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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.

Clostridium perfringens type D 9867 produced enterotoxin immunologically identical to that produced by types A and C which are responsible for C. perfringens food poisoning. Enterotoxin from type C 5386 and type D 9867 was produced in Duncan og Strong sporulation medium (DS medium). The supernatant fluid from DS-cultures was used for purification of the enterotoxin by affinity chromatography on a monospecific anti-enterotoxin-coupled CNBr activated-Sepharose 4B and activated CH-Sepharose 4B column. The enterotoxin purified by this one-step procedure proved to be of a purity comparable to that obtained by conventional methods, and possessed lethal activity in mice.

We have established previously that the 67-kDa elastin-binding protein (EBP), identical to the spliced variant of beta-galactosidase, acts as a recyclable chaperone that facilitates secretion of tropoelastin. (Hinek, A., Keeley, F. W., and Callahan, J. W. (1995) Exp. Cell Res. 220, 312-324). We now demonstrate that EBP also forms a cell surface-targeted molecular complex with protective protein/cathepsin A and sialidase (neuraminidase-1), and provide evidence that this sialidase activity is a prerequisite for the subsequent release of tropoelastin. We found that treatment with sialidase inhibitors repressed assembly of elastic fibers in cultures of human skin fibroblasts, aortic smooth muscle cells, and ear cartilage chondrocytes and caused impaired elastogenesis in developing chick embryos. Fibroblasts derived from patients with congenital sialidosis (primary deficiency of neuraminidase-1) and galactosialidosis (secondary deficiency of neuraminidase-1) demonstrated impaired elastogenesis, which could be reversed after their transduction with neuraminidase-1 cDNA or after treatment with bacterial sialidase, which has a similar substrate specificity to human neuraminidase-1. We postulate that neuraminidase-1 catalyzes removal of the terminal sialic acids from carbohydrate chains of microfibrillar glycoproteins and other adjacent matrix glycoconjugates, unmasking their penultimate galactosugars. In turn, the exposed galactosugars interact with the galectin domain of EBP, thereby inducing the release of transported tropoelastin molecules and facilitating their subsequent assembly into elastic fibers.

The previous model for the action of Clostridium perfringens enterotoxin (CPE) proposed that (i) CPE binds to host cell receptor(s), forming a small ( approximately 90 kDa) complex, (ii) the small complex interacts with other eucaryotic protein(s), forming a large ( approximately 160 kDa) complex, and (iii) the large complex triggers massive permeability changes, thereby inducing enterocyte death. In the current study, Western immunoblot analysis demonstrated that CPE bound to CaCo-2 human intestinal cells at 37 degrees C forms multiple large complex species, with apparent sizes of approximately 200, approximately 155, and approximately 135 kDa. These immunoblot experiments also revealed that occludin, an approximately 65-kDa tight junction protein, is present in the approximately 200-kDa large complex but absent from the other large complex species. Immunoprecipitation studies confirmed that occludin physically associates with CPE in large complex material and also indicated that occludin is absent from small complex. These results strongly suggest that occludin becomes associated with CPE during formation of the approximately 200-kDa large complex. A postbinding association between CPE and occludin is consistent with the failure of rat fibroblast transfectants expressing occludin to bind CPE in the current study. Those occludin transfectants were also insensitive to CPE, strongly suggesting that occludin expression is not sufficient to confer CPE sensitivity. However, the occludin-containing, approximately 200-kDa large complex may contribute to CPE-induced cytotoxicity, because nontoxic CPE point mutants did not form any large complex species. By showing that large complex material is comprised of several species (one containing occludin), the current studies indicate that CPE action is more complicated than previously appreciated and also provide additional evidence for CPE interactions with tight junction proteins, which could be important for CPE-induced pathophysiology.

A gene coding for epsilon-toxin was isolated from a field isolate of Clostridium perfringens type D by PCR amplification and was cloned under the control of T5 promoter fused with six-histidine tag at the amino terminal end. Escherichia coli cells harbouring this construct expressed high levels of the recombinant protein in the form of inclusion bodies. The protein was purified using single step affinity chromatography on a Ni(2+)-nitrilotriacetic acid (NTA) agarose column. Upon immunization of rabbit with the purified recombinant protein, high antibody titre was detected. The antibodies raised against the recombinant protein were able to recognize the recombinant as well as the native toxin. Anti epsilon-toxin monoclonal antibody was able to detect the recombinant protein in a Western blot. N-terminal sequence of the recombinant protein matched with the known sequence of the toxin. At the shake flask level, up to 20 mg of pure epsilon-prototoxin was produced per litre of culture.

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens . In this study, the expression of the epsilon toxin ( etx ) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene ( cpa ) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni 2+ -chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42 kDa (rCPA) or 31 kDa (rETX) in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.

