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The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated. Neutralization experiments with monoclonal antibodies have shown that the entities responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin preparations are identical. However, the cytopathic effects of the same preparations are caused by other entities. Therefore, titrations based upon lethal and dermonecrotic indicators of beta toxin are equally valid but those based on cytopathic effects are not. Similar experiments with Cl perfringens epsilon preparations have shown that their lethal, dermonecrotic and cytopathic activities are all caused by the same entity. It follows that all three activities can be valid indicators for toxin neutralization tests. Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the levels of test prescribed by the European Pharmacopoeia have produced consistent results which agree closely with the dermonecrotic test. This test has, in turn, been shown to reflect the results of the mouse lethal test accurately. Titrations of cattle and sheep sera at lower levels of test have also produced results in close agreement with the in vivo test. It is concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in the course of potency tests and field trials of Cl perfringens epsilon vaccines.

Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin belong to a novel family of actin ADP-ribosylating toxins. ADP-ribosylation of actin inhibits actin polymerization and G-actin-associated ATPase activity. The ADP-form of actin is ADP-ribosylated at a higher rate than actin with bound ATP. ADP-ribosylation of actin is reversible, a reaction, which is accompanied by reconstitution of actin ATPase activity.

The tryptophan content of Clostridium perfringens epsilon toxin was investigated. When the tryptophan content was determined by amino acid analysis after the hydrolysis of epsilon prototoxin with methanesulfonic acid containing 3-(2-aminoethyl)indole and by the spectrophotometric method with N-bromosuccinimide, the number of tryptophan residues was calculated at 1/mol of the protein. Cleavage of the prototoxin or the toxin with N-bromosuccinimide in the presence of urea gave two new fragments on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was only a tyrosine residue as the new N-terminal amino acid after the cleavage of the prototoxin or the toxin with N-bromosuccinimide. The data showed that epsilon prototoxin or epsilon toxin contained only one tryptophan residue.

Spin-labeling studies were conducted to elucidate the viscosity and phase transition temperatures of lipids isolated from psychrophilic, psychrotrophic, and mesophilic clostridia. Electron spin resonance spectroscopy indicated that the lipids, for all the growth temperatures tested, were in a fluid state and from 13 to 24 C higher than the corresponding lipid transition temperatures. When the organisms were grown at different temperatures, a psychrotropic and two mesophilic clostridia were shown to be able to adjust their lipid-phase transition temperature to the growth temperature. A psychrophilic Clostridium strain, when grown at different temperatures, synthesized lipids that had the same phase transition temperature. It is suggested that this lack of growth temperature-inducible regulation of lipid-phase transition temperature may be a molecular determinant for the psychrophily of this organism. It is proposed that the growth temperature range of an organism is dependent upon the ability of the organism to regulate its lipid fluidity within a specific range.

A possibility was shown of purification of the concentrated toxoids of the Cl. perfringens and Cl. oedematiens species in examination of KSK-2, KSK-2,5; MCA-2 silicagels. The specific activity of the preparations obtained and the yield approached the analogous indices obtained in stratification of Sephadex G-75. The effect of preliminary silicagel treatment and also of the nature of buffer solutions on the efficacy of the process of purification of the gangrenous toxoids was established.

We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes platelet activation possibly by inducing turnover of phosphoinositides and phosphorylation of a 47,000 Dalton protein (P47). Moreover, only 15 microM and 11 microM flurazepam inhibits PLC-induced platelet aggregation and serotonin secretion by 50% respectively. This study was conducted to better understand the mechanism of platelet activation by PLC and its inhibition by flurazepam. Incubation of (14C)-arachidonic acid labelled platelets with PLC produced diacylglycerol in a time- and concentration-dependent manner. Flurazepam did not inhibit diacylglycerol production by PLC. Paranitrophenolphosphorylcholine and prostaglandin E1 inhibited diacylglycerol production by 75% and 20% respectively. In a platelet-free system PLC hydrolyzed 14C-choline-phosphatidylcholine (14C-PC) in a time- and calcium ions-dependent manner. Flurazepam had no effect on PLC-induced hydrolysis of 14C-PC. Platelet cytosolic fraction (PCF), containing phosphatidylinositol-specific PLC (PI-PLC), hydrolyzed (3H-inositol)-phosphatidylinositol (3H-PI) in a platelet-free system. Flurazepam did not inhibit hydrolysis of 3H-PI by PCF. Phospholipase C caused phosphorylation of P47 in 32P-labelled platelets. Flurazepam did not block phosphorylation of P47 in the first three minutes and had very little inhibitory effect by five minutes. However, flurazepam completely blocked phosphorylation of P47 by seven minutes. Platelet aggregation induced by ionomycin, a calcium ionophore, was completely inhibited by 100 microM flurazepam whereas platelet aggregation induced by 12-O-Tetradecanoylphorbol-13-acetate (TPA), which mimics the action of diacylglycerol, was partially inhibited by 300 microM flurazepam. These findings suggest that PLC induced platelet activation depends, at least in part, on diacylglycerol production and phosphorylation of P47. These data also suggest that flurazepam does not inhibit PLC-induced platelet activation by inhibiting: (a) the production of diacylglycerol from phosphatidylcholine; and (b) the action of PI-PLC on phosphatidylinositol. The ability of flurazepam to inhibit ionomycin-induced platelet aggregation indicates that flurazepam is able to block platelet activation by inhibiting the increase in free cytosolic calcium ions in platelets or by inhibiting a step subsequent to the rise in intraplatelet calcium ions.

It was shown in experiments on guinea pigs that the injection of C1. perfringens type A toxoid induced an increase in acid phosphatase activity of animal blood serum. The action of the toxoid increased under the effect of C1. butyricum cultural filtrate, which gave rise to an earlier enhancement of the specific activity of the enzyme as compared to the injection of the toxoid alone. Increased activity of acid phosphatase may play a pathogenetic role in cases of anaerobic infection caused by association of C1. perfringens and C1. butyricum.

Preparative polyacrylamide gel electrophoresis has been used to purify the enterotoxin of Clostridium perfringens from Sephadex G-100 extracts. Purified toxin of high specific activity was eluted in 1 to 3 h, depending upon the length of the acrylamide gel used. Recovery of biological activity with this technique ranged from 80 to 90%. The purity and physical characteristics of the toxin were similar to those previously reported for the protein purified by other methods. Use of preparative electrophoresis will enable the production of larger amounts of high-specific-activity toxin in a shorter time than other currently available procedures. This method was also used to isolate a form of enterotoxin that has a mobility, relative to bromophenol blue tracking dye, of 0.87 to 0.90 in 7% acrylamide gels.

A diarrheic syndrome linked to Clostridium perfringens was observed in fattening pigs. A good correlation was observed between the onset and the severity of diarrhea and the fecal passage of C perfringens enterotoxin. Intestinal fluids from affected pigs had activity comparable with that detected in a purified C perfringens enterotoxin. Seven pigs excreting enterotoxin were shown to develop serum antibodies to C perfringens enterotoxin.

1. The effect of pH on the buoyant density of the complexes of Ag+ with DNA has been studied using 3H-labeled human DNA and several bacterial DNAs to determine the conditions necessary for the maximum resolution of compositional heterogeneity. In neutral CS2SO4 density gradients, Ag+ complexes with (G - C)-rich components are always denser than those with (A - T)-rich components, since (G - C)rich DNAs have a larger affinity for Ag+ than (A - T)-rich DNAs and their complexes are denser than (A - T)-rich complexes. In alkaline (pH greater than 9) CS2SO4 gradients, the buoyant density of the Ag+ - DNA complex is not a simple function of base composition. The Ag+ affinity of (A - T)-rich DNA is larger than that of (G - C)-rich DNA but the density of a (G - C)-rich complex is larger. Thus the ordering of the buoyant density changes depends on the amount of added Ag+. 2. The problem of resolving the density heterogeneity within a tracer DNA, and minor components of DNA, is explored and useful fractionation techniques are developed.

Clostridium perfringens type E iota toxin, a dermonecrotic and lethal binary toxin, was purified to homogeneity. Each protein component of the toxin, iota a (ia) or iota b (ib), appeared as a single band by gradient or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and yielded a single immunoprecipitin arc by crossed immunoelectrophoresis with homologous antiserum. Individually, ia (Mr 47,500) or ib (Mr 71,500) had little biological activity. However, when combined in equimolar amounts, there was a 64-fold increase in the guinea pig dermonecrotic titer. The biological activity of ia was heat stable (85 degrees C for 15 min), whereas ib was inactivated at 55 degrees C. Our results demonstrated that C. perfringens iota toxin required two different, nonlinked protein components for biological activity.

Highly purified alpha-toxin (phospholipase C) of Clostridium perfringens prepared by affinity chromatography on agarose-linked egg-yolk lipoprotein induced the in vitro aggregation of platelets of an irreversible type. The aggregation started after a time lag, the length of which depended on the concentration of the toxin; the reciprocal of the time lag was found to be directly proportional to the toxin concentration. Using this assay method, we demonstrated that the platelet-aggregating activity of alpha-toxin reached minimum at around 70 C but heating at higher temperatures inactivated it to a lesser extent; the same anomaly in heat inactivation was observed with phospholipase C activity possessed by the toxin. By subjecting purified alpha-toxin to isoelectric focusing, four molecular forms were isolated, all of which were associated with both the platelet-aggregating and phospholipase C activities. From all these results we concluded that the entity responsible for the platelet-aggregating activity is identical with alpha-toxin (phospholipase C).

