Perfringens Alpha-toxin Research Articles

Mechanism of membrane damage by Clostridium perfringens alpha-toxin.

The effect of Clostridium perfringens alpha-toxin on liposomes prepared from phosphatidylcholine (PC) containing the fatty acyl residues of 18 carbon atoms was investigated. The toxin-induced carboxyfluorescein (CF) leakage and phosphorylcholine release from multilamellar liposomes increased as the phase transition temperature of the phosphatidylcholines containing unsaturated fatty acyl residues decreased. However, there was no difference between the sensitivity of the different phosphatidylcholines solubilized by deoxycholate to the phospholipase C (PLC) activity of the toxin. However, the toxin did not hydrolyze solubilized distearoyl-L-alpha-phosphatidylcholine (DSPC) or phosphatidylcholine containing saturated fatty acyl residue, and caused no effect on liposomes composed of DSPC. These results suggest that the activity of the toxin is closely related to the membrane fluidity and double bond in PC. The N-terminal domain of alpha-toxin (AT1-246) and variant H148G did not induce CF leakage from liposomes composed of dioleoyl-L-alpha-phosphatidylcholine (DOPC). H148G bound to the liposomes, but AT1-246 did not. However, the C-terminal domain (AT251-370) conferred binding to liposomes and the membrane-damaging activity on AT1-246. These observations suggest that the membrane-damaging action of alpha-toxin is due to the binding of the C-terminal domain of the toxin to the double bond in the PC in the bilayer and hydrolysis of the PC by the N-terminal domain.

Cooperative Haemolysis Between Weakly-Beta Haemolytic Human Intestinal Spirochaetes and Clostridium perfringens

Interactions between human intestinal spirochaetes (HIS) related to intestinal spirochaetosis and intestinal pathogenic anaerobic bacteria were investigated by searching for the presence of cooperative haemolysis among 39 strains of weakly beta-haemolytic human intestinal spirochaetes and Clostridium perfringens alpha-toxin producers on plates carrying six different sheep blood agar media. An area of intense cooperative haemolysis (about 3-10 mm) was observed between all tested spirochaetal strains and C. perfringens where the clostridial alpha-toxin diffused toward the colonies of the spirochaetes overlapping part of their growth zone. The cooperative haemolysis was a potentiation of the haemolysis due to the single cultivation of human intestinal spirochaetes and C. perfringens and was observed after anaerobic incubation for 24-48 hours when both bacteria at a concentration range of 10(8)-10(3) CFU/ml were streaked at a distance of 3-10 mm to each other. A cooperative haemolysis was also observed between C. perfringens and weakly beta-haemolytic spirochaetes related to porcine and avian intestinal spirochaetosis and the spirochaete causing swine dysentery. The present study indicated that the damage produced in vitro by the clostridial alpha-toxin was enhanced only on the red blood cells which were in proximity to the HIS colonies causing the complete lysis of the erythrocytes. It is hence possible that the potentiation of the damage to red blood cells observed in vitro mimics an in vivo damage on the membranes of enterocytes to which HIS are attached when intestinal spirochaetosis occurs and when cytolysins similar to the alpha-toxin are available in the intestine of the host.

Alpha toxin from Clostridium perfringens induces proinflammatory changes in endothelial cells.

Alpha toxin from Clostridium perfringens type A, a phospholipase C, has been implicated in many of the localized and systemic features of gas gangrene. We demonstrated that human endothelial cells synthesize two vasoactive lipids, platelet-activating factor (PAF) and prostacyclin, in response to alpha toxin treatment. The stimulated synthesis of PAF required the enzymatic activity of the toxin and subsequent protein kinase C activation. Alpha toxin-treated endothelial cells accumulated the products of the phospholipase C reaction, diacylglycerol and ceramide, and exhibited a decrease in the enzymatic precursors phosphatidylcholine and sphingomyelin. Furthermore, the temporal accumulation of PAF depended on the concentration of the toxin in the overlying medium and was blocked in the presence of a neutralizing antibody. The cultured endothelial cells also exhibited enhanced neutrophil adhesion in response to alpha toxin which was mediated through the PAF receptor and P-selectin. P-selectin expression by endothelial cells and extravascular neutrophil accumulation were also observed in tissue sections from alpha toxin-injected Sprague-Dawley rats. These endothelial cell-mediated processes are important in maintaining vascular homeostasis and, when activated in a dysregulated manner by C. perfringens alpha toxin, may contribute to localized and systemic manifestations of gas gangrene including enhanced vascular permeability, localized neutrophil accumulation, and myocardial dysfunction.

