Analysis of protection afforded by a Clostridium perfringens alpha-toxoid against heterologous clostridial phospholipases C

The major virulence determinant in clostridial myonecrosis caused by Clostridium perfringens is a phospholipase C (PLC), the alpha-toxin. Previously, mice have been protected against challenge with heterologous alpha-toxin or Clostridium perfringens spores by immunisation with the C-domain (known as Cpa(247-370) or alpha-toxoid) of the alpha-toxin. In this study, we have determined the ability of the alpha-toxoid to protect against the lethal effects of a divergent C. perfringens alpha-toxin and against the PLCs of C. absonum or C. bifermentans, species which have been isolated from cases of clostridial myonecrosis. Protection against the C. perfringens alpha-toxin variant, the C. absonum alpha-toxin or the C. bifermentans PLC was elicited by immunisation with the alpha-toxoid in vivo.     

Differences in the Carboxy-Terminal (Putative Phospholipid Binding) Domains of Clostridium perfringens and Clostridium bifermentans Phospholipases C Influence the Hemolytic and Lethal Properties of These Enzymes

The phospholipases C of C. perfringens (alpha-toxin) and C. bifermentans (Cbp) show >50% amino acid homology but differ in their hemolytic and toxic properties. We report here the purification and characterisation of alpha-toxin and Cbp. The phospholipase C activity of alpha-toxin and Cbp was similar when tested with phosphatidylcholine in egg yolk or in liposomes. However, the hemolytic activity of alpha-toxin was more than 100-fold that of Cbp. To investigate whether differences in the carboxy-terminal domains of these proteins were responsible for differences in the hemolytic and toxic properties, a hybrid protein (NbiCalpha) was constructed comprising the N domain of Cbp and the C domain of alpha-toxin. The hemolytic activity of NbiCalpha was 10-fold that of Cbp, and the hybrid enzyme was toxic. These results confirm that the C-terminal domain of these proteins confers different properties on the enzymatically active N-terminal domain of these proteins.     

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.     

Regulation of Clostridium perfringens alpha-toxin-activated phospholipase C in rabbit erythrocyte membranes.

The rapid phosphatidic acid (PA) formation induced by Clostridium perfringens alpha-toxin was stimulated by AlF4- in rabbit erythrocyte membranes. GTP[gamma S] [guanosine 5'-O-(3-thiotriphosphate)] stimulated the rapid 1,2-diacylglycerol formation and inositol 1,4,5-trisphosphate release induced by the toxin. On the other hand, treatment of erythrocyte lysates with phorbol 12-myristate 13-acetate (PMA) resulted in inhibition of toxin-induced PA production, and long-term PMA or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) treatment of the lysates led to stimulation of PA formation. Furthermore, treatment of erythrocytes with the toxin caused an increase of protein kinase C activity in membrane fractions. The results suggest that toxin-induced PA formation is mediated by endogenous phospholipase C regulated through GTP-binding protein and protein kinase C in rabbit erythrocytes.     

Oral toxicities of Clostridium botulinum type C and D toxins of different molecular sizes.

Clostridium botulinum type C progenitor toxins of different molecule sizes, C-L (16S) and C-M (12S), were purified from cultures of strains 573, Stockholm, and CB-19. C-L toxin showed some hemaggglutinin activity, whereas C-M toxin did not. Neither C-L nor C-M toxin was activated upon trypsinization. Molecular dissociation of purified type C-L and C-M toxins into toxic and nontoxic components was demonstrated by sucrose density gradient ultracentrifugation and diethylaminoethyl-Sephadex chromatography at pH 8.0. The molecular construction of type C progenitor toxin appears to be analogous to that reported for botulinum toxins of other types. C-L and D-L toxins showed higher oral toxicities to mice than did C-M or D-M toxin. Such higher oral toxicities were ascribed to the higher stabilities of these toxins in gastric and intestinal juices.     

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.     

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.     

DNase production by Clostridium septicum.

Sixty-two Clostridium septicum isolates were assayed for extracellular DNase activity. All of the C. septicum isolates tested produced greater DNase activity than did the other DNase-producing clostridial isolates tested. The molecular weight of the DNase of C. septicum was determined to be approximately 45,000. DNase is a major extracellular protein produced by this organism.     

Identification of Clostridium botulinum, Clostridium argentinense, and related organisms by cellular fatty acid analysis.

