Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1991 May;59(5):1846–1852. doi: 10.1128/iai.59.5.1846-1852.1991

Characterization of monoclonal antibodies against alpha-hemolysin of Escherichia coli.

R L Oropeza-Wekerle 1, P Kern 1, D Sun 1, S Muller 1, J P Briand 1, W Goebel 1
PMCID: PMC257925  PMID: 1708360

Abstract

Monoclonal antibodies (MAbs) were raised against native and denatured alpha-hemolysin (HlyA) of Escherichia coli. Binding of the MAbs to native, denatured, and erythrocyte-complexed active wild-type hemolysin and mutant derivatives was tested. All 15 MAbs analyzed bound to native hemolysin, even when the toxin was complexed with human erythrocytes. While some MAbs were unable to bind to a specific native mutant hemolysin, others could not even bind to mutant hemolysin carrying deletions remote from their actual binding sites. A rough determination of the binding sites of 15 MAbs on HlyA was performed by Western immunoblot analysis using CNBr fragments of HlyA and mutant hemolysin proteins. Interestingly, the binding sites of the MAbs against native hemolysin seem to be more randomly distributed on HlyA than are those of MAbs against denatured hemolysin. Three MAbs inhibited the hemolytic activity significantly. Two of these MAbs bound to the hydrophobic region, and the other one bound to the repeat domain of HlyA. The use of synthetic peptides from these regions allowed determination of the linear epitopes for two of these MAbs.

Full text

PDF
1846

Images in this article

1848Image
on p.1848
1849Image
on p.1849
1849Image
on p.1849

Selected References

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

  1. Benz R., Schmid A., Wagner W., Goebel W. Pore formation by the Escherichia coli hemolysin: evidence for an association-dissociation equilibrium of the pore-forming aggregates. Infect Immun. 1989 Mar;57(3):887–895. doi: 10.1128/iai.57.3.887-895.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhakdi S., Tranum-Jensen J. Damage to cell membranes by pore-forming bacterial cytolysins. Prog Allergy. 1988;40:1–43. [PubMed] [Google Scholar]
  3. 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]
  4. Boehm D. F., Welch R. A., Snyder I. S. Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes. Infect Immun. 1990 Jun;58(6):1959–1964. doi: 10.1128/iai.58.6.1959-1964.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Erb K., Vogel M., Wagner W., Goebel W. Alkaline phosphatase which lacks its own signal sequence becomes enzymatically active when fused to N-terminal sequences of Escherichia coli haemolysin (HlyA). Mol Gen Genet. 1987 Jun;208(1-2):88–93. doi: 10.1007/BF00330427. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Felmlee T., Welch R. A. Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5269–5273. doi: 10.1073/pnas.85.14.5269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. GROSS E., WITKOP B. Nonenzymatic cleavage of peptide bonds: the methionine residues in bovine pancreatic ribonuclease. J Biol Chem. 1962 Jun;237:1856–1860. [PubMed] [Google Scholar]
  9. Grimminger F., Walmrath D., Birkemeyer R. G., Bhakdi S., Seeger W. Leukotriene and hydroxyeicosatetraenoic acid generation elicited by low doses of Escherichia coli hemolysin in rabbit lungs. Infect Immun. 1990 Aug;58(8):2659–2663. doi: 10.1128/iai.58.8.2659-2663.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gustafsson B., Rosén A., Holme T. Monoclonal antibodies against Vibrio cholerae lipopolysaccharide. Infect Immun. 1982 Nov;38(2):449–454. doi: 10.1128/iai.38.2.449-454.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Hacker J., Schröter G., Schrettenbrunner A., Hughes C., Goebel W. Hemolytic Escherichia coli strains in the human fecal flora as potential urinary pathogens. Zentralbl Bakteriol Mikrobiol Hyg A. 1983 May;254(3):370–378. [PubMed] [Google Scholar]
  13. 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]
  14. Ji G. E., O'Hanley P. Epitopes of Escherichia coli alpha-hemolysin: identification of monoclonal antibodies that prevent hemolysis. Infect Immun. 1990 Sep;58(9):3029–3035. doi: 10.1128/iai.58.9.3029-3035.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kraig E., Dailey T., Kolodrubetz D. Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family. Infect Immun. 1990 Apr;58(4):920–929. doi: 10.1128/iai.58.4.920-929.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. König B., König W., Scheffer J., Hacker J., Goebel W. Role of Escherichia coli alpha-hemolysin and bacterial adherence in infection: requirement for release of inflammatory mediators from granulocytes and mast cells. Infect Immun. 1986 Dec;54(3):886–892. doi: 10.1128/iai.54.3.886-892.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lally E. T., Kieba I. R., Demuth D. R., Rosenbloom J., Golub E. E., Taichman N. S., Gibson C. W. Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene. Biochem Biophys Res Commun. 1989 Feb 28;159(1):256–262. doi: 10.1016/0006-291x(89)92431-5. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Ludwig A., Jarchau T., Benz R., Goebel W. The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes. Mol Gen Genet. 1988 Nov;214(3):553–561. doi: 10.1007/BF00330494. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Nicaud J. M., Mackman N., Gray L., Holland I. B. Characterisation of HlyC and mechanism of activation and secretion of haemolysin from E. coli 2001. FEBS Lett. 1985 Aug 5;187(2):339–344. doi: 10.1016/0014-5793(85)81272-2. [DOI] [PubMed] [Google Scholar]
  24. Noegel A., Rdest U., Goebel W. Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli. J Bacteriol. 1981 Jan;145(1):233–247. doi: 10.1128/jb.145.1.233-247.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oropeza-Wekerle R. L., Müller E., Kern P., Meyermann R., Goebel W. Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins. J Bacteriol. 1989 May;171(5):2783–2788. doi: 10.1128/jb.171.5.2783-2788.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Oropeza-Wekerle R. L., Speth W., Imhof B., Gentschev I., Goebel W. Translocation and compartmentalization of Escherichia coli hemolysin (HlyA). J Bacteriol. 1990 Jul;172(7):3711–3717. doi: 10.1128/jb.172.7.3711-3717.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pellett S., Boehm D. F., Snyder I. S., Rowe G., Welch R. A. Characterization of monoclonal antibodies against the Escherichia coli hemolysin. Infect Immun. 1990 Mar;58(3):822–827. doi: 10.1128/iai.58.3.822-827.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wagner W., Kuhn M., Goebel W. Active and inactive forms of hemolysin (HlyA) from Escherichia coli. Biol Chem Hoppe Seyler. 1988 Jan;369(1):39–46. doi: 10.1515/bchm3.1988.369.1.39. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES