Identification and Characterization of Clostridium perfringens Beta Toxin Variants with Differing Trypsin Sensitivity and In Vitro Cytotoxicity Activity

By producing toxins, Clostridium perfringens causes devastating diseases of both humans and animals. C. perfringens beta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin.

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title: Enterotoxic Clostridia: Clostridium perfringens Type A and Clostridium difficile
abstract: This chapter discusses two enterotoxin-producing clostridia that rank among the most important enteric pathogens of humans, Clostridium difficile and enterotoxin-positive type A strains of C. perfringens. The major lethal toxins (LTs) are not the only biomedically important C. perfringens toxins; some C. perfringens isolates, mostly belonging to type A, express C. perfringens enterotoxin (CPE). Recognized outbreaks of C. perfringens type A food poisoning are usually very large, averaging about 100 cases. C. perfringens type A food poisoning is acquired by ingestion of a food item containing vegetative cells of a CPE-positive C. perfringens type A strain. C. perfringens isolates associated with non-food-borne human gastrointestinal (GI) disease consistently carry a plasmid-borne cpe gene, which distinguishes them from food poisoning isolates carrying a chromosomal cpe gene. C. difficile is an opportunistic pathogen that causes nosocomial diarrhea and colitis after the normal GI flora has been altered, most typically by antibiotics. C. difficile-mediated disease develops from the production of two toxins, toxin A and toxin B, which in some papers are referred to as the enterotoxin and cytotoxin, respectively. Toxin production occurs during the stationary phase, under conditions that limit the growth of the organism. Certain basic precautions should be taken to help control outbreaks of C. difficile disease. In most instances, the incidence of disease can be reduced simply by educating health care workers about the disease and how it is spread.

title: Clostridium perfringens type A-E toxin plasmids.
abstract: Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell.

title: Clostridial enteric diseases of domestic animals
abstract: Major Toxins 216 Disease and Pathogenesis by Toxin Type 217 Type A 218 Type B 219 Type C 219 Type D 220 Type E 220 Enterotoxigenic C. perfringens 221 Reports of untyped C. perfringens 221 Prophylaxis and Therapy 222 Diagnosis 222 CLOSTRIDIUM SEPTICUM 223 Introduction 223 Virulence Attributes and Pathogenesis of Enteric Disease 223 Prophylaxis and Therapy 224 Diagnosis 224 CLOSTRIDIUM DIFFICILE 224 CLOSTRIDIUM SPIROFORME 225 CLOSTRIDIUM COLINUM 226 REFERENCES 226

title: Chapter 8 – Enterotoxemic Infections
abstract: Publisher Summary 
This chapter deals with clostridial infections that cause intestinal disease in humans and in which diarrhea is a major or significant feature of the clinical syndrome. In addition to the distinct disease syndromes, clostridia are often involved in polymicrobial infections of the gastrointestinal tract and present without specific features except for the occasional association of gas at the infected site. The production of hydrogen gas by clostridia and C. perfringens accounts for radiographic and pathologic findings associated with emphysematous cholecystitis and pneumatosis cystoides intestinalis. Clostridia cause distinct gastrointestinal syndromes in humans as a result of the action of specific toxins on the intestinal mucosa. Host factors predispose to different clinical syndromes and include: antimicrobial exposure, trypsin deficiency, and neutropenia. Clostridia are widely dispersed in the environment and the risk of developing a particular syndrome relate to the relative predominance of a particular species in that environment, such as in hospitals (C. difficile diarrhea) or the Highlands of Papua New Guinea (C. perfringens type C enteritis).. Ingestion of contaminated foods in the case of C. perfringens type A diarrhea and C. perfringens type C enteritis may also determine the type of syndrome displayed.

title: Genotypic and Phenotypic Characterization of Clostridium perfringens Isolates from Darmbrand Cases in Post-World War II Germany
abstract: Clostridium perfringens type C strains are the only non-type-A isolates that cause human disease. They are responsible for enteritis necroticans, which was termed Darmbrand when occurring in post-World War II Germany. Darmbrand strains were initially classified as type F because of their exceptional heat resistance but later identified as type C strains. Since only limited information exists regarding Darmbrand strains, this study genetically and phenotypically characterized seven 1940s era Darmbrand-associated strains. Results obtained indicated the following. (i) Five of these Darmbrand isolates belong to type C, carry beta-toxin (cpb) and enterotoxin (cpe) genes on large plasmids, and express both beta-toxin and enterotoxin. The other two isolates are cpe-negative type A. (ii) All seven isolates produce highly heat-resistant spores with D 100 values (the time that a culture must be kept at 100°C to reduce its viability by 90%) of 7 to 40 min. (iii) All of the isolates surveyed produce the same variant small acid-soluble protein 4 (Ssp4) made by type A food poisoning isolates with a chromosomal cpe gene that also produce extremely heat-resistant spores. (iv) The Darmbrand isolates share a genetic background with type A chromosomal-cpe-bearing isolates. Finally, it was shown that both the cpe and cpb genes can be mobilized in Darmbrand isolates. These results suggest that C. perfringens type A and C strains that cause human food-borne illness share a spore heat resistance mechanism that likely favors their survival in temperature-abused food. They also suggest possible evolutionary relationships between Darmbrand strains and type A strains carrying a chromosomal cpe gene.

