Detection and diversity of a putative novel heterogeneous polymorphic proline-glycine repeat (Pgr) protein in the footrot pathogen Dichelobacter nodosus

Dichelobacter nodosus, a Gram-negative anaerobic bacterium, is the essential causative agent of footrot in sheep. Currently, depending on the clinical presentation in the field, footrot is described as benign or virulent; D. nodosus strains have also been classified as benign or virulent, but this designation is not always consistent with clinical disease. The aim of this study was to determine the diversity of the pgr gene, which encodes a putative proline-glycine repeat protein (Pgr). The pgr gene was present in all 100 isolates of D. nodosus that were examined and, based on sequence analysis had two variants, pgrA and pgrB. In pgrA, there were two coding tandem repeat regions, R1 and R2: different strains had variable numbers of repeats within these regions. The R1 and R2 were absent from pgrB. Both variants were present in strains from Australia, Sweden and the UK, however, only pgrB was detected in isolates from Western Australia. The pgrA gene was detected in D. nodosus from tissue samples from two flocks in the UK with virulent footrot and only pgrB from a flock with no virulent or benign footrot for >10 years. Bioinformatic analysis of the putative PgrA protein indicated that it contained a collagen-like cell surface anchor motif. These results suggest that the pgr gene may be a useful molecular marker for epidemiological studies

Isolation of the Bacteriophage DinoHI from Dichelobacter nodosus and its Interactions with other Integrated Genetic Elements

The Gram-negative anaerobic pathogen Dichelobacter nodosus carries several genetic elements that integrate into the chromosome. These include the intA, intB, intC and intD elements, which integrate adjacent to csrA and pnpA, two putative global regulators of virulence and the virulence-related locus, vrl, which integrates into ssrA. Treatment of D. nodosus strains with ultraviolet light resulted in the isolation of DinoHI, a member of the Siphoviridae and the first bacteriophage to be identified in D. nodosus. Part of the DinoHI genome containing the packaging site is found in all D. nodosus strains tested and is located at the end of the vrl, suggesting a role for DinoHI in the transfer of the vrl by transduction. Like the intB element, the DinoHI genome contains a copy of regA which has similarity to the repressors of lambdoid bacteriophages, suggesting that the maintenance of DinoHI and the intB element may be co-ordinately controlled

Type IV Fimbrial Biogenesis Is Required for Protease Secretion and Natural Transformation in Dichelobacter nodosus▿

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

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 (σ(54)) 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 σ(54)-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 σ(54), genome-wide transcriptional profiling was performed using a whole-genome oligonucleotide microarray. The results indicated that PilR and σ(54) regulated genes other than 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 σ(54)-dependent PilR/S system that also indirectly controls protease secretion

Twitching Motility Is Essential for Virulence in Dichelobacter nodosus▿ †

Type IV fimbriae are essential virulence factors of Dichelobacter nodosus, the principal causative agent of ovine foot rot. The fimA fimbrial subunit gene is required for virulence, but fimA mutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type IV fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. We showed that mutation of either the pilT or pilU gene eliminated the ability to carry out twitching motility. However, the pilT mutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas the pilU mutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway in D. nodosus. It was postulated that sufficient fimbrial retraction must occur in the pilU mutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in a pilU mutant of D. nodosus, aberrant motion was detected in an equivalent mutant of Pseudomonas aeruginosa. These observations explain how in D. nodosus protease secretion can occur in a pilU mutant but not in a pilT mutant. In addition, virulence studies with sheep showed that both the pilT and pilU mutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Schematic representation of the C-terminal deleted AprV5 derivatives.

<p>The number of amino acid residues in each derivative is indicated as are the pre-, pro-, mature and C-terminal extension (CTE) regions.</p

Extracellular protease zymograms of the <i>aprV5</i> C-terminal deletion strains.

<p>Cultures from 16 h, 25 h and 40 h TAS broth cultures of the wild-type strain VCS1703A (WT), the <i>aprV5</i>Δ<i>478–595</i> strain JIR3947 (<i>aprV5</i>Δ<i>C1</i>) and its complemented derivative JIR3968 (<i>aprV5</i>Δ<i>C1</i>(<i>V5⁺</i>)), the <i>aprV5</i>Δ<i>503–595</i> strain JIR3956 (<i>aprV5</i>Δ<i>C2</i>) and its complemented derivative JIR3965 (<i>aprV5</i>Δ<i>C2</i>(<i>V5⁺</i>)) and the <i>aprV5</i> mutant JIR3756 (<i>aprV5</i>) were examined. The profile of the <i>aprV5</i>Δ<i>560–595</i> strain JIR3969 (<i>aprV5</i>Δ<i>C3</i>) is not shown but was identical to that of <i>aprV5</i>Δ<i>C2</i>.</p

Identification of unprocessed proteases in the <i>aprV5</i> mutant and <i>aprV5</i>Δ<i>C</i> strains by mass spectrometry.

a<p>Coverage denotes the percentage of the full length protein sequence that has been matched to the MS data.</p>b<p>Best MASCOT score obtained between analyses from different gel pieces excised from identical gels.</p>c<p>MS value denotes the number of peptides matched to the MS data corresponding to sequences from within a specific region of the protein. MSMS value denotes the number of these peptides matches of which the sequences have been confirmed by MSMS analysis.</p

Quantitative analysis of protease activity of deletion mutants.

<p>Total protease activity in culture supernatants was determined with azocasein as the substrate. The culture supernatants from 16 h, 25 h and 40 h TAS broth cultures of the wild type strain VCS1703A (WT), the <i>aprV5</i> mutant JIR3756 (<i>aprV5</i>), the <i>aprV5</i>Δ<i>478–595</i> strain JIR3947 (<i>V5</i>Δ<i>C1</i>), the <i>aprV5</i>Δ<i>503–595</i> strain JIR3956 (<i>V5</i>Δ<i>C2</i>), the <i>aprV5</i>Δ<i>560–595</i> strain JIR3969 (<i>V5</i>Δ<i>C3</i>) were analysed as well as their complemented derivatives: JIR3968 (<i>V5</i>Δ<i>C1</i>(<i>V5⁺</i>)), JIR3965 (<i>V5</i>Δ<i>C2</i>(<i>V5⁺</i>)) and JIR3978 (<i>V5</i>Δ<i>C3</i>(<i>V5⁺</i>)). All values were obtained from three independent biological samples. Error bars represent SEM.</p