Site-specific mutagenesis of Clostridium perfringens alpha-toxin: replacement of Asp-56, Asp-130, or Glu-152 causes loss of enzymatic and hemolytic activities.

The current study has investigated the role of D-56, D-130, and E-152 in zinc ion binding properties, as well as the hemolytic, phospholipase C (PLC), and sphingomyelinase (SMase) activities of Clostridium perfringens alpha-toxin, based upon crystallography studies of the Bacillus cereus PLC, which had suggested these residues might be important for these functional activities. The replacement of D-56 in alpha-toxin resulted in complete loss of hemolytic, PLC, and SMase activities. The variant toxins at D-130 showed an approximately 100-fold reduction of biological activities compared to that of the wild-type toxin. The substitution of glutamine or glycine for E-152 caused complete loss of these activities, but substitution of aspartic acid for E-152 reduced but did not completely inhibit these activities. The variant toxins at D-56 and D-130, as well as the wild-type toxin, possessed approximately 2 mol of zinc atoms per mol of the protein, but E152G and E152Q contained approximately 1 mol of zinc metal per mol of the protein. On the other hand, the zinc content in E152D was calculated as about 1.4 mol in the toxin molecule. The replacement of D-56, D-130, or E-152 had no effect on binding to sheep erythrocytes and uptake of free zinc ion from the solution. The variant toxins at D-130 showed partial antigenic identity with the wild-type toxin on a double gel diffusion test. These observations suggest that D-56 in alpha-toxin is required for catalytic activity of alpha-toxin, D-130 is essential for maintenance of structure, and the carboxyl group of E-152 tightly ligands one zinc ion, which is essential for catalytic activity of the toxin.

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47.

Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.

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.

Monoclonal Antibody-Based ELISA for Detection of Clostridium perfringens Alpha-Toxin

A monoclonal antibody-based ELISA was developed to detect alpha-toxin present in Clostridium perfringens bacterial cell lysates and cell-free culture supernatants, Monoclonal antibodies against C. perfringens alpha-toxin were produced in hybridoma tissue culture supernatants and in BALB/c mice ascites fluid, The monoclonal antibodies obtained from hybridoma culture supernatant and ascites fluid showed identical antigen specificity, but the latter showed a higher titer, with a 50% endpoint at 1/4,000. The monoclonal antibodies were specific for phospholipase C produced by C. perfringens, but not by Bacillus cereus. The lower limit of phospholipase C detection was 16 ng/ml. The dose-dependent relationship between absorption at 490nm and concentration of phospholipase C diluted in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) or Trypticase glucose yeast broth fit a four-parameter and a quadratic model, respectively. The monoclonal antibody-based ELISA developed is a rapid, sensitive and specific detection method and can be used for quantitative characterization of C. perfringens alpha-toxin.

The potential role of Clostridium perfringens alpha toxin in the pathogenesis of acute pancreatitis.

Clostridium perfringens is a bowel commensal that can colonise the biliary tract. It produces the alpha toxin (phospholipase C), which can induce spontaneous tissue necrosis. To investigate whether there is any evidence that Clostridium perfringens alpha toxin can be detected in acute pancreatitis. Serum samples from 21 patients with acute pancreatitis and 22 controls were assayed for C perfringens phospholipase C as well as anti-phospholipase C IgG and IgM; IgG and IgM anti-toxins were measured by enzyme linked immunosorbent assay. In normal healthy controls there is a very high level of natural anti-toxin of both the IgG and IgM class. Of the 21 patients with acute pancreatitis alpha toxin was detected in five (23.8%). Levels of both IgG and IgM anti-toxin were significantly reduced in acute pancreatitis. The results suggest that there is an abnormality of the immune status to C perfringens alpha toxin in patients with acute pancreatitis. This may be the result of a release of alpha toxin, although it is difficult to state whether this is a primary or secondary phenomenon in these patients. These preliminary results merit further investigation.

Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes

The effect of Clostridium perfringens alpha-toxin on multilamellar liposomes prepared from various phospholipids and cholesterol was investigated. The toxin induced carboxyfluorescein leakage from liposomes composed of the choline-containing phospholipids such as egg-yolk phosphatidylcholine and bovine brain sphingomyelin in a dose-dependent manner, but did not induce leakage from those liposomes composed of bovine brain phosphatidylethanol amine, egg-yolk phosphatidylserine or phosphatidylglycerol. The toxin-induced carboxyfluorescein leakage from egg-yolk phosphatidylcholine liposomes was increased by addition of divalent cations. The toxin induced carboxyfluorescein release from liposomes composed of phosphatidylcholine containing unsaturated fatty acyl residues or shorter chain length saturated fatty acyl residues (12 or 14 carbon atoms), but did not induce such release from liposomes composed of phosphatidylcholine containing saturated fatty acyl residues of between 16 and 20 carbon atoms. Furthermore, the toxin-induced carboxyfluorescein release decreased with increasing chain length of the acyl residues of phosphatidylcholine used. The toxin bound to liposomes composed of phospholipids which are hydrolyzed by the toxin, but did not bind to those composed of phospholipids which are not attacked by the toxin. The toxin-induced carboxyfluorescein release from liposomes composed of dipalmitoleoyl- l-α-phosphatidyl-choline and cholesterol and the toxin binding to the liposomes decreased with decreasing cholesterol contents. These observations suggest that the specific binding site formed by the choline-containing phospholipids and cholesterol, and membrane fluidity in liposomes are essential for the membrane-damaging activity of alpha-toxin.

Threonine-74 is a key site for the activity of Clostridium perfringens alpha-toxin.

A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [65Zn]2+ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.

Synthesis and evaluation of a non-radioactive gene probe for the detection of C. perfringens alpha toxin

The synthesis and evaluation of a non-radioactive hybridization probe is described, specific detecting the Clostridium perfringens alphatoxin gene (plc) by colony blot hybridization assay. A vector free digoxigenin-dUTP-labelled probe was generated by polymerase chain reaction (PCR) targeting the cloned plc gene of C.perfringens strain ATCC 13124. In a colony blot hybridization assay 296 strains of C.perfringens were tested for plc. None of the strains failed in hybridization. Presence of plc was even demonstrated in C.perfringens strains reported to lack lecithinase activity. Specificity of the probe was shown with various strains of other bacterial species. None different Clostridia sp. tested, e.g. C.bifermentans, C.tertium, C.novyi, C.chauvoei, C.sporogenes, C.difficile, C.putrifucum, C.sordellii, C.botulinum, C. septicum and C.histolyticum, hybridized with the plc specific probe. Strains expressing an enzymatically related phospholipase like Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus gave also negative results. Comparing the results of conventionally used egg yolk turbidity assay and those gained with DNA hybridization, the plc probe proved to be a much more sensitive and specific diagnostic tool for the detection of C.perfringens plc.

Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin

Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.

Development of an ELISA assay for Clostridium perfringens phospholipase C (alpha toxin).

A new method for the assay of Clostridium perfringens alpha toxin (phospholipase C) is described using a sandwich ELISA. This assay has been shown to be quantitative, to have a high specificity for the toxin and is capable of detecting purified Clostridium perfringens phospholipase C at concentrations of as little as 0.005 units/ml in cooked meat culture medium.

Roles of the carboxy-terminal region of Clostridium perfringens alpha toxin

Roles of the carboxy-terminal region of Clostridium perfringens alpha toxin

Roles of the carboxy-terminal region of Clostridium perfringens alpha toxin.

Treatment of Clostridium perfringens alpha toxin with aminopeptidase resulted in no effect on various activities of the toxin. Aminopeptidase did not hydrolyze the native toxin or the toxin treated with urea in the presence of EDTA. Treatment with carboxypeptidase for 30 min resulted in a 75% decrease in these activities. Incubation of the native toxin with carboxypeptidase for 30 min released approximately 15 amino acids from the C-terminus of the toxin. The biological activities of a mutant toxin lacking 20 C-terminal residues of the toxin (AT1-350) showed about 59-87% of the activity of native toxin. The mutant toxin showed partial antigenic identity with the native toxin. These data suggest that the C-terminal domain contributes to maintaining the active form of the toxin.

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.

