Modulation of Virulence by Two Acidified Nitrite-Responsive Loci ofSalmonella entericaSerovar Typhimurium

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Abstract
Two acidified nitrite-inducible genes ofSalmonella entericaserovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designatednipABandnipC, which correspond tohcp-hcr(hybrid cluster protein) ofEscherichia coliandnorAofAlcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. ThenipABpromoter was regulated by oxygen in an Fnr-dependent manner. ThenipCpromoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although thenipABandnipCmutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.