Cloning and expression of the phospholipase C gene from Clostridium perfringens and Clostridium bifermentans.

The phospholipase C gene from Clostridium perfringens was isolated, and its sequence was determined. It was found that the structural gene codes for a protein of 399 amino acid residues. The NH2-terminal residues have the typical features of a signal peptide and are probably cleaved after secretion. Escherichia coli cells harboring the phospholipase C gene-containing plasmid expressed high levels of this protein in the periplasmic space. Phospholipase C purified from E. coli transformants was enzymatically active, hemolytic to erythrocytes, and toxic to animals when injected intravenously. The phospholipase C gene from a related organism, Clostridium bifermentans, was also isolated. The two phospholipase C genes were found to be 64% homologous in coding sequence. The C. bifermentans protein, however, was 50-fold less active enzymatically than the C. perfringens enzyme.     

Clostridium sordellii phospholipase C: gene cloning and comparison of enzymatic and biological activities with those of Clostridium perfringens and Clostridium bifermentans phospholipase C

The gene encoding Clostridium sordellii phospholipase C (Csp) was cloned and expressed as a histidine-tagged (His-tag) protein, and the protein was purified to compare its enzymatic and biological activities with those of Clostridium perfringens phospholipase C (Cpa) and Clostridium bifermentans phospholipase C (Cbp). Csp was found to consist of 371 amino acid residues in the mature form and to be more homologous to Cbp than to Cpa. The egg yolk phospholipid hydrolysis activity of the His-tag Csp was about one-third of that of His-tag Cpa, but the hemolytic activity was less than 1% of that of His-tag Cpa. His-tag Csp was nontoxic to mice. Immunization of mice with His-tag Cbp or His-tag Csp did not provide effective protection against the lethal activity of His-tag Cpa. These results indicate that Csp possesses similar molecular properties to Cbp and suggest that comparative analysis of toxic and nontoxic clostridial phospholipases is helpful for characterization of the toxic properties of clostridial phospholipases.     

Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens.

A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.     

Histopathological effect of Clostridium perfringens enterotoxin in the rabbit ileum.

Highly purified enterotoxin from Clostridium perfringens was found to have histopathological activity in the rabbit ileum. Unlike the action of cholera, Escherichia coli, and Shigella enterotoxins, epithelium was denuded from the tips of ileal villi at concentrations of the enterotoxin necessary to induce fluid accumulation in the rabbit. Whether or not this observed histopathology is essential for the diarrheal syndrome associated with Clostridium perfringens food poisoning remains unclear.     

Clostridium perfringens type C causing necrotising enteritis.

A rapidly fatal case of enteritis necroticans in a 24 year old man with diabetes was caused by Clostridium perfringens type C. The role of beta toxin in the disease is discussed. This type has not been previously described as a causative agent in necrotising bowel disease of man outside endemic areas.     

Purification of beta-toxin from Clostridium perfringens type C.

Beta-toxin was purified about 340-fold from culture supernatant fluid of Clostridium perfringens type C with a yield of about 24% in terms of biologically active beta-toxin. The purification involved ammonium sulfate fractionation, gel filtration through Sephadex G-100, isoelectrofocusing in a pH 3 to 6 gradient, and immunoaffinity chromatography. The purified beta-toxin gave a single band on polyacrylamide gel electrophoresis.     

Selective cytotoxicity of Clostridium perfringens delta toxin on rabbit leukocytes.

Clostridium perfringens delta toxin was selectively cytotoxic for various rabbit leukocyte populations. The sensitivity of these populations to the toxin varied, depending on the tissue from which they were derived, from 28% (appendix) to 41% (bone marrow) and 32% (spleen). Macrophages were uniformly killed by delta toxin, whereas thymocytes were essentially resistant. Selective cytotoxicity was correlated to the specific binding of the radiolabeled toxin by target cells. The relationship between sensitivity to the toxin and the presence of GM2 ganglioside in the cell membrane of rabbit leukocytes is discussed. Delta toxin might prove a useful new tool for separating leukocyte subpopulations based on their differential sensitivity to the cytolethal effect of this protein.     

Characterization of enterotoxin purified from Clostridium perfringens type C.

