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 hypersensitivity to the herbicide sulfometuron methyl, alpha-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. enterica serovar 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 on Salmonella 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.
Kaniga
K
K
Megan Health, Inc., St. Louis, Missouri 63110, USA. kkaniga@meganhealth.com
Compton
M S
MS
Curtiss
R
R
Sundaram
P
P
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001428:Bacterial Vaccines; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006090:Gram-Negative Bacteria / Q000201:enzymology / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000201:enzymology / Q000235:genetics; D008250:Lysine-tRNA Ligase / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D012481:Salmonella Infections, Animal / Q000517:prevention & control; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity*; D017422:Sequence Analysis, DNA; D014613:Vaccines, Attenuated; D014764:Viral Proteins
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D014613:Vaccines, Attenuated; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008250:Lysine-tRNA Ligase