Clostridium perfringens epsilon-toxin requires acid sphingomyelinase for cellular entry.

OBJECTIVES: Clostridium perfringens epsilon-toxin is considered to be a crucial agent in enterotoxemia in domestic animals. Epsilon-toxin enters host cells via endocytosis and results in the formation of late endosome/lysosome-derived vacuoles. In the present study, we found that acid sphingomyelinase promotes the internalization of epsilon-toxin in MDCK cells. METHODS: We measured the extracellular release of acid sphingomyelinase (ASMase) by epsilon-toxin. We examined the role of ASMase in epsilon-toxin-induced cytotoxicity using selective inhibitors and knockdown of ASMase. Production of ceramide after toxin treatment was determined by immunofluorescence technique. RESULTS: Blocking agents of ASMase and exocytosis of lysosomes inhibited this epsilon-toxin-induced vacuole formation. Lysosomal ASMase was liberated to extracellular space during treatment of the cells with epsilon-toxin in the presence of Ca2+. RNAi-mediated attenuation of ASMase blocked epsilon-toxin-induced vacuolation. Moreover, incubation of MDCK cells with epsilon-toxin led to production of ceramide. The ceramide colocalized with lipid raft-binding cholera toxin subunit B (CTB) in the cell membrane, indicating that conversion of lipid raft associated sphingomyelin to ceramide by ASMase facilitates lesion of MDCK cells and internalization of epsilon-toxin. CONCLUSIONS: Based on the present results, ASMase is required for efficient internalization of epsilon-toxin.

Clostridium perfringens α-toxin inhibits myogenic differentiation of C2C12 myoblasts.

Clostridium perfringens type A is the causative agent of clostridial myonecrosis, and α-toxin has been reported to be responsible for the pathogenesis. Recently, it was reported that regeneration of skeletal muscle after C. perfringens-induced muscle disorders is delayed, but the detailed mechanisms have not been elucidated. Here, we tested whether α-toxin impairs the differentiation of C2C12 myoblasts, a useful cell line to study muscle growth, maturation, and regeneration in vitro. α-Toxin dose-dependently inhibited myotube formation in C2C12 cultures after induction of their differentiation by horse serum. Also, immunoblot analysis revealed that α-toxin dose-dependently decreases the expressions of two skeletal muscle differentiation markers, myogenic differentiation 1 (MyoD) and myogenin. These results demonstrate that α-toxin impairs the myogenic differentiation of C2C12 myoblasts. To reveal the mechanism behind α-toxin-mediated impairment of myogenic differentiation, we focused on ceramide production since α-toxin is known to promote the formation of ceramide by its sphingomyelinase activity. Immunofluorescent analysis revealed that ceramide production is accelerated by treatment with α-toxin. Furthermore, a synthetic cell-permeable ceramide analog, C2-ceramide, inhibited myotube formation in C2C12 cells and decreased the expressions of MyoD and myogenin, suggesting that accelerated ceramide production is involved in the α-toxin-mediated blockage of myogenic differentiation. Together, our results illustrate that the impairment of myogenic differentiation by α-toxin might be crucial for the pathogenesis of C. perfringens to delay regeneration of severely damaged skeletal muscles.

Clostridium perfringens α-toxin specifically induces endothelial cell death by promoting ceramide-mediated apoptosis.

Clostridium perfringens type A-induced gas gangrene is characterized by severe myonecrosis, and α-toxin has been revealed to be a major virulence factor involved in the pathogenesis. However, the detailed mechanism is unclear. Here, we show that CD31+ endothelial cell counts decrease in muscles infected with C. perfringens in an α-toxin-dependent manner. In vitro experiments revealed that α-toxin preferentially and rapidly induces the death of human umbilical vein endothelial cells (HUVECs) compared with C2C12 murine muscle cells. The toxin induces apoptosis of HUVECs by increasing ceramide. Furthermore, the specificity might be dependent on differences in the sensitivity to ceramide between these cell lines. Together, our results suggest that α-toxin-induced endothelial cell death promotes severe myonecrosis and is involved in the pathogenesis of C. perfringens.