title: Some properties of beta-toxin produced by Clostridium perfringens type C
abstract: Purified beta-toxin from Clostridium perfringens type C was found to be a single polypeptide chain protein with a molecular weight of approximately 30,000. The toxin was heat labile, with 75% of its activity being inactivated by incubation at 50 degrees C for 5 min. Biological activity of the purified toxin was completely destroyed on exposure to trypsin for 30 min at 37 degrees C. The 50% lethal dose for mice was 1.87 microgram of purified toxin.

title: Freezing or adding trypsin inhibitor to equine intestinal contents extends the lifespan of Clostridium perfringens beta toxin for diagnostic purposes.
abstract: Clostridium perfringens type C causes necrotizing enteritis mostly in neonatal animals of several species, including horses. The virulence of C. perfringens type C is mostly mediated by beta toxin (CPB). This toxin is highly sensitive to the action of trypsin and other proteases, which explains the increased susceptibility of neonatal animals to type C infections. Final confirmation of type C disease diagnosis should be based on detection of CPB in the intestinal content of affected animals. However, because CPB is so sensitive to the action of proteases, it is believed that this toxin persists for only a limited period of time in specimens of intestinal content of animals collected for diagnostic purposes. This study was therefore performed to determine the stability of CPB in intestinal content of horses stored at different temperatures and to evaluate the use of trypsin inhibitor to extend the lifespan of CPB in intestinal content of horses. When the intestinal content of horses that had been spiked with different amounts of CPB was tested by a capture ELISA technique to detect CPB, 319 LD(50) of CPB per milliliter was the lowest amount that could be detected. When equine intestinal content spiked with 319 LD(50)/ml was stored at 4 °C, CPB was detected by ELISA until day 8 after spiking. Samples spiked with the same amount of CPB and stored at -20 °C were positive for at least 5 weeks after spiking. When intestinal samples spiked with 319 LD(50)/ml of CPB were mixed with 0.1 mg/ml or 1.0 mg/ml of trypsin inhibitor and stored at 4 °C, all the samples were positive for at least 5 weeks after spiking. This study demonstrates that C. perfringens CPB present in equine intestinal samples stored at 4 °C cannot be detected by ELISA for more than 8 days. Freezing the samples at -20 °C or adding trypsin inhibitor before storage at 4 °C preserves the lifespan of CPB for at least 5 weeks.

title: Development and Application of New Mouse Models To Study the Pathogenesis of Clostridium perfringens Type C Enterotoxemias
abstract: Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality.

title: Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model
abstract: Clostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log‐phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log‐phase vegetative cultures of wild‐type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron® technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre‐incubation of wild‐type CN3685 with a CPB‐neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild‐type CN3685 and that damage was prevented by pre‐incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685‐induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.

title: Dissecting the Contributions of Clostridium perfringens Type C Toxins to Lethality in the Mouse Intravenous Injection Model
abstract: The gram-positive anaerobe Clostridium perfringens produces a large arsenal of toxins that are responsible for histotoxic and enteric infections, including enterotoxemias, in humans and domestic animals. C. perfringens type C isolates, which cause rapidly fatal diseases in domestic animals and enteritis necroticans in humans, contain the genes for alpha toxin (plc), perfringolysin O (pfoA), beta toxin (cpb), and sometimes beta2 toxin (cpb2) and/or enterotoxin (cpe). Due to the economic impact of type C-induced diseases, domestic animals are commonly vaccinated with crude type C toxoid (prepared from inactivated culture supernatants) or bacterin/toxoid vaccines, and it is not clear which toxin(s) present in these vaccines actually elicits the protective immune response. To improve type C vaccines, it would be helpful to assess the contribution of each toxin present in type C supernatants to lethality. To address this issue, we surveyed a large collection of type C isolates to determine their toxin-producing abilities. When late-log-phase vegetative culture supernatants were analyzed by quantitative Western blotting or activity assays, most type C isolates produced at least three lethal toxins, alpha toxin, beta toxin, and perfringolysin O, and several isolates also produced beta2 toxin. In the mouse intravenous injection model, beta toxin was identified as the main lethal factor present in type C late-log-phase culture supernatants. This conclusion was based on monoclonal antibody neutralization studies and regression analyses in which the levels of alpha toxin, beta toxin, perfringolysin O, and beta2 toxin production were compared with lethality. Collectively, our results highlight the importance of beta toxin for type C-induced toxemia.

