Assay Methods for Clostridium perfringens Type A Enterotoxin

Enterotoxin produced by a sporulating culture of Clostridium perfringens type A NCTC 8798 was purified to a level of 3,500 mouse mean lethal doses per mg of nitrogen. High-titer sera were obtained from rabbits injected with enterotoxin and used to compare the sensitivity of serological tests and bioassays for C. perfringens enterotoxin. Reversed passive hemagglutination was by far the most sensitive test, followed by microslide diffusion, single gel diffusion and electroimmunodiffusion, guinea pig skin test, mouse test, and rabbit ileal loop test.     

Quantitation of Clostridium perfringens Type A Enterotoxin by Electroimmunodiffusion

Conditions for quantitation of Clostridium perfringens type A enterotoxin by electroimmunodiffusion are described. As little as 0.01 mug of enterotoxin could be detected. Electroimmunodiffusion was more sensitive than either single gel diffusion or quantitation based on erythemal activity of the toxin in guinea pig skin.     

Radioiodination of Enterotoxin from Clostridium perfringens Type A using Chloramine T

Procedures were examined for labelling enterotoxin isolated from Clostridium perfringens type A. with ¹²⁵I using chloramine T as the oxidizing agent. The iodination method was evaluated critically to establish the optimal conditions for the preparation of iodinated enterotoxin with a high specific radioactivity and without impairing the immunospecificity and biological activity. The use of 250 μg/ml of chloramine T in the reaction mixture. 500–1000 μCi of Na¹²⁵I/10 μg of enterotoxin and a reaction time of 40 s at pH 7–0 produced ¹²⁵I-enterotoxin of both high specific radioactivity and immunospecificity which retained its biological activity. No damage or aggregate formation due to the iodination process was observed. Enterotoxin labelled with high specific activity (135 μCi μg) showed extensive dissociation of ¹²⁵I when stored at 4°C and—20°C. In contrast, toxin labelled with low specific activity (7 μgCi/μg) was stable for as long as two months. The immunoreactivity of all labelled preparations was essentially unchanged after storage for one month.     

Scanning Isoelectric Focusing and Isotachophoresis of Clostridium perfringens Type A Enterotoxin

Fractionation of highly purified Cl. perfringens type A enterotoxin by scanning isoelectric focusing (SIF) and isotachophoresis (IT) in polyacrylamide gels is described for the first time. The use of 2% ampholytes pH 3–6 allowed the separation of enterotoxin into 2 species. The major component had an isoelectric point of 4·5 and possessed antigenic as well as functional activity. The minor component of enterotoxin, at equivalent concentrations, was devoid of any demonstrable biological activity had an isoelectric point of 4·6 and appeared to represent approximately 15% of the purified enterotoxin. With ampholytes pH 3·5–10 the minor and major components were focused at different times than when ampholine pH 3–6 was employed. Electrofocusing of enterotoxin in the presence of 6 M-urea did not alter the SIF pattern. During IT the major component of enterotoxin migrated ahead of the minor component. The 2 proteins were completely separated. Isotachophoretic separations required 0·023 M-phosphate pH 6·0 as the leading ion, 0·079 M-Tris as the counter-ion, 0·2 M-glycine (in Tris pH 8·1) as the terminating ion, 30 γ carrier ampholytes pH 3·5–10, 263 μg enterotoxin, 4% acrylamide and a current of 5 mA per gel column.     

Time of Enterotoxin Formation and Release During Sporulation of Clostridium perfringens Type A

Clostridium perfringens enterotoxin was detected intracellularly about 3 hr after the inoculation of vegetative cells into sporulation medium. The subsequent increase in intracellular enterotoxin concentration roughly paralleled but followed by 2.5 to 5 hr the increase in number of heat-resistant spores. The increase in biologically active toxin coincided with the increase in enterotoxin antigen. Enterotoxin was released from the sporangium with its lysis, concomitantly with the mature spore release.     

Use of Dogs as an Assay for Clostridium perfringens Enterotoxin

Three techniques for using the dog as an assay organism for Clostridium perfringens enterotoxin are described. These are believed to be more convenient than ligated ileal-loop procedures.     

Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens

Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C. The enterotoxin was detected by serological and biological assays. Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage. The culture maintained enterotoxigenicity throughout cultivation in a continuous system. The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium. No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.     

A Study of Rapid and Simplified Confirmatory Tests for Clostridium perfringens

Strains of Clostridium perfringens and culturally similar species which also may grow on selective isolation media for this organism were examined by conventional confirmatory tests, the API ZYM system and by individual tests for phosphatase and glutamic acid decarboxylase activity.
API ZYM tests, involving 19 different enzymes, confirmed the known similarity between Cl. perfringens, Cl. absonum, Cl. paraperfringens and Cl. sardiniensis but effectively distinguished this group from Cl. bifermentans, Cl. celatum, Cl. perenne and Cl. sordellii. A similar separation was achieved by a single test for acid phosphatase which could be applied to individual colonies on a plating medium.
Because the acid phosphatase test was found to be of greater value than nitrate reduction in distinguishing Cl. perfringens , it could replace the latter in the usual series of confirmatory tests. It is suggested that strains from Cl. perfringens isolation media should be screened for acid phosphatase activity at the purification stage and only positive strains subjected to further tests.
It was found that Cl. perfringens could not be distinguished from the other species on the basis of glutamate decarboxylase activity.     

Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens.

Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C. The enterotoxin was detected by serological and biological assays. Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage. The culture maintained enterotoxigenicity throughout cultivation in a continuous system. The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium. No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.     

Enterotoxin formation by Clostridium perfringens type A in a defined medium

Enterotoxin was produced by 9 of 10 strains of Clostridium perfringens type A when grown in a defined medium. Additional dextrin increased the amount of enterotoxin in extracts of sporulating cells of strain NCTC 10239.     

A Method for Purification of Clostridium perfringens Phospholipase C from Recombinant Bacillus subtilis Cells

We developed a method to purify Clostridium perfringens phospholipase C from a culture of recombinant Bacillus subtilis cells. This method consists of three purification steps, and it allowed us to obtain 6.2 mg of pure phospholipase C from 800 ml of culture.     

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.     

Clostridium perfringens Enterotoxin Interacts with Claudins via Electrostatic Attraction

Clostridium perfringens enterotoxin (CPE), a causative agent of food poisoning, is a pore-forming toxin disrupting the selective permeability of the plasma membrane of target cells, resulting in cell death. We previously identified claudin as the cell surface receptor for CPE. Claudin, a component of tight junctions, is a tetratransmembrane protein and constitutes a large family of more than 20 members, not all of which serve as the receptor for CPE. The mechanism by which the toxin distinguishes the sensitive claudins is unknown. In this study, we localized the region of claudin responsible for interaction with CPE to the C-terminal part of the second extracellular loop and found that the isoelectric point of this region in sensitive claudins was higher than insensitive claudins. Amino acid substitutions to lower the pI resulted in reduced sensitivity to CPE among sensitive claudins, whereas substitutions to raise the pI endowed CPE-insensitive claudins with sensitivity. The steric structure of the claudin-binding domain of CPE reveals an acidic cleft surrounded by Tyr³⁰⁶, Tyr³¹⁰, Tyr³¹², and Leu³¹⁵, which were reported to be essential for interaction with the sensitive claudins. These results imply that an electrostatic attraction between the basic claudin region and the acidic CPE cleft is involved in their interaction.     

Molecular Cloning and Functional Characterization of the Receptor for Clostridium perfringens Enterotoxin

A cDNA encoding the Clostridium perfringens enterotoxin receptor gene (CPE-R) was cloned from an expression library of enterotoxin-sensitive Vero cells. The nucleotide sequence of CPE-R showed that the enterotoxin receptor consists of 209 amino acids with a calculated molecular mass of 22,029 D. This receptor is highly hydrophobic, contains four putative transmembrane segments, and has significant similarity to the rat androgen withdrawal apoptosis protein RVP1 and the mouse oligodendrocyte specific protein, the functions of which are unknown. The expression of CPE-R was detected in the enterotoxin-sensitive Vero, Hep3B, and Intestine 407 cell lines, but not in the enterotoxin-insensitive K562 and JY cell lines. The CPE-R gene product expressed in enterotoxin-resistant L929 cells bound to enterotoxin specifically and directly and with high affinity and rendered the cells sensitive to the toxin, indicating that the cloned receptor is functional. Results showed that enterotoxin could not assemble into a complex with a defined structure unless it interacted with the receptor. From these results, it is proposed that the enterotoxin receptor is required for both target cell recognition and poreformation in the cell membrane.     

Enterotoxin formation by Clostridium perfringens type A in a defined medium.

Enterotoxin was produced by 9 of 10 strains of Clostridium perfringens type A when grown in a defined medium. Additional dextrin increased the amount of enterotoxin in extracts of sporulating cells of strain NCTC 10239.     

Glucose Degradation in Clostridium perfringens Type A

Cell-free extracts of Clostridium perfringens were found to contain all the enzymes of the Embden-Meyerhof pathway of glycolysis in addition to lactic acid dehydrogenase and the pyruvate-clastic system. Neither glucose-6-phosphate dehydrogenase nor 6-phosphogluconate dehydrogenase activities could be demonstrated, suggesting the absence of the conventional hexose-monophosphate pathway in this organism.     

反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合,共配成３６４份模拟标本,采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验,Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株）,ＲＰＬＡ与生物学试验结果完全一致,均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性,而各生物学试验却均为阴性,该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌,表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８,ＣｐＮＣＴＣ１０６１１,ＣｐＮＣＴＣ１０６１４,ＣｐＮＣＴＣ１０６１２,ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性,但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌,后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６,ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性,ＰＣＲ     

Enterotoxin of Clostridium perfringens type A forms ion-permeable channels in a lipid bilayer membrane

The enterotoxin of Clostridium perfringens type A was found to form ion-permeable channels in a lipid bilayer. A patch clamp technique was used to detect channel activities in an asolectin bilayer with incorporated enterotoxin. About 20% of the lipid bilayer patches examined showed rectangular or stepwise shift of membrane current. The shifts indicated the gating of ion-permeable channels in the patches. The channels showed high conductance (40-450 pS), no rectification in current-voltage curves and occasional long-lasting events. The significance of these findings is discussed in relation to the mechanism of action of the toxin.