The staphylococcal coagglutination technique has been used in recent years for detecting various bacterial antigens and toxins. It is a simple and rapid technique which requires no specialized equipment. This report describes a staphylococcal coagglutination procedure for assaying C. perfringens enterotoxin. Its sensitivity and specificity were studied with the aid of various enterotoxin preparations and culture filtrates of C. perfringens and other Clostridium species. This assay detects between 1.9 and 3.8 micrograms of enterotoxin per ml. Only the enterotoxin preparations and the C. perfringens culture fluids were positive.

Cyclic AMP was not detected (less than 5 X 10(-9) M intracellular concentration) at any stage of growth or sporulation of two strains of Clostridium perfringens grown with or without methylxanthines. Only Bacillus and Lactobacillus, genera belonging to the same phylogenetic cluster, have previously exhibited undetectable levels of cyclic AMP.

Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized. IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride. Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium. Denaturing and reducing agents were necessary to solubilize the IB. An alkylating agent was required to prevent reaggregation of the subunits. Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C. perfringens. IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly. Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C. perfringens, trypsin, or trypsin plus bile salts.

A method is described for isolating an enterotoxin from a coatless spore mutant (8-6) of Clostridium perfringens type A. The characteristics of this enterotoxin only slightly resembled those of previously isolated enterotoxins of C. perfringens. The type A (8-6) enterotoxin was found to be composed of two subunits of Mr 18 000 with isoelectric points of 3.8 and 4.3. The LD50 for mice was 39 micrograms/kg with 0.10 micrograms corresponding to one erythemal unit. The type A (8-6) enterotoxin was inactivated by heating for 10 min at 60 degrees C. The amino acid composition data of type A (8-6) and delta toxins was similar, but type A (8-6) and type A enterotoxins showed less similarity. This lack of similarity between type A and type A (8-6) enterotoxins was confirmed by the failure of anti-sera to type A enterotoxin to neutralize the type A (8-6) enterotoxin, in both the mouse and erythemal tests.

The surface charge against pH behaviour of spores of Clostridium perfringens type A was determined using a colloid titration method in the pH range 4-9. The native spores as well as various ionic forms of the spores loaded with divalent cations were found to be negatively charged. A positive colloid, MGCh, inhibited the ionic germination of the spores and this inhibition was highly dependent on pH. However, another positive colloid, GCh, did not inhibit the germination. Chemical modification of carboxyl groups on the spore surface by use of a water-soluble carbodiimide with two nucleophiles, glycine ethyl ester and taurine, caused spores not only to have little or no colloidal charge but also to lose the ability to germinate. The modification of carboxyl groups in the spore coat protein also resulted in elimination of, or marked reduction in its negative charge. These results suggest that carboxyl groups in the spore coat protein are the major negatively charged species on the spore surface. The role of surface charge in the ionic germination of the spores is discussed.

DNAs from the genomes of Clostridium perfringens and Cytophaga johnsonii display orthodox A-DNA and B-DNA structures despite their high (A+L) nucleotide content. Unique structures, such as those found for synthetic DNAs having specific special sequences, do therefore not necessarily occur for DNAs having more random base sequence even if these have unusual base compositions. Clostridium perfringens DNA exhibits unusual structural properties only prior to purification by gel filtration.

Gel filtration of supernatant fluids, from the wild-type Clostridium perfringens, strain CN3870, and several of the mutants and reverants derived from this strain, showed that these mutants failed to product detectable amounts of still produced sialidase III activity. The reverants tested had regained the ability to produce approximately wild-type levels of the I and II forms of both activities. These results showthat there is a direct relationship between the production of the I form and hemagglutinin and sialidase activities and the production of the II form of these biologically active proteins. Models that explain the genetic basis for these results are discussed.

The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure.

In the culture fluid of a hemagglutinin-positive strain of Clostridium botulinum type C, two toxins of different molecular size, hemagglutinin positive and negative, were separated by sucrose density gradient centrifugation.

