Next Article in Journal
Ochratoxin A in Roasted Coffee from French Supermarkets and Transfer in Coffee Beverages: Comparison of Analysis Methods
Next Article in Special Issue
Therapeutic Down-Modulators of Staphylococcal Superantigen-Induced Inflammation and Toxic Shock
Previous Article in Journal / Special Issue
The Systemic and Pulmonary Immune Response to Staphylococcal Enterotoxins
Review

NetB, a Pore-Forming Toxin from Necrotic Enteritis Strains of Clostridium perfringens

1
CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia
2
Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Victoria 3800, Australia
*
Author to whom correspondence should be addressed.
Received: 22 June 2010 / Revised: 9 July 2010 / Accepted: 22 July 2010 / Published: 23 July 2010
(This article belongs to the Special Issue Enterotoxins)

Abstract

The Clostridium perfringens necrotic enteritis B-like toxin (NetB) is a recently discovered member of the β-barrel pore-forming toxin family and is produced by a subset of avian C. perfringens type A strains. NetB is cytotoxic for avian cells and is associated with avian necrotic enteritis. This review examines the current state of knowledge of NetB: its role in pathogenesis, its distribution and expression in C. perfringens and its vaccine potential.
Keywords: NetB; necrotic enteritis; Clostridium perfringens; pore-forming toxin NetB; necrotic enteritis; Clostridium perfringens; pore-forming toxin

1. Introduction

C. perfringens is a Gram-positive anaerobe and is ubiquitous in the environment, being found in soil, in decaying organic matter, and as a member of the normal intestinal flora of many humans and animals [1]. Specific C. perfringens toxin types are associated with particular human and animal diseases, indicating that variations in toxin production influence the virulence properties of C. perfringens isolates [2,3,4]. C. perfringens strains are typed by the production of four major toxins (α, β, ε, ι) [3]. Type A strains are the most widespread and are commonly found in the intestines of warm blooded animals and in the environment. Type A strains cause gas gangrene in humans and enteric diseases in humans and animals [5,6,7,8]. Type C strains cause mucosal necrosis of the small intestine in humans and pigs, while type B and C strains cause enteric disease in small ruminants [5].
Necrotic enteritis in poultry is a global problem and it has been estimated that the disease costs the international poultry industry in excess of $US 2 billion per year in production losses and control measures [9,10,11]. Avian necrotic enteritis is an enteric disease that is characterized by necrotic lesions in the small intestinal mucosa [12]. The disease can be divided into two forms, clinical and subclinical. The clinical signs of acute necrotic enteritis can be seen from about two weeks of age and include marked depression, dehydration, apathy, diarrhea, ruffled feathers, and decreased feed consumption [13,14,15,16]. Birds showing clinical signs normally die within a few hours, with mortality rates reaching up to 1% per day [13]. The diagnosis of subclinical necrotic enteritis is based on a decrease in the feed conversion ratio and the detection of gross necrotic lesions in the small intestinal mucosa (Figure 1) followed by bacteriologic analysis and genotyping of isolates [12]. Macroscopic lesions can be seen in the small intestine and can sometimes be found in other organs, such as the liver, kidney and cecum [17]. In an affected bird, the small intestine can become enlarged due to gas accumulation and the wall of the intestine can become thin and delicate [18]. Subclinical necrotic enteritis is observed at varying ages of birds, but most commonly it is first detected in birds at 21 to 23 days of age [9]. The subclinical form of the disease is not only of significance to the animals’ welfare, but also to the producer through lost productivity and the added cost of disease intervention.
Figure 1. Gross pathology of the small intestine of infected birds. (ac) are three examples of necrotic enteritis lesions in the small intestines of broiler chickens challenged with C. perfringens.
Figure 1. Gross pathology of the small intestine of infected birds. (ac) are three examples of necrotic enteritis lesions in the small intestines of broiler chickens challenged with C. perfringens.
Toxins 02 01913 g001
All C. perfringens strains produce α-toxin, a major extracellular toxin that has been shown to be essential for human myonecrosis [19]. It was previously thought [14,20,21] that α-toxin was the major toxin involved in necrotic enteritis, but we have shown that an α-toxin null mutant is still virulent in a chicken necrotic enteritis disease model [22]. This result led us to postulate that virulent necrotic enteritis strains produce other toxins that are responsible for the pathogenesis of disease. Recently, we identified a new toxin, NetB, in avian C. perfringens type A strains [23]. NetB is a key virulence factor in C. perfringens strains that cause avian necrotic enteritis. Here we review our current knowledge of the biology, distribution, regulation, role in disease, and potential vaccine applications of this new toxin.

