DNA base excision repair potentiates the protective effect of Salmonella Pathogenicity Island 2 within macrophages

Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics and Department of Animal Sciences, University of Vermont, Burlington, VT 05405, USA Correspondence Akamol E. Suvarnapunya asuvarna{at}rockefeller.edu Reactive oxidants are a primary weapon of the macrophage antibacterial arsenal. The ability of virulent Salmonella to repair oxidative DNA lesions via the base-excision repair system (BER) enables its survival and replication within the macrophage, but is not required for extracellular growth. Salmonella also inhibits the targeting of oxidant generators to the Salmonella -containing vacuole (SCV) via Salmonella Pathogenicity Island 2 (SPI2). Accordingly, the relative contributions of these two discrete systems to Salmonella resistance to both oxidative mutagenesis and lethality within RAW 264.7 macrophages were investigated. A mutant unable to initiate BER was constructed by deleting all three BER bifunctional glycosylases ( fpg / nth / nei ), and was significantly impaired for early intramacrophage survival. Mutations in various SPI2 effector ( sifA and sseEFG ) and structural ( ssaV ) genes were then analysed in the BER mutant background. Loss of SPI2 function alone appeared to increase macrophage-induced mutation. Statistical analyses of the reduced intramacrophage survival of SPI2 mutants and the corresponding SPI2/BER mutants indicated a synergistic interaction between BER and SPI2, suggesting that SPI2 promotes intramacrophage survival by protecting Salmonella DNA from exposure to macrophage oxidants. Furthermore, this protection may involve the SseF and SseG effectors. In contrast, the SifA effector did not seem to play a major role in oxidant protection. It is speculated that Salmonella initially stalls oxidative killing by preserving its genomic integrity through the function of BER, until it can upregulate SPI2 to limit its exposure to macrophage oxidants. Abbreviations: AP, apurinic/apyrimidinic; BER, base excision repair; CRAMP, cathelicidin-related antimicrobial peptide; IFN- , interferon- ; iNOS, inducible nitric oxide synthase; SCV, Salmonella -containing vacuole; phox, phagocyte NADPH oxidase; RNI, reactive nitrogen intermediate; ROI, reactive oxygen intermediate; SPI2, Salmonella Pathogenicity Island 2; STE, Salmonella -translocated effector Present address: Laboratory of Infection Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. Present address: Department of Microbio