The bacteriocin N5 produced by Clostridium perfringens, strain BP6K-N5, after UV irradiation induction, was purified by ammonium sulfate precipitation, followed by ion-exchange chromatography on DEAE cellulose and gel filtration on Sephadex G100. By polyacrylamid-gel electrophoresis and immunodiffusion analysis the purified material was shown to be homogenous. The purified bacteriocin N5 is inactivated by proteolytic enzymes and by heat treatment at 50 degrees C for 15 minutes. It is a simple protein with a molecular weight of approximately 82.000. The protein appears to be a single polypeptide chain, as no dissociation is obtained by sodium dodecyl sulfate and beta-mercaptoethanol disc-gel-electrophoresis.

A passive bacteriocin typing system was developed for use as an epidemiologic tool to study outbreaks of food poisoning caused by Clostridium perfringens type A. The 90 strains tested were from England, the United States, India, Japan, France, and Canada. Forty of 74 strains produced bacteriocin when irradiated with ultraviolet light for 40-90 sec. Eight bacteriocins were concentrated with ammonium sulfate and titrated on a common indicator strain. These bacteriocins were found to be specific for C. perfringens type A. All of the 90 strains were typable, with 16 distinct bacteriocin types. Most (85.56%) of the strains were types 1-6. Strains of C. perfringens type A clustered into certain patterns of bacteriocin types according to their country of origin and source of isolation.

In gel-filtration of Cl. perfringens type A toxin on Sephadex F-100 and F-50 there was revealed relationship between the leukotoxic activity and a high-molecular component. A method of ion-exchange chromatography on a column with DEAE-Sephadex A-25 from the Cl. perfringens toxin there were obtained 8 fractions 3 of which possessed a marked leukotoxic activity: the capacity to destroy neutrophils in the Svejcar and Vancurik test and to-depress the phagocytic activity of leukocytes. Lecithinase and lethal activity was revealed in one of these fractions only. Leukotoxic fractions differed by the capacity to destroy neutrophils and to decrease their phagocytic activity.

The lethal activity of Clostridium perfringens epsilon toxin was inactivated by N-bromosuccinimide and N-chlorosuccinimide. Amino acid analysis of N-bromosuccinimide-treated prototoxin indicated that the one tryptophan residue present in the protein was abolished, and methionine and tyrosine reduced markedly. N-chloro-succinimide-treated prototoxin lost completely both tryptophan and methionine residues. The toxin was not inactivated by chloramine T, but all of methionine residues present in the protein was found to be oxidized by the agent. The data suggest that the one tryptophan residue present in the toxin is important for the lethal activity.

The sequence of the first 66 amino acids of the amino-terminal part of the enterotoxin from Clostridium perfringens type A is presented. The trypsin activation of the enterotoxin involves hydrolysis of Lys15-Glu16 and Lys25-Thr26 bonds. The N-terminal sequence of the trypsin-activated enterotoxin has limited homology with the sequence of the N-terminal region of the cholera toxin B subunit.

The effects of temperature, pH and sodium dodecyl sulfate on the conformation of the enterotoxin from Clostridium perfringens type A were followed by circular dichroism in both the peptide and aromatic regions. At near-physiological conditions (35 degrees C, pH 6.7) the enterotoxin exhibited a conformation consisting of approximately 60% pleated sheet, 40% non-periodic, and essentially no helix. The peptide region was relatively stable at temperatures up to 55 degrees C and at pH values ranging from 4-10. The aromatic region demonstrated profound, time-dependent changes at 55 degrees C. At temperatures greater than 55 degrees C, extremes of pH, and in the presence of SDS, the spectra in both regions showed major structural reorganization; in most cases a gain in helical content at the expense of sheet structure was observed. The conformational properties of the protein are very similar to those observed for the lectins, a group of carbohydrate-binding proteins.

Clostridium perfringens type A 125I-enterotoxin (125I-CPE) was bound to rabbit intestinal brush border membranes (BBMs) or Vero cells and then solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS). Solubilized radioactivity was analyzed by gel filtration chromatography on a Sepharose 4B column or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without sample boiling and autoradiography. Specifically bound 125I-CPE extracted from either BBMs or Vero cells was primarily associated with a complex of approximately 160,000 Mr. The CPE complex was partially purified by gel filtration or SDS-PAGE without sample boiling. SDS-PAGE analysis with sample boiling of the partially purified 125I-CPE complex from Vero cells or BBMs suggested that CPE complex contains both a 50,000-Mr protein and a 70,000-Mr protein in approximately equimolar amounts. This result is supported by affinity chromatography with CPE immobilized on Sepharose 4B, which showed the specific interaction of similar size proteins with CPE. The simplest explanation for these results is that CPE (Mr 35,000) interacts with 50,000-Mr and 70,000-Mr eucaryotic proteins to form a membrane-dependent complex of approximately 160,000 Mr. These results suggest that the receptor or target site(s) or both for CPE are similar in both BBMs and Vero cells. The significance of these findings in terms of CPE binding, insertion, and biologic action is discussed.