2. Identification and Analysis of NetB

NetB was discovered in an Australian strain of C. perfringens type A that was isolated from a chicken with necrotic enteritis [23]. Earlier work, aimed at defining the role of α-toxin, had indicated that secreted products from C. perfringens were able to cause lesions typical of necrotic enteritis in chickens [20]. Based on these results, and our finding that α-toxin was not required for disease, we tested culture supernatant from our α-toxin mutant against various mammalian and chicken cell lines to determine whether any novel cytotoxic factors could be identified. One cell line, LMH (ATCC CRL-2117), was found to be sensitive to an unknown protein in the supernatant. Subsequently, LMH cytotoxicity was used to follow this toxic activity during the purification process. The N-terminal sequence of the purified protein was determined and used to search the genome sequence of the parent C. perfringens strain, EHE-NE18. A novel toxin gene that encoded a 323 amino acid protein, including a 30 amino acid secretion signal sequence, was identified. Since this protein has similarity to C. perfringens β-toxin (38% identity) it was designated necrotic enteritis toxin, B-like (NetB) [23]. In addition to β-toxin, NetB has sequence identity to C. perfringens δ-toxin (40% identity), Staphylococcus aureus α-hemolysin (30% identity), the A, B and C components of S. aureus γ-toxin (25%, 22% and 23% identity, respectively), the S and F components of S. aureus leukocidin (25% and 22% identity, respectively) and to Bacillus cereus cytotoxin K (27% identity). Alignment of these sequences (Figure 2) reveals that only 14 amino acids are identical in these proteins and that 50 residues are conservatively substituted. The areas of similarity are dispersed throughout the toxin, with the weakest similarity in the N-terminal region.
At least 35% of the known protein toxins produced by bacteria belong to the pore-forming group of membrane damaging toxins [24]. These toxins form pores that disrupt the phospholipid membrane bilayer of both human and animal cells, causing an influx of ions (i.e., Na+, Cl, Ca2+, etc.) that may lead to osmotic cell lysis. Many of these toxins have been demonstrated to contribute to the virulence of bacteria and to play a key role in the pathogenesis of human and animal infections [25,26,27,28,29,30,31]. NetB has many of the conserved residues found in pore-forming toxins of the S. aureus toxin family [32]. For example, the α-hemolysin, or α-toxin, from S. aureus is a secreted, self-assembling, pore-forming toxin [33]. It has a signal peptide of 26 amino acids and in its mature form consists of a 293 amino acid polypeptide with a calculated molecular size of 33 kDa [34,35]. β-toxin also is a member of this pore‑forming family and is a lethal extracellular toxin produced by C. perfringens type B and C strains [36]. β-toxin is secreted in late exponential growth phase and is extremely susceptible to proteases and to heat [37,38]. Located on a plasmid [39], the β-toxin structural gene, cpb, encodes a 336 amino acid polypeptide that includes a 27-amino acid signal sequence, which is cleaved to form the mature β-toxin protein (34 kDa) [40].
Figure 2. ClustalW alignment of NetB. ClustalW alignment of the toxins C. perfringens NetB (EU143239), C. perfringens β-toxin (Cpb, AAA23284.1), C. perfringens δ-toxin (Cpd, ACF93710.1), B. cereus hemolysin II (Hly-II, NP_833256.1), cytotoxin K (CytK, CAC08441.1) and S. aureus α-toxin (Hla, NP_371687.1), γ-toxin A (HlgA, P0A074.1), B (HlgB, P0A077.1), C (HlgC, Q07227.1), Leukocidin S (LukS, NP_058465.1), F (LukF, NP_058466.1). Identical residues (*), conservative amino acid substitutions (:) and semi-conservative amino acid substitutions (.) are shown below the aligned sequences. Residues highlighted in bold represent the known or predicted signal peptide sequence of each protein and residue numbers begin following the signal peptide sequence. Red boxes indicate those residues that are known to be involved in the activity of at least one of these proteins, as discussed in the text. To compact the figure the Hly-II sequence was shortened.
Figure 2. ClustalW alignment of NetB. ClustalW alignment of the toxins C. perfringens NetB (EU143239), C. perfringens β-toxin (Cpb, AAA23284.1), C. perfringens δ-toxin (Cpd, ACF93710.1), B. cereus hemolysin II (Hly-II, NP_833256.1), cytotoxin K (CytK, CAC08441.1) and S. aureus α-toxin (Hla, NP_371687.1), γ-toxin A (HlgA, P0A074.1), B (HlgB, P0A077.1), C (HlgC, Q07227.1), Leukocidin S (LukS, NP_058465.1), F (LukF, NP_058466.1). Identical residues (*), conservative amino acid substitutions (:) and semi-conservative amino acid substitutions (.) are shown below the aligned sequences. Residues highlighted in bold represent the known or predicted signal peptide sequence of each protein and residue numbers begin following the signal peptide sequence. Red boxes indicate those residues that are known to be involved in the activity of at least one of these proteins, as discussed in the text. To compact the figure the Hly-II sequence was shortened.
Toxins 02 01913 g002
Site-directed mutagenesis has been used to characterize many amino acids critical for function in C. perfringens β-toxin and S. aureusα-toxin [41,42] and NetB contains many of these key residues including R200 (R212 from β-toxin). The corresponding residue in S. aureus α-toxin (R200) is important for binding and oligomerization of the protein as well as for hemolysis [43]. Mutation of the β-toxin residue Y203 (Y191 in NetB) to phenylalanine resulted in a 2.5-fold increase in the LD50. The corresponding residue in S. aureus α-toxin (Y191) is located at the predicted membrane binding surface of the protein [44]. Mutation of the β-toxin residue D167 (D156 in NetB) resulted in a complete loss of functional protein expression, suggesting that this region participates in crucial protomer-protomer interactions as well as being important for conformational rearrangements involved in multimer formation in the membrane bound form [44].
Since NetB had many of the residues essential for β-toxin and S. aureus α-toxin function we postulated that NetB was likely to be a pore-forming toxin. Subsequent studies demonstrated that NetB was found to be cytotoxic to LMH cells but not to other chicken cell lines such as DF1 and HD11 cells or the mammalian Vero cell line [23]. NetB (2.5 ng/µL) causes significant cell rounding and cell lysis in as little as 30 min. Incubation with PEG 1000 and PEG 1500 blocked these morphological changes and LDH release from the cells, indicating that NetB most likely forms plasma membrane pores with an estimated pore diameter of 1.6 to 1.8 nm [23]. This NetB pore diameter is slightly larger than the pore size predicted for β-toxin (1.36–1.6 nm) in HL 60 cells [45]. β-toxin has been shown to induce significant host cell swelling without blebbing, cell lysis, and the release of K+ from human leukemia cells (HL 60) [45]. Incubation of HL 60 cells with β-toxin resulted in the formation of toxin complexes of about 191 kDa (hexamer) and 228 kDa (heptamer). The 228 kDa complex was observed up to 30 min after incubation with the cells, while the 191 kDa complex remained after 60 min.
δ-toxin is another C. perfringens pore-forming toxin that has sequence identity with NetB [46]. This toxin is a hemolysin released by several C. perfringens type C and possibly type B strains [47]. It is cytotoxic for various cell types such as rabbit macrophages, human monocytes, and blood platelets from goats, rabbits, humans and guinea pigs [46,48,49,50]. The toxin forms pores in artificial lipid bilayers similar to β-toxin, however, these toxins do not share a similar receptor since δ-toxin binds to gangliosides (GM2) unlike β-toxin [46]. Some C. perfringens type B and C strains that are involved in necrotic enteritis in various animal species, mainly piglets, and also in humans, produce δ-toxin, but no direct link between this toxin and disease has been demonstrated.
The essential role of NetB in disease was determined by constructing a structural gene (netB) mutant in strain EHE-NE18 and assessing its virulence in a chicken disease model [23]. Virulence testing of an isogenic series of strains consisting of the wild type, netB mutant, and netB mutant complemented with the wild-type netB gene, revealed that the development of necrotic enteritis in chickens was dependent on the ability to produce functional NetB toxin. This result parallels studies on the α-toxin of S. aureus, which in a mouse model of S. aureus pneumonia has been shown to be an important virulence factor of methicillin-resistant S. aureus (MRSA) [51]. S. aureus α-toxin negative mutants were found to be 10-fold less virulent than α-toxin producing strains. Similar results have been obtained for β-toxin and it has been shown to be essential for hemorrhagic necrotizing enteritis in rabbit ileal loops [52]. In that study, mutations in the β-toxin structural gene, cpb, were constructed and shown to be avirulent in the ileal loop model. Complementation of a cpb mutant restored its ability to produce β-toxin and restored virulence in the loop model. In addition, highly purified β-toxin was able to reproduce intestinal damage that was similar to infection with the wild-type C. perfringens strain. Both NetB and β-toxin act in the gastrointestinal tract of animals, however, β-toxin has also been suggested to cause toxemia after absorption of the toxin from the intestines into the circulation [53,54]. However, no experiments aimed at determining if NetB enters the circulatory system of infected chickens have been reported.
Recent studies have revealed that in several necrotic enteritis isolates the netB gene is part of a large potential pathogenicity locus [55]. Comparative analysis of genomic sequences from seven independent disease-causing C. perfringens isolates and a range of non-necrotic enteritis strains showed that the netB gene is located on a 42 kilobase (kb) locus (NELoc-1). In addition, Southern blots suggest that the netB gene is located on an approximately 85 kb plasmid. From the genomic analysis, two other smaller loci (NELoc-2 and NELoc-3) also appear to be associated with necrotic enteritis strains. The identification of these pathogenicity loci may lead to the discovery of additional virulence factors that are involved in the pathogenesis of disease.

3. Strain Distribution

C. perfringens is commonly present within the normal flora of the gastrointestinal tract of chickens. However, under conditions that are still not fully understood, birds can develop necrotic enteritis, with disease progression correlating with the proliferation of C. perfringens cells in the intestine. To understand what types of C. perfringens isolates are involved in this changing ecological balance, several studies have examined the clonal variation of C. perfringens isolates. Epidemiological studies of strains from varied geographical locations have shown that healthy chickens harbor a range of genotypes, as determined by PCR, PFGE and MLST studies, while isolates from birds present in individual flocks that have acute necrotic enteritis tend to have only one genotype of C. perfringens [56,57,58,59,60,61,62,63,64]. In contrast to these findings, which involved acute disease outbreaks, in one study birds from a single flock that displayed mild necrotic enteritis symptoms were found to harbor a genetically varied range of isolates [64].
Since the discovery of NetB [23] several studies have screened for the presence of the netB gene within a wide variety of C. perfringens strains. Initial screening of a range of Australian poultry necrotic enteritis isolates found that the majority (77%) were netB positive [23]. In addition, in vitro expression analysis confirmed that all of the netB positive strains that were tested produced the NetB protein. Furthermore, several non-necrotic enteritis strains were analyzed and found to be netB negative. These netB-negative strains included representatives of each of the toxin types A to E, which had been isolated from cattle, sheep, pigs and humans. Subsequent studies that screened for the netB gene in strains of C. perfringens from other geographical regions showed that there is a good correlation between the isolation of C. perfringens from a diseased bird and the presence of the netB gene [59,64,65,66]. In combination, 60 to 90% of the strains isolated from birds with necrotic enteritis were found to have the netB gene. Chalmers et al. [59], studied Canadian C. perfringens strains, and found that the netB gene was detected only in isolates associated with necrotic enteritis outbreaks; it was not identified in isolates from healthy birds. Analysis of isolates from the USA showed that the netB gene was in 60% of necrotic enteritis strains (7/12) [65]. Examination of a single broiler flock from Sweden found that more than 90% of all isolates from necrotic enteritis-affected poultry were netB positive [64]. The same trend was found in an extended study of strains from three continents [66], where 70% of isolates from necrotic enteritis affected birds were netB positive. Together, these results suggest that the NetB toxin is necrotic enteritis specific, since the netB gene is strongly associated with necrotic enteritis-derived strains from poultry. Until recently, the netB gene had only been found in poultry related strains, however, the first non-poultry based recovery of a netB positive C. perfringens isolate has now been reported. A netB positive strain was isolated from liver abscesses from a cow that had died with gastrointestinal disease, although this particular C. perfringens strain was not considered to be the causative agent [65]. In a chicken disease model, this bovine isolate has also been shown to cause lesions characteristic of avian necrotic enteritis [67].
Not all C. perfringens strains isolated from birds that clearly displayed signs of necrotic enteritis were netB positive. In nearly every study, there was a minor fraction of netB negative isolates from diseased birds [23,59,64,65,66,68]. In one particular study [65] a netB negative isolate was recovered from a chicken displaying clear signs of necrotic enteritis. To show that there were no sampling errors involved, 25 isolates were recovered from different lesion areas of the same chicken; they were all netB negative. These results imply that although there is a clear association between production of NetB toxin and development of the pathology of necrotic enteritis, there may be other—yet to be determined—virulence factors that are produced by these netB negative disease-producing strains. By contrast, several netB negative strains isolated from lesion material have been tested for their ability to cause disease in necrotic enteritis animal models and found to induce little or no signs of disease [66,67,69]. Importantly, one netB negative strain, JGS4104, induced a significant level of disease (43.8%) [69], again suggesting that there may be other factors, besides NetB, that allow C. perfringens type A to cause tissue damage similar to necrotic enteritis. In this study the strains re-isolated from the diseased birds were analyzed by PFGE. Given the recent demonstration that the netB structural gene is plasmid borne [55], it is unclear if it is possible using PFGE to detect a difference between a strain that remains netB negative and one that has obtained the NELoc-1-containing plasmid. Clearly, more research needs to be done to test and assess the virulence of additional isolates of different origins and genotypes in the chicken disease model.
Whilst the netB gene has been found mainly in association with poultry with necrotic enteritis, it has also been identified in C. perfringens strains from healthy birds. In one study [65], 8.8% of isolates (7/80) from normal chickens were netB positive. In another study, analysis of strains from a single broiler flock that contained birds affected with mild necrotic enteritis, revealed that 25% of the isolates from apparently healthy birds were netB positive [64]. Nowell et al. 2009 [70] used an enrichment process to recover 88 C. perfringens isolates from retail grocery chicken samples and found that the netB gene could be detected in 21% of these isolates. Therefore, as previously suggested, it appears that the presence of a netB positive strain is not sufficient to cause disease, predisposing conditions are required. Analysis of strains from various Danish broiler flocks found that 61% of isolates recovered from healthy birds were netB positive [68]. This was markedly different from the results obtained from previous studies on strains from healthy birds [59,65,66]. When these Danish netB positive isolates were tested for in vitro expression of the NetB protein, a marked difference in the NetB expression rates was observed [68]. The NetB protein was found in 12 out of 13 of the isolates from the necrotic enteritis diseased birds. By comparison, only four out of 14 PCR positive isolates from healthy birds produced detectable NetB protein. These results are in stark contrast to our own findings, where every strain that carries netB produces NetB protein. It would be interesting to test these NetB producing and NetB non-producing netB positive strains isolated from healthy birds for NetB expression in vivo and to determine if they are equally capable of disease production.

