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.

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SUMMARYIn various human intracellular bacterial diseases, an increase of the proportion of circulating Vg9Vd 2 T cells has been observed. The prevalence of the finding among infected subjects and the time course of the elevation remain to be investigated. In the present study, comprising blood samples from a large number of cases of ulceroglandular tularaemia, the percentage of Vg9Vd 2 T cells within the first week of onset of disease (5´3^0´7% (mean^s.e.m.)) did not differ from that of control subjects (5´3^0´8%). Thereafter, percentages increased rapidly and within the interval of 8±40 days mean levels were . 20% (P , 0´001). Of 45 individuals sampled within 3 months of onset, 42 showed a percentage of Vg9Vd 2 T cells of . 10%. Significantly increased levels were still recorded at 18 months (13´8^2´4%; P , 0´05) but not at 24 months (10´2^2´1%; P . 0´10). Thus, a consistent increase of circulating Vg9Vd 2 T cells was demonstrated in tularaemia. The initial delay and the prolonged course of elevation may suggest a role in immunoregulation and/or immunological memory. Furthermore, the percentage of gd T cells expressing tumour necrosis factor-alpha in response to phorbol myristate acetate was decreased during the first week and up to 40 days after onset, possibly reflecting the modulation of an inflammatory response.

Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis. Here we demonstrate that during the first weeks of infection, a significant increase in levels of Vγ9Vδ2 cells occurred in peripheral blood: in 13 patients analyzed 7 to 18 days after the onset of disease, these lymphocytes represented, on average, 30.5% of CD3+ cells and nearly 100% of γδ+ T cells. By contrast, after vaccination with the live vaccine strain (LVS) of F. tularensis, only a minor increase occurred. Eleven days after vaccination, γδ T cells represented an average of 6.7% and Vγ9Vδ2 cells represented an average of 5.3% of T cells, as in control subjects. Since derivatives of nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 cells, this observation prompted an investigation of phosphoantigens in F. tularensis strains. The F. tularensis phosphoantigens triggered in vitro a proliferative response of human Vγ9Vδ2 peripheral blood leukocytes as well as a cytotoxic response and tumor necrosis factor release from a Vγ9Vδ2 T-cell clone. Quantitatively similar phosphoantigenic activity was detected in acellular extracts from two clinical isolates (FSC171 and Schu) and from LVS. Taken together, the chemical nature of the stimulus from the clinical isolates and the significant increase in levels of Vγ9Vδ2 cells in peripheral blood of tularemia patients indicate that phosphoantigens produced by virulent strains of F. tularensis trigger in vivo expansion of γδ T cells in tularemia.