Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene.

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.     

The role of histidine residues in the alpha toxin of Clostridium perfringens

The α -toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.     

Tyrosine 331 and phenylalanine 334 in Clostridium perfringens alpha-toxin are essential for cytotoxic activity

Differences in the biological properties of the Clostridium perfringens phospholipase C (alpha-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of alpha-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of alpha-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.     

Biochemical and immunological properties of the C-terminal domain of the alpha-toxin of Clostridium perfringens

Abstract The C-terminal domain of the alpha-toxin (cpa_247–370) of Clostridium perfringens has been expressed in Escherichia coli and purified. Antiserum raised against cpa_247–370 reacted in an identical manner to anti-alpha-toxin serum when used to map epitopes in the C-terminal domain, suggesting that cpa_247–370 was immunologically and structurally identical to this region in the alpha-toxin. The isolated cpa_247–370 was devoid of sphingomyelinase activity or haemolytic activity and was not cytotoxic for mouse lymphocytes. Haemolytic activity was detected when cpa_247–370 was tested with the N-terminal domain of the alpha-toxin (cpa_1–249), confirming that cpa_247–370 confers haemolytic properties on the phospholipase C activity of the alpha-toxin. Haemolytic activity was not detected if cpa_247–370 was tested with the Bacillus cereus phosphatidylcholine phospholipase C, nor if cpa_1–249 and cpa_247–370 were incubated sequentially with erythrocytes.     

Detection of α- and ε-toxigenic Clostridium perfringens Type D in sheep and goats using a DNA amplification technique (PCR)

Clostridium perfringens isolated from sheep and goat with enterotoxaemia at necropsy and from healthy animals at slaughter were typed using specific PCR assays for the detection of the α-, β- and ε-toxin genes. Clostridium perfringens isolated from all 52 animals with pathological signs of enterotoxaemia showed the presence of the α- and ε-toxin genes but were devoid of the β-toxin gene. These strains could therefore be identified as type D, characteristic for clostridial enterotoxaemia of sheep, lambs and goats. In contrast, Cl. perfringens isolated from 11 of 13 healthy animals only contained the α-toxin gene which is typical for type A. Two of the healthy animals contained Cl. perfringens with the α- and ε-toxin genes. However, when several individual Cl. perfringens colonies were analysed from each of these two animals, only a small percentage was found to contain the ε-toxin gene, whereas the majority of the colonies were of type A with the α-toxin gene only. This is in contrast to the findings from the diseased animals which contained practically only type D Cl. perfringens . The β-toxin gene was not found in any Cl. perfringens isolate from goat and sheep. Comparison of the PCR data with results obtained by the classical biological toxin assay using the mouse model showed a good correlation.     

The Level of Expression of α-toxin by Different Strains ofClostridium perfringensis Dependent on Differences in Promoter Structure and Genetic Background

The control of expression of the α-toxin gene (cpaorplc) ofClostridium perfringenshas been studied in three strains shown to have high (NCTC8237), intermediate (strain 13) and low (NCTC8533) phospholipase C activity in the culture supernatant. The phospholipase C activity was shown to be related tocpamRNA levels. Primer extension studies were performed to locate thecpapromoter regions in strains NCTC8237 and 13. Differences in promoter sequences could account for the differences in α-toxin production between strains 13 and NCTC8237. In contrast, the differences in α-toxin production between strains NCTC8237 and NCTC8533 were unlikely to be due to promoter differences because the upstream promoter-containing sequences were identical in these strains. The recombinant plasmid carrying the NCTC8237cpagene was introduced into strains 13 and NCTC8533. The level of production of the α-toxin was 16-fold higher in strain 13, indicating the presence of strain-dependant regulatory systems.     

The solution structure of the C-terminal modular pair from Clostridium perfringens mu-toxin reveals a noncellulosomal dockerin module