title: Effects of Clostridium perfringens Beta-Toxin on the Rabbit Small Intestine and Colon
abstract: Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 μg of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates.

title: Bacterial toxins: a table of lethal amounts.
abstract: ............................................................. SELECTION OF DATA AND SOURCES OF ERROR......................... THE TABLE .............................................................  .. ......................................................... Relevance to Possible Cloning of Genes Coding for Toxins.................... Recommended Containment Levels for Cloning ............................. (i) Proteins with an expected 50% lethal dose for humans of 100 ng/kg or less. (ii) Proteins with an expected 50% lethal dose for humans of >100 ng/kg and <1 agfkg .......................................................... (iii) Proteins with an expected 50% lethal dose for humans of 1 to 100 ag/kg . (iv) Proteins of low toxicity ............................................. Risks Associated with Cloning Toxin Genes in Escherichia coli ................ Literature Cited ........................................................... 86 86 90 90 90 91 91

title: Purification and characterization of Clostridium perfringens beta toxin.
abstract: A new procedure for the purification of beta toxin from culture supernatant fluid of Clostridium perfringens was established. The procedure consists of ammonium sulfate fractionation, affinity chromatography on zinc-chelate Sepharose and gel filtration on Toyopearl HW 60. Beta toxin was purified about 460-fold from the ammonium sulfate fraction with a yield of about 60% in terms of lethality of the toxin. The molecular weight of the toxin, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose gradient centrifugation, was approximately 40,000. The isoelectric point was 5.6. The minimal necrotic dose for guinea pigs was approximately 2 ng. The 50% lethal doses for adult mice were 310 ng/kg and 4.5 micrograms/kg, when injected i.v. and i.p., respectively.

title: Clostridium perfringens Delta Toxin Is Sequence Related to Beta Toxin, NetB, and Staphylococcus Pore-Forming Toxins, but Shows Functional Differences
abstract: Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside GM2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to GM2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes GM2 as receptor and forms anion-selective channels.

title: NetB, a New Toxin That Is Associated with Avian Necrotic Enteritis Caused by Clostridium perfringens
abstract: For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.

title: Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus
abstract: Oligonucleotide probes designed on the basis of the N-terminal sequence of Clostridium perfringens beta-toxin were used to isolate the encoding gene (cpb). The nucleotide sequence of cpb was determined, and on the basis of DNA hybridization experiments it was shown that the gene is found only in type B and C strains of C. perfringens. The deduced amino acid sequence of the beta-toxin revealed homology with the alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus. The beta-toxin purified from C. perfringens appeared to exist in monomeric and multimeric forms. Recombinant beta-toxin, produced in Escherichia coli, appeared to be mainly in the multimeric form.

title: Site-directed mutagenesis of Clostridium perfringens beta-toxin: expression of wild-type and mutant toxins in Bacillus subtilis.
abstract: Recombinant beta-toxin has been expressed and secreted from Bacillus subtilis. Biological activity was tested in vivo and in vitro. The lethal dose in mice was determined. Hemolysis of rabbit and sheep erythrocytes was tested but no effect was observed. Seven mutant proteins were produced. Targets for mutagenesis were mostly selected on the basis of the similarity between beta-toxin and alpha-toxin from Staphylococcus aureus, a pore-forming toxin. Mutations of two amino acids affected the lethal dose in mice. Both residues have counterparts in the membrane binding region of alpha-toxin. Alteration of the single cysteine residue did not affect protein function, contrary to previous suggestions.

