Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1992 Feb;60(2):353–359. doi: 10.1128/iai.60.2.353-359.1992

Influence of calcium on secretion and activity of the cytolysins of Actinobacillus pleuropneumoniae.

L A van Leengoed 1, H W Dickerson 1
PMCID: PMC257635  PMID: 1730467

Abstract

In vitro production of a secreted hemolytic cytolysin of Actinobacillus pleuropneumoniae has been reported to be dependent on the presence of calcium in culture media. This is not the case with Escherichia coli hemolysins, however, where calcium has been shown to be required only for activation and binding to target cells. Because the cytolysins of A. pleuropneumoniae have structural and functional similarities to those of hemolytic E. coli, we sought to reexamine the role that calcium plays in the secretion and activity of A. pleuropneumoniae cytolysins. A. pleuropneumoniae hemolytic strain S4074 secreted two major proteins into culture supernatants independent of the presence of calcium in growth medium. These proteins were identified with murine monoclonal antibodies as the 105-kDa cytolysin I and the 103-kDa cytolysin II. It was found that both cytolysins required calcium for binding to erythrocyte membranes. Culture fluids from bacteria grown with calcium lysed porcine erythrocytes even after free calcium in the fluid was removed prior to the hemolytic assay. When bacteria were grown in medium depleted of calcium, no lysis of erythrocytes was detected unless calcium was added to assay buffers. Culture supernatants from A. pleuropneumoniae nonhemolytic strain 1421 grown with or without calcium contained two predominant proteins, which were identified with mouse monoclonal antibodies as the 103-kDa cytolysin II and the 120-kDa cytolysin III. Binding to erythrocytes (without hemolysis) by cytolysin II was dependent on calcium. Cytolysin III did not bind to erythrocytes. These results indicate that the ability of strain S4074 to lyse swine erythrocytes (and the inability of strain 1421 to do so) was directly correlated with the presence of cytolysin I. Cytolysins I, II, and III bound to swine neutrophils and purified neutrophil membranes only in the presence of calcium. When calcium was depleted, cytolysin I of strain S4074 had a reduced binding and cytolysis II and III of strain 1421 did not bind at all. The data suggest that regardless of the target cell involved, calcium plays an integral role in the function but not the production of A. pleuropneumoniae cytolysins.

Full text

PDF
353

Images in this article

356Image
on p.356
356Image
on p.356
357Image
on p.357
357Image
on p.357

Selected References

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

  1. Boehm D. F., Welch R. A., Snyder I. S. Calcium is required for binding of Escherichia coli hemolysin (HlyA) to erythrocyte membranes. Infect Immun. 1990 Jun;58(6):1951–1958. doi: 10.1128/iai.58.6.1951-1958.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Chang Y. F., Young R., Struck D. K. Cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae. DNA. 1989 Nov;8(9):635–647. doi: 10.1089/dna.1.1989.8.635. [DOI] [PubMed] [Google Scholar]
  4. Devenish J., Rosendal S., Johnson R., Hubler S. Immunoserological comparison of 104-kilodalton proteins associated with hemolysis and cytolysis in Actinobacillus pleuropneumoniae, Actinobacillus suis, Pasteurella haemolytica, and Escherichia coli. Infect Immun. 1989 Oct;57(10):3210–3213. doi: 10.1128/iai.57.10.3210-3213.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eberspächer B., Hugo F., Bhakdi S. Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin. Infect Immun. 1989 Mar;57(3):983–988. doi: 10.1128/iai.57.3.983-988.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frey J., Meier R., Gygi D., Nicolet J. Nucleotide sequence of the hemolysin I gene from Actinobacillus pleuropneumoniae. Infect Immun. 1991 Sep;59(9):3026–3032. doi: 10.1128/iai.59.9.3026-3032.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frey J., Nicolet J. Hemolysin patterns of Actinobacillus pleuropneumoniae. J Clin Microbiol. 1990 Feb;28(2):232–236. doi: 10.1128/jcm.28.2.232-236.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frey J., Nicolet J. Regulation of hemolysin expression in Actinobacillus pleuropneumoniae serotype 1 by Ca2+. Infect Immun. 1988 Oct;56(10):2570–2575. doi: 10.1128/iai.56.10.2570-2575.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gangola P., Rosen B. P. Maintenance of intracellular calcium in Escherichia coli. J Biol Chem. 1987 Sep 15;262(26):12570–12574. [PubMed] [Google Scholar]
  10. Jacobson B. S. Imporved method for isolation of plasma membrane on cationic beads. Membranes from Dictyostelium discoideum. Biochim Biophys Acta. 1980 Aug 14;600(3):769–780. doi: 10.1016/0005-2736(80)90479-4. [DOI] [PubMed] [Google Scholar]
  11. Kamp E. M., Popma J. K., Anakotta J., Smits M. A. Identification of hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae by use of monoclonal antibodies. Infect Immun. 1991 Sep;59(9):3079–3085. doi: 10.1128/iai.59.9.3079-3085.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kamp E. M., van Leengoed L. A. Serotype-related differences in production and type of heat-labile hemolysin and heat-labile cytotoxin of Actinobacillus (Haemophilus) pleuropneumoniae. J Clin Microbiol. 1989 Jun;27(6):1187–1191. doi: 10.1128/jcm.27.6.1187-1191.1989. [DOI] [PMC free article] [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. Lian C. J., Rosendal S., MacInnes J. I. Molecular cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae. Infect Immun. 1989 Nov;57(11):3377–3382. doi: 10.1128/iai.57.11.3377-3382.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. SHOPE R. E. PORCINE CONTAGIOUS PLEUROPNEUMONIA. I. EXPERIMENTAL TRANSMISSION, ETIOLOGY, AND PATHOLOGY. J Exp Med. 1964 Mar 1;119:357–368. doi: 10.1084/jem.119.3.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Van Leengoed L. A., Kamp E. M., Pol J. M. Toxicity of Haemophilus pleuropneumoniae to porcine lung macrophages. Vet Microbiol. 1989 Apr;19(4):337–349. doi: 10.1016/0378-1135(89)90099-0. [DOI] [PubMed] [Google Scholar]
  18. van Leengoed L. A., Kamp E. M. Endobronchial inoculation of various doses of Haemophilus (Actinobacillus) pleuropneumoniae in pigs. Am J Vet Res. 1989 Dec;50(12):2054–2059. [PubMed] [Google Scholar]

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

RESOURCES