The 58-kilodalton major virulence factor of Francisella tularensis is required for efficient utilization of iron

Abstract

We investigated the role of the 58-kDa FTT0918 protein in the iron metabolism of Francisella tularensis. The phenotypes of SCHU S4, a prototypic strain of F. tularensis subsp. tularensis, and the Delta FTT0918 and Delta fslA isogenic mutants were analyzed. The gene product missing in the Delta fslA mutant is responsible for synthesis of a siderophore. When grown in broth with various iron concentrations, the two deletion mutants generally reached lower maximal densities than SCHU S4. The Delta FTT0918 mutant, but not the Delta fslA mutant, upregulated the genes of the F. tularensis siderophore locus (fsl) operon even at high iron concentrations. A chrome azurol sulfonate plate assay confirmed siderophore production by all strains except the Delta fslA strain. In a cross-feeding experiment using medium devoid of free iron, SCHU S4 promoted growth of the Delta fslA strain but not of the Delta FTT0918 strain. The sensitivity of SCHU S4 and the Delta FTT0918 and Delta fslA strains to streptonigrin demonstrated that the Delta FTT0918 strain contained a smaller free intracellular iron pool and that the Delta fslA strain contained a larger one than SCHU S4. In contrast to the marked attenuation of the Delta FTT0918 strain, the Delta fslA strain was as virulent as SCHU S4 in a mouse model. Altogether, the data demonstrate that the FTT0918 protein is required for F. tularensis to utilize iron bound to siderophores and that it likely has a role also in siderophore-independent iron acquisition. We suggest that the FTT0918 protein be designated Fe utilization protein A, FupA.

FIG. 1.

The fsl operon of Francisella tularensis SCHU S4.

FIG. 2.

Western blot. Bacterial lysates in Laemmli sample buffer were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. Each lane was loaded with lysate corresponding to 3 × 10⁷ bacteria of the indicated strain, and the filter was probed with a polyclonal antiserum directed against the FTT0918 protein.

FIG. 3.

Growth of F. tularensis strains in C-CDM supplemented with 0.2 (filled symbols) or 2.0 (open symbols) μg/ml FeSO₄. The strains were grown to logarithmic growth phase in CDM (A) or overnight in C-CDM (B) prior to subculturing in C-CDM. SCHU S4 (squares), the ΔfslA strain (triangles), the ΔFTT0918 strain (circles), and FUU075 (diamonds) were grown. Similar growth curves were obtained in at least two additional experiments.

FIG. 4.

Maximal density of SCHU S4 (squares) and the ΔfslA (triangles) and ΔFTT0918 (circles) strains in C-CDM supplemented with the indicated concentrations of FeSO₄. Values represent the averages of three observations obtained on separate days, and the error bars are the standard errors of means.

FIG. 5.

Regulation of the fsl operon by SCHU S4 (black bars), the ΔFTT0918 strain (light gray bars), FUU075 (white bars), and the ΔfslA strain (dark gray bars) after 10 h of growth in C-CDM with 2.0 or 0.2 μg/ml FeSO₄. Values are expressed as ratios relative to the expression level of SCHU S4 grown in C-CDM with 2.0 μg/ml of FeSO₄. The bars represent the averages of six (FUU075 and the ΔfslA strain) or nine (SCHU S4 and the ΔFTT0918 strain) observations obtained from two or three samples from experiments performed on separate occasions. The error bars indicate standard errors of means.

FIG. 6.

Siderophore production by the F. tularensis strains as determined by the CAS assay (A) and a cross-feeding experiment (B), with data presented as the increases of the halo between days 1 and 3 (A) and the increases in the growth radius of the ΔfslA receiver strain between days 2 and 3 (B). The bars represent the averages of values obtained from experiments performed on three separate occasions. The error bars indicate standard errors of means.

FIG. 7.

Siderophore utilization tested by a cross-feeding experiment. The data indicate the increases in growth radius of the receiver strain after 72 h. The bars represent the averages of values obtained from experiments performed on three separate occasions. The error bars indicate standard errors of means.

FIG. 8.

The iron pools of SCHU S4 (black bars) and the ΔFTT0918 (white bars) and ΔfslA (gray bars) strains were measured by the ferrozine assay during growth in 2.0 μg/ml (A) or 0.2 μg/ml FeSO₄ (B). The values represent the averages of data from six samples obtained from experiments performed on two separate occasions. The error bars indicate standard errors of means.