Clostridium Perfringens Alpha-toxin Research Articles

Role of tryptophan-1 in hemolytic and phospholipase C activities of Clostridium perfringens alpha-toxin.

Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the N-terminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.

Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin

Alpha-toxin is the key determinant in gas-gangrene. The toxin, a phospholipase C, cleaves phosphatidylcholine in eukaryotic cell membranes. Calcium ions have been shown to be required for the specific binding of toxin to membranes prior to phospholipid cleavage. Reported X-ray crystallographic structures of the toxin show that the C-terminal domain has a fold that is analogous to the eukaryotic calcium and membrane-binding C2 domains. We report the binding sites for three calcium ions that have been identified, by crystallographic methods, in the C-terminal domain of the protein close to the postulated membrane-binding surface. The position of these ions at the tip of the domain, and their function (to facilitate membrane binding) is similar to that of calcium ions observed bound to C2 domains. Using the optical spectroscopic techniques of circular dichroism (CD) and fluorescence spectroscopy, pronounced changes to both near and far-UV CD and tryptophan emission fluorescence upon addition of calcium to the C-terminal domain of α-toxin have been observed. The changes in near-UV CD, fluorescence enhancement and a 2 nm blue-shift in the fluorescence emission spectrum are consistent with tryptophan residue(s) becoming more immobilised in a hydrophobic environment. Calcium binding appears to be low-affinity: Kd≈175–250 μM at pH 8 assuming a 1:1 stoichiometry. as measured by spectroscopic methods.

Recombinant vaccinia viruses protect against Clostridium perfringens alpha-toxin.

Recombinant vaccinia viruses that expressed the nontoxic C-domain of Clostridium perfringens alpha-toxin were constructed. The J2R (thymidine kinase [TK] gene) and B13R (serpin 2 [SPI-2] gene) loci were used as insertion sites for the clostridial DNA, and expression of the foreign protein was measured in each case. A double recombinant that encoded the alpha-toxin truncate at the B13R locus and the protective antigen of Bacillus anthracis at the J2R locus was also constructed. Although differences in expression of the alpha-toxin C-domain were recorded, all of the vaccinia recombinants protected mice against a lethal challenge with alpha-toxin demonstrating that a recombinant vaccinia virus can be used to provide protection against a toxin challenge that is known to be solely antibody mediated.

Modulation of enzymatic activity and biological function of Listeria monocytogenes broad-range phospholipase C by amino acid substitutions and by replacement with the Bacillus cereus ortholog.

The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium's ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens alpha-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells.

Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed

A multiplex polymerase chain reaction (PCR) assay, developed to detect the alpha-toxin and enterotoxin genes ( cpa and cpe, respectively) of Clostridium perfringens, was used to identify enterotoxigenic isolates of this organism from feces and intestinal contents of pigs and from feed samples from pig farms in Iowa. The organism was grown on tryptose-sulfite-cycloserine (TSC) agar, TSC agar without egg-yolk, sheep blood agar, or in brain heart infusion broth or cooked meat medium. DNA was extracted by boiling and the PCR assay was carried out using reagents from a commercial kit. The 319 bp amplification product of cpa and the 364 bp product of cpe were visualized under UV light after electrophoresis in a 2% agarose gel containing ethidium bromide. The average sensitivity of the assay, determined on artificially contaminated feces, was 9.2×10 4 colony forming units per gram. Assay of 97 isolates from feces and intestinal contents revealed cpa in 89, but all were negative for cpe. While 28% of the 442 total samples cultured yielded C. perfringens, only 5% of 298 fecal or intestinal contents samples were positive upon direct examination by the PCR assay. Ninety-one and eight-tenths % of isolates with the phenotype of C. perfringens were cpa positive by PCR. Forty-three percent of feed samples were culture positive, while 48.3% were PCR positive for cpa. None of these were cpe positive. We conclude that PCR is a useful assay for rapid detection of C. perfringens in feed, and for confirmation of the identity of isolates presumed to be C. perfringens.

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.

The carboxy-terminal C2-like domain of the alpha-toxin from Clostridium perfringens mediates calcium-dependent membrane recognition.

The lethal, cytolytic alpha-toxin (phospholipase C) of Clostridium perfringens consists of two distinct modules: the larger N-terminal domain catalyses phospholipid hydrolysis, and its activity is potentiated by a smaller C-terminal domain. Calcium ions are essential for the binding of alpha-toxin to lipid films. Sixteen alpha-toxin variants with single amino acid substitutions in the C-terminal region were obtained using site-directed mutagenesis and T7 expression technology. Five of these variants showed reduced phospholipase C activity and were considerably less active than native alpha-toxin under calcium-limiting conditions. Replacement of Thr-272 by Pro diminished phospholipase C activity, severely affected haemolysis and platelet aggregation and perturbed a surface-exposed conformational epitope. The results of sequence comparisons and molecular modelling indicate that the C-terminal region probably belongs to the growing family of C2 beta-barrel domains, which are often involved in membrane interactions, and that the functionally important substitutions are clustered at one extremity of the domain. The combined findings suggest that the C-terminal region of alpha-toxin mediates interactions with membrane phospholipids in a calcium-dependent manner. Mutations to this domain may account for the natural lack of toxicity of the alpha-toxin homologue, phospholipase C of Clostridium bifermentans.

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.

An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter.

The plc gene, which encodes phospholipase C (alpha-toxin) of Clostridium perfringens, possesses three poly(A) tracts forming an intrinsically curved DNA region immediately upstream of the promoter. The in vivo transcriptional activity of the plasmid-borne plc gene was stimulated by this curved-DNA-containing sequence, depending on its proper linear and rotational orientation. The in vitro transcriptional activity of the plc gene was also stimulated by the upstream sequence. In addition, the stimulatory effect of the sequence and the degree of DNA bending were greater at lower temperature, as was demonstrated by both in vitro and in vivo transcription assays, and a gel-mobility assay, respectively. A similar temperature effect was also observed with the chromosomal plc gene. These observations suggest that the upstream DNA curvature per se stimulates the initiation of transcription of the plc gene, possibly through direct contact with RNA polymerase.

Use of site-directed mutagenesis to probe structure-function relationships of alpha-toxin from Clostridium perfringens.

The NH2-terminal domain of the alpha-toxin of Clostridium perfringens is highly homologous to the complete phospholipase C from Bacillus cereus (PC-PLC), for which a high-resolution crystal structure is available. This structural information was used as the basis of a site-directed mutagenesis strategy in which critical amino acid residues of alpha-toxin involved in zinc binding, interaction with substrate, or catalysis were replaced. Biochemical studies with the corresponding toxin variants indicate that there is probably a single active site endowed with lecithinase, sphingomyelinase, and hemolytic activities. By using a highly purified variant in which the catalytic aspartate residue at position 56 was replaced by asparagine, it was shown that phospholipase activity was essential for lethality in vivo and for mediating platelet aggregation in vitro.

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.

Purification, Crystallization and Preliminary X-ray Diffraction Studies of Alpha-Toxin of Clostridium perfringens

Alpha-toxin of Clostridium perfringes, cloned in Escherichia coli, has been purified and crystallized from ammonium sulphate using the hanging drop vapour diffusion method at 20°C. The crystals diffract to a minimum Bragg spacing of 2.7 Å, belong to the space group R32 (with a = b = 153.3 Å, c = 95.4 Å, α = β = 90° and γ = 120°) and contain a single polypeptide chain in the crystallographic unit.