In Vivo Effects of Clostridium perfringens Enteropathogenic Factors on the Rat Ileum

An experimental model was established using the terminal ileum of the rat for characterizing and studying the effects of crude cell-free extract from Clostridium perfringens upon physiological and histological parameters involved in the transport process. Further work was done with the model system using purified enterotoxin (protein) from the cell extract. Using an in vivo perfusion technique it was found that crude extract induces a reversal of net transport, from absorption in controls to secretion, of water, sodium, and chloride. Glucose absorption was greatly inhibited, whereas potassium and bicarbonate transports were unaffected. Crude extract also caused histological damage to the villus epithelium by denuding the villus tips, thereby leaving the lamina propria exposed. Similar responses in transport of water, sodium, chloride, bicarbonate, and glucose were caused by purified toxin. Little or no histological damage resulted from the pure toxin activity. However, the toxin was shown to have the capacity to denude villus tips under the proper experimental conditions.     

Evidence for antibiotic induced Clostridium perfringens diarrhoea

Clostridium difficile is a well documented cause of antibiotic associated diarrhoea in hospitalised patients, but may account for only approximately 20% of all cases. This leader reviews the current knowledge and understanding of the pathogenesis, epidemiology, and diagnosis of non-food borne Clostridium perfringens diarrhoea. Although enterotoxigenic C perfringens has been implicated in some C difficile negative cases of antibiotic associated diarrhoea, C perfringens enterotoxin detection methods are not part of the routine laboratory investigation of such cases. Testing for C perfringens enterotoxin in faecal samples from patients with antibiotic associated diarrhoea and sporadic diarrhoea on a routine basis would have considerable resource implications. Therefore, criteria for initiating investigations and optimum laboratory tests need to be established. In addition, establishing the true burden of C perfringens antibiotic associated diarrhoea is important before optimum control and treatment measures can be defined.     

Deletion analysis of the Clostridium perfringens enterotoxin.

To further our knowledge of the structure-function relationship and mechanism of action of the Clostridium perfringens enterotoxin (CPE), a series of recombinant CPE (rCPE) species containing N- and C-terminal CPE deletion fragments was constructed by recombinant DNA approaches. Each rCPE species was characterized for its ability to complete the first four early steps in the action of CPE, putatively ordered as specific binding, a postbinding physical change to bound CPE, large-complex formation, and induction of alterations in small-molecule membrane permeability. These studies demonstrated that (i) at least 44 amino acids can be removed from the N terminus of CPE without loss of cytotoxicity, (ii) removal of the first 53 amino acids from the N terminus of CPE produces a fragment that appears to be noncytotoxic because it cannot undergo the post-binding physical change step in CPE action, (iii) removal of as few as five amino acids from the C terminus of CPE produces a noncytotoxic fragment lacking receptor binding activity, and (iv) a fragment lacking the first 44 N-terminal amino acids of native CPE formed twice as much large complex and was twice as cytotoxic as native CPE. From these structure-function results, it appears that the minimum-size cytotoxic CPE fragment comprises approximately residues 45 to 319 of native CPE. Results from these deletion fragment studies have also contributed to our understanding of CPE action by (i) independently supporting previous suggestions that binding, the postbinding physical change step, and large-complex formation represent important steps in CPE cytotoxicity and (ii) providing independent evidence confirming the putative sequential order of these early events in CPE action.     