Delta-toxin from Clostridium perfringens perturbs intestinal epithelial barrier function in Caco-2 cell monolayers.

Clostridium perfringens delta-toxin is a ß-barrel-pore-forming toxin (ß-PFT) and a presumptive virulence factor of type B and C strains, which are causative organisms of fatal intestinal diseases in animals. We showed previously that delta-toxin causes cytotoxicity via necrosis in sensitive cells. Here, we examined the effect of delta-toxin on intestinal membrane integrity. Delta-toxin led to a reduction in transepithelial electrical resistance (TEER) and increased the permeability of fluorescence isothiocyanate-conjugated dextran in human intestinal epithelial Caco-2 cells without changing the tight junction proteins, such as zonula occludens-1 (ZO-1), occludin, and claudin-1. On the other hand, delta-toxin reduced the cellular levels of adherence junction protein E-cadherin before cell injury. A disintegrin and metalloprotease (ADAM) 10 facilitates E-cadherin cleavage and was identified as the cellular receptor for alpha-toxin, a ß-PFT produced by Staphylococcus aureus. ADAM10 inhibitor (GI254023X) blocked the toxin-induced decrease in TEER and cleavage of E-cadherin. Delta-toxin enhanced ADAM10 activity in a dose- and time-dependent manner. Furthermore, delta-toxin colocalized with ADAM10. These results indicated that ADAM10 plays a key role in delta-toxin-induced intestinal injury.

Role of pannexin 1 in Clostridium perfringens beta-toxin-caused cell death.

BACKGROUND: Beta-toxin produced by Clostridium perfringens is a key virulence factor of fatal hemorrhagic enterocolitis and enterotoxemia. This toxin belongs to a family of ß-pore-forming toxins (PFTs). We reported recently that the ATP-gated P2X7 receptor interacts with beta-toxin. The ATP-release channel pannexin 1 (Panx1) is an important contributor to P2X7 receptor signaling. Hence, we investigated the involvement of Panx1 in beta-toxin-caused cell death. METHODS: We examined the effect of Panx1 in beta-toxin-induced cell death utilizing selective antagonists, knockdown of Panx1, and binding using dot-blot analysis. Localization of Panx1 and the P2X7 receptor after toxin treatment was determined by immunofluorescence staining. RESULTS: Selective Panx1 antagonists (carbenoxolone [CBX], probenecid, and Panx1 inhibitory peptide) prevented beta-toxin-caused cell death in THP-1 cells. CBX did not block the binding of the toxin to cells. Small interfering knockdown of Panx1 blocked beta-toxin-mediated cell death through inhibiting the oligomer formation of the toxin. Beta-toxin triggered a transient ATP release from THP-1 cells, but this early ATP release was blocked by CBX. ATP scavengers (apyrase and hexokinase) inhibited beta-toxin-induced cytotoxicity. Furthermore, co-administration of ATP with beta-toxin enhanced the binding and cytotoxicity of the toxin. CONCLUSIONS: Based on our results, Panx1 activation is achieved through the interaction of beta-toxin with the P2X7 receptor. Then, ATP released by the Panx1 channel opening promotes oligomer formation of the toxin, leading to cell death. GENERAL SIGNIFICANCE: Pannexin 1 is a novel candidate therapeutic target for beta-toxin-mediated disease.

Oligomer formation of Clostridium perfringens epsilon-toxin is induced by activation of neutral sphingomyelinase.

BACKGROUND: Clostridium perfringens epsilon-toxin is responsible for fatal enterotoxemia in ungulates. The toxin forms a heptamer in the lipid rafts of Madin-Darby Canine Kidney (MDCK) cells, leading to cell death. Here, we showed that epsilon-toxin requires neutral sphingomyelinase (nSMase) activity during oligomerization. METHODS: We tested the role of nSMase in the oligomerization of epsilon-toxin using specific inhibitors, knockdown of nSMase, formation of ceramide, and localization of epsilon-toxin and ceramide by immunofluorescence staining. RESULTS: Epsilon-toxin induced the production of ceramide is a dose- and time-dependent manner in ACHN cells. GW4869, an inhibitor of nSMase, inhibited ceramide production induced by the toxin. GW4869 and knockdown of nSMase blocked toxin-induced cell death and oligomer formation of epsilon-toxin. Confocal microscopy images showed that the toxin induced ceramide clustering and colocalized with ceramide. CONCLUSIONS: These results demonstrated that oligomer formation of epsilon-toxin is facilitated by the production of ceramide through activation of nSMase caused by the toxin. GENERAL SIGNIFICANCE: Inhibitors of nSMase may confer protection against infection.