title: Biological Activities and Pore Formation of Clostridium perfringens Beta Toxin in HL 60 Cells*
abstract: Clostridium perfringens beta toxin is an important agent of necrotic enteritis. Of the 10 cell lines tested, only the HL 60 cell line was susceptible to beta toxin. The toxin induced swelling and lysis of the cell. Treatment of the cells with the toxin resulted in K+ efflux from the cells and Ca2+, Na+, and Cl- influxes. These events reached a maximum just before the cells were lysed by the toxin. Incubation of the cells with the toxin showed the formation of toxin complexes of about 191 and 228 kDa, which were localized in the domains that fulfilled the criteria of lipid rafts. The complex of 228 kDa was observed until 30 min after incubation, and only the complex of 191 kDa was remained after 60 min. Treatment of the cells with methyl-beta-cyclodextrin or cholesterol oxidase blocked binding of the toxin to the rafts and the toxin-induced K+ efflux and swelling. The toxin-induced Ca2+ influx and morphological changes were inhibited by an increase in the hydrodynamic diameter of polyethylene glycols from 200 to 400 and markedly or completely inhibited by polyethylene glycol 600 and 1000. However, these polyethylene glycols had no effect on the toxin-induced K+ efflux. The toxin induced carboxyfluorescein release from phosphatidyl-choline-cholesterol liposomes containing carboxyfluorescein and formed an oligomer with 228 kDa in a dose-dependent manner but did not form an oligomer with the 191-kDa complex. We conclude that the toxin acts on HL 60 cells by binding to lipid rafts and forming a functional oligomer with 228 kDa.

title: Clostridium perfringens beta-toxin forms multimeric transmembrane pores in human endothelial cells.
abstract: Beta-toxin is one of the lethal toxins of Clostridium perfringens. It shares sequence homology with the pore-forming alpha-toxin of Staphylococcus aureus and structural homology has been indicated by mutagenesis studies. Human endothelial cells are sensitive to the toxic effect of alpha-toxin and in order to investigate the function of beta-toxin we have looked at the effect of the protein on human umbilical vein endothelial cells. We show that like alpha-toxin beta-toxin induces release of arachidonic acid in a dose dependent manner. In addition we show that both toxins cause leakage of inositol from the cells, consistent with the formation of transmembrane pores. The effect of toxin mutants on endothelial cells correlates with the lethal dose of each mutant in mice. Furthermore, we demonstrate the formation of heat stable toxin multimers in the cell membrane. Multimer formation was not observed on other cell types tested. We conclude that beta-toxin is a cell specific pore-forming toxin, structurally and functionally related to alpha-toxin of Staphylococcus aureus.

title: Clostridium perfringens Beta-Toxin Induces Necrostatin-Inhibitable, Calpain-Dependent Necrosis in Primary Porcine Endothelial Cells
abstract: Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca2+]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis (“necroptosis”).

title: Structural and Functional Analysis of the Pore-Forming Toxin NetB from Clostridium perfringens
abstract: Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin.  Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers. Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.

title: Cysteine-Scanning Mutagenesis Supports the Importance of Clostridium perfringens Enterotoxin Amino Acids 80 to 106 for Membrane Insertion and Pore Formation
abstract: Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal symptoms of the second most common bacterial food-borne illness. Previous studies suggested that a region named TM1, which has amphipathic characteristics and spans from amino acids 81 to 106 of the native CPE protein, forms a β-hairpin involved in β-barrel pore formation. To further explore the potential role of TM1 in pore formation, the single Cys naturally present in CPE at residue 186 was first altered to alanine by mutagenesis; the resultant rCPE variant, named C186A, was shown to retain cytotoxic properties. Cys-scanning mutagenesis was then performed in which individual Cys mutations were introduced into each TM1 residue of the C186A variant. When those Cys variants were characterized, three variants were identified that exhibit reduced cytotoxicity despite possessing binding and oligomerization abilities similar to those of the C186A variant from which they were derived. Pronase challenge experiments suggested that the reduced cytotoxicity of those two Cys variants, i.e., the F91C and F95C variants, which model to the tip of the β-hairpin, was attributable to a lessened ability of these variants to insert into membranes after oligomerization. In contrast, another Cys variant, i.e., the G103C variant, with impaired cytotoxicity apparently inserted into membranes after oligomerization but could not form a pore with a fully functional channel. Collectively, these results support the TM1 region forming a β-hairpin as an important step in CPE insertion and pore formation. Furthermore, this work identifies the first amino acid residues specifically involved in those two steps in CPE action.

title: Clostridium perfringens beta-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity.
abstract: The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. The purified GST-beta-toxin fusion protein from the E. coli transformant cells was not lethal. The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C. perfringens type C. Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not. These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin. Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue.

