Regulation of type IV fimbrial biogenesis in Dichelobacter nodosus

Type IV fimbriae are expressed by several bacterial pathogens and are essential for virulence in Dichelobacter nodosus, which causes ovine footrot. We have identified a two-component signal transduction system (PilR/S) and an alternative sigma factor (σ⁵⁴) that were shown by insertional inactivation to be required for the regulation of fimbrial biogenesis in D. nodosus. Western blots showed that in both pilR and rpoN mutants, fimbrial subunit production was significantly reduced by a process that was shown to occur at a PilR- and σ⁵⁴-dependent promoter. The mutants lacked surface fimbriae, which were shown to be required for the adherence of D. nodosus cells to tissue culture monolayers. The reduction in fimbrial subunit production in these mutants also resulted in a concomitant loss of the ability to secrete extracellular proteases. A maltose binding protein-PilR fusion protein was purified and was shown to bind specifically to a region located 234 to 594 bp upstream of the fimA transcriptional start point. To determine additional targets of PilR and σ⁵⁴, genome-wide transcriptional profiling was performed using a whole-genome oligonucleotide microarray. The results indicated that PilR and σ⁵⁴ regulated genes other fimA; these genes appear to encode surface-exposed proteins whose role in virulence is unknown. In conclusion, this study represents a significant advancement in our understanding of how the ability of D. nodosus to cause ovine footrot is regulated, as we have shown that the biogenesis of type IV fimbriae in D. nodosus is regulated by a σ⁵⁴-dependent PilR/S system that also indirectly controls protease secretion.