Evidence for coupling of Clostridium perfringens alpha-toxin-induced hemolysis to stimulated phosphatidic acid formation in rabbit erythrocytes

When rabbit erythrocytes were exposed to low concentrations of Clostridium perfringens alpha-toxin, hot-cold hemolysis was observed. The toxin induced production of phosphatidic acid (PA) in a dose-dependent manner when incubated with erythrocytes at 37 degrees C. When erythrocyte membranes were incubated with the toxin and [gamma-32P]ATP in the presence or absence of ethanol, [32P]PA formation was maximal within 30 s, then sharply decreased, and began again after 5 min of incubation. Ethanol had no effect on the early appearance (at approximately 5 min) of PA formation induced by the toxin but significantly inhibited formation of PA over 10 min of incubation. Treatment of erythrocyte membranes with alpha-toxin resulted in the biphasic formation of 1,2-diacylglycerol and PA as well as an increase of inositol-1,4,5-trisphosphate (IP3) and decrease of phosphatidylinositol-4,5-bisphosphate (PIP2) within 30 s. Neomycin inhibited the toxin-induced increase in turbidity of egg yolk suspensions but did not inhibit the toxin-induced hemolysis of intact erythrocytes. On the other hand, neomycin inhibited the toxin-induced hemolysis of saponin-treated erythrocytes. In addition, neomycin inhibited PA formation induced by the toxin in erythrocyte membranes. IP3 was released by incubation of PIP2 with erythrocyte membranes but not by incubation of PIP2 with the toxin. The toxin stimulated the membrane-induced release of IP3 from PIP2. These data suggest that the toxin-induced hemolysis is dependent on the action of phospholipase C in erythrocyte membranes.

Enterotoxigenic bacteria in the sudden infant death syndrome.

Faecal samples from 123 infants who died with sudden infant death syndrome (SIDS) and from a comparative group of 52 age-matched babies were analysed for toxigenic bacteria and their toxins. Serum samples from the SIDS infants were also analysed for these toxins. A significantly higher proportion of toxigenic bacteria and their toxins were found in faecal samples of SIDS babies than in samples from the comparative group. These toxins were also found in serum from the SIDS babies. Clostridium perfringens was found in 54 (45.4%) of 119 SIDS cases compared with 10 (19.6%) of 51 healthy babies (chi 2 = 10.1, p < 0.01); C. difficile in 33 (27.7%) of 119 SIDS cases compared with 8 (14.8%) of 54 healthy babies (chi 2 = 3.43ns, p < 0.1); Staphylococcus aureus in 12 (27.3%; 66.7% enterotoxigenic) of 44 SIDS cases compared with 12 (85.7%; non-enterotoxigenic) of 14 healthy babies (chi 2 = 14.9, p < 0.001); C. botulinum in 6 (5.0%) of 120 SIDS cases compared with 0 of 53 healthy babies (chi 2 = 2.74, p < 0.1). Campylobacter jejuni, Yersinia enterocolitica, Vibrio parahaemolyticus, salmonellae and Bacillus cereus were not detected. Heat-labile toxin, lethal to mice (HLML) was found in 32 (27.1%) of 118 SIDS faecal samples compared with 5 (10.6%) of 47 healthy babies (chi 2 = 5.24, p < 0.05); cytotoxins in 38 (30.9%) of 123 SIDS faecal samples compared with 0 of 21 of healthy babies (chi 2 = 8.8, p < 0.01) and 24 (27.6%) of 87 SIDS serum samples. C. perfringens enterotoxin was detected in 33 (34.4%) of 96 SIDS faecal extracts compared with 0 of 23 of healthy babies (chi 2 = 10.94, p < 0.001), and in 27 (24.5%) of 110 SIDS serum samples. C. perfringens alpha-toxin (presumptive) was detected in 14 (17.5%) of 80 SIDS faecal extracts compared with 0 of 17 from healthy babies (chi 2 = 3.5ns, p congruent to 0.05) and in 2 (2.3%) of 87 SIDS serum samples. C. difficile toxin was detected in four SIDS faecal samples and two serum samples. C. botulinum toxin was detected in only one of 120 SIDS faecal samples compared with none of 49 from healthy babies. Staphylococcal enterotoxins were detected in 8 (19.5%) of 41 SIDS faecal samples compared with 0 of 19 from healthy babies (chi 2 = 4.278, p < 0.05), and in 4 (10.8%) of 37 SIDS serum samples.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacterial phospholipases C

A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.