Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1984 Nov;46(2):318–323. doi: 10.1128/iai.46.2.318-323.1984

Correlation between toxin binding and hemolytic activity in membrane damage by staphylococcal alpha-toxin.

S Bhakdi, M Muhly, R Füssle
PMCID: PMC261533  PMID: 6500692

Abstract

The binding of Staphylococcus aureus alpha-toxin to rabbit and human erythrocytes was studied by hemolytic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. Hemolytic assays showed that toxin binding to 10% cell suspensions at neutral pH was very ineffective in the concentration range 3 X 10(-8) to 3 X 10(-7) M (1 to 10 micrograms/ml), and less than 5% of added toxin became cell bound. However, binding was augmented as toxin levels were raised, abruptly increasing to 50 to 60% at 2 X 10(-6) to 3 X 10(-6) M (60 to 100 micrograms/ml). When rabbit erythrocytes were lysed with 1 to 5 micrograms of toxin per ml, both monomeric and hexameric forms of the toxin could be detected on the membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. In contrast, human erythrocytes treated with 1 to 6 micrograms of toxin per ml did not lyse, and membrane-bound toxin was not detectable. When toxin concentrations were raised to 30 to 100 micrograms/ml, human erythrocytes also lysed and toxin hexamers became membrane bound in comparable amounts as on rabbit cell membranes. Lowering the pH led to a marked increase in susceptibility of human, but not rabbit erythrocytes towards alpha-toxin. When human cells were lysed at pH 5.0 with 5 micrograms of toxin per ml, membrane-bound hexameric toxin became detectable. The demonstrated correlation between the presence of hexameric, cell-bound toxin and hemolytic activity supports the channel concept of toxin-mediated cytolysis. The results also show that toxin binding does not exhibit overall characteristics of a simple receptor-ligand interaction.

Full text

PDF

Images in this article

319Image
on p.319
320Image
on p.320
321Image
on p.321

Selected References

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

  1. Arbuthnott J. P., Freer J. H., Bernheimer A. W. Physical states of staphylococcal alpha-toxin. J Bacteriol. 1967 Oct;94(4):1170–1177. doi: 10.1128/jb.94.4.1170-1177.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arbuthnott J. P., Freer J. H., Billcliffe B. Lipid-induced polymerization of staphylococcal -toxin. J Gen Microbiol. 1973 Apr;75(2):309–319. doi: 10.1099/00221287-75-2-309. [DOI] [PubMed] [Google Scholar]
  3. Bernheimer A. W. Cytolytic toxins of bacterial origin. The nature and properties of cytolytic proteins are discussed with emphasis on staphylococcal alpha-toxin. Science. 1968 Feb 23;159(3817):847–851. doi: 10.1126/science.159.3817.847. [DOI] [PubMed] [Google Scholar]
  4. Bhakdi S., Füssle R., Tranum-Jensen J. Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5475–5479. doi: 10.1073/pnas.78.9.5475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bhakdi S., Tranum-Jensen J. Membrane damage by complement. Biochim Biophys Acta. 1983 Aug 11;737(3-4):343–372. doi: 10.1016/0304-4157(83)90006-0. [DOI] [PubMed] [Google Scholar]
  6. Bhakdi S., Tranum-Jensen J., Utermann G., Füssle R. Binding and partial inactivation of Staphylococcus aureus alpha-toxin by human plasma low density lipoprotein. J Biol Chem. 1983 May 10;258(9):5899–5904. [PubMed] [Google Scholar]
  7. Buckelew A. R., Jr, Colacicco G. Lipid monolayers. Interactions with staphylococcal alpha-toxin. Biochim Biophys Acta. 1971 Mar 9;233(1):7–16. doi: 10.1016/0005-2736(71)90352-x. [DOI] [PubMed] [Google Scholar]
  8. Cassidy P., Harshman S. Iodination of a tyrosyl residue in staphylococcal alpha-toxin. Biochemistry. 1976 Jun 1;15(11):2342–2348. doi: 10.1021/bi00656a015. [DOI] [PubMed] [Google Scholar]
  9. Cassidy P., Harshman S. Studies on the binding of staphylococcal 125I-labeled alpha-toxin to rabbit erythrocytes. Biochemistry. 1976 Jun 1;15(11):2348–2355. doi: 10.1021/bi00656a016. [DOI] [PubMed] [Google Scholar]
  10. Freer J. H., Arbuthnott J. P., Bernheimer A. W. Interaction of staphylococcal alpha-toxin with artificial and natural membranes. J Bacteriol. 1968 Mar;95(3):1153–1168. doi: 10.1128/jb.95.3.1153-1168.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Freer J. H., Arbuthnott J. P., Billcliffe B. Effects of staphylococcal -toxin on the structure of erythrocyte membranes: a biochemical and freeze-etching study. J Gen Microbiol. 1973 Apr;75(2):321–332. doi: 10.1099/00221287-75-2-321. [DOI] [PubMed] [Google Scholar]
  12. Füssle R., Bhakdi S., Sziegoleit A., Tranum-Jensen J., Kranz T., Wellensiek H. J. On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin. J Cell Biol. 1981 Oct;91(1):83–94. doi: 10.1083/jcb.91.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. GREEN H., BARROW P., GOLDBERG B. Effect of antibody and complement on permeability control in ascites tumor cells and erythrocytes. J Exp Med. 1959 Nov 1;110:699–713. doi: 10.1084/jem.110.5.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harboe N., Ingild A. Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol Suppl. 1973;1:161–164. doi: 10.1111/j.1365-3083.1973.tb03798.x. [DOI] [PubMed] [Google Scholar]
  15. Kato I., Naiki M. Ganglioside and rabbit erythrocyte membrane receptor for staphylococcal alpha-toxin. Infect Immun. 1976 Jan;13(1):289–291. doi: 10.1128/iai.13.1.289-291.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. SEARS D. A., WEED R. I., SWISHER S. N. DIFFERENCES IN THE MECHANISM OF IN VITRO IMMUNE HEMOLYSIS RELATED TO ANTIBODY SPECIFICITY. J Clin Invest. 1964 May;43:975–985. doi: 10.1172/JCI104983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Thelestam M., Möllby R. Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. Infect Immun. 1975 Aug;12(2):225–232. doi: 10.1128/iai.12.2.225-232.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Thelestam M., Möllby R., Wadström T. Effects of staphylococcal alpha-, beta-, delta-, and gamma-hemolysins on human diploid fibroblasts and HeLa cells: evaluation of a new quantitative as say for measuring cell damage. Infect Immun. 1973 Dec;8(6):938–946. doi: 10.1128/iai.8.6.938-946.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES