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. 1997 Mar;65(3):1014–1022. doi: 10.1128/iai.65.3.1014-1022.1997

Deletion analysis of the Clostridium perfringens enterotoxin.

J F Kokai-Kun 1, B A McClane 1
PMCID: PMC175083  PMID: 9038311

Abstract

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.

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Selected References

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  1. Czeczulin J. R., Collie R. E., McClane B. A. Regulated expression of Clostridium perfringens enterotoxin in naturally cpe-negative type A, B, and C isolates of C. perfringens. Infect Immun. 1996 Aug;64(8):3301–3309. doi: 10.1128/iai.64.8.3301-3309.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Czeczulin J. R., Hanna P. C., McClane B. A. Cloning, nucleotide sequencing, and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli. Infect Immun. 1993 Aug;61(8):3429–3439. doi: 10.1128/iai.61.8.3429-3439.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Granum P. E., Whitaker J. R., Skjelkvåle R. Trypsin activation of enterotoxin from Clostridium perfringens type A: fragmentation and some physicochemical properties. Biochim Biophys Acta. 1981 May 29;668(3):325–332. doi: 10.1016/0005-2795(81)90165-3. [DOI] [PubMed] [Google Scholar]
  4. Hanna P. C., McClane B. A. A recombinant C-terminal toxin fragment provides evidence that membrane insertion is important for Clostridium perfringens enterotoxin cytotoxicity. Mol Microbiol. 1991 Jan;5(1):225–230. doi: 10.1111/j.1365-2958.1991.tb01843.x. [DOI] [PubMed] [Google Scholar]
  5. Hanna P. C., Mietzner T. A., Schoolnik G. K., McClane B. A. Localization of the receptor-binding region of Clostridium perfringens enterotoxin utilizing cloned toxin fragments and synthetic peptides. The 30 C-terminal amino acids define a functional binding region. J Biol Chem. 1991 Jun 15;266(17):11037–11043. [PubMed] [Google Scholar]
  6. Hanna P. C., Wieckowski E. U., Mietzner T. A., McClane B. A. Mapping of functional regions of Clostridium perfringens type A enterotoxin. Infect Immun. 1992 May;60(5):2110–2114. doi: 10.1128/iai.60.5.2110-2114.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hanna P. C., Wnek A. P., McClane B. A. Molecular cloning of the 3' half of the Clostridium perfringens enterotoxin gene and demonstration that this region encodes receptor-binding activity. J Bacteriol. 1989 Dec;171(12):6815–6820. doi: 10.1128/jb.171.12.6815-6820.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hauser D., Eklund M. W., Boquet P., Popoff M. R. Organization of the botulinum neurotoxin C1 gene and its associated non-toxic protein genes in Clostridium botulinum C 468. Mol Gen Genet. 1994 Jun 15;243(6):631–640. doi: 10.1007/BF00279572. [DOI] [PubMed] [Google Scholar]
  9. Horiguchi Y., Akai T., Sakaguchi G. Isolation and function of a Clostridium perfringens enterotoxin fragment. Infect Immun. 1987 Dec;55(12):2912–2915. doi: 10.1128/iai.55.12.2912-2915.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hulkower K. I., Wnek A. P., McClane B. A. Evidence that alterations in small molecule permeability are involved in the Clostridium perfringens type A enterotoxin-induced inhibition of macromolecular synthesis in Vero cells. J Cell Physiol. 1989 Sep;140(3):498–504. doi: 10.1002/jcp.1041400314. [DOI] [PubMed] [Google Scholar]
  11. Kokai-Kun J. F., McClane B. A. Evidence that a region(s) of the Clostridium perfringens enterotoxin molecule remains exposed on the external surface of the mammalian plasma membrane when the toxin is sequestered in small or large complexes. Infect Immun. 1996 Mar;64(3):1020–1025. doi: 10.1128/iai.64.3.1020-1025.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kokai-Kun J. F., Songer J. G., Czeczulin J. R., Chen F., McClane B. A. Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens. J Clin Microbiol. 1994 Oct;32(10):2533–2539. doi: 10.1128/jcm.32.10.2533-2539.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Matsuda M., Ozutsumi K., Iwahashi H., Sugimoto N. Primary action of Clostridium perfringens type A enterotoxin on HeLa and Vero cells in the absence of extracellular calcium: rapid and characteristic changes in membrane permeability. Biochem Biophys Res Commun. 1986 Dec 15;141(2):704–710. doi: 10.1016/s0006-291x(86)80229-7. [DOI] [PubMed] [Google Scholar]