In an assay procedure, purified Clostridium perfringens enterotoxin induced the accumulation of fluid in the ileal loop of the axenic pig. The smallest amount of enterotoxin causing a positive response was 25 micrograms (1:32 titer by counterimmunoelectrophoresis [CIEP] ), and 100 micrograms (1:128 CIEP titer) caused a marked response. Possibly, diarrhea in pigs with 1:32 CIEP titer or more of enterotoxin in the feces may be associated with enterotoxigenic C perfringens.

Two plasmids were found and studied in the bacteriocinogenic strain N5 of Clostridium perfringens: one is a bacteriocinogenic factor (MW=5.7 X 10(6)) and the other a cryptic plasmid (MW 32.4 X 10(6)). Simultaneous loss of the ability to produce bacteriocin and of the bacteriocin resistance in a "cured" variant corresponds to the loss of the bacteriocinogenic plasmid DNA. The syntheses of haemolysin 0 and phospholipase C do not seem to be coded for by the cryptic plasmid.

The amino-terminal region of the enterotoxin of Clostridium perfringens was investigated by automated sequence analysis. The primary structure results revealed that the enterotoxin is composed of a single polypeptide amino acid sequence. Computer comparison of a 20-residue sequence with a sequence library of reported proteins revealed no significant chemical similarities, indicating that the enterotoxin represents a unique polypeptide primary structure.

The complete primary structure of rubredoxin (Rd) isolated from Clostridium perfringens was sequenced to be: MKKFICDVCGYIYDPAVGDPDNGVEPGTEFKDIPDDWVCPLCGVDKSQFSETEE. The sequence was highly homologous to that of C. pasteurianum Rd but was different at 13 sites out of the total 54 amino acid residues (76% homology). It contained 1 Fe atom, 4 cysteine residues, and no labile sulfur, had a molecular weight of 6,056, and shared the general properties of classical anaerobic Rds. The pI was 4.4. The Rd was reduced with NADH in the presence of a specific NAD(P)H oxidoreductase preparation from the bacterium. The Km value of nitrate reductase for Rd as an electron-donor was 12 microM, a value comparable to that of the 13 microM for ferredoxin (Fd). These results taken together provide additional support for its role as the electron carrier in the nitrate reductase system [Seki, S., Ikeda, A., and Ishimoto, M. (1988) J. Biochem. 103, 583-584].

Teta-hemolysine was purified from Cl. perfringens strain BP6K 28 as follows: reprecipitation in the isoelectric point of the enzyme with 2 N H2SO4 containing 15% NaCl, DEAE cellulose chromatography, gel filtration on Sephadex G-100-G75 or Bio Gel P-60--P-100, rechromatography on DEAE-Sephadex A-50 and affinity chromatography. The biological activity of homogenous teta-hemolysine, estimated by complete hemolysis of human erythrocytes, exceeded 100, 000 theta E over mg protein but the enzyme was highly labile. Molecular weight of the enzyme was 53,000 daltons.

The reactivity of perfringolysin O with thiol and disulfide compounds was studied. The activation potency of thiols was roughly proportional to the reaction rate constants of 5,5'-dithiobis-(2-nitrobenzoic acid) with thiols, which should be inversely proportional to their oxidation-reduction potentials. 1,2-Dimercaptoethane, which had the highest rate constant, most potently activated the toxin among the thiols tested and 4,4'-dipyridyl disulfide, which is known to be one of the most potent thiol-disulfide exchanging reagents, strongly inhibited toxin activity. Toxin activity was also inhibited by other thiol inhibitors.

Polyacrylamide gel electrophoresis of highly purified Clostridium perfringens enterotoxin revealed electrophoretic microheterogeneity of the enterotoxin, apparently because of slight charge differences in the peptides. Detergent gel electrophoresis showed that purified enterotoxin formed high molecular weight aggregates in the presence of both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide. No conditions capable of inhibiting this phenomenon were found. Although a molecular weight of 35 000 daltons has been reported in the literature, the experimentally determined molecular weight values in the presence of detergents corresponded to multiples of a theoretical subunit molecular weight of 17 500 daltons. Binding studies performed by equilibrium dialysis and ultracentrifugation methods revealed that the enterotoxin bound very small amounts of SDS per gram of protein. The evidence presented indicates possible detergent induced structural alterations of the protein.

A method for the determination of the A-T content of DNA based on second-derivative ultraviolet spectra is presented. It allows measurement in a wide range of pH values, ionic strengths, and buffer media. It is nondestructive for the sample and requires not more than 10 micrograms of DNA.