Cellular Uptake of Clostridium botulinum C2 Toxin Requires Acid Sphingomyelinase Activity.

Clostridium botulinum C2 toxin consists of an enzyme component (C2I) and a binding component (C2II). Activated C2II (C2IIa) binds to a cell receptor, giving rise to lipid raft-dependent oligomerization, and it then assembles with C2I. The whole toxin complex is then endocytosed into the cytosol, resulting in the destruction of the actin cytoskeleton and cell rounding. Here, we showed that C2 toxin requires acid sphingomyelinase (ASMase) activity during internalization. In this study, inhibitors of ASMase and lysosomal exocytosis blocked C2 toxin-induced cell rounding. C2IIa induced Ca2+ influx from the extracellular medium to cells. C2 toxin-induced cell rounding was enhanced in the presence of Ca2+ ASMase was released extracellularly when cells were incubated with C2IIa in the presence of Ca2+ Small interfering RNA (siRNA) knockdown of ASMase reduced C2 toxin-induced cell rounding. ASMase hydrolyzes sphingomyelin to ceramide on the outer leaflet of the membrane at acidic pH. Ceramide was detected in cytoplasmic vesicles containing C2IIa. These results indicated that ASMase activity is necessary for the efficient internalization of C2 toxin into cells. Inhibitors of ASMase may confer protection against infection.

Peptidoglycan accelerates granulopoiesis through a TLR2- and MyD88-dependent pathway.

Granulopoiesis is accelerated during Gram-negative bacterial infection through activation of toll-like receptor 4 (TLR4). In this study, we tested whether activation of TLR2 promotes granulopoiesis by using the well-known TLR2 agonist, peptidoglycan (PGN). Neutrophils in bone marrow and spleen, and plasma granulocyte colony-stimulating factor (G-CSF) were increased in mice that had received intraperitoneal PGN administration. Incorporation of BrdU into bone marrow neutrophils increased, demonstrating that PGN accelerated granulopoiesis. Treatment of bone marrow cells (BMCs) with PGN increased neutrophils in vitro and promoted the secretion of G-CSF from Ly-6G-Ly-6C+ monocytes. The accelerated granulopoiesis caused by PGN was not seen in TLR2-deficient and MyD88-deficient BMCs. Additionally, PGN induced G-CSF production in human umbilical vein endothelial cells. These findings demonstrate that PGN promotes the secretion of G-CSF from monocytes and endothelial cells, leading to the acceleration of granulopoiesis. Our results illustrate that bacterial recognition by TLR2 facilitates granulopoiesis during Gram-positive bacterial infection.

Role of P2X7 receptor in Clostridium perfringens beta-toxin-mediated cellular injury.

BACKGROUND: Clostridium perfringens beta-toxin is a pore-forming toxin (PFT) and an important agent of necrotic enteritis and enterotoxemia. We recently reported that beta-toxin strongly induced cell death in THP-1 cells via the formation of oligomers. We here describe that the P2X(7) receptor, which is an ATP receptor, interacts with beta-toxin. METHODS: We tested the role of P2X(7) receptor in beta-toxin-induced toxicity using specific inhibitors, knockdown of receptor, expression of the receptor and interaction by dot-blot assay. The potency of P2X(7) receptor was further determined using an in vivo mouse model. RESULTS: Selective P2X(7) receptor antagonists (oxidized ATP (o-ATP), oxidized ADP, and Brilliant Blue G (BBG)) inhibited beta-toxin-induced cytotoxicity in THP-1 cells. o-ATP also blocked the binding of beta-toxin to cells. The P2X(7) receptor and beta-toxin oligomer were localized in the lipid rafts of THP-1 cells. siRNA for the P2X(7) receptor inhibited toxin-induced cytotoxicity and binding of the toxin. In contrast, the siRNA knockdown of P2Y(2) or P2Y(6) had no effect on beta-toxin-induced cytotoxicity. The addition of beta-toxin to P2X(7)-transfected HEK-293 cells resulted in binding of beta-toxin oligomer. Moreover, beta-toxin specifically bound to immobilized P2X(7) receptors in vitro and colocalized with the P2X(7) receptor on the THP-1 cell surface. Furthermore, beta-toxin-induced lethality in mice was blocked by the preadministration of BBG. CONCLUSIONS: The results of this study indicate that the P2X(7) receptor plays a role in beta-toxin-mediated cellular injury. GENERAL SIGNIFICANCE: P2X(7) receptor is a potential target for the treatment of C. perfringens type C infection.

Vizantin inhibits endotoxin-mediated immune responses via the TLR 4/MD-2 complex.

Vizantin has immunostimulating properties and anticancer activity. In this study, we investigated the molecular mechanism of immune activation by vizantin. THP-1 cells treated with small interfering RNA for TLR-4 abolished vizantin-induced macrophage activation processes such as chemokine release. In addition, compared with wild-type mice, the release of MIP-1ß induced by vizantin in vivo was significantly decreased in TLR-4 knockout mice, but not in TLR-2 knockout mice. Vizantin induced the release of IL-8 when HEK293T cells were transiently cotransfected with TLR-4 and MD-2, but not when they were transfected with TLR-4 or MD-2 alone or with TLR-2 or TLR-2/MD-2. A dipyrromethene boron difluoride-conjugated vizantin colocalized with TLR-4/MD-2, but not with TLR-4 or MD-2 alone. A pull-down assay with vizantin-coated magnetic beads showed that vizantin bound to TLR-4/MD-2 in extracts from HEK293T cells expressing both TLR-4 and MD-2. Furthermore, vizantin blocked the LPS-induced release of TNF-α and IL-1ß and inhibited death in mice. We also performed in silico docking simulation analysis of vizantin and MD-2 based on the structure of MD-2 complexed with the LPS antagonist E5564; the results suggested that vizantin could bind to the active pocket of MD-2. Our observations show that vizantin specifically binds to the TLR-4/MD-2 complex and that the vizantin receptor is identical to the LPS receptor. We conclude that vizantin could be an effective adjuvant and a therapeutic agent in the treatment of infectious diseases and the endotoxin shock caused by LPS.

Clostridium perfringens α-Toxin Impairs Lipid Raft Integrity in Neutrophils.

Clostridium perfringens type A, a Gram-positive, anaerobic bacterium, causes gas gangrene. Recently, we reported that C. perfringens α-toxin blocked neutrophil differentiation in an enzyme activity-dependent manner to impair host innate immunity, which should be crucial for the pathogenesis of C. perfringens. However, the detailed mechanism remains unclear. Lipid rafts have been reported to be platforms for signaling molecules involved in the regulation of cell differentiation in many different cell types. In this study, we found that cell surface expression of a lipid raft marker, GM1 ganglioside, decreased in association with neutrophil differentiation by flow cytometry analysis and morphological observation. In vitro treatment of isolated mouse bone marrow cells with α-toxin or an α-toxin variant lacking phospholipase C and sphingomyelinase activities revealed that α-toxin increased the cell surface expression of GM1 ganglioside in an enzyme activity-dependent manner. C. perfringens infection also increased GM1 ganglioside levels in bone marrow myeloid cells. Moreover, treatment of bone marrow cells with methyl-ß-cyclodextrin, a lipid raft-disrupting agent, impaired neutrophil differentiation. Together, our results suggest that the integrity of lipid rafts should be properly maintained during granulopoiesis, and α-toxin might perturb lipid raft integrity leading to the impairment of neutrophil differentiation.

Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex.

Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD(+) analog ßTAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD(+)-bound form (NAD(+)-Ia-actin) and the ADP ribosylated form [Ia-ADP ribosylated (ADPR)-actin] remain unclear. Accidentally, we found that ethylene glycol as cryo-protectant inhibits ADP ribosylation and crystallized the NAD(+)-Ia-actin complex. Here we report high-resolution structures of NAD(+)-Ia-actin and Ia-ADPR-actin obtained by soaking apo-Ia-actin crystal with NAD(+) under different conditions. The structures of NAD(+)-Ia-actin and Ia-ADPR-actin represent the pre- and postreaction states, respectively. By assigning the ßTAD-Ia-actin structure to the transition state, the strain-alleviation model of ADP ribosylation, which we proposed previously, is experimentally confirmed and improved. Moreover, this reaction mechanism appears to be applicable not only to Ia but also to other ADP ribosyltransferases.

Identification of the replication region in pBCNF5603, a bacteriocin-encoding plasmid, in the enterotoxigenic Clostridium perfringens strain F5603.

BACKGROUND: Most recent studies of Clostridium perfringens plasmids have focused on toxin-encoding or antibiotic resistance plasmids. To cause intestinal disease, a toxigenic strain must grow in the intestines to levels allowing for sufficient toxin production and this in vivo growth often involves overcoming the normal intestinal microbial population. For this purpose, bacteriocin production might be important. RESULTS: In this study, as the first step in the genetic analysis of a co-existing plasmid with an enterotoxin gene (cpe)-encoding plasmid, the bacteriocin gene-encoding plasmid, pBCNF5603, was completely sequenced. This plasmid has some homology with two previously sequenced C. perfringens plasmids, namely, pCP13 carrying a cpb2 gene and pIP404 carrying a bcn gene. Using recombinant plasmids, the rep gene homologous to the PCP63 gene on pCP13 appeared to be functional. Comparative genomics indicated that the identified rep gene homologs were found on two additional toxin plasmids, pCP-OS1 and pCP-TS1. While functional analysis using recombinant plasmids indicated that pBCNF5603 and pCP13 are likely to be incompatible, the plasmid replication and partitioning region of pBCNF5603 alone was insufficient for stable maintenance of this plasmid. CONCLUSIONS: These findings suggest that pBCNF5603 evolved from recombination events between C. perfringens plasmids and inter-species mobile genetic element(s). In addition, the bcn-encoding plasmid, pBCNF5603, is likely to be included in the Inc family, which includes pCP13 and two variant iota-encoding plasmids. Furthermore, the bcn gene on pBCNF5603 could contribute to gastrointestinal disease induced by enterotoxigenic C. perfringens.

Clostridium perfringens TpeL Induces Formation of Stress Fibers via Activation of RhoA-ROCK Signaling Pathway.

Clostridium perfringens TpeL belongs to a family of large clostridial glucosylating cytotoxins. TpeL modifies Rac1 and Ras subfamily proteins. Herein we report TpeL-induced formation of stress fibers via RhoA-Rho kinase (ROCK) signaling. A recombinant protein (TpeL1-525) derived from the TpeL N-terminal catalytic domain in the presence of streptolysin O (SLO) induced the formation of actin stress fibers in Madin-Darby canine kidney (MDCK) cells in a dose-dependent manner. The RhoA/ROCK pathway is known to control the formation of stress fibers. We examined the role of the RhoA/ROCK pathway in TpeL-induced formation of stress fibers. TpeL1-525-induced formation of stress fibers was inhibited by the ROCK inhibitor, Y27632 and Rho protein inhibitor, C3 transferase. TpeL1-525 activated RhoA and ROCK in a dose-dependent manner. C3 transferase blocked TpeL1-525-induced activation of RhoA and ROCK whereas Y27632 inhibited TpeL-induced activation of ROCK. These results demonstrate for the first time that TpeL induces the formation of stress fibers by activating the RhoA/ROCK signaling pathway.

Novel inhibitor of bacterial sphingomyelinase, SMY-540, developed based on three-dimensional structure analysis.