Enterotoxin produced by a sporulating culture of Clostridium perfringens type C, which had been isolated from a case of severe necrotic enteritis, was purified. The molecular weight was estimated to be 36,000 by gel chromatography on Sephadex G-100 and 33,400 by ultracentrifugation. The sedimentation coefficient S20,W was 2.92. The toxin protein exhibited unusual behavior on sodium dodecyl sulfate gels, and toxin aggregates having molecular weights of 68,000, 85,000, 105,000, and 140,000 were obtained. The purified enterotoxin often separated, apparently due to slight charge differences, into two protein bands on 7% polyacrylamide gels. Electrofocusing of enterotoxin on polyacrylamide gels gave an approximate isoelectric point of 4.3, with the enterotoxin being fractionated into four distinct protein bands. The specific toxicity of the enterotoxin was about 1,900 mouse mean lethal doses per mg of calculated nitrogen. The data obtained indicate that the enterotoxin from C. perfringens type C is identical in most respects to that obtained from type A strains. Whether or not this toxin plays a role in the necrotic enteritis caused by type C strains is unknown at present.     

Comparison of receptors for Clostridium perfringens type A and cholera enterotoxins in isolated rabbit intestinal brush border membranes

The rabbit intestinal brush border membrane (BBM) receptors for Clostridium perfringens type A (CPE) and cholera (CT) enterotoxins were compared. Initial studies characterized binding of 125I-CPE to isolated BBMs as specific, saturable, and irreversible. BBMs appear to contain a single type of CPE binding site. Protease pretreatment of BBMs strongly reduced subsequent specific binding of 125I-CPE but not 125I-CT, while neuraminidase pretreatment had little effect on binding of either enterotoxin. Proteases did not significantly release pre-bound 125I-CPE. Preincubation of CPE with an affinity-purified preparation containing a previously identified (Biochem. Biophys. Res. Commun. 112, 1099-105) CPE-binding protein resulted in reduced specific binding of 125I-CPE and an inhibition of CPE biologic activity. Similar experiments showed that CPE-binding protein had no effect on CT binding or biologic activity. Gangliosides had no significant effect on specific binding or biologic activity of CPE but reduced CT binding and biologic activity. Lipids, including gangliosides, separated by thin layer chromatography specifically bound CT but not CPE. Preincubation of BBMs with CT did not reduce subsequent binding of 125I-CPE; conversely, prebound CPE did not affect subsequent 125I-CT binding. These results strongly suggest that CPE does not share the CT BBM receptor ganglioside GM1, and support a role for the CPE-binding protein in CPE binding.     

Lipopolysaccharide interaction with rabbit erythrocyte membranes

In this study we have characterized the association of a polysaccharide-deficient, lipid-rich lipopolysaccharide (LPS) with rabbit erythrocytes (RaRBC). With polymyxin B sulfate-mediated hemolysis as a probe, we have shown that Salmonella minnesota R595 LPS interacts with RaRBC in two distinguishable steps. The first step whereby RaRBC exposed to LPS are rendered sensitive to polymyxin B-initiated lysis probably represents absorption of LPS to the RaRBC membrane. We investigated two possible mechanisms for the subsequent time-dependent decrease in response of LPS-treated RaRBC to polymyxin B. We found that the decay in polymyxin B susceptibility of LPS-treated RaRBC cannot be attributed to a decrease in binding of LPS to the RaRBC. On the other hand, our results are consistent with a time-dependent rearrangement of the amphipathic LPS within the lipid bilayer of the RaRBC membrane. In particular, at lower incubation temperatures of RaRBC and LPS, the decay in polymyxin B-induced hemolysis is slower, presumably, because the increased membrane viscosity allows less rapid rearrangement of LPS within the lipid bilayer. A putative hydrophobic intercalation of LPS into a mammalian cell membrane may be of importance in LPS stimulation of responsive cells.     

Some properties of beta-toxin produced by Clostridium perfringens type C.

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.     

Role of alpha-toxin in Clostridium perfringens infection determined by using recombinants of C. perfringens and Bacillus subtilis.

