Cross-complementation of Clostridium perfringens PLC and Clostridium septicum α-toxin mutants reveals PLC is sufficient to mediate gas gangrene

Clostridium perfringens and Clostridium septicum are the most common causes of clostridial myonecrosis or gas gangrene. Although they mediate a similar disease pathology, they elaborate functionally very different α-toxins. We used a reciprocal complementation approach to assess the contribution of the primary toxin of each species to disease and found that C. perfringens α-toxin (PLC) was able to mediate the gross pathology of myonecrosis even in a C. septicum background, although it could not induce vascular leukostasis. Conversely, while C. septicum α-toxin restored some virulence to a C. perfringens plc mutant, it was less active than in its native background.     

Characterization of a Toxin-Deficient Clostridium perfringens Strain,KZ1340

Clostridium perfringens KZ1340, previously classified as Clostridium plagarum, is an isolate from Antarctic soil, and was identified as an α, θ-, and κ-toxin non-producing variant. On Southern hybridization, the variant was found to be defective in the pfoA (θ-toxin) gene, but the plc (α-toxin) and colA (κ-toxin) genes were present on the same EcoRI fragment as in the standard strain, NCTC8237. Northern analysis revealed that mature plc mRNA was transcribed in KZ1340 though less efficiently than in NCTC8237, while no mature colA mRNA was present in KZ1340. After transformation of the pfoA and plc genes into the KZ1340 via shuttle vector, pJIR418, the pfoA gene was successfully expressed but the plc gene was not efficiently expressed, suggesting that in KZ1340 there is negative regulation of plc gene expression. Toxin-deficient C. perfringens KZ1340 might be a suitable host for expression analysis of the pfoA gene and other clostridial virulence genes, if expressed efficiently, because it produces a small amount of extracellular toxins.     

The cysteine protease α-clostripain is not essential for the pathogenesis of Clostridium perfringens-mediated myonecrosis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.     

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.     

Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb Hindlll fragment in the C. perfringens chromosome. A 4.3 kb Pstl fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb Hindlll fragment, or the 4.3 kb Pstl fragment resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The Pstl fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51 274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved In virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.     

Sequencing and analysis of the gene encoding the α-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile

A library of total Clostridium novyi DNA was established and screened for the α-toxin gene (tcnα) by hybridization with oligonucleotides derived from a partial N-terminal sequence and by using specific antisera. Overlapping subgenic tcnα fragments were isolated and subsequently the total sequence of tcnα was determined. The 6534 nucleotide open reading frame encodes a polypeptide of M_r 250 166 and pI 5.9. The N-terminal α-toxin (Tcnα) sequence MLITREQLMKIASIP determined by Edman degradation confirmed the identity of the reading frame and the assignment of the translation start point. The toxin is not modified posttranslationally at its N-terminus nor does it consist of different subunits. Overall the amino acid sequence shows 48% homology between the Tcnα and both toxins A (TcdA) and B (TcdB) of Clostridium difficile. The C-terminal 382 residues of Tcnα constitute a repetitive domain similar to those reported for TcdA and TcdB of C. difficile. The individual repeat motifs of these three toxins consist of oligopeptides some 19–52 amino acids in length, arranged in four to five different groups. Genetic, biochemical and pharmacological data thus confirm that the three toxins belong to one subgroup, designated large clostridial cytotoxins (LCT). Further definition of their structure and detailed molecular action should allow the LCTs to be used tools for the analysis of microfilament assembly and function.     

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.     

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.     

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.     

Genome mapping ofClostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne

The intron-encoded endonuclease I-CeuI fromChlamydomonas eugametos was shown to cleave the circular chromosomes of allClostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins,β, ε, and?, in addition to the enterotoxin andλ-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins,? andμ, were missing.     

The alpha-toxin of Clostridium septicum is essential for virulence

Clostridium septicum is the causative agent of spontaneous gas gangrene or atraumatic myonecrosis, a sudden and frequently fatal infection that is increasingly associated with malignancy of the colon. Little is known about the disease process although the focus of virulence studies has been the alpha-toxin, a pore-forming cytolysin that is encoded by the csa gene and secreted as an inactive protoxin. Until now a lack of techniques for the genetic manipulation of C. septicum has hindered the use of molecular approaches to understand pathogenesis. By introducing plasmids by conjugation from Escherichia coli, we have developed methods for the genetic manipulation of C. septicum and constructed a chromosomal csa mutant by allelic exchange. Virulence testing of an isogenic series of strains consisting of the wild type, the csa mutant, and a csa mutant complemented with the wild-type csa gene revealed that the development of fulminant myonecrosis in mice was dependent on the ability to produce a functional haemolytic alpha-toxin. Furthermore, the inhibition of leukocyte influx into the lesion, which is very typical of clostridial myonecrosis, was also dependent on the ability to produce alpha-toxin. This study represents the first definitive identification of a virulence factor in this organism and opens the way for further studies that will delineate the role of other putative virulence factors in this significant pathogen.     

The enteric toxins of Clostridium perfringens

The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract. The enteric toxins of C. perfringens share two common features: (1) they are all single polypeptides of modest (~25–35 kDa) size, although lacking in sequence homology, and (2) they generally act by forming pores or channels in plasma membranes of host cells. These enteric toxins include C. perfringens enterotoxin (CPE), which is responsible for the symptoms of a common human food poisoning and acts by forming pores after interacting with intestinal tight junction proteins. Two other C. perfringens enteric toxins, -toxin (a bioterrorism select agent) and -toxin, cause veterinary enterotoxemias when absorbed from the intestines; - and -toxins then apparently act by forming oligomeric pores in intestinal or extra-intestinal target tissues. The action of a newly discovered C. perfringens enteric toxin, 2 toxin, has not yet been defined but precedent suggests it might also be a pore-former. Experience with other clostridial toxins certainly warrants continued research on these C. perfringens enteric toxins to develop their potential as therapeutic agents and tools for cellular biology.

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Perfringolysin O expression in Clostridium perfringens is independent of the upstream pfoR gene

The pathogenesis of Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O. Previous studies (T. Shimizu, A. Okabe, J. Minami, and H. Hayashi, Infect. Immun. 59:137-142, 1991) carried out with Escherichia coli suggested that the perfringolysin O structural gene, pfoA, was positively regulated by the product of the upstream pfoR gene. In an attempt to confirm this hypothesis in C. perfringens, a pfoR-pfoA deletion mutant was complemented with isogenic pfoA(+) shuttle plasmids that varied only in their ability to encode an intact pfoR gene. No difference in the ability to produce perfringolysin O was observed for C. perfringens strains carrying these plasmids. In addition, chromosomal pfoR mutants were constructed by homologous recombination in C. perfringens. Again no difference in perfringolysin O activity was observed. Since it was not possible to alter perfringolysin O expression by mutation of pfoR, it was concluded that the pfoR gene product is unlikely to have a role in the regulation of pfoA expression in C. perfringens.     

Identification and characterization of a putative endolysin encoded by episomal phage phiSM101 of <Emphasis Type="Italic">Clostridium perfringens</Emphasis>

Clostridium perfringens produces potent toxins and histolytic enzymes, causing various diseases including life-threatening fulminant diseases in humans and other animals. Aiming at utilizing a phage endolysin as a therapeutic alternative to antibiotics, we surveyed the genome and bacteriophage sequences of C. perfringens. A phiSM101 muramidase gene (psm) revealed by this study can be assumed to encode an N-acetylmuramidase, since the N-terminal catalytic domain deduced from the gene shows high homology of those of N-acetylmuramidases. The psm gene is characteristic in that it is present in phiSM101, an episomal phage of enterotoxigenic C. perfringens type A strain, SM101, and also in that homologous genes are present in the genomes of all five C. perfringens toxin types. The psm gene was cloned and expressed in Escherichia coli as a protein histidine-tagged at the N-terminus (Psm-his). Psm-his was purified to homogeneity by nickel-charged immobilized metal affinity chromatography and anion-exchange chromatography. The purified enzyme lysed cells of all C. perfringens toxin types but not other clostridial species tested, as was shown by a turbidity reduction assay. These results indicate the Psm-his is useful as a cell-wall lytic enzyme and also suggest that it is potentially useful for biocontrol of this organism.     

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.     

Effect of continuous sub-culturing on infectivity of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> ATCC13124 in mouse gas gangrene model

Clostridium perfringens is a Validated Biological Agent and a pathogen of medical, veterinary, and military significance. Gas gangrene is the most destructive of all the clostridial diseases and is caused by C. perfringens type A strains wherein the infection spreads quickly (several inches per hour) with production of gas. Influence of repeated in vitro cultivation on the infectivity of C. perfringens was investigated by comparing the surface proteins of laboratory strain and repository strains of the bacterium using 2DE-MS approach. In order to optimize host-pathogen interaction during experimental gas gangrene infection, we also explored the role of particulate matrix on ability of C. perfringens to cause gas gangrene.