Molecular analysis of the role of polynucleotide phosphorylase in the virulence of 'Dichelobacter nodosus'

Abstract

'Dichelobacter nodosus' is the key causative agent of ovine footrot. 'D. nodosus' strains display a range of virulence, from benign to virulent. In general, virulent strains secrete higher levels of thermostable proteases and have increased twitching motility compared to benign strains. Four integrated genetic elements have been identified, the 'intA', 'intB', 'intC' and 'intD' elements that integrate into 'tRNA-ser' genes next to 'pnpA' (polynucleotide phosphorylase) and 'glpA' in 'D. nodosus'. PnpA acts as a global regulator of virulence in 'Salmonella enterica' and GlpA is closely related to CsrA and RsmA, which act as global repressors of virulence in 'Helicobacter pylori' and 'Erwinia carotovora'. Whittle 'et al.' (1999) proposed that genetic elements next to 'glpA' and 'pnpA' alter the expression of 'glpA' and 'pnpA', thus regulating virulence in D. nodosus. The hypothesis that PnpA is a virulence repressor in 'D. nodosus' was investigated by using a suicide plasmid containing two segments from 'pnpA' flanking the 'tetM' gene to produce 'pnpA' knockouts in virulent and benign strains. Benign strains with 'pnpA' knockouts had a significant increase in twitching motility, supporting the hypothesis that PnpA is a virulence repressor in 'D. nodosus'. Complementation experiments confirmed that the increase in twitching motility was due to the 'pnpA' knockout. Secreted proteases from virulent strains were found to be considerably more thermostable than proteases from benign strains, but the 'pnpA' knockout in benign strains did not increase protease thermostability. A PnpA specific phosphorolytic activity assay showed that the virulent strains had a slightly lower level of PNPase activity than benign strains. A qPCR assay was developed to measure mRNA levels from 'pnpA' and 'aprV2', which encodes a thermostable protease. Differences in mRNA levels between strains were not large, and there was no correlation with virulence. However, there may be post-transcriptional regulation of 'pnpA' and 'aprV2' expression. Amino acid differences between both PnpA and AprV2 in benign and virulent strains were identified. Although these changes do not appear likely to alter the functions of PnpA and AprV2, their role in virulence should be investigated further.