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.

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We have started the construction of a two-dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient in the first dimension and 9-16% gradient or tricine SDS-PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Accelerated proliferation of the tick-borne bacterial pathogen Francisella tularensis was demonstrated in mice when the bacterium was injected together with salivary gland extract from Ixodes ricinus ticks. A significant increase in the numbers of bacteria was recorded in the dermal site of infection,the draining lymph nodes, and the spleen. Analysis of the expression of cytokine messenger ribonucleic acids showed polarization toward a Th2 profile. Salivary gland extract-mediated suppression of interleukin-12 and interferon-gamma, the cytokines required for the expression of the protective immunity against tularemic infection, apparently contributed to the decreased resistance against this tick-transmitted pathogen.

In humans, expansion of circulating V␥9V␦2 T cells seems to be a pathophysiological denominator shared by protozoan and intracellular bacterial diseases. The assumption was tested here on legionellosis, a condition conforming to the category but not yet described with respect to ␥␦ T cells. Levels of V␥9V␦2 T cells in peripheral blood were measured at various intervals in 14 subjects undergoing a Pontiac fever-like disease, shown by serological investigation to be caused by Legionella micdadei. In samples obtained 4 to 6 days after the onset of the disease, the mean percentage (؎ the standard deviation) of V␥9V␦2 ؉ T cells among CD3 ؉ cells was 1.0% ؎ 0.5%, compared to 5.0% ؎ 3.9% in healthy control subjects (P < 0.001). Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset, mean peak levels were as high as Ϸ15%. During the next 6 months, values slowly declined, although without reaching the normal range. Percentages of ␥␦ ؉ T cells expressing tumor necrosis factor alpha or gamma interferon in response to phorbol myristate acetate were assayed in vitro. At 14 to 16 days after the onset of disease, the expression of both cytokines was increased (P < 0.01), whereas at 5 to 7 weeks, the expression of tumor necrosis factor alpha was decreased (P < 0.05), possibly reflecting modulation of an inflammatory response. In conclusion, Pontiac fever was found to be associated with a pronounced and long-lasting expansion of V␥9V␦2 T cells, implying that the subset may also be pathophysiologically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, the expansion was preceded by a depletion of circulatory V␥9V␦2 T cells. Possibly, V␥9V␦2 T cells are initially recruited to a site of infection before they expand in response to antigen and occur in high numbers in blood.