B-228. Role of a Copper-Zinc Superoxide Dismutase (SodC) of Francisella tularensis in Intramacrophage Survival, Resistance to Oxidative Stress and Virulence in Mice

C. S. Bakshi, M. Mahawar, D. W. Metzger;
Albany Med. Coll., Albany, NY.

Background: Francisella tularensis; the causative agent of tularemia is an intracellular pathogen. F. tularensis genome encodes various antioxidant systems, which include catalase/peroxidase, alkyl hydroperoxide reductases, and Fe- and CuZn- containing superoxide dismutases (sodB and sodC, respectively). In numerous bacterial pathogens, SodC has been demonstrated to be an important virulence factor that protects pathogens from host-derived superoxide (O2-) and nitric oxide (NO) radicals. However, the role of SodC in intramacrophage survival and virulence of Francisella is not completely understood. Methods: We took a genetic approach to generate deletion mutant of sodCsodC) and a sodBC double mutant (sodBFt/ΔsodC). These mutants and the isogenic wild type strain were evaluated in oxidative susceptibility studies, macrophage survival assays and mouse virulence experiments to assess the functional importance of F. tularensis SodC. Results: ΔsodC and sodBFt/ΔsodC mutants were found to be highly sensitive to oxidative stress under acellular growth conditions. When tested for intramacrophage survival, ΔsodC and sodBFt/ΔsodC mutants replicated at a rate similar to the wild type strain in resting macrophages however; they were killed rapidly when the macrophages were activated by IFN-γ treatment. Inhibition of NADPH oxidase or iNOS in IFN-γ activated macrophages restored replication of ΔsodC and sodBFt/ΔsodC mutants. Virulence testing in C57BL/6 mice revealed that 50-80% of the mice infected with ΔsodC and sodBFt/ΔsodC mutants survived the infection. The virulence of ΔsodC mutant was completely restored in mouse strains deficient for IFN-γ or iNOS, recapitulating the results observed in macrophages under in vitro conditions. Conclusions: The results demonstrate that SodC provides resistance against oxidative stress by dismutating the O2- and also contributes to resistance against NO derived byproducts by limiting the production of highly toxic peroxynitrite radicals. The results also suggest that SodC act in concert with SodB to offer resistance against oxidative stress resulting in an enhanced intracellular survival and virulence of F. tularensis.