4. Regulation of NetB Production and NetB Sequence Variation

Expression of the netB gene is regulated by VirSR, the global regulator of C. perfringens toxin production [71]. In the human gas gangrene-causing C. perfringens type A strain 13, the VirSR system is part of a complex signal transduction pathway that directly regulates the production of extracellular toxins and enzymes such as perfringolysin O and α-clostripain and regulates the expression of α-toxin, collagenase and many housekeeping genes by controlling the expression of the regulatory RNA molecule, VR-RNA [72]. In two separate necrotic enteritis derived C. perfringens strains, mutation of the virR gene greatly reduced the production of NetB toxin [71]. This effect was reversed when the virR mutants were complemented with the wild-type virR gene. VirR acts on its target genes by binding to two directly repeated sequences, or VirR boxes, that are located immediately upstream of the promoter. Similar repeats are located upstream of the putative netB promoter. These VirR boxes were shown to be functional in a C. perfringens-based bioassay and in vitro studies showed that purified VirR protein could bind to the netB-associated VirR boxes. Other workers have shown that in strain 13 the VirSR system appears to be regulated by an Agr-dependent quorum sensing system that leads to the controlled expression of specific genes in response to changes in cell density [73]. These findings suggest that in the gastrointestinal tract of infected birds the production of NetB is activated when the C. perfringens population reaches a threshold level as a result of predisposing factors, as reviewed previously [17,74], and nutrients start to become rate-limiting for growth.
Any differences in necrotic enteritis disease progression could potentially be explained by sequence differences in the NetB proteins produced by different strains. To examine the variation of NetB proteins, the nucleotide sequence of the netB gene from a range of isolates was determined [66]. The poultry isolates examined were from various geographical locations including Australia, Belgium, Denmark and Canada. The netB gene and its promoter region displayed very little sequence variation; most of these strains had virtually identical nucleotide sequences in this region. Within the netB coding sequence there was only one amino acid change, A168T, which was observed in six strains. Biological analysis of the NetBA168T derivative showed that it was as active as the wild-type NetB protein and that strains carrying the variant sequence produced similar levels and types of disease when used in infection studies. The observation that NetB is very well conserved was supported by a separate study [68], which found that most of the 27 netB positive isolates examined contained wild-type coding and promoter regions. The only amino acid sequence changes observed were NetBA168T, the variant previously reported, and a single example of a novel variant, NetBA166V.

5. NetB and the Development of a Necrotic Enteritis Vaccine

Since NetB is essential for disease pathogenesis, and is a secreted toxin that should be readily accessible to the host immune system, it has considerable potential for vaccine development [23]. Vaccines against C. perfringens or its toxins have been successfully used to prevent enteric diseases in various mammalian species, including humans [75,76,77]. Vaccine development for necrotic enteritis in chickens has previously focused on α-toxin, but the experimental vaccines produced have not provided the level of vaccine efficacy seen with clostridial vaccines in other animal species, presumably because α-toxin is not essential for virulence. It has been shown that chicken flocks with high levels of maternal antibodies against α-toxin have lower mortality levels than flocks with lower titers [78]. Vaccines based on C. perfringens type A and C have been experimentally tested [79]. Layer hens vaccinated with these toxoids had high levels of anti-α-toxin antibodies in their progeny compared to unvaccinated hens. Both toxoid vaccines showed levels of protection against subclinical necrotic enteritis, with the type C toxoid vaccine giving higher levels of protection than the type A toxoid. α-toxin has also been delivered as a subunit vaccine and by live delivery in Salmonella vectors, where it has been shown to provide some protection [80,81,82]. Recently, a commercial vaccine (NETVAX®, Schering-Plough) has been developed based on a C. perfringens type A toxoid. However, the NetB status of the organisms used to develop this vaccine is not known.
Other studies aimed at the development of a necrotic enteritis vaccine have been reported. Analysis of serum from birds infected with virulent C. perfringens isolates identifiedseveralimmunogenic secreted proteins [80]. These proteins included α-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, fructose 1,6-biphosphate aldolase, and a hypothetical protein. These proteins significantly protected broiler chickensagainst a relatively mild challenge, with α-toxin, pyruvate:ferredoxinoxidoreductase and the hypothetical protein offering protection against a more severe challenge [81]. This study provided the first evidence that a subunit vaccine could be useful in controlling necrotic enteritis in chickens. Of interest is that the chicken serum did not react strongly with NetB; possibly because NetB is not highly immunogenic in chickens or the levels of NetB produced in vivo are not high enough to promote reliable immune recognition.
There are no reports in the literature addressing the use of NetB in vaccine formulations, although there are reports of related proteins from other bacteria being successfully used in experimental vaccines. For example, vaccine-based targeting of S. aureus α-toxin provided protection against staphylococcal pneumonia [83,84]. In a murine model system, monoclonal antibody-basedtherapy was efficacious for preventionand treatment of this disease [84]. Two distinct anti-α-toxin monoclonal antibodies that blocktoxin activity prevented human lung cell injury in vitro and protected experimental animals against lethal S. aureus pneumonia. Active immunization with the first 50 amino acids of α-toxin also conferred protection against S. aureus pneumonia. This study revealed that both passive and active immunization strategies for prevention or therapy of staphylococcal pneumonia were feasible; similar approaches could be attempted using NetB to protect against necrotic enteritis. A genetically toxoided version of S. aureus α-toxin, which was produced by site-directed mutagenesis, has been shown to generate an antigen-specific immunoglobulin G response and to be protective against staphylococcal pneumonia [83]. A similar genetically toxoided version of NetB may have value for necrotic enteritis vaccine design.

6. Conclusions

The recently identified pore-forming toxin NetB is a key virulence determinant in C. perfringens strains that cause necrotic enteritis in chickens. The dimensions of the pore formed by the toxin have been estimated, but the determination of its exact structure and its comparison to other pore-forming toxins awaits crystallization studies. The toxin is produced by most strains isolated from necrotic lesions, but is less commonly found in C. perfringens isolates from healthy birds. As with many of the toxins encoded by C. perfringens, NetB production is regulated by the VirSR two-component regulatory system, potentially by a quorum sensing-dependent mechanism. NetB is cytotoxic to chicken LMH cells but not to several other chicken cell lines or Vero cells. Further studies aimed at determining its cellular specificity and identifying its receptor should help to elucidate the precise role that NetB plays in disease pathogenesis. Since NetB is involved in virulence, and is a secreted protein that should be readily accessible to the host immune system, it represents a promising target for vaccine development. Our growing knowledge of the importance and function of NetB may also lead to the development of useful therapeutic agents and biomarkers. The discovery that netB is part of a predicted pathogenicity locus that encodes other potential virulence factors indicates that further research is required to further elucidate the mechanism of pathogenesis of this important disease.

Acknowledgements

Research in the authors’ laboratories was supported by the Australian Poultry Cooperative Research Centre and the Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics.

References

  1. Hatheway, C.L. Toxigenic clostridia. Clin. Microbiol. Rev. 1990, 3, 66–98. [Google Scholar]
  2. Petit, L.; Gibert, M.; Popoff, M.R. Clostridium perfringens: toxinotype and genotype. Trends Microbiol. 1999, 7, 104–110. [Google Scholar]
  3. Rood, J.I. Virulence genes of Clostridium perfringens. Annu. Rev. Microbiol. 1998, 52, 333–360. [Google Scholar]
  4. Smedley, J.G., III; Fisher, D.J.; Sayeed, S.; Chakrabarti, G.; McClane, B.A. The enteric toxins of Clostridium perfringens. Rev. Physiol. Biochem. Pharmacol. 2004, 152, 183–204. [Google Scholar] [PubMed]
  5. Songer, J.G. Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev. 1996, 9, 216–234. [Google Scholar]
  6. Brynestad, S.; Granum, P.E. Clostridium perfringens and foodborne infections. Int. J. Food Microbiol. 2002, 74, 195–202. [Google Scholar]
  7. Rood, J.I.; McClane, B.A. Clostridium perfringens: enterotoxaemic Diseases. In Molecular Medical Microbiology; Sussman, M., Ed.; Academic: London, UK, 2002; Volume 1, pp. 1117–1139. [Google Scholar]
  8. Rood, J.I. Clostridium perfringens and histotoxic disease. In The Prokaryotes: A Handbook on the Biology of Bacteria, 3rd; Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.H., Stackebrandt, E., Eds.; Springer: New York, NY, USA, 2007; Volume 4, pp. 753–770. [Google Scholar]
  9. Van der Sluis, W. Clostridial enteritis—a syndrome. World Poultry 2000, 16, 56–57. [Google Scholar]
  10. Van der Sluis, W. Clostridial enteritis is an often underestimated problem. World Poultry 2000, 16, 42–43. [Google Scholar]
  11. Lovland, A.; Kaldhusdal, M. Severely impaired production performance in broiler flocks with high incidence of Clostridium perfringens-associated hepatitis. Avian Pathol. 2001, 30, 73–81. [Google Scholar]
  12. Kaldhusdal, M.; Hofshagen, M. Barley inclusion and avoparcin supplementation in broiler diets. 2. Clinical, pathological, and bacteriological findings in a mild form of necrotic enteritis. Poult. Sci. 1992, 71, 1145–1153. [Google Scholar] [PubMed]
  13. Helmboldt, C.F.; Bryant, E.S. The pathology of necrotic enteritis in domestic fowl. Avian Dis. 1971, 15, 775–780. [Google Scholar]
  14. Al-Sheikhly, F.; Truscott, R.B. The pathology of necrotic enteritis of chickens following infusion of broth cultures of Clostridium perfringens into the duodenum. Avian Dis. 1977, 21, 230–240. [Google Scholar]
  15. Al-Sheikhly, F.; Al-Saieg, A. Role of Coccidia in the occurrence of necrotic enteritis of chickens. Avian Dis. 1980, 24, 324–333. [Google Scholar]
  16. Gazdzinski, P.; Julian, R.J. Necrotic enteritis in turkeys. Avian Dis. 1992, 36, 792–798. [Google Scholar]
  17. Van Immerseel, F.; De Buck, J.; Pasmans, F.; Huyghebaert, G.; Haesebrouck, F.; Ducatelle, R. Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathol. 2004, 33, 537–549. [Google Scholar]
  18. Broussard, C.T.; Hofacre, C.L.; Page, R.K.; Fletcher, O.J. Necrotic enteritis in cage-reared commercial layer pullets. Avian Dis. 1986, 30, 617–619. [Google Scholar]
  19. Awad, M.M.; Bryant, A.E.; Stevens, D.L.; Rood, J.I. Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringens-mediated gas gangrene. Mol. Microbiol. 1995, 15, 191–202. [Google Scholar]
  20. Al-Sheikhly, F.; Truscott, R.B. The pathology of necrotic enteritis of chickens following infusion of crude toxins of Clostridium perfringens into the duodenum. Avian Dis. 1977, 21, 241–255. [Google Scholar]
  21. Fukata, T.; Hadate, Y.; Baba, E.; Uemura, T.; Arakawa, A. Influence of Clostridium perfringens and its toxin in germ-free chickens. Res. Vet. Sci. 1988, 44, 68–70. [Google Scholar]
  22. Keyburn, A.L.; Sheedy, S.A.; Ford, M.E.; Williamson, M.M.; Awad, M.M.; Rood, J.I.; Moore, R.J. Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens. Infect. Immun. 2006, 74, 6496–6500. [Google Scholar]
  23. Keyburn, A.L.; Boyce, J.D.; Vaz, P.; Bannam, T.L.; Ford, M.E.; Parker, D.; Di Rubbo, A.; Rood, J.I.; Moore, R.J. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog. 2008, 4, e26. [Google Scholar]
  24. Alouf, J.E. Pore-forming bacterial protein toxins: an overview. Curr. Top. Microbiol. Immunol. 2001, 257, 1–14. [Google Scholar]
  25. Bhakdi, S.; Tranum-Jensen, J. Alpha-toxin of Staphylococcus aureus. Microbiol. Rev. 1991, 55, 733–751. [Google Scholar]
  26. Paton, J.C. The contribution of pneumolysin to the pathogenicity of Streptococcus pneumoniae. Trends Microbiol. 1996, 4, 103–106. [Google Scholar]
  27. Rubins, J.B.; Charboneau, D.; Fasching, C.; Berry, A.M.; Paton, J.C.; Alexander, J.E.; Andrew, P.W.; Mitchell, T.J.; Janoff, E.N. Distinct roles for pneumolysin's cytotoxic and complement activities in the pathogenesis of pneumococcal pneumonia. Am. J. Respir. Crit. Care Med. 1996, 153, 1339–1346. [Google Scholar]
  28. Ellemor, D.M.; Baird, R.N.; Awad, M.M.; Boyd, R.L.; Rood, J.I.; Emmins, J.J. Use of genetically manipulated strains of Clostridium perfringens reveals that both alpha-toxin and theta-toxin are required for vascular leukostasis to occur in experimental gas gangrene. Infect. Immun. 1999, 67, 4902–4907. [Google Scholar]
  29. Schmiel, D.H.; Miller, V.L. Bacterial phospholipases and pathogenesis. Microbes Infect. 1999, 1, 1103–1112. [Google Scholar]
  30. Titball, R.W. Membrane-damaging and cytotoxic phospholipases. In The Comprehensive Sourcebook of Bacterial Protein Toxins; Alouf, J.E., Freer, J.H., Eds.; Academic: London, UK, 1999; pp. 311–329. [Google Scholar]
  31. Kennedy, C.L.; Krejany, E.O.; Young, L.F.; O'Connor, J.R.; Awad, M.M.; Boyd, R.L.; Emmins, J.J.; Lyras, D.; Rood, J.I. The alpha-toxin of Clostridium septicum is essential for virulence. Mol. Microbiol. 2005, 57, 1357–1366. [Google Scholar]
  32. Prevost, G.; Mourey, L.; Colin, D.A.; Menestrina, G. Staphylococcal pore-forming toxins. Curr. Top. Microbiol. Immunol. 2001, 257, 53–83. [Google Scholar]
  33. Gouaux, E. alpha-Hemolysin from Staphylococcus aureus: an archetype of beta-barrel, channel-forming toxins. J. Struct. Biol. 1998, 121, 110–122. [Google Scholar]
  34. Tweten, R.K.; Christianson, K.K.; Iandolo, J.J. Transport and processing of staphylococcal alpha-toxin. J. Bacteriol. 1983, 156, 524–528. [Google Scholar]
  35. Gray, G.S.; Kehoe, M. Primary sequence of the alpha-toxin gene from Staphylococcus aureus wood 46. Infect. Immun. 1984, 46, 615–618. [Google Scholar]
  36. Shatursky, O.; Bayles, R.; Rogers, M.; Jost, B.H.; Songer, J.G.; Tweten, R.K. Clostridium perfringens beta-toxin forms potential-dependent, cation-selective channels in lipid bilayers. Infect. Immun. 2000, 68, 5546–5551. [Google Scholar]
  37. Sakurai, J.; Duncan, C.L. Some properties of beta-toxin produced by Clostridium perfringens type C. Infect. Immun. 1978, 21, 678–680. [Google Scholar]
  38. Vidal, J.E.; McClane, B.A.; Saputo, J.; Parker, J.; Uzal, F.A. Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon. Infect. Immun. 2008, 76, 4396–4404. [Google Scholar]
  39. Katayama, S.; Dupuy, B.; Daube, G.; China, B.; Cole, S.T. Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne. Mol. Gen. Genet. 1996, 251, 720–726. [Google Scholar]
  40. Hunter, S.E.; Brown, J.E.; Oyston, P.C.; Sakurai, J.; Titball, R.W. Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus. Infect. Immun. 1993, 61, 3958–3965. [Google Scholar]
  41. Steinthorsdottir, V.; Fridriksdottir, V.; Gunnarsson, E.; Andresson, O.S. Site-directed mutagenesis of Clostridium perfringens beta-toxin: expression of wild-type and mutant toxins in Bacillus subtilis. FEMS Microbiol. Lett. 1998, 158, 17–23. [Google Scholar]
  42. Nagahama, M.; Kihara, A.; Miyawaki, T.; Mukai, M.; Sakaguchi, Y.; Ochi, S.; Sakurai, J. Clostridium perfringens beta-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity. Biochim. Biophys. Acta 1999, 1454, 97–105. [Google Scholar]
  43. Walker, B.; Bayley, H. Key residues for membrane binding, oligomerization, and pore forming activity of staphylococcal alpha-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification. J. Biol. Chem. 1995, 270, 23065–23071. [Google Scholar]
  44. Song, L.; Hobaugh, M.R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J.E. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 1996, 274, 1859–1866. [Google Scholar]
  45. Nagahama, M.; Hayashi, S.; Morimitsu, S.; Sakurai, J. Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells. J. Biol. Chem. 2003, 278, 36934–36941. [Google Scholar]
  46. Manich, M.; Knapp, O.; Gibert, M.; Maier, E.; Jolivet-Reynaud, C.; Geny, B.; Benz, R.; Popoff, M.R. Clostridium perfringens delta toxin is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences. PLoS One 2008, 3, e3764. [Google Scholar]
  47. Alouf, J.E.; Jolivet-Reynaud, C. Purification and characterization of Clostridium perfringens delta-toxin. Infect. Immun. 1981, 31, 536–546. [Google Scholar]
  48. Jolivet-Reynaud, C.; Cavaillon, J.M.; Alouf, J.E. Selective cytotoxicity of Clostridium perfringens delta toxin on rabbit leukocytes. Infect. Immun. 1982, 38, 860–864. [Google Scholar]
  49. Cavaillon, J.M.; Jolivet-Reynaud, C.; Fitting, C.; David, B.; Alouf, J.E. Ganglioside identification on human monocyte membrane with Clostridium perfringens delta-toxin. J. Leukoc. Biol. 1986, 40, 65–72. [Google Scholar]
  50. Jolivet-Reynaud, C.; Launay, J.M.; Alouf, J.E. Damaging effects of Clostridium perfringens delta toxin on blood platelets and their relevance to ganglioside GM2. Arch. Biochem. Biophys. 1988, 262, 59–66. [Google Scholar]
  51. Patel, A.H.; Nowlan, P.; Weavers, E.D.; Foster, T. Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement. Infect. Immun. 1987, 55, 3103–3110. [Google Scholar]
  52. Sayeed, S.; Uzal, F.A.; Fisher, D.J.; Saputo, J.; Vidal, J.E.; Chen, Y.; Gupta, P.; Rood, J.I.; McClane, B.A. Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model. Mol. Microbiol. 2008, 67, 15–30. [Google Scholar]
  53. Bergeland, M.E. Pathogenesis and immunity of Clostridium perfringens type C enteritis in swine. J. Am. Vet. Med. Assoc. 1972, 160, 568–571. [Google Scholar]
  54. Fisher, D.J.; Fernandez-Miyakawa, M.E.; Sayeed, S.; Poon, R.; Adams, V.; Rood, J.I.; Uzal, F.A.; McClane, B.A. Dissecting the contributions of Clostridium perfringens type C toxins to lethality in the mouse intravenous injection model. Infect. Immun. 2006, 74, 5200–5210. [Google Scholar]
  55. Lepp, D.; Roxas, B.; Parreira, V.R.; Marri, P.R.; Rosey, E.L.; Gong, J.; Songer, J.G.; Vedantam, G.; Prescott, J.F. Identification of novel pathogenicity loci in Clostridium perfringens strains that cause avian necrotic enteritis. PLoS One 2010, 5, e10795. [Google Scholar]
  56. Engstrom, B.E.; Fermer, C.; Lindberg, A.; Saarinen, E.; Baverud, V.; Gunnarsson, A. Molecular typing of isolates of Clostridium perfringens from healthy and diseased poultry. Vet. Microbiol. 2003, 94, 225–235. [Google Scholar]
  57. Nauerby, B.; Pedersen, K.; Madsen, M. Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens. Vet. Microbiol. 2003, 94, 257–266. [Google Scholar]
  58. Gholamiandekhordi, A.R.; Ducatelle, R.; Heyndrickx, M.; Haesebrouck, F.; Van Immerseel, F. Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status. Vet. Microbiol. 2006, 113, 143–152. [Google Scholar]
