Resistance of Francisella tularensis strains against reactive nitrogen and oxygen species with special reference to the role of KatG

Abstract

Francisella tularensis is a facultative intracellular bacterial pathogen capable of proliferating within host macrophages. The mechanisms that explain the differences in virulence between various strains of the species are not well characterized. In the present study, we show that both attenuated (strain LVS) and virulent (strains FSC200 and SCHU S4) strains of the pathogen replicate at similar rates in resting murine peritoneal exudate cells (PEC). However, when PEC were activated by exposure to gamma interferon (IFN-gamma), they killed LVS more rapidly than virulent strains of the pathogen. Addition of N(G)-monomethyl-l-arginine, an inhibitor of inducible nitric oxide synthase, to IFN-gamma-treated PEC, completely inhibited killing of the virulent strains, whereas it only partially blocked the killing of LVS. Similarly, in a cell-free system, SCHU S4 and FSC200 were more resistant to killing by H(2)O(2) and ONOO(-) than F. tularensis LVS. Catalase encoded by katG is a bacterial factor that can detoxify bactericidal compounds such as H(2)O(2) and ONOO(-). To investigate its contribution to the virulence of F. tularensis, katG deletion-containing mutants of SCHU S4 and LVS were generated. Both mutants demonstrated enhanced susceptibility to H(2)O(2) in vitro but replicated as effectively as the parental strains in unstimulated PEC. In mice, LVS-DeltakatG was significantly attenuated compared to LVS whereas SCHU S4-DeltakatG, despite slower replication, killed mice as quickly as SCHU S4. This implies that clinical strains of the pathogen have katG-independent mechanisms to combat the antimicrobial effects exerted by H(2)O(2) and ONOO(-), the loss of which could have contributed to the attenuation of LVS.

FIG. 1.

Growth and survival of SCHU S4, FSC200, and LVS in PEC after different treatments. (A) Nontreated. (B) Stimulated with IFN-γ (10 μg/ml) 15 h before infection and thereafter. (C) Stimulated with IFN-γ (10 μg/ml) and N-MMLA (1 mM) 15 h before infection and thereafter. The values shown represent the average of triplicates ± the standard deviation. The experiment in panel A was repeated four times and the experiments in panels B and C were repeated three times with similar results.

FIG. 2.

Survival of SCHU S4, FSC200, and LVS grown to the logarithmic phase in Chamberlain medium and thereafter diluted to approximately 10⁶ CFU/ml in PBS and exposed to H₂O₂ (1 mM) (A), SIN-1 (0.8 mM) that generates ONOO⁻ (B), or the indicated concentration of H₂O₂ for 2 h (C) or SIN-1 for 4 h (D). The values shown represent the average of triplicates ± the standard deviation. This experiment was performed three times with similar results.

FIG. 3.

Survival of SCHU S4, SCHU S4-katG, LVS, and LVS-katG grown to the logarithmic phase in Chamberlain medium, diluted to approximately 10⁶ CFU/ml in PBS, and thereafter exposed to hydrogen peroxide (A) or SIN-1 that generates ONOO⁻ (B) for 2 h. The values shown represent the average of triplicates ± the standard deviation. This experiment was performed three times with similar results.

FIG. 4.

Growth and survival of SCHU S4-ΔkatG and SCHU S4 (A) and LVS-ΔkatG and LVS (B) in PEC after different treatments. IFN-γ (10 μg/ml) or IFN-γ and N-MMLA (1 mM) were supplied to the cultures 15 h before infection and thereafter. The values shown represent the average of triplicates ± the standard deviation. This experiment was performed three times with similar results.