Cellular uptake of the Clostridium perfringens binary iota-toxin

The binary iota-toxin is produced by Clostridium perfringens type E strains and consists of two separate proteins, the binding component iota b (98 kDa) and an actin-ADP-ribosylating enzyme component iota a (47 kDa). Iota b binds to the cell surface receptor and mediates the translocation of iota a into the cytosol. Here we studied the cellular uptake of iota-toxin into Vero cells. Bafilomycin A1, but not brefeldin A or nocodazole, inhibited the cytotoxic effects of iota-toxin, indicating that toxin is translocated from an endosomal compartment into the cytoplasm. Acidification (pH < or = 5.0) of the extracellular medium enabled iota a to directly enter the cytosol in the presence of iota b. Activation by chymotrypsin induced oligomerization of iota b in solution. An average mass of 530 +/- 28 kDa for oligomers was determined by analytical ultracentrifugation, indicating heptamer formation. The entry of iota-toxin into polarized CaCo-2 cells was studied by measuring the decrease in transepithelial resistance after toxin treatment. Iota-toxin led to a significant decrease in resistance when it was applied to the basolateral surface of the cells but not following application to the apical surface, indicating a polarized localization of the iota-toxin receptor.     

Molecular basis for the pathological actions of Clostridium perfringens iota toxin

Clostridium perfringens type E iota toxin is composed of two separate and independent polypeptide chains that act synergistically in mouse lethal assays. The light chain is an enzyme that mono(ADP-ribosyl)ates certain amino acids. The enzyme displays substantial activity when homopoly-L-arginine is used as a substrate, but it shows little activity when polyasparagine, polylysine or polyglutamic acid are used. In keeping with the properties of an ADP-ribosylating enzyme, the toxin possesses the following characteristics. It produces incorporation of radioactivity into polyarginine when adenine-labeled NAD is used, but radioactivity is not incorporated when nicotinamide-labeled NAD is used. Irrespective of labeling, enzymatic activity is accompanied by the release of free nicotinamide. After incorporation of ADP-ribose groups into polyarginine, enzymatic and chemical techniques can be used to release the incorporated material. Snake venom phosphodiesterase releases mainly AMP; hydroxylamine releases AMP and ADP-ribose. The heavy chain of iota toxin has little or no enzyme activity, and it does not substantially affect the enzyme activity of the light chain. The heavy chain may be a binding component that directs the toxin to vulnerable cells. The data suggest that iota toxin is a representative of a novel class of ADP-ribosylating toxins.     

Epitope mapping of the alpha-toxin of Clostridium perfringens.

A panel of monoclonal antibodies specific for the Clostridium perfringens alpha-toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid. The toxin-binding activity of each monoclonal antibody was evaluated. The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities. In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures. Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays. Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule. This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin.     

Detection of fibronectin-binding proteins in Clostridium perfringens

Clostridium perfringens is an anaerobic spore-forming pathogen of humans and animals. C. perfringens type A strains, 13, CPN50, and NCTC8237, isolated from human gas gangrene, bound specifically to human fi bronectin (Fn). The trypsin-treatment of the bacterial cells significantly reduced the Fn-binding. A ligand blotting analysis of all three C. perfringens strains revealed that 5 protein bands of 34 kDa, 29 kDa, 26 kDa, 17 kDa, and 12 kDa specifically bound to biotinylated Fn. These results suggest that C. perfringens possesses certain Fn-binding proteins on the cell surface.     

Capsular Polysaccharide of Clostridium perfringens Hobbs 10

A capsular polysaccharide was isolated from a strain of Clostridium perfringens Hobbs 10 type A by cold-water extraction of whole, heavily encapsulated cells. The water-soluble polymer was isolated by alcohol precipitation and purified by treatment with chloroform-butanol, cetytrimethylammonium bromide, and column gel permeation chromatography by using Bio-Gel A-5m agarose. The formation of a single precipitin line, when the isolated polysaccharide was reacted with its homologous antisera by double diffusion in gel, was considered a criterion of immunochemical purity. The purified polymer appeared as a single peak when eluted from diethylaminoethyl-Sephadex with a linear gradient of NaCl. The polysaccharide was composed of glucose, galactose, galactosamine, and iduronic acid in a molar ratio of 4.1:5.1.7:1, respectively. These constituents accounted for 83% of the dry weight. The polysaccharide appeared to have a molecular weight of 40,000 and exhibited aggregation up to 120,000. A trace of peptide material could not be removed during purification.     