CONTEXT: Bacterial sphingomyelinase (SMase) is thought to play a crucial role in bacterial evasion of the immune response during the early stages of infections. OBJECTIVE: The objective of this study was to predict the chemical structure required for competitive SMase inhibition, then synthesize and test the effect of potential inhibitors on the hydrolysis of sphingomyelin (SM) and protection against infection by Bacillus cereus. MATERIALS AND METHODS: We synthesized 10 potential SMase inhibitors, derivatives of RY221B-a analogues, based on predictions from three-dimensional structural analysis. We then tested the effect of these compounds on the inhibition of SM hydrolysis and protection of mice inoculated with B. cereus. RESULTS: One compound, SMY-540, displayed a strong inhibitory effect (IC50 = 0.8 µM) upon SMase and prevented mortality in mice. CONCLUSION: SMY-540 is an effective inhibitor of Bc-SMase and has potential for use in the development of drugs to treat infectious diseases caused by bacteria that produce SMase.

Clostridium perfringens alpha-toxin recognizes the GM1a-TrkA complex.

Clostridium perfringens alpha-toxin is the major virulence factor in the pathogenesis of gas gangrene. Alpha-toxin is a 43-kDa protein with two structural domains; the N-domain contains the catalytic site and coordinates the divalent metal ions, and the C-domain is a membrane-binding site. The role of the exposed loop region (72-93 residues) in the N-domain, however, has been unclear. Here we show that this loop contains a ganglioside binding motif (H … SXWY … G) that is the same motif seen in botulinum neurotoxin and directly binds to a specific conformation of the ganglioside Neu5Acα2-3(Galß1-3GalNAcß1-4)Galß1-4Glcß1Cer (GM1a) through a carbohydrate moiety. Confocal microscopy analysis using fluorescently labeled BODIPY-GM1a revealed that the toxin colocalized with GM1a and induced clustering of GM1a on the cell membranes. Alpha-toxin was only slightly toxic in ß1,4-N-acetylgalactosaminyltransferase knock-out mice, which lack the a-series gangliosides that contain GM1a, but was highly toxic in α2,8-sialyltransferase knock-out mice, which lack both b-series and c-series gangliosides, similar to the control mice. Moreover, experiments with site-directed mutants indicated that Trp-84 and Tyr-85 in the exposed alpha-toxin loop play an important role in the interaction with GM1a and subsequent activation of TrkA. These results suggest that binding of alpha-toxin to GM1a facilitates the activation of the TrkA receptor and induces a signal transduction cascade that promotes the release of chemokines. Therefore, we conclude that GM1a is the primary cellular receptor for alpha-toxin, which can be a potential target for drug developed against this pathogen.

The p38 MAPK and JNK pathways protect host cells against Clostridium perfringens beta-toxin.

Clostridium perfringens beta-toxin is an important agent of necrotic enteritis and enterotoxemia. Beta-toxin is a pore-forming toxin (PFT) that causes cytotoxicity. Two mitogen-activated protein kinase (MAPK) pathways (p38 and c-Jun N-terminal kinase [JNK]-like) provide cellular defense against various stresses. To investigate the role of the MAPK pathways in the toxic effect of beta-toxin, we examined cytotoxicity in five cell lines. Beta-toxin induced cytotoxicity in cells in the following order: THP-1 = U937 > HL-60 > BALL-1 = MOLT-4. In THP-1 cells, beta-toxin formed oligomers on lipid rafts in membranes and induced the efflux of K(+) from THP-1 cells in a dose- and time-dependent manner. The phosphorylation of p38 MAPK and JNK occurred in response to an attack by beta-toxin. p38 MAPK (SB203580) and JNK (SP600125) inhibitors enhanced toxin-induced cell death. Incubation in K(+)-free medium intensified p38 MAPK activation and cell death induced by the toxin, while incubation in K(+)-high medium prevented those effects. While streptolysin O (SLO) reportedly activates p38 MAPK via reactive oxygen species (ROS), we showed that this pathway did not play a major role in p38 phosphorylation in beta-toxin-treated cells. Therefore, we propose that beta-toxin induces activation of the MAPK pathway to promote host cell survival.

