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.

Francisella tularensis is a gram-negative intracellular bacterium and the causative agent of the zoonotic disease tularemia. F. tularensis is a category A select agent and thus a potential agent of bioterrorism. Whereas an F. tularensis live, attenuated vaccine strain (LVS) is the basis of an investigational vaccine, this vaccine is not licensed for human use because of efficacy and safety concerns. In the present study, we immunized mice with isolated native outer membrane proteins (OMPs), ethanol-inactivated LVS (iLVS), or purified LVS lipopolysaccharide (LPS) and assessed the ability of each vaccine preparation to protect mice against pulmonary challenge with the virulent type A F. tularensis strain SchuS4. Antibody isotyping indicated that both Th1 and Th2 antibody responses were generated in mice after immunization with OMPs or iLVS, whereas LPS immunization resulted in only immunoglobulin A production. In survival studies, OMP immunization provided the greatest level of protection (50% survival at 20 days after infection with SchuS4), and there were associated 3-log reductions in the spleen and liver bacterial burdens (compared to nonvaccinated mice). Cytokine quantitation for the sera of SchuS4-challenged mice indicated that OMP and iLVS immunizations induced high levels of tumor necrosis factor alpha and interleukin-2 (IL-2) production, whereas only OMP immunization induced high levels of IL-10 production. By comparison, high levels of proinflammatory cytokines, including RANTES, granulocyte colony-stimulating factor, IL-6, IL-1␣, IL-12p40, and KC, in nonvaccinated mice indicated that these cytokines may facilitate disease progression. Taken together, the results of this study demonstrate the potential utility of an OMP subunit (acellular) vaccine for protecting mammals against type A F. tularensis.

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Syphilis, a sexually transmitted infection caused by the spirochetal bacterium Treponema pallidum, remains a global public health problem. T. pallidum is believed to be an extracellular pathogen and, as such, the identification of T. pallidum outer membrane proteins that could serve as targets for opsonic or bactericidal antibodies has remained a high research priority for vaccine development. However, the identification of T. pallidum outer membrane proteins has remained highly elusive. Recent studies and bioinformatics have implicated four treponemal proteins as potential outer membrane proteins (TP0155, TP0326, TP0483 and TP0956). Indirect immunofluorescence assays performed on treponemes encapsulated within agarose gel microdroplets failed to provide evidence that any of these four molecules were surface-exposed in T. pallidum. Second, recombinant fusion proteins corresponding to all four candidate outer membrane proteins were used separately, or in combination, to vaccinate New Zealand White rabbits. Despite achieving high titers (>1:50,000) of serum antibodies, none of the rabbits displayed chancre immunity after intradermal challenge with viable T. pallidum.