Identification, molecular cloning and analysis of thetufA,rpsJ, andvirA genes of Serpulina hyodysenteriae

Serpulina hyodysenteriae produces a number of putative virulence factors attributed to the pathogenesis of the disease such as adhesins, motility, chemotaxis and hemolysin/cytotoxin. To examine the role virulence factors in the pathogenesis of the disease a plasmid library of S. hyodysenteriae genomic DNA was constructed and screened. Colony immunoblot screening, using monoclonal antibodies 10G6 and 3D1, generated against S. hyodysenteriae serotype 2 strain B204, identified two immunopositive clones. The DNA insert, from pRED3C6, hybridized with HindIII digested genomic DNA of all known S. hyodysenteriae serotypes/serogroups but not with the intestinal spirochetes S. innocens, S. pilosicoli, and Treponema succinifaciens. We report here the DNA and deduced amino acid sequence, characterization and specific gene inactivated mutants of S. hyodysenteriae. Analysis of the deduced amino acid sequence revealed the typical features of a N-terminal signal sequence for signal peptidase II cleavage. Serpulina hyodysenteriae isogenic mutants were generated by allelic exchange. The mice orally inoculated with the S. hyodysenteriae mutants had reduced lesions when compared to the S. hyodysenteriae parent strain. Hence, this gene was designated virulence-associated gene A (virA). Our in vitro and in vivo results suggest other pathogenic mechanisms, besides hemolysin and cytotoxin, are involved in the development of intestinal lesions and the progression of swine dysentery.;The insert DNA from clone two, designated pRED3D1, was identified by reacting with monoclonal antibody 3D1. The insert DNA had nucleotide sequence similar to the tufA and rpsJ genes from other bacterial taxons. The deduced EF-Tu and S10 proteins consisted of 410 and 101 amino acids with predicted molecular masses of 44.7 and 11.4-kDa, respectively. Hybridization using the tufA and rpsJ genes as probes revealed only one copy of each gene in S. hyodysenteriae, while the related intestinal spirochetes, S. pilosicoli had two copies and S. innocens did not hybridize. The organization of the fus, tufA, and rpsJ genes of S. hyodysenteriae was similar to that of the archaebacterium Methanococcus vannielii.