Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.

The objective of this study was to develop an understanding of the molecular mechanisms by which type IV fimbrial biogenesis, natural transformation, and protease secretion are linked in the ovine foot rot pathogen, Dichelobacter nodosus. We have shown that like the D. nodosus fimbrial subunit FimA, the pilin-like protein PilE and the FimN, FimO, and FimP proteins, which are homologs of PilB, PilC, and PilD from Pseudomonas aeruginosa, are essential for fimbrial biogenesis and natural transformation, indicating that transformation requires an intact type IV fimbrial apparatus. The results also showed that extracellular protease secretion in the fimN, fimO, fimP, and pilE mutants was significantly reduced, which represents the first time that PilB, PilC, and PilE homologs have been shown to be required for the secretion of unrelated extracellular proteins in a type IV fimbriate bacterium. Quantitative real-time PCR analysis of the three extracellular protease genes aprV2, aprV5, and bprV showed that the effects on protease secretion were not mediated at the transcriptional level. Bioinformatic analysis did not identify a classical type II secretion system, and the putative fimbrial biogenesis gene pilQ was the only outer membrane secretin gene identified. Based on these results, it is postulated that in D. nodosus, protease secretion occurs by a type II secretion-related process that directly involves components of the type IV fimbrial biogenesis machinery, which represents the only type II secretion system encoded by the small genome of this highly evolved pathogen.