Beta2 toxin, a novel toxin produced by Clostridium perfringens

doi:10.1016/S0378-1119(97)00493-9

Gene

Volume 203, Issue 1, 5 December 1997, Pages 65-73

https://doi.org/10.1016/S0378-1119(97)00493-9 Get rights and content

Abstract

A novel toxin (Beta2) and its gene were characterized from a Clostridium perfringens strain isolated from a piglet with necrotic enteritis. At the amino-acid level, Beta2 toxin (27 670 Da) has no significant homology with the previously identified Beta toxin (called Beta1) (34 861 kDa) from C. perfringens type B NCTC8533 ( Hunter, S.E.C., Brown, J.E., Oyston, P.C.F., Sakurai, J., Titball, R.W., 1993. Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus. Infect. Immun. 61, 3958–3965). Both Beta1 and Beta2 toxins were lethal for mice and cytotoxic for the cell line I407, inducing cell rounding and lysis without affecting the actin cytoskeleton. The genes encoding Beta1 and Beta2 toxins have been localized in unlinked loci in large plasmids of C. perfringens. In addition, Beta2 toxin-producing C. perfringens strains were found to be associated with animal diseases such as necrotic enteritis in piglets and enterocolitis in horses.

Introduction

Clostridium perfringens strains are divided into five toxinotypes (A–E), according to the type of the major toxins that they produce. C. perfringens types B and C are reported to synthesize the beta toxin (Hatheway, 1990). This toxin has been implicated in necrotic enteritis in man and animals, a disease that is characterized by a sudden acute onset with lethal hemorrhagic mucosal ulceration or severe mucosal necrosis of the small intestine. The human disease is termed `pig bel' or `Darmbrand'. Malnutrition and the presence of trypsin inhibitors in food such as sweet potatoes have been involved as risk factors for the onset of the disease (Lawrence and Walker, 1976; Lawrence and Cooke, 1980). In addition, sporadic cases of necrotizing bowel disease due to C. perfringens C have been reported in humans in Europe and the United States (Schwartz et al., 1980; Severin et al., 1984). Necrotic enteritis due to C. perfringens type C and occasionally type B occur frequently in young piglets and more rarely in lambs, foals and calves (Findlay and Buntain, 1968; Bergeland, 1972; Niilo et al., 1974; Dickie et al., 1978; Vaissaire et al., 1983; Howard-Martin et al., 1986; Niilo, 1987).

In the literature, the characteristics of the Beta toxin are not clear, and molecular masses of 20–42 kDa have been determined by various investigators (Worthington and Mulders, 1975; Sakurai and Duncan, 1977; Sakurai and Fujii, 1987). The beta toxin gene (cpb) from C. perfringens type B NCTC8533 has been cloned and sequenced. The deduced product is a secreted protein of 34 861 Da that displays significant homology with alpha-toxin, gamma-toxin, and leucocidin of Staphylococcus aureus (Hunter et al., 1993). We have previously purified a toxin from a C. perfringens type C CWC245 strain isolated from a piglet that died of necrotizing enterocolitis, and we found that the purified protein (28 kDa) was cytotoxic for CHO cells and induced hemorrhagic necrosis of the intestinal mucosa in the guinea-pig ligated intestinal loop test (Jolivet-Reynaud et al., 1986). Since this toxin is different in size from the previous identified Beta toxin, we decided to identify and characterize its gene.

Here, we report the identification of the beta2 toxin gene (cpb2) from C. perfringens CWC245 and show that the deduced protein corresponds to a new C. perfringens toxin (referred as Beta2 toxin) that is distinct from the Beta toxin produced by strain NCTC8533 (called Beta1). We also investigated the distribution of cpb2 and cpb1 in several clinical and food isolates of C. perfringens.

Section snippets

Bacterial strains and DNA

The reference strains are in the text, and the isolates used in this study are listed in Table 1. C. perfringens strains were grown in broth containing trypticase (30 g/l), yeast extract (20 g/l), glucose (5 g/l), and cysteine–HCl (0.5 g/l) (pH 7.2) (TGY) at 37°C under anaerobic conditions. Clostridium total and plasmid DNAs were extracted and purified as previously described (Perelle et al., 1993).

Plasmids pUC18 and pUC19 (Appligene, Strasbourg, France) were used for cloning in E. coli TG1, and

Cloning of the beta2 toxin gene (cpb2)

The primers P279 and P280 were designed, based on the protein sequences of the N-terminal part and internal peptide no. 13 of Beta2 toxin, and used to PCR-amplify a fragment of 676 bp from C. perfringens CWC245 DNA (Fig. 1). This DNA fragment was cloned into the vector pUC18 at the SmaI site yielding the plasmid pMRP109. To obtain the 3′ part of the gene, a Sau3A DNA library of C. perfringens CWC245 into pUC19 was screened with P280, and the corresponding fragment of 2.5 kb was isolated (pMRP126)

Discussion

The classification of C. perfringens strains in five toxinotypes (A, B, C, D, and E) was based primarily on the presence of toxin(s) lethal for mice in culture supernatants, and on neutralization of this lethality by antibodies raised against crude culture supernatants (Sterne and Warrack, 1964). It was later found that C. perfringens type A strains from food intoxication differed from type A strains isolated from gangrena by production of an enterotoxin (McDonell, 1986). This toxin is produced

Acknowledgements

We would like to thank G. Daube, P. Fach, J. Frey, P. Hogh and C. Legardinier for kind gifts of C. perfringens strains, J. D'Alayer for protein sequencing and K. Ireton for preparing the manuscript. This work was supported by a grant (R93/09) from the Ministère de l'Agriculture.

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Beta2 toxin, a novel toxin produced by Clostridium perfringens

Abstract

Introduction

Section snippets

Bacterial strains and DNA

Cloning of the beta2 toxin gene (cpb2)

Discussion

Acknowledgements

Plasmid

Toxicon

Syst. Appl. Microbiol.

Lancet

Toxicon

J. Pediatr.

Gene

FEBS Lett.

Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins

Mol. Cel. Biochem.

Pathogenesis and immunity of Clostridium perfringens type C enteritis in swine

J. Am. Vet. Med. Assoc.

The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid-borne

Mol. Microbiol.

Enterotoxemia in two foals

J. Am. Vet. Med. Assoc.

Enterotoxemia due to Clostridium welchii type C in young lambs

Vet. Rec.

Transient expression of RhoA, -B, and -C GTPases in Hela cells potentiates resistance to Clostridium difficile toxins A and B but not to Clostridium sordellii lethal toxin

Infect. Immun.

Toxigenic clostridia

Clin. Microbiol. Rev.

Necrotizing infectious enteritis in piglets caused by Clostridium perfringens type C I. Biochemical and toxigenic properties of the Clostridium

Acta Vet. Scand.

Clostridium perfringens type C enterotoxemia in a newborn foal

J. Am. Vet. Med. Assoc.