The pathogenesis of poultry NE has been the subject of considerable investigation in recent years, following reassessment of the role of CPA and subsequent identification of the novel pore-forming toxin NetB 
. We show here for the first time that netB
resides on a ~42 kb plasmid-encoded pathogenicity locus (NELoc-1) harbored specifically by NE strains. In addition, we identified two other loci associated with NE, NELoc-2 and -3, the latter of which also resides on a plasmid that is similar to, but distinct from, that carrying netB
. Sequence alignments of NELoc-1 to -3 from seven strains revealed their striking uniformity among diverse NE isolates (Figure S4
). The high level of sequence conservation was surprising and suggests that these plasmids arose from a recent evolutionary event. Southern blotting results with a tcpF
probe, together with the detection of the tcp
locus sequence in the CP4 Solexa reads, indicate that the plasmids carrying NELoc-1 and -3 also carry this conjugation locus. The observation that these unrelated NE strains carry nearly identical plasmid loci is consistent with the hypothesis that the plasmids were acquired through conjugative transfer. This study provides further evidence of the remarkable contribution of closely related multiple plasmids to the infection biology of C. perfringens
in its ability to cause distinct and serious enteric infections in different animal species 
While functional studies are ultimately required to elucidate the contribution of these genes to NE pathogenesis, sequence analysis can assist greatly in this effort. NELoc-1 encodes 37 putative proteins, of which 25 have no apparent counterpart in any of the sequenced C. perfringens
strains. An internalin-like protein is located immediately upstream from netB
containing a leucine-rich repeat (LRR) domain (CP4_3446) with 30–35% identity to Internalin F, D and A of Listeria monocytogenes
and putative internalins of Bacillus
spp.. Originally identified in L. monocytogenes
as surface-anchored proteins required for attachment and invasion 
, internalins now constitute a multi-gene family, with 25 members identified in the L. monocytogenes
genome alone 
. Genome sequencing projects have also revealed internalin-like proteins in C. botulinum
, C. perfringens and C. tetani
, though none have been functionally characterized 
. The hallmark LRR domain, responsible for the horseshoe-like conformation of the protein, is found in a functionally diverse set of proteins, and is typically involved in protein-protein interactions 
. The presence of a VirR-box upstream of the gene suggests that it may be co-regulated with netB
, and therefore also involved in NE pathogenesis.
Immediately downstream from netB
is a gene encoding a 391 aa protein (CP4_3450) containing a beta-trefoil domain similar to that of the carbohydrate-binding ricin B subunit 
. BLAST searches revealed similarity to the ricin-like domains of a diverse set of proteins, including pierisin from Pieris rapae
(27% identity over 334 aa) and MTX from Bacillus sphaericus
(30% over 286 aa), which are related ADP-ribosylating toxins 
, the Cyt1Ca insecticidal pore-forming toxin from Bacillus thuringiensis
(28% over 264 aa) 
, and the HA-1 non-toxin hemagglutinin of C. botulinum
(33% identity over 279 aa) 
. These proteins share a similar secondary structure consisting of the C-terminal ricin-like domain responsible for carbohydrate binding and, with the exception of HA1, a distinct N-terminal active-site domain. The ~130 aa N-terminal region predicted in CP4_3450 did not share sequence similarity with any protein found in GenBank, and a putative function could therefore not be assigned.
and CP4_3450 are oriented divergently from the majority of the other genes in NELoc-1. Given this common orientation and evidence for co-regulation, it is plausible that these three genes are co-transcribed, and may act in unison. One possible scenario is that CP4_3446 and CP4_3450 act directly with NetB to enhance its pore-forming activity, although this seems unlikely as the cytotoxicity of purified recombinant NetB was found to be comparable to that of the native protein 
. Alternatively, they may instead be involved in recognition of eukaryotic cell surface receptors, as suggested by the presence of carbohydrate- (CP4_3450) and protein- (CP4_3446) binding domains and similarity to proteins known to function in this capacity.
The presence of two putative chitinases (CP4_3454 and CP4_3455) is intriguing and suggests a possible role for chitin hydrolysis in NE pathogenesis. This abundant polysaccharide is an insoluble homopolymer of N-acetylglucosamine (GlcNac) found in the exoskeletons of invertebrates and the cell walls of fungi. Chitinases have been detected in other clostridial species, including C. paraputrificum 
and C. botulinum 
, both of which have chitinolytic activity, but to our knowledge this is the first example of a chitinase gene identified in C. perfringens
. These enzymes are typically produced by bacteria that use chitin as a carbon source, or are pathogens of insects or fungi 
. The incorporation of chitin in the diets of chickens has been shown in some cases to improve growth performance 
. The putative chitin synthase (CP4_0468) in NELoc-3 may work in conjunction with the two putative chitinases (CP4_3454 and CP4_3455) in NELoc-1 to take advantage of the chitin as an extra carbon source. The degradation of chitin into GlcNac may also confer a survival advantage to the NE strains since it inhibits the adhesion of probiotic lactobacilli to chicken intestinal mucus 
. Chitin synthase may be needed to convert excess GlcNac back into chitin if the need arises. Intriguingly, in the intestinal bacterial pathogen V. cholerae
the product of a putative chitinase gene with GlcNAc-binding activity has been shown to act as a common adhesion molecule for both chitinous and intestinal surfaces 
. GlcNAc forms the bulk of the carbohydrates present in intestinal mucin and this protein has been shown not only to be involved in intestinal colonization in mice but also to increase the quantity of intestinal mucin produced 
, suggesting a possible role for putative chitinases in intestinal colonization in NE.
Also of interest were two putative leukocidins (CP4_3458 and CP4_3459) that exhibited close similarity to NetB, and to a lesser extent to the related C. perfringens
pore-forming beta and delta toxins 
, hemolysin II of Bacillus spp.
, and the bi-component leukocidins of S. aureus
. The two genes are in the same reading frame; elimination of the intervening stop codon forms a single gene with an intact leukocidin domain, and the predicted protein forms a nearly full-length alignment with NetB. These genes may therefore represent an evolutionary relic of a previous netB
duplication event, or even a now-defunct second member of an original bi-component hetero-oligomeric pore-forming toxin.
The discovery of a potential novel c-di-GMP signaling system, composed of putative PDE (CP4_3469) and DGC (CP4_3478) genes, as well as several nearby genes for putative cell surface proteins (CP4_3471, CP4_3472 and CP4_3473) was of particular interest. This signaling system has recently been shown to play a central role in governing adhesion, motility, and virulence in pathogenic bacteria such as P. aeruginosa
Typhimurium, S. aureus
and V. cholerae 
. The c-di-GMP nucleotide acts as a second messenger by binding and regulating downstream effectors that are only now being elucidated 
. Intracellular c-di-GMP levels are modulated through the opposing activities of a DGC and PDE, responsible for its synthesis and degradation, respectively 
. Both CP4_3469 and CP4_3478 contain the GGDEF domain (responsible for DGC activity), while CP4_3469 also contains an EAL domain (responsible for PDE activity). It is therefore likely that CP4_3478 functions as a DGC, while CP4_3469 may have both PDE and DGC activities, although it is common for PDEs to also carry a non-functional GGDEF domain 
. CP4_3478 also contains a signal peptide and several N-terminal transmembrane regions, making it a good candidate for an integral membrane DGC. The cluster of four surface-associated protein genes found between CP4_3469 and CP4_3478 may encode adhesins under the control of this system. Two of the genes were also found in avirulent strain JGS1473, however, suggesting a limited role in virulence. It is well-recognized that large numbers of C. perfringens
will coat the damaged intestinal epithelial surface of chickens with NE 
, but little to no information exists on the specificity of this adherence.
The recognition of a homolog of the large hypothetical protein CPE1281 from Strain 13 was among many of the unexpected discoveries reported here. Earlier work based on mass spectrometry of proteins expressed by virulent but not avirulent C. perfringens
recovered from birds with NE 
mis-identified the protein as CPE1281, instead of the NE-specific homolog. Immunization of chickens with the most immunogenic part of the CE1281 protein gave excellent protection of birds against experimentally-induced NE 
Epitope-mapping of the CPE1281 protein (not the NE 1281 homolog described here) identified immunodominant regions of the CPE1281 protein, which include the possible zinc-binding signature region of this hypothetical protease in both proteins (G—HELGHNF), which were used in immunization when expressed from a Salmonella
vaccine vector 
. Further work is required to determine whether the efficacy of this protein in protecting birds against NE is the result of cross-protection against the NE CPE1281 homologue.
Two homologs of tubulin/FtsZ (CP4_0461 and CP4_0464) are found in NELoc-2 sharing 33% and 36% protein identity, respectively, with tubulin/FtsZ common to other C. perfringens
strains. The homologs might be similar in function to the Bacillus thuringiensis
tubulin homolog TubZ, which is proposed to facilitate plasmid segregation by coupling with plasmid DNA, via DNA-binding proteins, and causing plasmids separation 
. In this case, NELoc-2 may not be specifically involved in virulence, but might instead contribute to the preservation of the NELoc-1 and -3 virulence plasmids via the two tubulin/FtsZ homologs. This may help explain the frequency of plasmid occurrence in NE stains from different sources.