On the basis of 686 analyses of 285 strains of Clostridium botulinum, Clostridium argentinense (formerly C. botulinum type G), and phenotypically related organisms, 14 cellular fatty acid (CFA) groups of toxic organisms and 6 CFA groups of nontoxic organisms were delineated. The CFA groups of toxic strains included two of type A, three of proteolytic strains of type B, two of proteolytic strains of type F, one each of nonproteolytic strains of types B, E, and F, and one each of types C alpha, C beta, and D and C. argentinense. The groups of phenotypically similar nontoxic strains included Clostridium sporogenes, Clostridium putrificum, nontoxic strains with phenotypic characteristics similar to those of nonproteolytic strains of C. botulinum types B, E, and F (BEF-like), two groups of nontoxigenic organisms with phenotypic characteristics similar to those of C. botulinum types C and D and Clostridium novyi (CDN-like), and Clostridium subterminale, which has phenotypic characteristics similar to those of C. argentinense. Within the toxin types, 89 to 100% of the strains were correctly identified by CFA analysis, and 74 to 100% of the analyses were correct. Of 36 strains of C. sporogenes, 30 (83%) were correctly identified; 17% of the strains of C. sporogenes were incorrectly identified as C. botulinum type A or B. All analyses of C. putrificum and C. subterminale were correctly identified. There was no significant level of similarity between strains of C. botulinum and phenotypically similar organisms and 85 other species of clostridia or 407 other taxa of gram-positive and gram-negative bacteria. Additionally, the one strain each of Clostridium baratii and Clostridium butyricum previously reported to produce C. botulinum toxin could be differentiated from C.botulinum types as well as from strains of C. baratii and C. butyricum that did not produce neurotoxin.     

C2 toxicity in extract of Clostridium botulinum type C spores

Toxic protein(s) neutralized with anti-C2 toxic serum was extracted from Clostridium botulinum type C spores treated with an alkaline mercaptoethanol solution. C2 toxicity in the spores was located in the spore coat fraction and was more heat stable than that found in culture fluid.     

Oral toxicities of Clostridium botulinum type A and B toxins from different strains

The production and the oral toxicity for mice of Clostridium botulinum type A and B toxins of different strains were studied. All five type B strains produced both 16S (large or L) and 12S (medium or M) toxins, although the relative amounts varied with the strains. The culture supernatant of type B Okra strain was the most potent in oral toxicity. The L toxin of this culture was about 700 times more toxic in feeding tests with mice than the L toxin from type B strain NH-2, whereas the M toxins of the two strains had the same oral toxicity. These results indicate that the oral toxicity of type B toxin varies with the culture strain. Oral toxicities of L toxin produced by type A strains 62A and 97 were comparable but were 10 times higher than those of their M toxins. Hybrids of toxic and nontoxic components separated from L toxins of type B strains Okra and NH-2 revealed that the high oral toxicity of the B-L toxin of strain Okra is attributable not to the toxic but to the nontoxic component of the toxin. The present study suggests that the 16S molecular-sized toxin elaborated by a certain strain of C. botulinum type B is implicated in the high fatality rate in type B human botulism.     

Oral toxicities of Clostridium botulinum toxins in response to molecular size.

Clostridium botulinum type A, B, and F toxins of different molecular sizes were fed to mice to compare the oral toxicities. The progenitor toxin, a complex of a toxic and nontoxic component, of any type was higher in oral toxicity to mice than the dissociated toxic component or the derivative toxin. The former may no doubt play a more important role in the pathogenesis of food-borne botulism. The higher oral toxicity possessed by the progenitor toxin, including the exceptionally high one found with type B-L toxin, can be explained solely by the protection afforded by the nontoxic component attached to the toxic component. The possibility of the highest oral toxicity of type B-L toxin to humans is discussed.     

Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes.

We purified and characterized an extracellular phospholipase produced by Listeria monocytogenes. This enzyme was separated as a homogeneous protein of 29 kDa by chromatography on DEAE-52 cellulose and Bio-Gel P100 columns. It is a zinc-dependent phospholipase C (PLC) that is mainly active at pH 6 to 7 and expresses lecithinase activity and a weaker sphingomyelinase activity. The exoenzyme also hydrolyzed phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin but not phosphatidylinositol. It was distinct from the 36-kDa phosphatidylinositol PLC produced by L. monocytogenes and from the L. ivanovii sphingomyelinase. The pure protein expressed a weak, calcium-independent hemolytic activity and was not toxic in mice. Western immunoblot analysis using a rabbit immune serum raised against the enzyme showed that all virulent strains of L. monocytogenes tested produced in the culture supernatant a 29-kDa PLC. In contrast, no proteins antigenically related to the 29-kDa PLC were detected in supernatants of L. ivanovii, L. seeligeri, L. innocua, or L. welshimeri. The role in virulence of the 29-kDa PLC specifically produced by L. monocytogenes remains to be established.     