  59. Chalmers, G.; Bruce, H.L.; Hunter, D.B.; Parreira, V.R.; Kulkarni, R.R.; Jiang, Y.F.; Prescott, J.F.; Boerlin, P. Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations. J. Clin. Microbiol. 2008, 46, 3957–3964. [Google Scholar]
  60. Chalmers, G.; Martin, S.W.; Hunter, D.B.; Prescott, J.F.; Weber, L.J.; Boerlin, P. Genetic diversity of Clostridium perfringens isolated from healthy broiler chickens at a commercial farm. Vet. Microbiol. 2008, 127, 116–127. [Google Scholar]
  61. Chalmers, G.; Martin, S.W.; Prescott, J.F.; Boerlin, P. Typing of Clostridium perfringens by multiple-locus variable number of tandem repeats analysis. Vet. Microbiol. 2008, 128, 126–135. [Google Scholar]
  62. Drigo, I.; Agnoletti, F.; Bacchin, C.; Bettini, F.; Cocchi, M.; Ferro, T.; Marcon, B.; Bano, L. Toxin genotyping of Clostridium perfringens field strains isolated from healthy and diseased chickens. Ital. J. Anim. Sci. 2008, 7, 397–400. [Google Scholar]
  63. Timbermont, L.; Lanckriet, A.; Gholamiandehkordi, A.R.; Pasmans, F.; Martel, A.; Haesebrouck, F.; Ducatelle, R.; Van Immerseel, F. Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers. Comp. Immunol. Microbiol. Infect. Dis. 2008, 32, 503–512. [Google Scholar]
  64. Johansson, A.; Aspan, A.; Kaldhusdal, M.; Engstrom, B.E. Genetic diversity and prevalence of netB in Clostridium perfringens isolated from a broiler flock affected by mild necrotic enteritis. Vet. Microbiol. 2009, 144, 87–92. [Google Scholar]
  65. Martin, T.G.; Smyth, J.A. Prevalence of netB among some clinical isolates of Clostridium perfringens from animals in the United States. Vet. Microbiol. 2009, 136, 202–205. [Google Scholar]
  66. Keyburn, A.L.; Yan, X.X.; Bannam, T.L.; Van Immerseel, F.; Rood, J.I.; Moore, R.J. Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin. Vet. Res. 2010, 41, 21. [Google Scholar]
  67. Smyth, J.A.; Martin, T.G. Disease producing capability of netB positive isolates of C. perfringens recovered from normal chickens and a cow, and netB positive and negative isolates from chickens with necrotic enteritis. Vet. Microbiol. 2010. [Google Scholar]
  68. Abildgaard, L.; Sondergaard, T.E.; Engberg, R.M.; Schramm, A.; Hojberg, O. In vitro production of necrotic enteritis toxin B, NetB, by netB-positive and netB-negative Clostridium perfringens originating from healthy and diseased broiler chickens. Vet. Microbiol. 2010, 144, 231–235. [Google Scholar]
  69. Cooper, K.K.; Songer, J.G. Virulence of Clostridium perfringens in an experimental model of poultry necrotic enteritis. Vet. Microbiol. 2010, 142, 323–328. [Google Scholar]
  70. Nowell, V.J.; Poppe, C.; Parreira, V.R.; Jiang, Y.F.; Reid-Smith, R.; Prescott, J.F. Clostridium perfringens in retail chicken. Anaerobe 2010, 16, 314–315. [Google Scholar]
  71. Cheung, J.K.; Keyburn, A.L.; Carter, G.P.; Lanckriet, A.L.; Van Immerseel, F.; Moore, R.J.; Rood, J.I. The VirSR two-component signal transduction system regulates NetB toxin production in Clostridium perfringens. Infect. Immun. 2010, 78, 3064–3072. [Google Scholar]
  72. Ohtani, K.; Hirakawa, H.; Tashiro, K.; Yoshizawa, S.; Kuhara, S.; Shimizu, T. Identification of a two-component VirR/VirS regulon in Clostridium perfringens. Anaerobe 2010, 16, 258–264. [Google Scholar]
  73. Ohtani, K.; Yuan, Y.; Hassan, S.; Wang, R.; Wang, Y.; Shimizu, T. Virulence gene regulation by the agr system in Clostridium perfringens. J. Bacteriol. 2009, 191, 3919–3927. [Google Scholar]
  74. McDevitt, R.M.; Brooker, J.D.; Acamovic, T.; Sparks, N.H.C. Necrotic enteritis; a continuing challenge for the poultry industry. Worlds Poult. Sci. J. 2006, 62, 221–247. [Google Scholar]
  75. Lawrence, G.W.; Lehmann, D.; Anian, G.; Coakley, C.A.; Saleu, G.; Barker, M.J.; Davis, M.W. Impact of active immunisation against enteritis necroticans in Papua New Guinea. Lancet 1990, 336, 1165–1167. [Google Scholar]
  76. Uzal, F.A.; Kelly, W.R. Protection of goats against experimental enterotoxaemia by vaccination with Clostridium perfringens type D epsilon toxoid. Vet. Rec. 1998, 142, 722–725. [Google Scholar]
  77. Springer, S.; Selbitz, H.J. The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine. FEMS Immunol. Med. Microbiol. 1999, 24, 333–336. [Google Scholar]
  78. Heier, B.T.; Lovland, A.; Soleim, K.B.; Kaldhusdal, M.; Jarp, J. A field study of naturally occurring specific antibodies against Clostridium perfringens alpha toxin in Norwegian broiler flocks. Avian Dis. 2001, 45, 724–732. [Google Scholar]
  79. Lovland, A.; Kaldhusdal, M.; Redhead, K.; Skjerve, E.; Lillehaug, A. Maternal vaccination against subclinical necrotic enteritis in broilers. Avian Pathol. 2004, 33, 83–92. [Google Scholar]
  80. Kulkarni, R.R.; Parreira, V.R.; Sharif, S.; Prescott, J.F. Clostridium perfringens antigens recognized by broiler chickens immune to necrotic enteritis. Clin. Vaccine Immunol. 2006, 13, 1358–1362. [Google Scholar]
  81. Kulkarni, R.R.; Parreira, V.R.; Sharif, S.; Prescott, J.F. Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis. Clin. Vaccine Immunol. 2007, 14, 1070–1077. [Google Scholar]
  82. Zekarias, B.; Mo, H.; Curtiss, R., III. Recombinant attenuated Salmonella enterica serovar typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens. Clin. Vaccine Immunol. 2008, 15, 805–816. [Google Scholar]
  83. Wardenburg, J.B.; Schneewind, O. Vaccine protection against Staphylococcus aureus pneumonia. J. Exp. Med. 2008, 205, 287–294. [Google Scholar]
  84. Ragle, B.E.; Wardenburg, J.B. Anti-alpha-hemolysin monoclonal antibodies mediate protection against Staphylococcus aureus pneumonia. Infect. Immun. 2009, 77, 2712–2718. [Google Scholar]
Back to TopTop