title: Crystallization and preliminary X-ray diffraction studies of delta-toxin from Clostridium perfringens.
abstract: Clostridium perfringens is a Gram-positive anaerobic bacterium that is responsible for a wide range of diseases in humans and both wild and domesticated animals, including birds. C. perfringens is notable for its ability to produce a plethora of toxins, e.g. phospholipases C (alpha-toxin), pore-forming toxins (epsilon-toxin, beta-toxin and enterotoxin) and binary toxins (iota-toxin). Based on alpha-, beta-, epsilon- and iota-toxin production, the bacterium is classified into five different toxinotypes (A-E). Delta-toxin, which is a 32.6 kDa protein with 290 amino acids, is one of three haemolysins released by type C and possibly by type B strains of C. perfringens. This toxin is immunogenic and lytic to erythrocytes from the even-toed ungulates sheep, goats and pigs, and is cytotoxic to other cell types such as rabbit macrophages, human monocytes and blood platelets from goats, rabbits, guinea pigs and humans. The recombinant delta-toxin has been cloned, expressed, purified and crystallized in two different crystal forms by the hanging-drop vapour-diffusion method. Of these two different crystal forms, only the form II crystal diffracted to atomic resolution (dmin=2.4 Å), while the form I crystal diffracted to only 15 Å resolution. The form II crystals belonged to space group P2(1)2(1)2, with one molecule in the crystallographic asymmetric unit and unit-cell parameters a=49.66, b=58.48, c=112.93 Å.

title: Molecular Architecture and Functional Analysis of NetB, a Pore-forming Toxin from Clostridium perfringens*
abstract: Clostridium perfringens toxin NetB is a key factor in avian necrotic enteritis.  NetB forms heptameric pores structurally similar to Staphylococcus aureus toxins but lacks a phosphocholine binding pocket. NetB activity is enhanced by cholesterol.  NetB has distinct binding specificity, and cholesterol may act as a receptor.  The structure of NetB will facilitate development of control measures against necrotic enteritis. NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.

title: Rapid Cytopathic Effects of Clostridium perfringens Beta-Toxin on Porcine Endothelial Cells
abstract: Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C β-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

title: Clostridium perfringens beta-toxin targets endothelial cells in necrotizing enteritis in piglets.
abstract: Beta-toxin (CPB) is known to be the major virulence factor of Clostridium perfringens type C strains, which cause necrotizing enteritis in pigs, sheep, goats, calves, and humans. The exact mode of action, in particular the cellular targets of CPB in the intestine of naturally affected species, is however still not resolved. To investigate localization of CPB in naturally occurring necrotizing enteritis, we evaluated 52 piglets with spontaneously acquired C. perfringens type C enteritis and 14 control animals by immunohistochemistry. Our results consistently revealed binding of CPB to vascular endothelial cells in peracute to acute lesions of necrotizing enteritis. Subacute cases, in contrast, demonstrated reduced or no CPB staining at the endothelium, mainly due to widespread vascular necrosis. From these results we conclude, that the pathogenesis of C. perfringens type C induced necrotizing enteritis involves binding of CPB to endothelial cells in the small intestine during the early phase of the disease. Thus, by targeting endothelial cells, CPB might specifically induce vascular necrosis, hemorrhage and subsequent hypoxic tissue necrosis.

title: Clostridium perfringens beta-toxin binding to vascular endothelial cells in a human case of enteritis necroticans.
abstract: Clostridium perfringens type C-induced enteritis necroticans is a rare but often fatal disease in humans. A consistent histopathological finding is an acute, deep necrosis of the small intestinal mucosa associated with acute vascular necrosis and massive haemorrhage in the lamina propria and submucosa. Retrospective immunohistochemical investigations of tissues from a diabetic adult who died of enteritis necroticans revealed endothelial localization of C. perfringens beta-toxin in small intestinal lesions. Our results indicate that vascular necrosis might be induced by a direct interaction between C. perfringens beta-toxin and endothelial cells and that targeted disruption of endothelial cells plays a role in the pathogenesis of enteritis necroticans.

title: Characterization of Membrane-Associated Clostridium perfringens Enterotoxin following Pronase Treatment
abstract: After binding, Clostridium perfringens enterotoxin (CPE) initially localizes in a small (∼90-kDa) complex in plasma membranes. This event is followed by formation of a second membrane complex, referred to as large (160-kDa) complex. Contrary to a previous hypothesis proposing that CPE inserts into intestinal brush border membranes (BBMs) when this toxin is localized in the small complex, this study shows that BBMs do not offer CPE localized in the small complex protection from pronase. However, our experiments indicate that BBMs do substantially protect CPE from pronase when this toxin is localized in large complex. Since the onset of CPE-induced permeability alterations closely coincides with large-complex formation, these new results suggest that CPE-induced alterations in permeability may result from pore formation due to the partial membrane insertion of CPE when this toxin is present in large complex.

