RESUMO
Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.
Assuntos
Proteínas de Bactérias , Francisella tularensis , Proteínas de Membrana , Isomerases de Dissulfetos de Proteínas , Proteoma/análise , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Cromatografia Líquida/métodos , Francisella tularensis/genética , Francisella tularensis/metabolismo , Francisella tularensis/patogenicidade , Humanos , Focalização Isoelétrica , Macrófagos/citologia , Macrófagos/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteômica/métodos , Taxa de Sobrevida , Tularemia/metabolismo , Tularemia/mortalidade , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Immunity to Francisella tularensis is largely mediated by T lymphocytes but an important role of B lymphocytes in early stage of infection was previously uncovered. We wanted to find out if F. tularensis is able to infect B cells and/or influence them by direct contact. To investigate this possibility we infected B cell lines from mouse (A20) or humans (Ramos RA-1), or primary mouse spleen cells, with F. tularensis LVS and F. tularensis FSC200 in vitro. In all cases, we detected bacteria on the cell surface and inside the B cells using transmission electron microscopy. More than 20% cells were infected by microbes after 24h. The number of bacteria, determined by CFU, increased about 1 log during 24h. Infection with live bacteria led to apoptosis of Ramos cells and mouse CD19(+) spleen cells. Approximately 30% of cells were apoptotic after 24h and 70% after 48 h, independently of the F. tularensis strain, while only 10% of non-infected cell were apoptotic at either time point. Apoptosis was confirmed by Western blot using anti-PARP antibodies. Thus, this study demonstrates unique phenomenon - namely, the ability of the intracellular pathogen F. tularensis to invade and induce apoptosis in B cells.