Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1989 Mar;57(3):983–988. doi: 10.1128/iai.57.3.983-988.1989

Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin.

B Eberspächer 1, F Hugo 1, S Bhakdi 1
PMCID: PMC313209  PMID: 2465275

Abstract

Mono- and polyclonal antibodies were used to construct a sandwich enzyme-linked immunosorbent assay that permitted quantitation of Escherichia coli hemolysin in soluble and membrane-bound forms. Toxin concentrations of 4 to 14 micrograms/ml were measured in culture supernatants of E. coli LE 2001 at times of peak hemolytic activity. Quantitative studies on the binding of E. coli hemolysin to rabbit erythrocytes were conducted at 0 and 37 degrees C. At 37 degrees C, 85 to 95% of bindable toxin was cell bound after 60 min, and no saturability of binding was observed in the studied range of concentrations, which resulted in deposition of approximately 100 to 50,000 toxin molecules per cell. Binding was slower and less effective at 0 degrees C; however, hemolysis did occur at low temperature. The number of cell-bound toxin molecules required to generate a hemolytic lesion within 60 min was estimated to be approximately 100 molecules per cell at 37 degrees C and 800 to 1,000 molecules per cell at 0 degrees C. Upon prolonged incubation (5 to 20 h, 37 degrees C), the number of molecules evoking a functional lesion decreased to approximately 5 to 20 per cell. These results are compatible with the concept that E. coli hemolysin first adsorbs to the cell surface, with membrane insertion and pore formation following in a second step that may be temporally dissociated from that of binding. The data support the pore concept of toxin action by showing that attachment of a low and finite number of toxin molecules to an erythrocyte will ultimately generate a cytolytic lesion.