The genome of the opportunistic pathogen Clostridium perfringens encodes a large number of secreted glycoside hydrolases. Their predicted activities indicate that they are involved in the breakdown of complex carbohydrates and other glycans found in the mucosal layer of the human gastrointestinal tract, within the extracellular matrix, and on the surface of host cells. One such group of these enzymes is the family 84 glycoside hydrolases, which has predicted hyaluronidase activity and comprises five members [C. perfringens glycoside hydrolase family 84 (CpGH84) A-E]. The first identified member, CpGH84A, corresponds to the μ-toxin whose modular architecture includes an N-terminal catalytic domain, four family 32 carbohydrate-binding modules, three FIVAR modules of unknown function, and a C-terminal putative calcium-binding module. Here, we report the solution NMR structure of the C-terminal modular pair from the μ-toxin. The three-helix bundle FIVAR module displays structural homology to a heparin-binding module within the N-terminal of the a C protein from group B Streptoccocus. The C-terminal module has a typical calcium-binding dockerin fold comprising two anti-parallel helices that form a planar face with EF-hand calcium-binding loops at opposite ends of the module. The size of the helical face of the μ-toxin dockerin module is approximately equal to the planar region recently identified on the surface of a cohesin-like X82 module of CpGH84C. Size-exclusion chromatography and heteronuclear NMR-based chemical shift mapping studies indicate that the helical face of the dockerin module recognizes the CpGH84C X82 module. These studies represent the structural characterization of a noncellulolytic dockerin module and its interaction with a cohesin-like X82 module. Dockerin/X82-mediated enzyme complexes may have important implications in the pathogenic properties of C. perfringens.     

Effects of Clostridium perfringens phospholipase C in mammalian cells

Clostridium perfringens phospholipase C (Cp-PLC), the major virulence factor in the pathogenesis of gas gangrene, is a Zn(2+) metalloenzyme with lecithinase and sphingomyelinase activities. Its structure shows an N-terminal domain containing the active site, and a C-terminal Ca(2+) binding domain required for membrane interaction. Although the knowledge of the structure of Cp-PLC and its interaction with aggregated phospholipids has advanced significantly, an understanding of the effects of Cp-PLC in mammalian cells is still incomplete. Cp-PLC binds to artificial bilayers containing cholesterol and sphingomyelin or phosphatidylcholine (PC) and degrades them, but glycoconjugates present in biological membranes influence its binding or positioning toward its substrates. Studies with Cp-PLC variants harboring single amino-acid substitutions have revealed that the active site, the Ca(2+) binding region, and the membrane interacting surface are required for cytotoxic and haemolytic activity. Cp-PLC causes plasma membrane disruption at high concentrations, whereas at low concentrations it perturbs phospholipid metabolism, induces DAG generation, PKC activation, Ca(2+) mobilization, and activates arachidonic acid metabolism. The cellular susceptibility to Cp-PLC depends on the composition of the plasma membrane and the capacity to up-regulate PC synthesis. The composition of the plasma membrane determines whether Cp-PLC can bind and acquire its active conformation, and thus the extent of phospholipid degradation. The capacity of PC synthesis and the availability of precursors determine whether the cell can replace the degraded phospholipids. Whether the perturbations of signal transduction processes caused by Cp-PLC play a role in cytotoxicity is not clear. However, these perturbations in endothelial cells, platelets and neutrophils lead to the uncontrolled production of intercellular mediators and adhesion molecules, which inhibits bacterial clearance and induces thrombotic events, thus favouring bacterial growth and spread in the host tissues.     

Clostridium perfringens α-toxin up-regulates plasma membrane CD11b expression on murine neutrophils by changing intracellular localization

Gas gangrene caused by Clostridium perfringens type A infection is a highly lethal infection of soft tissue characterized by rapid spread of tissue necrosis. This tissue destruction is related to profound attenuation of blood flow accompanied by formation of platelet-leukocyte aggregates in the blood vessels. Several studies have identified α-toxin, which has both sphingomyelinase and phospholipase C activities, as a major virulence factor in the aggregate formation via activation of the platelet gpIIbIIIa. Here, we show that α-toxin greatly and rapidly increases plasma membrane localization of CD11b, which binds to the platelet gpIIbIIIa via fibrinogen, in mouse neutrophils. Interestingly, short-term treatment of α-toxin has little effect on gene expression profiles in neutrophils, and the toxin does not change the total protein expression levels of CD11b in whole cell lysates. The following analysis demonstrated that CD11b localizes to intracellular vesicles in intact cells, but the localization changed to the cytoplasmic membrane in α-toxin-treated cells. These results suggest that CD11b is recruited to the cytoplasmic membrane by α-toxin. Previously, we reported that α-toxin promotes the formation of ceramide by its sphingomyelinase activity in mouse neutrophils. Interestingly, a synthetic cell-permeable ceramide analog, C₂-ceramide, increases plasma membrane localization of CD11b, suggesting that ceramide production by α-toxin recruits CD11b to the cytoplasmic membrane to promote platelet-leukocyte aggregation. Together, our results illustrate that the increase of cell membrane CD11b expression by α-toxin might be crucial for the pathogenesis of C. perfringens to promote formation of platelet-leukocyte aggregates, leading to rapid tissue necrosis due to ischemia.     

Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B

Strain 1470 is the standard typing strain for serogroup F of Clostridium difficile containing both toxin genes, toxA-1470 and toxB-1470 . A polymerase chain reaction (PCR)-based approach to the sequencing of the total toxB-1470 gene identified an open reading frame (ORF) of 7104 nucleotides. In comparison with the previously sequenced toxB of C. difficile VPI10463, the toxB-1470 gene has 16 additional nucleotides, 13 within the 5'-untranslated region and three within the coding region. The M _r of ToxB-1470 is 269 262, with an isoelectric point (IP) of 4.16. The equivalent values for ToxB are M _r 269 709 and IP 4.13. In comparison with ToxB, ToxB-1470 differs primarily in the N-terminal region between positions 1 and 868 where 148 amino acids residues are changed. The C-terminal region between residues 869–2367 is highly conserved with only six amino acid alterations. Dot matrix comparison of ToxB-1470 with ToxA and ToxB reveals the highest homology between ToxB-1470 and ToxB. Thus ToxB-1470 did not originate from recombination between ToxA and ToxB. On cultured endothelial cells, from porcine pulmonary artery, purified ToxB-1470 is less potent than ToxB. The cytopathic effects of ToxB-1470 are indistinguishable from those caused by the lethal toxin (LT) of Clostridium sordellii , but are clearly different from the patterns observed after exposure of endothelial cells to ToxA and ToxB of C. difficile (VPI10463) or α-toxin (Tcnα) of Clostridium novyi . The LT-like action of ToxB-1470 was not due to altered internalization processes, as microinjection and addition to the medium induced identical effects on the cells. Since the differences between ToxB and ToxB-1470 are clustered within the N-terminal third of the respective proteins, we conclude that these domains carry the toxic determinants. A three-domain structure is proposed for the family of large clostridal cytotoxins.     

Unexpected wide substrate specificity of C. perfringens α-toxin phospholipase C

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the main virulence factor for gas gangrene in humans. The lipase activity serves the bacterium to generate lipid signals in the host eukaryotic cell, and ultimately to degrade the host cell membranes. Several previous reports indicated that CpPLC was specific for phosphatidylcholine and sphingomyelin. Molecular docking studies described in this paper predict favorable interactions of the CpPLC active site with other phospholipids, e.g. phosphatidylethanolamine, phosphatidylinositol and, to a lesser extent, phosphatidylglycerol. On the basis of these predictions, we have performed experimental studies showing α-toxin to degrade all the phospholipids mentioned above. The molecular docking data also provide an explanation for the observed lower activity of CpPCL on sphingomyelin as compared to the glycerophospholipids.     

Regulatory function of the Saccharomyces cerevisiae RAS C-terminus.

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.     

Typing of Clostridium perfringens by in vitro amplification of toxin genes

G. DAUBE, B. CHINA, P. SIMON, K. HVALA AND J. MAINIL. 1994. The strains of Clostridium perfringens are classified according to major toxins produced. Classically, this determination involves the seroneutralization of their lethal effect in mice. However, this method requires specific antisera and a large number of mice. In this work, a new typing method was developed based on the amplification of toxin genes by polymerase chain reaction (PCR). By combination of several pairs of primers, the toxinotype of a Cl. perfringens strain was determined by looking at the pattern of bands on an agarose gel electrophoresis. This mixture contained primers amplifying simultaneously a part of α-toxin, α-toxin, β-toxin and enterotoxin genes. In order to distinguish between toxinotype A and E, the *** l -toxin gene fragment must be amplified in a separate PCR reaction. Moreover, with the primers combination, in most cases, a PCR product corresponding to the α-toxin gene was obtained from direct enrichments of animal intestinal contents.     

Bypass suppression analysis maps the signalling pathway within a multidomain protein: the RsbP energy stress phosphatase 2C from Bacillus subtilis

The network controlling the general stress response in Bacillus subtilis requires both the RsbP phosphatase and the RsbQ α/β hydrolase to convey signals of energy stress. RsbP contains three domains: an N-terminal PAS, a central coiled-coil and a C-terminal PP2C phosphatase. We report here a genetic analysis that established the functional interactions of the domains and their relationship to RsbQ. Random mutagenesis of rsbP yielded 17 independent bypass suppressors that had activity in an rsbQ null strain background. The altered residues clustered in three regions of RsbP: the coiled-coil and two predicted helices of the phosphatase domain. One helix (α0) is unique to a subfamily of bacterial PP2C phosphatases that possess N-terminal sensing domains. The other (α1) is distinct from the active site in all solved PP2C structures. The phenotypes of the suppressors and directed deletions support a model in which the coiled-coil negatively controls phosphatase activity, perhaps via the α0-α1 helices, with RsbQ hydrolase activity and the PAS domain jointly comprising a positive sensing module that counters the coiled-coil. We propose that the α0 helix characterizes an extended PP2C domain in many bacterial signalling proteins, and suggest it provides a means to communicate information from diverse input domains.     

Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi alpha-toxin

Clostridium sordellii lethal toxin and Clostridium novyi α-toxin, which are virulence factors involved in the toxic shock and gas gangrene syndromes, are members of the family of clostridial glucosylating toxins. The toxins inactivate Rho/Ras proteins by glucosylation or attachment of GlcNAc (α-toxin). Here, we studied the activation of the autoproteolytic processing of the toxins by inositol hexakisphosphate (InsP(6)) and compared it with the processing of Clostridium difficile toxin B. In the presence of low concentrations of InsP(6) (<1 μM), toxin fragments consisting of the N-terminal glucosyltransferase (or GlcNAc-transferase) domains and the cysteine protease domains (CPDs) of C. sordellii lethal toxin, C. novyi α-toxin, and C. difficile toxin B were autocatalytically processed. The cleavage sites of lethal toxin (Leu-543) and α-toxin (Leu-548) and the catalytic cysteine residues (Cys-698 of lethal toxin and Cys-707 of α-toxin) were identified. Affinity of the CPDs for binding InsP(6) was determined by isothermal titration calorimetry. In contrast to full-length toxin B and α-toxin, autocatalytic cleavage and InsP(6) binding of full-length lethal toxin depended on low pH (pH 5) conditions. The data indicate that C. sordellii lethal toxin and C. novyi α-toxin are InsP(6)-dependently processed. However, full-length lethal toxin, but not its short toxin fragments consisting of the glucosyltransferase domain and the CPD, requires a pH-sensitive conformational change to allow binding of InsP(6) and subsequent processing of the toxin.     

Crystal structure of the C. perfringens alpha-toxin with the active site closed by a flexible loop region

Clostridium perfringens biotype A strains are the causative agents of gas-gangrene in man and are also implicated as etiological agents in sudden death syndrome in young domestic livestock. The main virulence factor produced by these strains is a zinc-dependent, phosphatidylcholine-preferring phospholipase C (alpha-toxin). The crystal structure of alpha-toxin, at pH 7.5, with the active site open and therefore accessible to substrate has previously been reported, as has calcium-binding to the C-terminal domain of the enzyme at pH 4.7. Here we focus on conformation changes in the N-terminal domain of alpha-toxin in crystals grown at acidic pH. These changes result in both the obscuring of the toxin active site and the loss of one of three zinc ions from it. Additionally, this "closed" form contains a small alpha helix, not present in the open structure, which hydrogen bonds to both the N and C-terminal domains. In conjunction with the previously reported findings that alpha-toxin can exist in active and inactive forms and that Thr74Ile and Phe69Cys substitutions markedly reduced the haemolytic activity of the enzyme, our work suggests that these loop conformations play a critical role in the activity of the toxin.     

Large clostridial cytotoxins

The large clostridial cytotoxins are a family of structurally and functionally related exotoxins from Clostridium difficile (toxins A and B), C. sordellii (lethal and hemorrhagic toxin) and C. novyi (-toxin). The exotoxins are major pathogenicity factors which in addition to their in vivo effects are cytotoxic to cultured cell lines causing reorganization of the cytoskeleton accompanied by morphological changes. The exotoxins are single-chain protein toxins, which are constructed of three domains: receptor-binding, translocation and catalytic domain. These domains reflect the self-mediated cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and execution of their cytotoxic activity by an inherent enzyme activity. Enzymatically, the toxins catalyze the transfer of a glucosyl moiety from UDP-glucose to the intracellular target proteins which are the Rho and Ras GTPases. The covalent attachment of the glucose moiety to a conserved threonine within the effector region of the GTPases renders the Rho-GTPases functionally inactive. Whereas the molecular mode of cytotoxic effects is fully understood, the mechanisms leading to inflammatory processes in the context of disease (e.g., antibiotic-associated pseudomembranous colitis caused by Clostridium difficile) are less clear.     

The rate assay of alpha-toxin assembly in membrane

Abstract A rapid and easy method to determine the 'rate' of the assembly of α-toxin from Staphylococus aureus in erythrocyte membrane was described. Upon addition of a small amount of α-toxins into erythrocyte suspension, absorbance at 700 nm decreased linearly after a short period of lag time. From the linear portion of the record the rate of the assembly of α-toxin was calculated. An optimum temperature and an optimum pH for the assembly of the toxin on erythrocyte membranes were found to be 25–30°C and pH 5.