strain. Streptolysin O treatment of infected human macrophages indicated that catalase-peroxidase and the heat shock protein GroEL are released intracellularly by actively growing F. tularensis. Mice immunized with F. tularensis developed significant cellmediated immune responses to catalase-peroxidase, the heat shock protein GroEL, and bacterioferritin as measured by splenic lymphocyte proliferation and gamma interferon production. Finally, we expressed the major culture filtrate proteins that are promising vaccine candidates in Escherichia coli at high levels in soluble form to facilitate study of their immunobiology and potential role in vaccines.

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“…6). Previous Francisella proteomics studies have catalogued LVS protein profiles [29,30], immune reactive proteins [31], or stress response proteins [32]. A proteomic comparison of three Francisella strains has been performed, identifying many protein differences [33].…”

Section: Discussionmentioning

confidence: 99%

Modulation of virulence factors in Francisella tularensis determines human macrophage responses

Carlson

Carroll

O’Dee

et al. 2007

Microbial Pathogenesis

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Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis Live Vaccine Strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially-expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a σ 54 modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically-defined media. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses.

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Towards proteome database of Francisella tularensis

Cited by 31 publications

References 88 publications

Characterization ofFrancisella tularensisOuter Membrane Proteins

Characterization ofFrancisella tularensisOuter Membrane Proteins

Identification, Recombinant Expression, Immunolocalization in Macrophages, and T-Cell Responsiveness of the Major Extracellular Proteins of Francisella tularensis

Modulation of virulence factors in Francisella tularensis determines human macrophage responses

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