Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Aug;65(8):3489–3492. doi: 10.1128/iai.65.8.3489-3492.1997

Site-specific mutagenesis of Clostridium perfringens alpha-toxin: replacement of Asp-56, Asp-130, or Glu-152 causes loss of enzymatic and hemolytic activities.

M Nagahama 1, T Nakayama 1, K Michiue 1, J Sakurai 1
PMCID: PMC175496  PMID: 9234819

Abstract

The current study has investigated the role of D-56, D-130, and E-152 in zinc ion binding properties, as well as the hemolytic, phospholipase C (PLC), and sphingomyelinase (SMase) activities of Clostridium perfringens alpha-toxin, based upon crystallography studies of the Bacillus cereus PLC, which had suggested these residues might be important for these functional activities. The replacement of D-56 in alpha-toxin resulted in complete loss of hemolytic, PLC, and SMase activities. The variant toxins at D-130 showed an approximately 100-fold reduction of biological activities compared to that of the wild-type toxin. The substitution of glutamine or glycine for E-152 caused complete loss of these activities, but substitution of aspartic acid for E-152 reduced but did not completely inhibit these activities. The variant toxins at D-56 and D-130, as well as the wild-type toxin, possessed approximately 2 mol of zinc atoms per mol of the protein, but E152G and E152Q contained approximately 1 mol of zinc metal per mol of the protein. On the other hand, the zinc content in E152D was calculated as about 1.4 mol in the toxin molecule. The replacement of D-56, D-130, or E-152 had no effect on binding to sheep erythrocytes and uptake of free zinc ion from the solution. The variant toxins at D-130 showed partial antigenic identity with the wild-type toxin on a double gel diffusion test. These observations suggest that D-56 in alpha-toxin is required for catalytic activity of alpha-toxin, D-130 is essential for maintenance of structure, and the carboxyl group of E-152 tightly ligands one zinc ion, which is essential for catalytic activity of the toxin.

Full Text

The Full Text of this article is available as a PDF (344.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Awad M. M., Bryant A. E., Stevens D. L., Rood J. I. Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringens-mediated gas gangrene. Mol Microbiol. 1995 Jan;15(2):191–202. doi: 10.1111/j.1365-2958.1995.tb02234.x. [DOI] [PubMed] [Google Scholar]
  2. Bhamidipati S. P., Hamilton J. A. NMR studies of phospholipase C hydrolysis of phosphatidylcholine in model membranes. J Biol Chem. 1993 Feb 5;268(4):2431–2434. [PubMed] [Google Scholar]
  3. Fujii Y., Nomura S., Oshita Y., Sakurai J. Excitatory effect of Clostridium perfringens alpha toxin on the rat isolated aorta. Br J Pharmacol. 1986 Jul;88(3):531–539. doi: 10.1111/j.1476-5381.1986.tb10233.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fujii Y., Sakurai J. Contraction of the rat isolated aorta caused by Clostridium perfringens alpha toxin (phospholipase C): evidence for the involvement of arachidonic acid metabolism. Br J Pharmacol. 1989 May;97(1):119–124. doi: 10.1111/j.1476-5381.1989.tb11931.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Guillouard I., Garnier T., Cole S. T. Use of site-directed mutagenesis to probe structure-function relationships of alpha-toxin from Clostridium perfringens. Infect Immun. 1996 Jul;64(7):2440–2444. doi: 10.1128/iai.64.7.2440-2444.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hough E., Hansen L. K., Birknes B., Jynge K., Hansen S., Hordvik A., Little C., Dodson E., Derewenda Z. High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus. Nature. 1989 Mar 23;338(6213):357–360. doi: 10.1038/338357a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Nagahama M., Iida H., Nishioka E., Okamoto K., Sakurai J. Roles of the carboxy-terminal region of Clostridium perfringens alpha toxin. FEMS Microbiol Lett. 1994 Jul 15;120(3):297–301. doi: 10.1111/j.1574-6968.1994.tb07049.x. [DOI] [PubMed] [Google Scholar]
  9. Nagahama M., Michiue K., Sakurai J. Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes. Biochim Biophys Acta. 1996 Apr 3;1280(1):120–126. doi: 10.1016/0005-2736(95)00288-x. [DOI] [PubMed] [Google Scholar]
  10. Nagahama M., Okagawa Y., Nakayama T., Nishioka E., Sakurai J. Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin. J Bacteriol. 1995 Mar;177(5):1179–1185. doi: 10.1128/jb.177.5.1179-1185.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nagahama M., Sakurai J. Threonine-74 is a key site for the activity of Clostridium perfringens alpha-toxin. Microbiol Immunol. 1996;40(3):189–193. doi: 10.1111/j.1348-0421.1996.tb03333.x. [DOI] [PubMed] [Google Scholar]
  12. Ochi S., Hashimoto K., Nagahama M., Sakurai J. Phospholipid metabolism induced by Clostridium perfringens alpha-toxin elicits a hot-cold type of hemolysis in rabbit erythrocytes. Infect Immun. 1996 Sep;64(9):3930–3933. doi: 10.1128/iai.64.9.3930-3933.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sakurai J., Fujii Y., Shirotani M. Contraction induced by Clostridium perfringens alpha toxin in the isolated rat ileum. Toxicon. 1990;28(4):411–418. doi: 10.1016/0041-0101(90)90079-m. [DOI] [PubMed] [Google Scholar]
  14. Sakurai J., Ochi S., Tanaka H. Evidence for coupling of Clostridium perfringens alpha-toxin-induced hemolysis to stimulated phosphatidic acid formation in rabbit erythrocytes. Infect Immun. 1993 Sep;61(9):3711–3718. doi: 10.1128/iai.61.9.3711-3718.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sakurai J., Ochi S., Tanaka H. Regulation of Clostridium perfringens alpha-toxin-activated phospholipase C in rabbit erythrocyte membranes. Infect Immun. 1994 Feb;62(2):717–721. doi: 10.1128/iai.62.2.717-721.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Titball R. W. Bacterial phospholipases C. Microbiol Rev. 1993 Jun;57(2):347–366. doi: 10.1128/mr.57.2.347-366.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Vallee B. L., Auld D. S. New perspective on zinc biochemistry: cocatalytic sites in multi-zinc enzymes. Biochemistry. 1993 Jul 6;32(26):6493–6500. doi: 10.1021/bi00077a001. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES