Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Nov;64(11):4655–4658. doi: 10.1128/iai.64.11.4655-4658.1996

In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin (pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease of V. cholerae, trypsin, and other proteases.

K Nagamune 1, K Yamamoto 1, A Naka 1, J Matsuyama 1, T Miwatani 1, T Honda 1
PMCID: PMC174427  PMID: 8890221

Abstract

Vibrio cholerae produces a cytolytic toxin named El Tor cytolysin/hemolysin which is encoded by the hlyA gene. This cytolysin is produced as a 79-kDa precursor form (pro-HlyA) into the culture supernatant after cleavage of the signal peptide of the hlyA product (prepro-HlyA). The pro-HlyA is then processed to a 65-kDa mature cytolysin (mature HlyA) after cleavage of the 15-kDa N-terminal peptide (pro region) of the 79-kDa precursor, usually at the bond between Ala-157 and Asn-158. We investigated whether proteases could process the recombinant 79-kDa pro-HlyA to the 65-kDa mature HlyA. We observed that the soluble hemagglutinin/ protease (HA/protease; a major protease of V. cholerae), trypsin, alpha-chymotrypsin, subtilisin BPN', papain, and thermolysin all processed the pro-HlyA to the 65-kDa mature form of the protein. Along with this, the protease-processed HlyA showed drastically increased hemolytic activity. The N-terminal amino acid of the mature form of cytolysin generated by HA/protease was Phe-151, and that due to trypsin was Ser-149. Other proteases also cleaved the pro-HlyA at a nearby site, between Leu-146 and Ser-153, and all the processed cytolysins showed increased hemolytic activity. These data suggest that the active El Tor cytolysin of V. cholerae could be derived from the C-terminal region of a pro-HlyA following proteolytic cleavage of the bonds in the vicinity of Leu-146 to Asn-158 by any of a wide variety of proteases.

Full Text

The Full Text of this article is available as a PDF (216.8 KB).

Selected References

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

  1. Blake P. A., Weaver R. E., Hollis D. G. Diseases of humans (other than cholera) caused by vibrios. Annu Rev Microbiol. 1980;34:341–367. doi: 10.1146/annurev.mi.34.100180.002013. [DOI] [PubMed] [Google Scholar]
  2. Booth B. A., Boesman-Finkelstein M., Finkelstein R. A. Vibrio cholerae hemagglutinin/protease nicks cholera enterotoxin. Infect Immun. 1984 Sep;45(3):558–560. doi: 10.1128/iai.45.3.558-560.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Braun V., Schönherr R., Hobbie S. Enterobacterial hemolysins: activation, secretion and pore formation. Trends Microbiol. 1993 Sep;1(6):211–216. doi: 10.1016/0966-842x(93)90134-d. [DOI] [PubMed] [Google Scholar]
  4. Finkelstein R. A., Boesman-Finkelstein M., Holt P. Vibrio cholerae hemagglutinin/lectin/protease hydrolyzes fibronectin and ovomucin: F.M. Burnet revisited. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1092–1095. doi: 10.1073/pnas.80.4.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Finkelstein R. A., Hanne L. F. Purification and characterization of the soluble hemagglutinin (cholera lectin)( produced by Vibrio cholerae. Infect Immun. 1982 Jun;36(3):1199–1208. doi: 10.1128/iai.36.3.1199-1208.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hall R. H., Drasar B. S. Vibrio cholerae HlyA hemolysin is processed by proteolysis. Infect Immun. 1990 Oct;58(10):3375–3379. doi: 10.1128/iai.58.10.3375-3379.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hanne L. F., Finkelstein R. A. Characterization and distribution of the hemagglutinins produced by Vibrio cholerae. Infect Immun. 1982 Apr;36(1):209–214. doi: 10.1128/iai.36.1.209-214.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Honda T., Finkelstein R. A. Purification and characterization of a hemolysin produced by Vibrio cholerae biotype El Tor: another toxic substance produced by cholera vibrios. Infect Immun. 1979 Dec;26(3):1020–1027. doi: 10.1128/iai.26.3.1020-1027.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Häse C. C., Finkelstein R. A. Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain. J Bacteriol. 1991 Jun;173(11):3311–3317. doi: 10.1128/jb.173.11.3311-3317.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ichinose Y., Yamamoto K., Nakasone N., Tanabe M. J., Takeda T., Miwatani T., Iwanaga M. Enterotoxicity of El Tor-like hemolysin of non-O1 Vibrio cholerae. Infect Immun. 1987 May;55(5):1090–1093. doi: 10.1128/iai.55.5.1090-1093.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Iwanaga M., Ichinose Y. An aberrant hemolysin of Vibrio cholerae non-O1. Microbiol Immunol. 1991;35(9):705–715. doi: 10.1111/j.1348-0421.1991.tb01604.x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Naka A., Honda T., Miwatani T. A simple purification method of Vibrio cholerae non-O1 hemagglutinin/protease by immunoaffinity column chromatography using a monoclonal antibody. Microbiol Immunol. 1992;36(4):419–423. doi: 10.1111/j.1348-0421.1992.tb02040.x. [DOI] [PubMed] [Google Scholar]
  15. Neurath H. Evolution of proteolytic enzymes. Science. 1984 Apr 27;224(4647):350–357. doi: 10.1126/science.6369538. [DOI] [PubMed] [Google Scholar]
  16. Tang G. Q., Iida T., Yamamoto K., Honda T. A mutant toxin of Vibrio parahaemolyticus thermostable direct hemolysin which has lost hemolytic activity but retains ability to bind to erythrocytes. Infect Immun. 1994 Aug;62(8):3299–3304. doi: 10.1128/iai.62.8.3299-3304.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Yamamoto K., Al-Omani M., Honda T., Takeda Y., Miwatani T. Non-O1 Vibrio cholerae hemolysin: purification, partial characterization, and immunological relatedness to El Tor hemolysin. Infect Immun. 1984 Jul;45(1):192–196. doi: 10.1128/iai.45.1.192-196.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Yamamoto K., Ichinose Y., Nakasone N., Tanabe M., Nagahama M., Sakurai J., Iwanaga M. Identity of hemolysins produced by Vibrio cholerae non-O1 and V. cholerae O1, biotype El Tor. Infect Immun. 1986 Mar;51(3):927–931. doi: 10.1128/iai.51.3.927-931.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yamamoto K., Ichinose Y., Shinagawa H., Makino K., Nakata A., Iwanaga M., Honda T., Miwatani T. Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products. Infect Immun. 1990 Dec;58(12):4106–4116. doi: 10.1128/iai.58.12.4106-4116.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Young D. B., Broadbent D. A. Biochemical characterization of extracellular proteases from Vibrio cholerae. Infect Immun. 1982 Sep;37(3):875–883. doi: 10.1128/iai.37.3.875-883.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES