|Rosey, Everett - PHARMACIA & UPJOHN, MI|
|Kennedy, Michael - PHARMACIA & UPJOHN, MI|
|Jensen, Neil - FORMER, USDA/ARS/NADC|
|Bosworth, Brad - FORMER, USDA/ARS/NADC|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 27, 1999
Publication Date: N/A
Interpretive Summary: Brachyspira (Serpulina) hyodysenteriae, a corkscrew shaped bacterium, causes swine dysentery. This worldwide disease is estimated to cost the U.S. port industry $100 million per year. There are no vaccines for swine dysentery. Standard methods for diagnosing the disease are based on culturing the bacterium. These methods are slow and sometimes imprecise. Unfortunately, we don't know how B. hyodysenteriae cells are able to damag swine intestinal tissues, producing the severe bloody diarrhea typical of swine dysentery. Previously we identified a protein, known as Nox, that we thought might protect B. hyodysenteriae cells when they were in the pig's intestine. In these recent studies, we found that genetically engineered B. hyodysenteriae cells that could not make Nox were killed by oxygen. We also discovered that these cells were "attenuated" for virulence. This means that their ability to cause disease was significantly reduced. These eresults are important because we have shown that inhibiting a particular protein of B. hyodysenteriae will inhibit swine dysentery. These results also are important because the genetically engineered strains can be tested as vaccine strains.
Technical Abstract: The B. hyodysenteriae NADH (nox) oxidase gene on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or for kanamycin resistance (kan). The resulting plasmids, respectively, pCm-delta-NOX and pKm-delta-NOX, were used to transform wild type B. hyodysenteriae B204 cells and generate antibiotic resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild type nox gene in these strains had been replaced through allelic exchange by the inactivated nox gene containing cat or kan. Soluble NADH oxidase levels in cell lysates of Nox-Cm and Nox-Km were reduced 92-96% compared to parent strain B204 activity. SDS-PAGE and Western immunoblot analysis revealed that both nox- cell lysates were missing the 48 kDa Nox protein. In an aerotolerance test, cells of both nox- strains were at least 100-fold more sensitive to oxygen exposure than were cells of the wild type B204. In swine experimental infections, both nox- strains were less virulent than strain B204, that is, fewer animals became 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, in that role, contributes to the pathogenic ability of the spirochete.