Molecular and Functional Characterization of Salmonella enterica Serovar Typhimurium poxA Gene: Effect on Attenuation of Virulence and Protection

Abstract

Salmonella enterica poxA mutants exhibit a pleiotropic phenotype, including reduced pyruvate oxidase activity; reduced growth rate; and hypersensitivity to the herbicide sulfometuron methyl, α-ketobutyrate, and amino acid analogs. These mutants also failed to grow in the presence of the host antimicrobial peptide, protamine. In this study, PoxA− mutants of S. entericaserovar Typhimurium (S. typhimurium) were found to be 10,000-fold attenuated in orally inoculated BALB/c mice and 1,000-fold attenuated in intraperitoneally inoculated BALB/c mice, compared to wild-type S. typhimurium UK-1. In addition,poxA mutants were found to be capable of colonizing the spleen, mesenteric lymph nodes, and Peyer’s patches; to induce strong humoral immune responses; and to protect mice against a lethal wild-type Salmonella challenge. A 2-kb DNA fragment was isolated from wild-type S. typhimurium UK-1 based on its ability to complement an isogenic poxA mutant. The nucleotide sequence of this DNA fragment revealed an open reading frame of 325 amino acids capable of encoding a polypeptide of 36.8 kDa that was confirmed in the bacteriophage T7 expression system. Comparison of the translated sequence to the available databases indicated high homology to a family of lysyl-tRNA synthetases. Our results indicate that a mutation of poxA has an attenuating effect onSalmonella virulence. Further, poxA mutants are immunogenic and could be useful in designing live vaccines with a variety of bacterial species. To our knowledge, this is the first report on the effect of poxA mutation on bacterial virulence.

A bacterial ortholog of class II lysyl‐tRNA synthetase activates lysine

“…Transposon insertion in genX was shown to significantly decrease PoxB expression in Salmonella typhimurium and in E. coli [20]. Inactivation of genX was also shown to provoke a severe attenuation of S. typhimurium virulence, making such a strain a possible candidate for vaccine development [21]. An E. coli GenX-inactivated mutant shows a pleiotropic phenotype, it is more sensitive than the wild type counterpart to treatment with antibiotics, a variety of amino acid analogs and dyes [20].…”

Elongation factor P: Function and effects on bacterial fitness

“…51,57,59 The mutations lead to reduced growth rate and motility, 49,[51][52][53][54]56,57,59 susceptibility to hyperosmotic conditions and different stress factors, such as acids, detergents and antibiotics, 27,49,53,55,60 enhanced metabolism, 27,55 and reduced virulence. [54][55][56][57]59 While there seem to be no influence on transcription, the translation appears to be affected, 51,55 resulting in an altered expression of a number of proteins. 27,53,55 Frequently, metabolic proteins, membraneassociated proteins, transporters and two-component regulatory systems, especially those involved in chemotaxis and motility, display changed expression levels in the deletion strains, whereas proteins belonging to the basal transcriptiontranslation machinery are rarely affected.…”

“…67,68 Interestingly, the poxAstrains are still immunogenic and can therefore be used to generate effective antibodies against their wild-type counterpart. 54 Another protein overexpressed in the poxAstrain is the virulence factor PhoP, 55 which belongs to the two-component system PhoP/PhoQ. Constitutively, expression of PhoP impairs virulence and survival of S. typhimurium in mammalian hosts.…”

The prpZ gene cluster encoding eukaryotic-type Ser/Thr protein kinases and phosphatases is repressed by oxidative stress and involved in Salmonella enterica serovar Typhi survival in human macrophages

“…A control strain, DEF071, containing the prpZ promoter in the opposite orientation was also constructed. Strains DEF070 and DEF071 were grown up to an OD 600 nm = 0.6 in N-minimal medium containing Kaniga et al (1998) pSIF012 pMEG-375 with flanking regions of prpZ and prkX, used for deletion of prpZ-prkY-prkX…”