Molecular and functional characterization of Salmonella enterica serovar typhimurium poxA gene: Effect on attenuation of virulence and protection

Salmonella enterica poxA mutants exhibit a pleiotropic phenotype, including reduced pyruvate oxidase activity; reduced growth rate; and hypersensitivi ty to the herbicide sulfometuron methyl, alpha-ketobutyrate, and amino acid analogs, These mutants also failed to grow in the presence of the host ant imicrobial peptide, protamine. In this study, PoxA(-) mutants of S. enteric a serovar Typhimurium (S, typhimurium) were found to be 10,000-fold attenua ted in orally inoculated BALB/c mice and 1,000-fold attenuated in intraperi toneally inoculated BALB/c mice, compared to wild-type S. typhimurium UK-1. In addition, poxA mutants were found to be capable of colonizing the splee n, mesenteric lymph nodes, and Peyer's patches; to induce strong humoral im mune responses; and to protect mice against a lethal wild-type Salmonella c hallenge. A 2-kb DNA fragment was isolated from wild-type S. typhimurium UK -I based on its ability to complement an isogenic poxA mutant. The nucleoti de sequence of this DNA fragment revealed an open reading frame of 325 amin o acids capable of encoding a polypeptide of 36.8 kDa that was confirmed in the bacteriophage T7 expression system. Comparison of the translated seque nce to the available databases indicated high homology to a family of lysyl -tRNA synthetases, Our results indicate that a mutation of poxA has an atte nuating effect on Salmonella virulence. Further, poxA mutants are immunogen ic and could be useful in designing live vaccines with a variety of bacteri al species. To our knowledge, this is the first report on the effect of pox A mutation on bacterial virulence.