Identification of novel pathogenicity loci in Clostridium perfringens strains that cause avian necrotic enteritis

Abstract

Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes approximately 85 and approximately 70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne.

Figure 1. Genetic organization of NE-specific loci.

The genetic organization of (A) NELoc-1, (B) NELoc-2 and (C) NELoc-3 is shown, each arrow representing a predicted gene and the total size given below each locus. Predicted functional annotations and locus tags are shown above and below each gene, respectively. Genes are colour-coded by their putative role based upon sequence analyses.

Figure 2. Identification of VirR boxes in NE strain genomes.

(A). A modified VirR-box consensus sequence was produced based on the alignment of known VirR-boxes with the VirR-box upstream from netB. The VirR-box consensus previously reported by Cheung et al. (2004) is shown in bold, and absolutely conserved nucleotides are in boxes. (B) VirR-boxes identified in CP4, including one novel element upstream of the internalin-like protein gene. Nucleotides that conform with the previously reported VirR-box consensus are in green, and those that do not conform in red.

Figure 3. PFGE and Southern blot analyses of plasmids from C. perfringens poultry strains.

(A). Agarose plugs containing DNA isolated from the eight sequenced poultry strains were digested with NotI and subjected to PFGE. (B). Southern blotting was performed with DIG-labelled probes for netB and hdhA. Results from both netB and hdhA probes are shown overlayed. In all lanes with two bands, the upper band represents netB and the lower band hdhA. Both probes hybridized to the same band in CP2. In CP6, only the hdhA probe hybridized, while in JGS4143, only the netB probe hybridized.

Figure 4. Plasmid location of NELoc-1 and -3.

Nucleotide sequence alignments of pCPF5603, pCP8533etx and (A) NELoc-1 or (B) NELoc-3 were generated with Artemis Comparison Tool (ACT). The small boxes represent ORFs, with toxin genes colored in red and IS1151 or IS1469 genes in yellow. Grey boxes connecting the different sequences represent regions with sequence similarity. Colored bars underneath each sequence represent different loci as follows: blue, tcp; green, cpe; red, cpb2; black, pCPF5603-specific; light blue, etx. Orange bars represent the sequence found duplicated in pCP8533etx and NELoc-1 and -3 sequences.