Laboratory diagnosis of Clostridium perfringens antibiotic-associated diarrhoea

Clostridium perfringens has been reported as the cause of up to 15% of cases of antibiotic-associated diarrhoea (AAD) and may be diagnosed by detection of enterotoxin (CPEnt) in faeces. The performance of a commercial ELISA method for CPEnt, with culture and PCR methods to confirm the presence of enterotoxigenic C. perfringens, was evaluated in 200 consecutive specimens from patients with clinical details suggestive of AAD: 8% of the specimens were positive for CPEnt, 16% were positive for C. difficile cytotoxin and 2% gave positive test results for both C. perfringens and C. difficile toxins. Culture and PCR results confirmed the majority of ELISA results, although 2 (12.5%) reactive specimens were only weakly positive. C. perfringens is a potentially important cause of infective AAD and can be detected with the C. perfringens enterotoxin ELISA kit, although weak positive results should be considered with caution.     

Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca(2+) concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes.     

Clostridium perfringens Type A Enterotoxin Damages the Rabbit Colon

Clostridium perfringens enterotoxin causes the gastrointestinal (GI) symptoms of C. perfringens type A food poisoning and CPE-associated non-food-borne human GI diseases. It is well established that CPE induces fluid accumulation and severe tissue damage in ligated small intestinal loops of rabbits and other animals. However, a previous study had also reported that CPE binds to rabbit colonic cells yet does not significantly affect rabbit colonic loops. To the contrary, the current study determined that treatment with 50 or 100 μg/ml of CPE causes significant histologic lesions and luminal fluid accumulation in rabbit colonic loops. Interestingly, a CPE-neutralizing monoclonal antibody blocked the development of CPE-induced histologic damage but not luminal fluid accumulation in these loops. Similar luminal fluid accumulation, without significant histologic damage, also occurred after treatment of colonic loops with heat-inactivated CPE, antibody alone, or bovine serum albumin (BSA), indicating that increased osmolarity was causing or contributing to fluid accumulation in CPE-treated colonic loops. Comparative studies revealed the similar development of histologic damage and luminal fluid accumulation in both small intestinal loops and colonic loops after as little as a 1-h treatment with 50 μg/ml of CPE. Consistent with the CPE sensitivity of the small intestine and colon, Western blotting detected CPE binding and large-complex formation in both organs. In addition, Western blotting demonstrated the presence of the high-affinity CPE receptors claudin-3 and -4 in both organs of rabbits, consistent with the observed toxin binding. Collectively, these results offer support for the possible involvement of the colon in CPE-mediated GI disease.     

Clostridium perfringens enterotoxin damages the human intestine in vitro

In vitro, Clostridium perfringens enterotoxin (CPE) binds to human ileal epithelium and induces morphological damage concurrently with reduced short-circuit current, transepithelial resistance, and net water absorption. CPE also binds to the human colon in vitro but causes only slight morphological and transport changes that are not statistically significant.     

Capsular polysaccharide of Clostridium perfringens Hobbs 9.

Several closely related capsular polysaccharides were isolated from a strain of Clostridium perfringens Hobbs 9 type A by extraction of encapsulated cells with cold 0.85% NaCl. The soluble polymers were precipitated with alcohol and purified by (NH4)2SO4 fractionation, enzymatic digestion with papain and ribonuclease, and chromatography on diethylaminoethyl-Sephadex A25. The polysaccharides were composed mainly of glucose, galactose, and galactosamine. The major fraction contained these constituents (representing 77% of the dry weight) in a molar ratio of 1:1.6:1.1. All of the fractions contained phosphate and peptide material that was not removed during purification. The polysaccharides were closely related but not identical as indicated by double-diffusion-in-gel experiments. Immunoelectrophoresis in agarose demonstrated that the polysaccharides had identical mobilities and that no resolution into additional fractions occurred. The immunological activity of all the purified polysaccharides was destroyed by periodate oxidation but was unaffected by protease.     