Extracellular solute-binding proteins (ESBs) of bacteria may serve as chemoreceptors, recognition constituents of transport systems, and initiators of signal transduction pathways, suggesting several possible roles for the conserved ESB gene in NELoc-3 
. The NADP-dependent 7-alpha-hydroxysteroid dehydrogenase found in NELoc-3 shares 64.3% protein identity with the 7-HSDH of C. difficile
. It may similarly be responsible for converting the primary bile acid, chenodeoxycholic acid, into the secondary bile acid, 7-keto-lithocholic acid, which is potentially toxic to other members of the microbiota 
., though it is not clear how NE strains are protected.
PFGE analyses revealed the presence of two to five plasmids, ranging in size from ~45 to 150 kb, in ten NE isolates of known virulence. Southern blotting of pulsed-field gels revealed netB
(NELoc-1) on a ~80 kb–90 kb plasmid in virulent isolates and hdhA
(NELoc-3) on a separate ~70 kb–80 kb plasmid. Only in CP2 were netB
apparently co-localized to the same plasmid. Almost all C. perfringens
toxins are now known to be transcribed from genes on large plasmids. These plasmids share a conserved backbone carrying a tcp
conjugation locus, and it has been suggested that they be referred to as the pCPF5603-like toxin plasmids 
. Alignment of NELoc-1 and -3 and flanking sequences with members of this family reveal a shared structure and site of insertion, most closely resembling pCP8533etx, suggesting that the NELoc-1 and -3 plasmids are genuine members of this growing plasmid family. Toxin genes present in other pCPF5603-like toxin plasmids, including cpe 
, etx 
and iap/ibp 
, are in close proximity to IS1151
sequences, and the detection of circular transposition intermediates carrying both IS1151
and toxin gene sequences 
has led to the suggestion that these elements mobilize many of the C. perfringens
toxin genes 
. Contrary to this, related sequences were not found near either NELoc-1 or NELoc-3, which may indicate that these loci were acquired through a different mechanism, possibly involving a distinct transposable element.
Interestingly, the plasmid on which NELoc-3 resided also contained cpb2
, which has been previously identified in NE isolates 
. The role of the Cpb2 toxin in enteric disease including NE is unclear. Since cpb2
is widespread in C. perfringens
, the sequencing approach taken here failed to identify this gene as part of the “NE signature”, but does not preclude the involvement of it, or other toxins such as CPA, in NE.
Overlapping PCR studies of NELoc-1, -2 and -3 demonstrated a conserved organization and site of insertion among 10 type A NE strains (Figures S6
, and S8
). Only CP2 had a different profile, with the absence of ~6kb encompassing the chitinase B and leukocidin I and II genes. This may be indicative of the stepwise evolution of the virulence loci of C. perfringens
plasmids through addition or deletion of specific segments bordered by IS elements.
In contrast to NELoc-1, both NELoc-2 and -3 were also found in the avirulent strain CP6. Interestingly, this strain was originally recovered from a chicken with NE, but was subsequently avirulent in experimental challenge studies 
. The presence of intact NELoc-2 and -3 in this strain suggests that it may have lost at least part of NELoc-1; anecdotally, loss of virulence is not uncommon in NE isolates and the recognition of the plasmid basis of virulence provides a simple explanation for this well-recognized phenomenon. The conservation of the NELoc-3 plasmid in NE isolates suggests that both plasmids contribute to the virulence of NE strains. Further work is required to characterize this plasmid.
In conclusion, this study has contributed significantly to our understanding of the pathogenetic basis of NE in chickens. The majority of virulence-associated genes identified here are carried on different plasmids in virulent NE strains of C. perfringens, and loss of the locus encoding netB appears to attenuate virulence. The findings of this study also support previous evidence for a common ancestor of the C. perfringens toxin plasmids. Functional characterization of putative virulence genes identified here may provide significant insights into the mechanism of NE pathogenesis.
Genes identified as potential virulence factors can be cloned into suitable vectors for expression of recombinant proteins. It may be determined that codon optimization is necessary to improve levels of protein expressed. Following isolation and purification, the proteins could be combined with an appropriate adjuvant, and used to immunize chickens, providing protection against infection caused by Clostridium perfringens. Alternatively, they could be cloned into vectors suitable for direct vaccination of birds as nucleic acid vaccines. They could also be cloned into viral vectors, which could be transfected into host cells for the production and purification of recombinant virus, which could then be used to immunize birds. Finally, they could be cloned into bacterial expression systems, which could then be isolated as recombinant bacteria, which could also be used to immunize birds.