Isolation, oxygen sensitivity, and virulence of NADH oxidase mutants of the anaerobic spirochete Brachyspira (Serpulina) hyodysenteriae, etiologic agent of swine dysentery

Brachyspira (Serpulina) hyodysenteriae, the etiologic agent of swine dysent ery, uses the enzyme NADH oxidase to consume oxygen. To investigate possibl e roles for NADH oxidase in the growth and virulence of this anaerobic spir ochete, mutant strains deficient in oxidase activity were isolated and char acterized. The cloned NADH oxidase gene (nox; GenBank accession no. U19610) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence w ith either a gene for chloramphenicol resistance (cat) or a gene for kanamy cin resistance (kan), The resulting plasmids, respectively, pCm Delta NOX a nd pKm Delta NOX, were used to transform wild-type B, hyodysenteriae B204 c ells and generate the antibiotic resistant strains Nox-Cm and Nox-Km, PCR a nd Southern hybridization analyses indicated that the chromosomal wild-type nor genes in these strains had been replaced, through allelic exchange, by the inactivated nor gene containing cat or kan, Sodium dodecyl sulfate-pol yacrylamide gel electrophoresis and Western immunoblot analysis revealed th at both nor mutant cell lysates were missing the 48-kDa Nor protein. Solubl e NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were re duced 92 to 96% compared to the activity level in parent strain B204, In an aerotolerance test, cells of both nor mutants were at least 100-fold more sensitive to oxygen exposure than were cells of the wild-type parent strain B204, In swine experimental infections, both nor mutants were less virulen t than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no d eaths. These results provide evidence that NADH oxidase serves to protect B , hyodysenteriae cells against oxygen toxicity and that the enzyme, in that role, contributes to the pathogenic ability of the spirochete.