Biological Characteristics of Clostridium perfringens Type A Enterotoxin

An enterotoxin with the ability to induce fluid accumulation in rabbit ileal loops, erythema in the skin of guinea pigs, and lethality in mice appears in cell extracts (CE) and culture filtrates (CF) of sporulating cells of some Clostridium perfringens type A strains. All activities in CE and CF were eluted simultaneously from a Sephadex G-200 column. Different elution patterns were obtained for these activities present in CE and CF. Rabbit immune serum against CF and the active CE fractions eliminated the three biological activities in CE and CF. These activities present in CF and CE were not eliminated by any of the known antitoxins present in diagnostic serum against C. perfringens types A, B, C, D, and E. Immunodiffusion studies with immune serum against active CE fractions and CF indicated a precipitin line of identity between CF and CE of NCTC 8798 and other enterotoxin-positive strains but not enterotoxin-negative strains. Disc electrophoresis of active G-200 fractions on 7.0% polyacrylamide gels revealed a single area containing erythemal activity and mouse lethality. Immunodiffusion with acrylamide gels, containing crude fractionated enterotoxin, and immune serum against partially purified enterotoxin revealed a single precipitin band in the same area as the biological activities. Immunoelectrophoresis of CE of enterotoxin-positive and enterotoxin-negative strains also showed one precipitin band which occurred only with enterotoxin-positive strains. These findings suggest that one component is responsible for the biological activities attributed to the enterotoxin.     

Purification and characterization of Clostridium perfringens delta-toxin.

Delta-toxin, an extracellular hemolysin released by Clostridium perfringens type C, was purified from culture supernatant fluid by sequential ammonium sulfate precipitation, thiol-Sepharose gel chromatography, isoelectric focusing, and Sephadex G-75 gel filtration. The purified preparation had a specific activity of 320,000 hemolytic units per mg of protein and was homogeneous, as determined by immunochemical and electrophoretic tests. This toxin was characterized as a single polypeptide chain composed of 391 amino acid residues, 30% of which were hydrophobic. The molecular weight was found to be 42,000, and the isoelectric point was pH 9.1. Delta-toxin appeared to be amphiphilic by charge shift electrophoresis in a three-detergent system. It was immunogenic in rabbits and lethal to mice at a dose of 0.12 micrograms. The lytic activity of delta-toxin was restricted to erythrocytes of even-toed ungulates (sheep, goats, and pigs). This activity was inhibited by GM2 ganglioside but not by other gangliosides, cholesterol, lecithin, or sphingomyelin.     

Epidemiology of diarrhoea caused by enterotoxigenic Clostridium perfringens

Enterotoxigenic strains of Clostridium perfringens have recently been implicated in some cases of antibiotic-associated diarrhoea. We present here the results of an epidemiological study of this disease. Five cases of diarrhoea caused by C. perfringens serotype 41 occurred during a 9-week period, and then during a 6-week period there were three cases due to serotype 27 and two due to serotype 24; in all but one case two geriatric wards were involved. In total there were 16 cases in 22 months. All cases were identified by the detection of C. perfringens enterotoxin in the faeces. The mean number of C. perfringens in these cases was 10(8.8) cfu/g of faeces. Of 37 patients who had negative test results for C. perfringens enterotoxin, 18 had positive cultures for C. perfringens, with mean faecal counts of 10(5.3) cfu/g, and nine of these patients had diarrhoea. Thirteen different serotypes were isolated from these 18 patients, including type 41 from seven patients and type 27 from one. Hand carriage of the offending serotype was demonstrated in three of four infected patients, none of four controls and two of 14 ward staff. C. perfringens of serotypes causing disease was isolated from 59% of environmental areas where there was active disease, 27% of areas where there had been disease which had since resolved and 9% of areas where there was no history of disease. The findings imply that cross infection may occur.