Sadayuki Ochi

Characterization of the Enzymatic Component of Clostridium perfringens Iota-Toxin

The iota(a) component (i(a)) of Clostridium perfringens ADP ribosylates nonmuscle beta/gamma actin and skeletal muscle alpha-actin. Replacement of Arg-295 in i(a) with alanine led to a complete loss of NAD(+)-glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD(+) to i(a), that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i(a) on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i(a) are dependent on ARTase activity.

Role of the C-domain in the biological activities of Clostridium perfringens alpha-toxin.

Clostridium perfringens alpha‐toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC). In this study we examined the role of the C‐domain (251–370 residues; CP251–370) in biological activities of the toxin. The N‐domain (1–250 residues; CP1–250) of the alpha‐toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them. A hybrid protein (BC‐CP251–370) consisting of BcPLC and CP251–370 bound to the red cells and lysed them. Incubation of CP1–250 with CP251–370 completely complemented hemolytic and PLC activities. CP251–370 also conferred hemolytic activity on BcPLC. CP251–340 (251–340 residues) significantly stimulated PLC activity of CP1–250, but did not confer hemolytic activity on CP1–250. Kinetic analysis suggested that CP251–370 increased affinity toward the substrate of CP1–250. The results suggested that CP251–370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1–250. Acrylodan‐labeled CP251–370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N′‐dimethyl‐N‐(iodoacetyl)‐N′‐(7‐nitrobenz‐2‐oxa‐1,3‐diazolyl)ethylene diamine (NBD)‐labeled CP251–370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251–370 to a hydrophobic environment. These observations suggest that interaction of CP251–370 of alpha‐toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1–250.

Clostridium perfringens β-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity

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

Clostridium perfringens alpha-toxin-induced hemolysis of horse erythrocytes is dependent on Ca2+ uptake.

Clostridium perfringens alpha-toxin is able to lyse various erythrocytes. Exposure of horse erythrocytes to alpha-toxin simultaneously induced hot-cold hemolysis and stimulated production of diacylglycerol and phosphorylcholine. When A23187-treated erythrocytes were treated with the toxin, these events were dependent on the concentration of extracellular Ca2+ . Incubation with the toxin of BAPTA-AM-treated horse erythrocytes caused no hemolysis or production of phosphorylcholine, but that of the BAPTA-treated erythrocytes did. When Quin 2-AM-treated erythrocytes were incubated with the toxin in the presence of 45Ca2+, the cells accumulated 45Ca2+ in a dose- and a time-dependent manner. These results suggest that the toxin-induced hemolysis and hydrolysis of phosphatidylcholine are closely related to the presence of Ca2+ in the cells. Flunarizine, a T-type Ca2+ channel blocker, and tetrandrine, an L- and T-type Ca2+ channel blocker, inhibited the toxin-induced hemolysis and Ca2+ uptake. However, L-type Ca2+ channel blockers, nifedipine, verpamil and diltiazem, an N-type blocker, omega-conotoxin SVIB, P-type blockers, omega-agatoxin TK and omega-agatoxin IVA, and a Q-type blocker, omega-conotoxin MVII C, had no such inhibitory effect. The observation suggests that Ca2+ taken up through T-type Ca2+ channels activated by the toxin plays an important role in hemolysis induced by the toxin.

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‐α‐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‐α‐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.

Recent Insights into Clostridium perfringens Beta-Toxin

Clostridium perfringens beta-toxin is a key mediator of necrotizing enterocolitis and enterotoxemia. It is a pore-forming toxin (PFT) that exerts cytotoxic effect. Experimental investigation using piglet and rabbit intestinal loop models and a mouse infection model apparently showed that beta-toxin is the important pathogenic factor of the organisms. The toxin caused the swelling and disruption of HL-60 cells and formed a functional pore in the lipid raft microdomains of sensitive cells. These findings represent significant progress in the characterization of the toxin with knowledge on its biological features, mechanism of action and structure-function having been accumulated. Our aims here are to review the current progresses in our comprehension of the virulence of C. perfringens type C and the character, biological feature and structure-function of beta-toxin.

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 WIG 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.

The Relationship between Histidine Redidues and Various Biological Activities of Clostridium perfringens Alpha Toxin

Clostridium perfringens alpha toxin is known to possess various biological activities (hemolysis, lethality, necrosis) and, in addition, phospholipase C (PLC) activity (4). It has been reported that these biological activities of the toxin are due to the PLC activity (4). We have reported that the toxin-induced contraction of isolated rat ileum or aorta and the toxin-induced hemolysis of rabbit erythrocytes are closely related to phospholipid metabolism stimulated by the toxin (1, 6, 7, 8). However, little is known about the relationship between these biological activities and PLC activity. The gene encoding alpha toxin has been isolated from chromosome of the organism and characterized (9). The genes encoding PLC’s of C. bifermentans and Bacillus cereus also have been isolated. From these findings, the deduced amino acid sequences of alpha toxin and these enzymes were found to have significant homology up to approximately 250 residues from N-terminus. Hough et al. have reported the three-dimensional structure of the B. cereus PLC, which binds three Zn2+(2). Little and Otnass reported that the B. cereus PLC has two tightly bound zinc ions and one loosely bound zinc ion (3). It is reported that these ions are coordinated to fixed histidine residues in the enzyme (2). Alpha toxin is known to be one of zinc-enzymes. Vallee and Auld (10) reported the possibility that the ligand spacers and vicinal amino acids of the B. cereus PLC resemble the likely location of the metal binding sites in the toxin (2). Recently, it has been reported that histidine residues in zinc-proteins such proteases and botulinum toxin are ligands to Zn2+. Therefore, to clarify the mode-of-action of alpha toxin, we replaced all of histidine residues in the toxin to glycine by site-directed mutagenesis, and then investigated the relationship between histidine residues and Zn2+ contents or the biological and PLC activities.

Effect of Clostridium perfringens Alpha Toxin on Contraction of Isolated Guinea‐Pig Diaphragm

The effect of Clostridium perfringens alpha toxin on contraction induced by‐electric stimulation of isolated guinea‐pig diaphragm was investigated. The toxin inhibited electrically stimulated contraction of the tissue in a dose‐ and incubation time‐dependent manner. Tetrodotoxin resulted in no effect of the action of the toxin. Nifedipine dose‐dependently delayed the action of the toxin, but verapamil and diltiazem did not. On the other hand, treatment of the toxin with N‐acetylimidazole caused significant reduction of the inhibitory activity of the toxin on contraction, but did not cause significant loss of phospholipase C activity (PN activity) as measured by hydrolysis of p‐nitrophenylphosphorylcholine. The data showed that the toxin impairs contraction of isolated guinea‐pig diaphragm.