  15. McClane B. A. Clostridium perfringens enterotoxin acts by producing small molecule permeability alterations in plasma membranes. Toxicology. 1994 Feb 28;87(1-3):43–67. doi: 10.1016/0300-483x(94)90154-6. [DOI] [PubMed] [Google Scholar]
  16. McClane B. A., Hanna P. C., Wnek A. P. Clostridium perfringens enterotoxin. Microb Pathog. 1988 May;4(5):317–323. doi: 10.1016/0882-4010(88)90059-9. [DOI] [PubMed] [Google Scholar]
  17. McClane B. A., McDonel J. L. Characterization of membrane permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin. Biochim Biophys Acta. 1980 Aug 14;600(3):974–985. doi: 10.1016/0005-2736(80)90499-x. [DOI] [PubMed] [Google Scholar]
  18. McClane B. A., McDonel J. L. Protective effects of osmotic stabilizers on morphological and permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin. Biochim Biophys Acta. 1981 Mar 6;641(2):401–409. doi: 10.1016/0005-2736(81)90496-x. [DOI] [PubMed] [Google Scholar]
  19. McClane B. A. Osmotic stabilizers differentially inhibit permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin. Biochim Biophys Acta. 1984 Oct 17;777(1):99–106. doi: 10.1016/0005-2736(84)90501-7. [DOI] [PubMed] [Google Scholar]
  20. McClane B. A., Wnek A. P., Hulkower K. I., Hanna P. C. Divalent cation involvement in the action of Clostridium perfringens type A enterotoxin. Early events in enterotoxin action are divalent cation-independent. J Biol Chem. 1988 Feb 15;263(5):2423–2435. [PubMed] [Google Scholar]
  21. McClane B. A., Wnek A. P. Studies of Clostridium perfringens enterotoxin action at different temperatures demonstrate a correlation between complex formation and cytotoxicity. Infect Immun. 1990 Sep;58(9):3109–3115. doi: 10.1128/iai.58.9.3109-3115.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McDonel J. L. Binding of Clostridium perfringens [125I]enterotoxin to rabbit intestinal cells. Biochemistry. 1980 Oct 14;19(21):4801–4807. doi: 10.1021/bi00562a014. [DOI] [PubMed] [Google Scholar]
  23. McDonel J. L., McClane B. A. Binding versus biological activity of Clostridium perfringens enterotoxin in Vero cells. Biochem Biophys Res Commun. 1979 Mar 30;87(2):497–504. doi: 10.1016/0006-291x(79)91823-0. [DOI] [PubMed] [Google Scholar]
  24. McDonel J. L., McClane B. A. Production, purification, and assay of Clostridium perfringens enterotoxin. Methods Enzymol. 1988;165:94–103. doi: 10.1016/s0076-6879(88)65018-x. [DOI] [PubMed] [Google Scholar]
  25. Sigrist H., Ronner P., Semenza G. A hydrophobic form of the small-intestinal sucrase-isomaltase complex. Biochim Biophys Acta. 1975 Oct 17;406(3):433–446. doi: 10.1016/0005-2736(75)90022-x. [DOI] [PubMed] [Google Scholar]
  26. Wieckowski E. U., Wnek A. P., McClane B. A. Evidence that an approximately 50-kDa mammalian plasma membrane protein with receptor-like properties mediates the amphiphilicity of specifically bound Clostridium perfringens enterotoxin. J Biol Chem. 1994 Apr 8;269(14):10838–10848. [PubMed] [Google Scholar]
  27. Wnek A. P., McClane B. A. Preliminary evidence that Clostridium perfringens type A enterotoxin is present in a 160,000-Mr complex in mammalian membranes. Infect Immun. 1989 Feb;57(2):574–581. doi: 10.1128/iai.57.2.574-581.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wnek A. P., Strouse R. J., McClane B. A. Production and characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin. Infect Immun. 1985 Nov;50(2):442–448. doi: 10.1128/iai.50.2.442-448.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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