Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O
2−). In the present study, mutants of
F.
tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion
F.
tularensis mutant of
sodC (Δ
sodC) and a
F.
tularensis Δ
sodC mutant with attenuated Fe-superoxide dismutase (
sodB) gene expression (
sodB Δ
sodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a mouse model of respiratory tularemia. The
F.
tularensis Δ
sodC and
sodB Δ
sodC mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type
F.
tularensis LVS. Transcomplementing the
sodC gene in the Δ
sodC mutant or inhibiting the IFN-γ-dependent production of O
2− or nitric oxide (NO) enhanced intramacrophage survival of the
sod mutants. The Δ
sodC and
sodB Δ
sodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type
F.
tularensis LVS. As observed for macrophages, the virulence of the Δ
sodC mutant was restored in
ifn-γ
−/−,
inos−/−, and
phox−/− mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of
F.
tularensis in mice.
Francisella tularensis is considered a potential biological threat due to its extreme infectivity, ease of artificial dissemination via aerosols, and substantial capacity to cause illness and death. A hallmark of all
F.
tularensis subspecies is their ability to survive and replicate within macrophages (
18) and other cell types (
6,
11,
25,
28). While recent work has furthered our understanding of
F.
tularensis virulence mechanisms, little is known with respect to its ability to resist the microbicidal production of reactive oxygen species (ROS) or reactive nitrogen species (RNS).Superoxide dismutases (SODs) are metalloproteins that are classified according to their coordinating active site metals. SODs catalyze the dismutation of the highly reactive superoxide (O
2−) anion to hydrogen peroxide (H
2O
2) and O
2 (
26). The dismutation of O
2− prevents accumulation of microbicidal ROS and RNS in infected macrophages. Three major categories of SODs have been identified in bacteria and include Mn-, Fe-, and CuZn-containing SODs (SodA, SodB, and SodC, respectively) and are required for aerobic survival (
27). The
F.
tularensis genome encodes SodB (FTL_1791) and SodC (FTL_0380). In several intracellular bacterial pathogens, SodC is an important virulence factor, and its localization to the periplasmic space protects bacteria from host-derived O
2− and NO radicals (
8,
9,
21,
32). Moreover, many virulent bacteria possess two copies of the
sodC gene (
4). The evolutionary maintenance of an extra
sodC gene copy suggests that it serves some essential function in survival (
4). As an intracellular pathogen,
F.
tularensis is exposed to ROS and RNS generated by inflammatory cells during the macrophage activation process, which suggests that SODs may play an important role in its intracellular survival and pathogenesis. We have demonstrated that decreases in SodB activity render
F.
tularensis sensitive to ROS and attenuate virulence in mice (
2). However, the contribution of
F.
tularensis SodC in virulence and intramacrophage survival has not been defined. In this study we have constructed a
F.
tularensis sodC mutant (Δ
sodC) and a
F.
tularensis sodBC double mutant (
sodB Δ
sodC) and determined that SodC in conjunction with SodB primarily protects the pathogen from host-derived ROS and is required for intramacrophage survival and virulence of
F.
tularensis in mice.
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