Isolation, Oxygen Sensitivity, and Virulence of NADH Oxidase Mutants of the Anaerobic SpirocheteBrachyspira(Serpulina)hyodysenteriae, Etiologic Agent of Swine Dysentery

Abstract

Brachyspira (Serpulina)hyodysenteriae, the etiologic agent of swine dysentery, uses the enzyme NADH oxidase to consume oxygen. To investigate possible roles for NADH oxidase in the growth and virulence of this anaerobic spirochete, mutant strains deficient in oxidase activity were isolated and characterized. The cloned NADH oxidase gene (nox; GenBank accession no. U19610 ) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or a gene for kanamycin resistance (kan). The resulting plasmids, respectively, pCmΔNOX and pKmΔNOX, were used to transform wild-type B. hyodysenteriae B204 cells and generate the antibiotic-resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild-type nox genes in these strains had been replaced, through allelic exchange, by the inactivatednox gene containing cat or kan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis revealed that both nox mutant cell lysates were missing the 48-kDa Nox protein. Soluble NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were reduced 92 to 96% compared to the activity level in parent strain B204. In an aerotolerance test, cells of both nox 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, bothnox mutants were less virulent than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no deaths. 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.

Fluoride and organic weak acids as respiration inhibitors for oral streptococci in acidified environments

“…Moreover, for S. pneumoniae, NADH oxidase has been found to be involved also in the development of genetic competence during growth (2). Many organisms previously considered strict anaerobes have been found to have NADH oxidase activity and are now considered more as microaerophiles, for example Treponema denticola (11) and Brachyspira hyodysenteriae (28). For the latter spirochete, the enzyme is involved in virulence in the pig gut, apparently by reducing O 2 toxicity.…”

Optimization of cholesterol removal, growth and fermentation patterns of Lactobacillus acidophilus ATCC 4962 in the presence of mannitol, fructo-oligosaccharide and inulin: a response surface methodology approach

“…While the utilization of FOS and inulin has been widely reported, the utilization of mannitol to produce high concentration of SCFA was less studied and was also found to be strain dependent. Lactic acid bacteria that produced NADH oxidase would have the alternative NADH-H + -oxidizing mechanism, resulting in higher ability to grow on substrates more chemically reduced than glucose, such as mannitol (Stanton et al 1999). This may contribute to the better growth of L. acidophilus ATCC 4962 in the presence of mannitol and subsequently produced higher amount of SCFA and higher cholesterol removal.…”

Isolation and Maintenance of Brachyspira Species

“…Although B. hyodysenteriae strain B204 cultured cells can withstand exposure to air for 2 hours or more in the laboratory (Stanton et al, 1999), the oxygen sensitivities of other brachyspire strains are largely unknown. To increase isolation success, intestinal contents or tissue samples should be protected from air exposure whenever possible.…”