The large clostridial toxins from Clostridium sordellii and C. difficile repress glucocorticoid receptor activity

We have previously shown that Bacillus anthracis lethal toxin represses glucocorticoid receptor (GR) transactivation. We now report that repression of GR activity also occurs with the large clostridial toxins produced by Clostridium sordellii and C. difficile. This was demonstrated using a transient transfection assay system for GR transactivation. We also report that C. sordellii lethal toxin inhibited GR function in an ex vivo assay, where toxin reduced the dexamethasone suppression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Furthermore, the glucocorticoid antagonist RU-486 in combination with C. sordellii lethal toxin additively prevented glucocorticoid suppression of TNF-alpha. These findings corroborate the fact that GR is a target for the toxin and suggest a physiological role for toxin-associated GR repression in inflammation. Finally, we show that this repression is associated with toxins that inactivate p38 mitogen-activated protein kinase (MAPK).     

Human neutrophil peptide receptors: mobilization mediated by phospholipase C.

Incubation of human neutrophils with phospholipase C from Clostridium perfringens caused an increase in the ability of the treated cells to bind the chemotactic peptide, F-Met-Leu-Phe. The increase in binding was related to an increase in specific binding of the ligand. The increase in specific binding was, in turn, related to an increased number of peptide receptors. The dissociation constant (KD) for the tripeptide was not altered, on the average, by enzyme treatment. The increase in peptide receptor number was related temporally, and possibly mechanistically, to enzyme-stimulated secretory function involving the secondary granules. Phospholipase C treatment did not similarly augment binding of the complement-derived attractant, C5a. Receptor numbers for different chemotactic ligands may therefore be controlled by different mechanisms. Supplementary experiments provided evidence that this phenomenon was attributable to phospholipase C activity and not to contaminating protease(s).     

Purification and characterization of the lethal toxin (alpha-toxin) of Clostridium septicum.

Clostridium septicum lethal (alpha-toxin) was purified and found to be a basic protein (pI 8.4) of approximately 48 kDa that is both lethal and hemolytic. The alpha-toxin had a hemolytic activity of approximately 2 x 10(7) hemolytic units per mg and a 50% lethal dose of approximately 10 micrograms/kg of body weight for mice. The alpha-toxin formed concentration-dependent, sodium dodecyl sulfate-resistant aggregates of approximately 230 kDa. Mice immunized with alpha-toxin showed a significant increase in survival time over mock-immunized mice when challenged with C. septicum. Rabbit polyclonal antibody was generated against the purified toxin and was used to confirm that toxin with the same molecular weight was present in seven different C. septicum isolates. No proteins in the supernatants from cultures of Clostridium perfringens, Clostridium histolyticum, Clostridium chauvoei, or Clostridium difficile were found to react with the C. septicum alpha-toxin-specific antibody.     

Detection of phospholipase C in nontuberculous mycobacteria and its possible role in hemolytic activity

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.     

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.     

Hemolytic and sphingomyelinase activities of Clostridium perfringens alpha-toxin are dependent on a domain homologous to that of an enzyme from the human arachidonic acid pathway.

The N-terminal domain of Clostridium perfringens alpha-toxin, homologous with the nontoxic phospholipase C of Bacillus cereus, was expressed in Escherichia coli and shown to retain all of the phosphatidylcholine hydrolyzing activity of the alpha-toxin, but not the sphingomyelinase, hemolytic, or lethal activities. The C-terminal domain of alpha-toxin showed sequence and predicted structural homologies with the N-terminal region of arachidonate 5-lipoxygenase, an enzyme from the human arachidonic acid pathway which plays a role in inflammatory and cardiovascular diseases in humans.     

Clostridium botulinum type D toxin: purification, molecular structure, and some immunological properties.

Clostridium botulinum type D progenitor toxin was purified. The addition of ribonucleic acid to the whole culture helped initial acid precipitation of the toxin. As with type B, both L (16S) and M toxins (12S) obtained from a hemagglutinin-positive strain, whereas M toxin only was produced by a hemagglutinin-negative strain. M toxin (molecular weight, 300,000) consisted of one molecule each of a toxic (molecular weight, 170,000) and a nontoxic component (molecular weight, 130,000); L toxin consisted of both components plus hemagglutinin. The specific toxicity of M toxin was 5 X 10(8) mean lethal doses per mg of N; that of L toxin was 2.4 X 10(8) mean lethal doses per mg of N. These toxins were fully or nearly fully active, but in un-nicked form. Trypsinization caused nicking in the toxic component, forming a molecule made up of two peptide chains with molecular weights of 110,000 and 60,000; there was little or no increase in toxicity. The toxic component of type D was not antigenically related to that of type C, whereas the nontoxic component was antigenically indistinguishable from that of type C. The toxicities of both L nad M toxins of the hemagglutinin-positive strain were increased twofold by trypsinization. Neither toxin contained the C2 toxic factor elaborated by C and D strain.