Clostridium perfringens alpha-toxin induces the release of IL-8 through a dual pathway via TrkA in A549 cells.

A characteristic feature of gas gangrene with Clostridium perfringens (C. perfringens) is the absence of neutrophils within the infected area and the massive accumulation of neutrophils at the vascular endothelium around the margins of the necrotic region. Intravenous injection of C. perfringens alpha-toxin into mice resulted in the accumulation of neutrophils at the vascular endothelium in lung and liver, and release of GRO/KC, a member of the CXC chemokine family with homology to human interleukin-8 (IL-8). Alpha-toxin triggered activation of signal transduction pathways causing mRNA expression and production of IL-8, which activates migration and binding of neutrophils, in A549 cells. K252a, a tyrosine kinase A (TrkA) inhibitor, and siRNA for TrkA inhibited the toxin-induced phosphorylation of TrkA and production of IL-8. In addition, K252a inhibited the toxin-induced phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). PD98059, an ERK1/2 inhibitor, depressed phosphorylation of ERK1/2 and nuclear translocation of nuclear factor kappa B (NF-κB) p65, but SB203580, a p38 MAPK inhibitor, did not. On the other hand, PD98059 and SB203580 suppressed the toxin-induced production of IL-8. Treatment of the cells with PD98059 resulted in inhibition of IL-8 mRNA expression induced by the toxin and that with SB203580 led to a decrease in the stabilization of IL-8 mRNA. These results suggest that alpha-toxin induces production of IL-8 through the activation of two separate pathways, the ERK1/2/NF-κB and p38 MAPK pathways.

Bacillus cereus sphingomyelinase recognizes ganglioside GM3.

Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin (SM) to phosphocholine and ceramide in a divalent metal ion-dependent manner, and is a virulence factor for septicemia. Bc-SMase has three characteristic sites, viz., the central site (catalytic site), side-edge site (membrane binding site), and ß-hairpin region (membrane binding site). Here, we show that the ß-hairpin directly binds to gangliosides, especially NeuAcα2-3Galß1-4Glcß1-1ceramide (GM3) through a carbohydrate moiety. Neuraminidase inhibited the binding of Bc-SMase to mouse peritoneal macrophages in a dose-dependent manner. SPR analysis revealed that the binding response of Bc-SMase to liposomes containing GM3 was about 15-fold higher than that to liposomes lacking GM3. Moreover, experiments with site-directed mutants indicated that Trp-284 and Phe-285 in the ß-hairpin play an important role in the interaction with GM3. The binding of W284A and F285A mutant enzymes to mouse macrophages decreased markedly in comparison to the binding by wild-type enzymes. Therefore, we conclude that GM3 is the primary cellular receptor for Bc-SMase, and that the ß-hairpin region is the tethering region for gangliosides.

A recombinant carboxy-terminal domain of alpha-toxin protects mice against Clostridium perfringens.

Clostridium perfringens alpha-toxin (CP, 370 residues) is one of the main agents involved in the development of gas gangrene. In this study, the immunogenicity and protective efficacy of the C-terminal domain (CP251-370) of the toxin and phospholipase C (PLC; CB, 372 residues) of Clostridum bifermentans isolated from cases of clostridium necrosis were examined. The recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins. Antibodies that cross-reacted with alpha-toxin were produced after immunization with recombinant proteins including GST-CP251-370, GST-CP281-370, GST-CP311-370, CB1-372 and GST-CB251-372. Anti-GST-CP251-370, anti-GST-CP281-370 and anti-GST-CP311-370 sera neutralized both the PLC and hemolytic activities of alpha-toxin, whereas anti-CB1-372 and anti-GST-CB251-372 weakly neutralized these activities. Immunization with GST-CP251-370 and GST-CP281-370 provided protection against the lethal effects of the toxin and C. perfringens type A NCTC8237. Partial protection from the toxin and C. perfringens was elicited by immunization with GST-CP311-370 and CB1-372. GST-CP251-370 and GST-CP281-370 are promising candidates for vaccines for clostridial-induced gas gangrene.