Clostridium perfringens type A strains which differed in alpha-toxin (phospholipase C [PLC]) productivity were inoculated intraperitoneally or intravenously into mice, and then their 50% mouse lethal doses (LD50) were determined. Strain NCTC 8237 produced ninefold higher PLC activity than strain 13. The mean LD50 for the former was 1 log unit lower than that for the latter. Two isogenic strains were constructed from strain 13: strain 13(pJIR418 alpha) (pJIR418 alpha contains the plc gene), which produced ninefold higher PLC activity than strain 13; and strain 13 PLC-, which showed no PLC productivity at all because of transformation-mediated gene disruption. The mean LD50 for strain 13(pJIR418 alpha) was 1 log unit lower than those for strain 13 PLC- and strain 13. These results indicate that PLC functions as a virulence-determining factor when it is produced in a sufficient amount. Such a difference in LD50 was also observed between Bacillus subtilis with and without the cloned plc gene. Inoculation of B. subtilis PLC+ intravenously into mice caused marked thrombocytopenia and leukocytosis. Mice inoculated with B. subtilis at 2 LD50 died because of circulatory collapse. Histological examination revealed that intravascular coagulation and vascular congestion occurred most prominently in the lungs. These results suggest that PLC plays a key role in the systemic intoxication of clostridial myonecrosis, probably by affecting the functions of platelets and phagocytes.     

Regulation of virulence by the RevR response regulator in Clostridium perfringens

Clostridium perfringens causes clostridial myonecrosis or gas gangrene and produces several extracellular hydrolytic enzymes and toxins, many of which are regulated by the VirSR signal transduction system. The revR gene encodes a putative orphan response regulator that has similarity to the YycF (WalR), VicR, PhoB, and PhoP proteins from other Gram-positive bacteria. RevR appears to be a classical response regulator, with an N-terminal receiver domain and a C-terminal domain with a putative winged helix-turn-helix DNA binding region. To determine its functional role, a revR mutant was constructed by allelic exchange and compared to the wild type by microarray analysis. The results showed that more than 100 genes were differentially expressed in the mutant, including several genes involved in cell wall metabolism. The revR mutant had an altered cellular morphology; unlike the short rods observed with the wild type, the mutant cells formed long filaments. These changes were reversed upon complementation with a plasmid that carried the wild-type revR gene. Several genes encoding extracellular hydrolytic enzymes (sialidase, hyaluronidase, and α-clostripain) were differentially expressed in the revR mutant. Quantitative enzyme assays confirmed that these changes led to altered enzyme activity and that complementation restored the wild-type phenotype. Most importantly, the revR mutant was attenuated for virulence in the mouse myonecrosis model compared to the wild type and the complemented strains. These results provide evidence that RevR regulates virulence in C. perfringens; it is the first response regulator other than VirR to be shown to regulate virulence in this important pathogen.     

Characterization of Toxin Plasmids in Clostridium perfringens Type C Isolates

Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence.Clostridium perfringens isolates are classified into five toxinotypes (A to E) based upon the production of four (α, β, ɛ, and ι) typing toxins (29). Each toxinotype is associated with different diseases affecting humans or animals (25). In livestock species, C. perfringens type C isolates cause fatal necrotizing enteritis and enterotoxemia, where toxins produced in the intestines absorb into the circulation to damage internal organs. Type C-mediated animal diseases result in serious economic losses for agriculture (25). In humans, type C isolates cause enteritis necroticans, which is also known as pigbel or Darmbrand (15, 17), an often fatal disease that involves vomiting, diarrhea, severe abdominal pain, intestinal necrosis, and bloody stools. Acute cases of pigbel, resulting in rapid death, may also involve enterotoxemia (15).By definition, type C isolates must produce alpha and beta toxins (24, 29). Alpha toxin, a 43-kDa protein encoded by the chromosomal plc gene, has phospholipase C, sphingomyelinase, and lethal properties (36). Beta toxin, a 35-kDa polypeptide, forms pores that lyse susceptible cells (28, 35). Recent studies demonstrated that beta toxin is necessary for both the necrotizing enteritis and lethal enterotoxemia caused by type C isolates (33, 37). Besides alpha and beta toxins, type C isolates also commonly express beta2 toxin, perfringolysin O, or enterotoxin (11).There is growing appreciation that naturally occurring plasmids contribute to both C. perfringens virulence and antibiotic resistance. For example, all typing toxins, except alpha toxin, can be encoded by genes carried on large plasmids (9, 19, 26, 30-32). Other C. perfringens toxins, such as the enterotoxin or beta2 toxin, can also be plasmid encoded (6, 8, 12, 34). Furthermore, conjugative transfer of several C. perfringens antibiotic resistance plasmids or toxin plasmids has been demonstrated, supporting a key role for plasmids in the dissemination of virulence or antibiotic resistance traits in this bacterium (2).Despite their pathogenic importance, the toxin-encoding plasmids of C. perfringens only recently came under intensive study (19, 26, 27, 31, 32). The first carefully analyzed C. perfringens toxin plasmids were two plasmid families carrying the enterotoxin gene (cpe) in type A isolates (6, 8, 12, 26). One of those cpe plasmid families, represented by the ∼75-kb prototype pCPF5603, has an IS1151 sequence present downstream of the cpe gene and also carries the cpb2 gene, encoding beta2 toxin. A second cpe plasmid family of type A isolates, represented by the ∼70-kb prototype pCPF4969, lacks the cpb2 gene and carries an IS1470-like sequence, rather than an IS1151 sequence, downstream of the cpe gene. The pCPF5603 and pCPF4969 plasmid families share an ∼35-kb region that includes transfer of a clostridial plasmid (tcp) locus (26). The presence of this tcp locus likely explains the demonstrated conjugative transfer of some cpe plasmids (5) since a similar tcp locus was shown to mediate conjugative transfer of the C. perfringens tetracycline resistance plasmid pCW3 (2).The iota toxin-encoding plasmids of type E isolates are typically larger (up to ∼135 kb) than cpe plasmids of type A isolates (19). Plasmids carrying iota toxin genes often encode other potential virulence factors, such as lambda toxin and urease, as well as a tcp locus (19). Many iota toxin plasmids of type E isolates share, sometimes extensively, sequences with cpe plasmids of type A isolates (19). It has been suggested that many iota toxin plasmids evolved from the insertion of a mobile genetic element carrying the iota toxin genes near the plasmid-borne cpe gene in a type A isolate, an effect that silenced the cpe gene in many type E isolates (3, 19).Plasmids carrying the epsilon toxin gene (etx) vary from ∼48 kb to ∼110 kb among type D isolates (32). In part, these etx plasmid size variations in type D isolates reflect differences in toxin gene carriage. For example, the small ∼48-kb etx plasmids present in some type D isolates lack both the cpe gene and the cpb2 gene. In contrast, larger etx plasmids present in other type D isolates often carry the cpe gene, the cpb2 gene, or both the cpe and cpb2 genes. Thus, the virulence plasmid diversity of type D isolates spans from carriage of a single toxin plasmid, possessing from one to three distinct toxin genes, to carriage of three different toxin plasmids.In contrast to the variety of etx plasmids found among type D isolates, type B isolates often or always share the same ∼65-kb etx plasmid, which is related to pCPF5603 but lacks the cpe gene (27). This common etx plasmid of type B isolates, which carries a cpb2 gene and the tcp locus, is also present in a few type D isolates. Most type B isolates surveyed to date carry their cpb gene, encoding beta toxin, on an ∼90-kb plasmid, although a few of those type B isolates possess an ∼65-kb cpb plasmid distinct from their ∼65-kb etx plasmid (31).To our knowledge, the cpb gene has been mapped to a plasmid (uncharacterized) in only a single type C strain (16). Furthermore, except for the recent localization of the cpe gene to plasmids in type C strains (20), plasmid carriage of other potential toxin genes in type C isolates has not been investigated. Considering the limited information available regarding the toxin plasmids of type C isolates, our study sought to systematically characterize the size, diversity, and toxin gene carriage of toxin plasmids in a collection of type C isolates. Also, to gain insight into possible mobilization of the cpb gene by insertion sequences or conjugative transfer, the presence of IS1151 sequences or the tcp locus on type C toxin plasmids was investigated.     

Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon

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.     

Phospholipid metabolism induced by Clostridium perfringens alpha-toxin elicits a hot-cold type of hemolysis in rabbit erythrocytes.

GTP and AIF4- significantly stimulated the late phosphatidic acid (PA) formation induced by Clostridium perfringens alpha-toxin in rabbit erythrocyte lysates. Pertussis toxin blocked the PA production. AIF4- markedly enhanced phosphatidylethanol production induced by alpha-toxin in the presence of ethanol. GTP[gamma S] stimulated the PA formation and hemolysis induced by alpha-toxin, and GDP[beta S] inhibited them. An H-to-G mutation at position 126 (H126G) induced the PA formation and hemolysis in a Co2+ concentration-dependent manner. H148G induced neither the PA formation nor hemolysis. These results suggest that the toxin-induced hemolysis is due to activation of phospholipid metabolism systems through GTP-binding protein.