Full text

PDF
983

Images in this article

985Image
on p.985

Selected References

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

  1. 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]
  2. Bhakdi S., Mackman N., Nicaud J. M., Holland I. B. Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores. Infect Immun. 1986 Apr;52(1):63–69. doi: 10.1128/iai.52.1.63-69.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhakdi S., Tranum-Jensen J. Damage to cell membranes by pore-forming bacterial cytolysins. Prog Allergy. 1988;40:1–43. [PubMed] [Google Scholar]
  4. Bhakdi S., Tranum-Jensen J. Damage to mammalian cells by proteins that form transmembrane pores. Rev Physiol Biochem Pharmacol. 1987;107:147–223. doi: 10.1007/BFb0027646. [DOI] [PubMed] [Google Scholar]
  5. Bhakdi S., Tranum-Jensen J., Sziegoleit A. Mechanism of membrane damage by streptolysin-O. Infect Immun. 1985 Jan;47(1):52–60. doi: 10.1128/iai.47.1.52-60.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brooks H. J., O'Grady F., McSherry M. A., Cattell W. R. Uropathogenic properties of Escherichia coli in recurrent urinary-tract infection. J Med Microbiol. 1980 Feb;13(1):57–68. doi: 10.1099/00222615-13-1-57. [DOI] [PubMed] [Google Scholar]
  7. Cavalieri S. J., Bohach G. A., Snyder I. S. Escherichia coli alpha-hemolysin: characteristics and probable role in pathogenicity. Microbiol Rev. 1984 Dec;48(4):326–343. doi: 10.1128/mr.48.4.326-343.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooke E. M., Ewins S. P. Properties of strains of Escherichia coli isolated from a variety of sources. J Med Microbiol. 1975 Feb;8(1):107–111. doi: 10.1099/00222615-8-1-107. [DOI] [PubMed] [Google Scholar]
  9. Cramer W. A., Dankert J. R., Uratani Y. The membrane channel-forming bacteriocidal protein, colicin El. Biochim Biophys Acta. 1983 Mar 21;737(1):173–193. doi: 10.1016/0304-4157(83)90016-3. [DOI] [PubMed] [Google Scholar]
  10. Evans D. J., Jr, Evans D. G., Höhne C., Noble M. A., Haldane E. V., Lior H., Young L. S. Hemolysin and K antigens in relation to serotype and hemagglutination type of Escherichia coli isolated from extraintestinal infections. J Clin Microbiol. 1981 Jan;13(1):171–178. doi: 10.1128/jcm.13.1.171-178.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Felmlee T., Pellett S., Welch R. A. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol. 1985 Jul;163(1):94–105. doi: 10.1128/jb.163.1.94-105.1985. [DOI] [PMC free article] [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. Goebel W., Hedgpeth J. Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol. 1982 Sep;151(3):1290–1298. doi: 10.1128/jb.151.3.1290-1298.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hacker J., Hughes C., Hof H., Goebel W. Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice. Infect Immun. 1983 Oct;42(1):57–63. doi: 10.1128/iai.42.1.57-63.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henkart P. A. Mechanism of lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 1985;3:31–58. doi: 10.1146/annurev.iy.03.040185.000335. [DOI] [PubMed] [Google Scholar]
  16. Hughes C., Hacker J., Roberts A., Goebel W. Hemolysin production as a virulence marker in symptomatic and asymptomatic urinary tract infections caused by Escherichia coli. Infect Immun. 1983 Feb;39(2):546–551. doi: 10.1128/iai.39.2.546-551.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hugo F., Arvand M., Reichwein J., Mackman N., Holland I. B., Bhakdi S. Identification with monoclonal antibodies of hemolysin produced by clinical isolates of Escherichia coli. J Clin Microbiol. 1987 Jan;25(1):26–30. doi: 10.1128/jcm.25.1.26-30.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jorgensen S. E., Hammer R. F., Wu G. K. Effects of a single hit from the alpha hemolysin produced by Escherichia coli on the morphology of sheep erythrocytes. Infect Immun. 1980 Mar;27(3):988–994. doi: 10.1128/iai.27.3.988-994.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jorgensen S. E., Mulcahy P. F., Wu G. K., Louis C. F. Calcium accumulation in human and sheep erythrocytes that is induced by Escherichia coli hemolysin. Toxicon. 1983;21(5):717–727. doi: 10.1016/0041-0101(83)90277-5. [DOI] [PubMed] [Google Scholar]
  20. Koronakis V., Cross M., Senior B., Koronakis E., Hughes C. The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli. J Bacteriol. 1987 Apr;169(4):1509–1515. doi: 10.1128/jb.169.4.1509-1515.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lo R. Y., Strathdee C. A., Shewen P. E. Nucleotide sequence of the leukotoxin genes of Pasteurella haemolytica A1. Infect Immun. 1987 Sep;55(9):1987–1996. doi: 10.1128/iai.55.9.1987-1996.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ludwig A., Vogel M., Goebel W. Mutations affecting activity and transport of haemolysin in Escherichia coli. Mol Gen Genet. 1987 Feb;206(2):238–245. doi: 10.1007/BF00333579. [DOI] [PubMed] [Google Scholar]
  23. Mackman N., Holland I. B. Functional characterization of a cloned haemolysin determinant from E. coli of human origin, encoding information for the secretion of a 107K polypeptide. Mol Gen Genet. 1984;196(1):129–134. doi: 10.1007/BF00334104. [DOI] [PubMed] [Google Scholar]
  24. Mackman N., Nicaud J. M., Gray L., Holland I. B. Secretion of haemolysin by Escherichia coli. Curr Top Microbiol Immunol. 1986;125:159–181. doi: 10.1007/978-3-642-71251-7_10. [DOI] [PubMed] [Google Scholar]
  25. Menestrina G. Escherichia coli hemolysin permeabilizes small unilamellar vesicles loaded with calcein by a single-hit mechanism. FEBS Lett. 1988 May 9;232(1):217–220. doi: 10.1016/0014-5793(88)80420-4. [DOI] [PubMed] [Google Scholar]
  26. Menestrina G., Mackman N., Holland I. B., Bhakdi S. Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes. Biochim Biophys Acta. 1987 Nov 27;905(1):109–117. doi: 10.1016/0005-2736(87)90014-9. [DOI] [PubMed] [Google Scholar]
  27. Minshew B. H., Jorgensen J., Counts G. W., Falkow S. Association of hemolysin production, hemagglutination of human erythrocytes, and virulence for chicken embryos of extraintestinal Escherichia coli isolates. Infect Immun. 1978 Apr;20(1):50–54. doi: 10.1128/iai.20.1.50-54.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schein S. J., Kagan B. L., Finkelstein A. Colicin K acts by forming voltage-dependent channels in phospholipid bilayer membranes. Nature. 1978 Nov 9;276(5684):159–163. doi: 10.1038/276159a0. [DOI] [PubMed] [Google Scholar]
  29. Seetharama S., Cavalieri S. J., Snyder I. S. Immune response to Escherichia coli alpha-hemolysin in patients. J Clin Microbiol. 1988 May;26(5):850–856. doi: 10.1128/jcm.26.5.850-856.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Strathdee C. A., Lo R. Y. Extensive homology between the leukotoxin of Pasteurella haemolytica A1 and the alpha-hemolysin of Escherichia coli. Infect Immun. 1987 Dec;55(12):3233–3236. doi: 10.1128/iai.55.12.3233-3236.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Waalwijk C., van den Bosch J. F., MacLaren D. M., de Graaff J. Hemolysin plasmid coding for the virulence of a nephropathogenic Escherichia coli strain. Infect Immun. 1982 Jan;35(1):32–37. doi: 10.1128/iai.35.1.32-37.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Welch R. A., Dellinger E. P., Minshew B., Falkow S. Haemolysin contributes to virulence of extra-intestinal E. coli infections. Nature. 1981 Dec 17;294(5842):665–667. doi: 10.1038/294665a0. [DOI] [PubMed] [Google Scholar]
  33. Welch R. A., Hull R., Falkow S. Molecular cloning and physical characterization of a chromosomal hemolysin from Escherichia coli. Infect Immun. 1983 Oct;42(1):178–186. doi: 10.1128/iai.42.1.178-186.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Welch R. A. Identification of two different hemolysin determinants in uropathogenic Proteus isolates. Infect Immun. 1987 Sep;55(9):2183–2190. doi: 10.1128/iai.55.9.2183-2190.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES