Novel role of the nitrite transporter NirC in Salmonella pathogenesis: SPI2-dependent suppression of inducible nitric oxide synthase in activated macrophages

Abstract

Activation of macrophages by interferon gamma (IFN-γ) and the subsequent production of nitric oxide (NO) are critical for the host defence againstSalmonella entericaserovar Typhimurium infection. We report here the inhibition of IFN-γ-induced NO production in RAW264.7 macrophages infected with wild-typeSalmonella. This phenomenon was shown to be dependent on thenirCgene, which encodes a potential nitrite transporter. We observed a higher NO output from IFN-γ-treated macrophages infected with anirCmutant ofSalmonella. ThenirCmutant also showed significantly decreased intracellular proliferation in a NO-dependent manner in activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the genein trans. Under acidified nitrite stress, a twofold more pronounced NO-mediated repression of SPI2 was observed in thenirCknockout strain compared to the wild-type. This enhanced SPI2 repression in thenirCknockout led to a higher level of STAT-1 phosphorylation and inducible nitric oxide synthase (iNOS) expression than seen with the wild-type strain. In iNOS knockout mice, the organ load of thenirCknockout strain was similar to that of the wild-type strain, indicating that the mutant is exclusively sensitive to the host nitrosative stress. Taken together, these results reveal that intracellularSalmonellaevade killing in activated macrophages by downregulating IFN-γ-induced NO production, and they highlight the critical role ofnirCas a virulence gene.