Characterization of a Unique Outer Membrane Protein Required for Oxidative Stress Resistance and Virulence of Francisella tularensis

Francisella tularensis, the causative agent of tularemia, lacks typical bacterial virulence factors and toxins but still exhibits extreme virulence. The bacterial multidrug efflux systems consist of an inner membrane, a transmembrane membrane fusion protein, and an outer membrane (OM) component that form a contiguous channel for the secretion of a multitude of bacterial products. Francisella contains three orthologs of the OM proteins; two of these, termed TolC and FtlC, are important for tularemia pathogenesis. The third OM protein, SilC, is homologous to the silver cation efflux protein of other bacterial pathogens. The silC gene (FTL_0686) is located on an operon encoding an Emr-type multidrug efflux pump of F. tularensis. The role of SilC in tularemia pathogenesis is not known. In this study, we investigated the role of SilC in secretion and virulence of F. tularensis by generating a silC gene deletion (Delta silC) mutant and its transcomplemented strain. Our results demonstrate that the Delta silC mutant exhibits increased sensitivity to antibiotics, oxidants, silver, diminished intramacrophage growth, and attenuated virulence in mice compared to wild-type F. tularensis. However, the secretion of antioxidant enzymes of F. tularensis is not impaired in the Delta silC mutant. The virulence of the Delta silC mutant is restored in NADPH oxidase-deficient mice, indicating that SilC resists oxidative stress in vivo. Collectively, this study demonstrates that the OM component SilC serves a specialized role in virulence of F. tularensis by conferring resistance against oxidative stress and silver. IMPORTANCE Francisella tularensis, the causative agent of a fatal human disease known as tularemia, is a category A select agent and a potential bioterror agent. The virulence mechanisms of Francisella are not completely understood. This study investigated the role of a unique outer membrane protein, SilC, of a multidrug efflux pump in the virulence of F. tularensis. This is the first report demonstrating that the OM component SilC plays an important role in efflux of silver and contributes to the virulence of F. tularensis primarily by providing resistance against oxidative stress. Characterization of these unique virulence mechanisms will provide an understanding of the pathogenesis of tularemia and identification of potential targets for the development of effective therapeutics and prophylactics for protection from this lethal disease.