title: Both Epsilon-Toxin and Beta-Toxin Are Important for the Lethal Properties of Clostridium perfringens Type B Isolates in the Mouse Intravenous Injection Model
abstract: Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the pathogenesis of C. perfringens type B infections in domestic animals.

title: Enteritis necroticans (pigbel) in a diabetic child.
abstract: Enteritis necroticans (pigbel), an often fatal illness characterized by hemorrhagic, inflammatory, or ischemic necrosis of the jejunum, occurs in developing countries but is rare in developed countries, where its occurrence is confined to adults with chronic illnesses. The causative organism of enteritis necroticans is Clostridium perfringens type C, an anaerobic gram-positive bacillus. In December 1998, enteritis necroticans developed in a 12-year-old boy with poorly controlled diabetes mellitus after he consumed pig intestines (chitterlings). He presented with hematemesis, abdominal distention, and severe diabetic ketoacidosis with hypotension. At laparotomy, extensive jejunal necrosis required bowel resection, jejunostomy, and ileostomy. Samples were obtained for histopathological examination. Polymerase-chain-reaction (PCR) assay was performed on paraffin-embedded bowel tissue with primers specific for the cpa and cpb genes, which code for the alpha and beta toxins produced by C. perfringens.



Histologic examination of resected bowel tissue showed extensive mucosal necrosis, the formation of pseudomembrane, pneumatosis, and areas of epithelial regeneration that alternated with necrotic segments--findings consistent with a diagnosis of enteritis necroticans. Gram's staining showed large gram-positive bacilli whose features were consistent with those of clostridium species. Through PCR amplification, we detected products of the cpa and cpb genes, which indicated the presence of C. perfringens type C. Assay of ileal tissue obtained during surgery to restore the continuity of the patient's bowel was negative for C. perfringens.



The preparation or consumption of chitterlings by diabetic patients and other chronically ill persons can result in potentially life-threatening infectious complications.

title: Fatal Enteritis Necroticans (Pigbel) in a Diabetic Adult
abstract: Enteritis necroticans is a segmental necrotizing infection of the jejunum and ileum caused by Clostridium perfringens, Type C. The disease occurs sporadically in parts of Asia, Africa, and the South Pacific, where it primarily affects children with severe protein malnutrition. The disease is extremely rare in developed countries, where it has been seen primarily in diabetics. Two cases have previously been reported in the United States, one in a child with poorly controlled Type 1 diabetes. A 66-year-old woman with a 12-year history of Type 2 diabetes mellitus developed severe abdominal pain and bloody diarrhea after eating a meal of turkey sausage. She died unattended at home. An autopsy showed peritonitis and segmental necrosis of the jejunum and ileum. Microscopic examination showed Gram-positive club-shaped bacilli consistent with Clostridia coating a necrotic mucosa. Products of cpa and cpb genes of C. perfringens, Type C were identified in the necrotic jejunum by polymerase chain reaction amplification.

title: Clostridium perfringens Beta-Toxin Forms Potential-Dependent, Cation-Selective Channels in Lipid Bilayers
abstract: Recombinant beta-toxin from Clostridium perfringenstype C was found to increase the conductance of bilayer lipid membranes (BLMs) by inducing channel activity. The channels exhibited a distribution of conductances within the range of 10 to 380 pS, with the majority of the channels falling into two categories of conductance at 110 and 60 pS. The radii of beta-toxin pores found for the conductance states of 110 and 60 pS were 12.7 and 11.1 Å, respectively. The single channels and the steady-state currents induced by beta-toxin across the BLMs exhibited ideal monovalent cation selectivity. Addition of divalent cations (Zn2+, Cd2+, or Mg2+) at a concentration of 2 mM increased the rate of beta-toxin insertion into BLMs and the single-channel conductance, while application of 5 mM Zn2+ to a beta-toxin-induced steady-state current decreased the inward current by approximately 45%. The mutation of arginine 212 of beta-toxin to aspartate, previously shown to increase the 50% lethal dose of beta-toxin for mice nearly 13-fold, significantly reduced the ability of beta-toxin to form channels. These data support the hypothesis that the lethal action of beta-toxin is based on the formation of cation-selective pores in susceptible cells.