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

Abstract

Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter 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 the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD 50 s) than did the wild type in C57BL/6J mouse infections. The lower LD 50 s 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.A hallmark of Salmonella enterica serovar Typhimurium (hereafter referred to as serovar Typhimurium) infection of the mouse is the presence of intracellular bacteria, primarily in macrophages, during all systemic phases of the disease (29,33). Macrophages ordinarily phagocytose particles and bacteria, which are then degraded in phagolysosomes by various means, including acid, cationic peptides, and oxidative radicals (18). In contrast, serovar Typhimurium is able to survive these challenges and exploit the macrophage as a site of intracellular replication and persistence. In order to circumvent or survive this hostile environment, serovar Typhimurium has evolved properties that allow it to avoid and persist in the presence of the innate and adaptive host immune systems.Reactive nitrogen intermediates (RNI) are also part of the arsenal of antibacterial measures produced by macrophages (13,18,35). Macrophages produce the inducible nitric oxide synthase (iNOS), which catalyzes the generation of nitric oxide (NO), upon stimulation with cytokines and/or mitogens. iNOS has been demonstrated to play a role in host defense from infection by a number of microbial agents, including serovar Typhimurium (25,34,45). While reactive oxygen intermediates seem to play a bactericidal role early during the infection process, NO displays a bacteriostatic effect at later time points (45), when most of the bacteria are intracellular. Although the localization of iNOS is not clear, recent work suggests that iNOS is present in multiple subcellular locations within macrophages (47). Furthermore, NO ...

The yjeB ( nsrR ) Gene of Escherichia coli Encodes a Nitric Oxide-Sensitive Transcriptional Regulator

“…One of the potential targets for the unidentified regulator was ytfE, which can also be strongly induced by aqueous NO in anaerobic cultures (21). The ytfE homologue of Salmonella enterica serovar Typhimurium (designated nipC) was identified in a screen for promoters inducible by acidified nitrite (23). Homologues of ytfE are also NO inducible in the denitrifying organisms Ralstonia eutropha and Pseudomonas stutzeri, which make NO as an intermediate of denitrification (29,44).…”

Hydroxylamine Assimilation by Rhodobacter capsulatus E1F1

“…A similar increase in the expression of hcp gene was observed during anaerobic growth with nitrate in Shewanella oneidensis (22), and a nitrite-inducible Fnr-dependent promoter was identified upstream of nipAB, the hcp-hcr homologues of S. enterica (21). However, in Clostridium perfringens HCP has been proposed to be involved in the adaptive response to oxidative stress (23).…”

“…A nitriteinducible HCP has also been described in Salmonella (21). The presence of the hcp gene in the nas gene cluster of R. capsulatus suggests that the HCP of this strain could be involved in assimilation and/or detoxification of NH 2 OH, which is a toxic compound that may be formed during nitrate/nitrite assimilation.…”

“…In E. coli (18), Thiobacillus ferrooxidans (20). and Salmonella enterica (21), a gene coding for a putative NADH oxidoreductase (hcr) is located immediately downstream of the hcp gene, and the HCR protein of E. coli catalyzes reduction of HCP in the presence of NADH (18). However, hcr genes are not found near the hcp genes present in the genomes of other bacteria and Archaea.…”

Genetic analysis of petrobactin transport inBacillus anthracis

“…Mice were monitored over a period of 14 days. LD50 values were calculated using the Moving Average Interpolation program available at (http:// falkow.stanford.edu/whatwedo/software/software.html) (Kim et al, 2003).…”