Clostripain (CLO) produced by Clostridium histolyticum is an arginine-specific endopeptidase with the potential for applicability to diverse medical and industrial uses. In this study, we developed an expression system allowing high-level production and efficient purification of recombinant CLO (rCLO). Our expression system comprises pCLO, an rCLO expressing vector, and Clostridium perfringens 13Δ6, an in-frame deletion strain as to six genes encoding major virulence factors and secretory proteins. rCLO was purified from the culture supernatant of C. perfringens 13Δ6/pCLO by ammonium sulfate precipitation, hydroxyapatite chromatography, and affinity chromatography on benzamidine-Sepharose. From 200 ml of culture supernatant 4.5 mg of purified rCLO was obtained. N-Terminal amino acid sequencing and molecular mass determination of the purified rCLO and commercially available CLO revealed that the two enzymes have identical subunits, a 38.1-kDa heavy chain and a 15.0-kDa light chain, indicating that rCLO is processed in the same manner as CLO. Analysis of the enzymatic activities toward N-benzoyl-L-arginine p-nitroanilide and acyl-L-lysine p-nitroanilide showed that rCLO and CLO exhibit strict specificity for arginine at the P1 position, and that the specific activity of the former is approximately 2-fold higher than that of the latter. These results indicate that the new method involving a virulence-attenuated C. perfringens strain is useful for preparing large amounts of high-grade rCLO.

Background Claudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with Clostridium perfringens enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes. Results Immunoblotting and mass spectrometry (MS) experiments demonstrate strong enrichment of the CPE-binding claudins -3, -4 and -7, indicating specific association with glutathione-S-transferase (GST)-CPE116–319 fusion protein. In parallel, the co-elution of (non-CPE-binding) claudin-1 and claudin-5 was observed. The complete set of co-enriched proteins was identified by MS after electrophoretic separation. Relative mass spectrometric protein quantification with stable isotope labeling with amino acids in cell culture (SILAC) made it possible to discriminate specific binding from non-specific association to GST and/or matrix material. Conclusion CPE116–319 provides an efficient tool for single step enrichment of different claudins from cell lysates. Numerous proteins were shown to be co-enriched with the CPE-binding claudins, but there are no indications (except for claudins -1 and -5) for an association with tight junctions.

A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.

Summary A complete structure of asparagine linked sugar chains of bovine IgG was determined to be as follows. This structure is inconsistent with the results obtained from myeloma proteins in that incomplete galactosylation occurred in the branch with α1→3 linked mannosyl residue.

Anti-enterotoxin immunoglobulins immobilized on CH-Sepharose or CNBr-Sepharose were used for affinity chromatography purification of Clostridium perfringens enterotoxin. Cell extracts containing enterotoxin or partially purified toxin preparations were applied to the column and nonspecifically-bound protein was eluted. NaOH was used to elute specifically bound toxin. The purity of enterotoxin purified by Sephadex G-100 chromatography followed by affinity chromatography appears similar to toxin highly purified by conventional means. The procedure can be used successfully for the rapid (less than 2 h) purification of small amounts of enterotoxin.

An affinity column for α- l -fucosidases was constructed by linking p -aminophenyl 1-thio-α- l -fucopyranoside to Sepharose 4B through linkers of succinyl 3,3′-diamino-dipropylamine. Excellent purification of α- l -fucosidase from rat epididymis, Clostridium perfringens and Limulus polyphemus (horse shoecrab) could be effected in one step with good yield. An affinity column purification step can be introduced at any point in published purification procedures. The purified enzyme is essentially free of other glycosidases and proteolytic enzymes. The column material is stable and can be reused for at least two years.

Alterations in the oligosaccharides of complex surface glycoproteins have been studied in wild-type rat skeletal myoblasts (L6-9/1) and two wheat germ agglutinin resistant mutants, selected for either high level of resistance (WGARI) or low level of resistance (WGARII). All three lines possessed high mannose oligosaccharides of the structure Mann GlcNAc, where n = 6, 7, or 8. L6-9/1 had complex oligosaccharides of the type (±NeuNAcα2-3GalβGlcNAcβ)nMan3GlcNAc(±Fuc)GlcNAc where n = 3 or 4, in addition to small amounts of biantennary and higher order structures. WGARII, the mutant with low resistance to wheat germ agglutinin, possessed similar complex oligosaccharides except that they had undergone some base-sensitive modification which rendered them resistant to Clostridium perfringens sialidase. WGARI, mutant of high resistance to wheat germ agglutinin, possessed complex oligosaccharides of the type (GlcNAcβ)nMan3GlcNAc(±Fuc)GlcNAc, with n = 2,3, or 4. Since the WGARI mutants were unaltered in differentiation, it is concluded that the terminal sialic acid and galactose residues on the protein-bound oligosaccharides are not required for differentiation.

A number of derivatives of Sepharose 4B gels were prepared containing tyramine, -Gly-Gly-Tyr and the tetrapeptide, Thr (Bu t )-Phe-Pro-Tyr-. A specific inhibitor of sialidase, p- diazo-phenyl oxamic acid, was also coupled to these phenol-substituted Sepharose gels. The effectiveness of these gels to purify sialidase by “affinity” chromatography was examined. The sialidase from Vibrio cholerae was adsorbed by these gels and subsequently re-eluted by a change in pH and temperature. The adsorption and elution of enzyme was independent of the presence or absence of the “specific inhibitor” of the sialidase, phenyl oxamic acid. A mechanism for the adsorption and subsequent elution of the sialidase is proposed. It is important to re-evaluate the biological data obtained with sialidase prepared by the affinity column recommended by Cuatrecasas and Illiano, Biochem. Biophys. Res. Commun. 44, 178–184 (1971), since glycosidases from Clostridium perfringens other than sialidase are also adsorbed and eluted under the conditions specified.

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.

Ten distinct monoclonal antibodies (MAbs) against alpha toxin of Clostridium perfringens were produced by the fusion of SP2/O with spleen cells of mice immunized with alpha toxoid, and alpha toxin mixed with or without ethylenediamine-tetraacetate (EDTA). The antibody activity was evaluated by antigen-binding activity in an enzyme linked immunosorbent assay (ELISA), by phospholipase C (PLC)-neutralizing activity using both egg yolk lecithin and p-nitrophenylphosphoryl-choline (PNPPC) hydrolysis reactions and by anti-lethal activity in mice. Since the toxin-neutralizing activities of each MAb were not parallel, it has been suggested that the three biological activities may not be located in the same site in the toxin molecule. This report also describes the development of a simple purification of the toxin by affinity chromatography and a sensitive immunoassay for quantitation of the toxin using the monoclonal antibody.

A new procedure for the purification of phospholipase C from Clostridium perfringens has been devised that results in essentially pure enzyme. The procedure consists of ammonium sulfate fractionation, ion-exchange chromatography on QAE-Sephadex, and affinity chromatography on phosphatidylcholine linked to Sepharose. The molecular weight of the enzyme, determined by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and gel filtration, is 43,000; and the isoelectric point is pH 5.4. The enzyme was optimally active with phosphatidylcholine dispersed in sodium deoxycholate, although appreciable activity was observed with either phosphatidylcholine or sphingomyelin dispersed with ethanol. The requirement for metal ions in the assay could be met by a number of different ions. The pure enzyme was found to contain 2 mol zinc per mol enzyme, thus implicating it as a zinc metalloenzyme.

The human plasma α-galactosidases which specifically hydrolyze galactosyl-(α1→4)galactosyl(β1→ 4)glucosylceramide consist of an A group with optimal enzymatic activity at pH 5.4, and a B group, which is characterized by optimal activity at pH 7.2. The relationship between the A and B groups of these α-galactosidases (ceramide trihexosidases) has been investigated with regard to their sialic acid content. Partial neuraminidase treatment of the most acidic (A-1) form of ceramide trihexosidase yields a complex mixture of 14 enzymatically active proteins separable by isoelectric focusing. Exposure to neuraminidase for a longer time causes an almost complete conversion of the A-1 form to a protein which has the same electrophoretic properties as the least acidic (B-V) form. Conversely, a crude kidney sialyltransferase preparation can be used to incorporate either CMP[1-14C]sialic acid or UDP-N-acetyl[1-14C]glucosamine into the B-V form of the enzyme. Sialyltransferase treatment causes the formation of a complex mixture of enzymatically active proteins, one of which has the same electrophoretic characteristics as the A-1 and A-2 forms of ceramide trihexosidase. On the basis of these studies it is suggested that the multiple forms of plasma ceramide trihexosidase are glycoproteins which differ primarily in their sialic acid content.

Beta-toxin was purified about 340-fold from culture supernatant fluid of Clostridium perfringens type C with a yield of about 24% in terms of biologically active beta-toxin. The purification involved ammonium sulfate fractionation, gel filtration through Sephadex G-100, isoelectrofocusing in a pH 3 to 6 gradient, and immunoaffinity chromatography. The purified beta-toxin gave a single band on polyacrylamide gel electrophoresis.

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.

Snail agglutinin (anti-AEc, anti-A1 of cold agglutinin type) from the albumin gland of Euhadra callizona amaliae was purified by affinity chromatography (meconium-A aminoethyl cellulose). Its physicochemicl properties are as follows: S20, w: 5.3S. Isoelectric point: pH 3.6. Molecular weight: 8.9 × 104. Hexose content: 5.1%. The amino acid composition is that of an acidic protein. The agglutination is inhibited by GalNAc, GNAc, raffinose, and melibiose at low concentration. It does not agglutinate B- and O-RDE-treated human red cells. Some blood group A-active substances (secretor A saliva and hog gastric mucin A + H) have low potency to inhibit the agglutination. Anti-ABC has no ability to release the cell-fixed anti-Eorssman haemolysin and the cell-fixed blood group A-decomposing enzyme. The significance of the antigen distribution on the limited area of the erythrocyte and protein surface is also discussed. Precipitin reaction of this agglutinin with various blood group-active substances has shown that anti-AEc is directed to a narrow-range spectrum of determining antigenicity.

Antibodies against epsilon toxin were isolated from hyperimmune horse serum by affinity chromatography. Purified epsilon prototoxin covalently bound to Affigel 202 was used as immunosorbent, and antibodies were eluted with 6.0 M guanidine chloride. In a single run 80 mg of antibody could be recovered from a 20 microliter column of immunosorbent. The antibody was shown to belong to the IgG(T) class of immunoglobulins.