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. 1989 Aug;57(8):2547–2552. doi: 10.1128/iai.57.8.2547-2552.1989

Pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae, does not require a thiol group for in vitro activity.

F K Saunders 1, T J Mitchell 1, J A Walker 1, P W Andrew 1, G J Boulnois 1
PMCID: PMC313484  PMID: 2744861

Abstract

The role of the single cysteine residue in the activity of the thiol-activated toxin pneumolysin was investigated using oligonucleotide-mediated, site-directed mutagenesis. Three modified toxins in which the cysteine residue was changed to an alanine, a serine, or a glycine residue were purified to homogeneity and examined for activity. The Cys-428----Ala modified toxin was indistinguishable from the wild-type recombinant toxin in terms of hemolytic activity and lytic and inhibitory effects on human polymorphonuclear leukocytes (PMN), indicating that the cysteine residue is not essential for toxin activity. The Cys-428----Ser and Cys-429----Gly modified toxins had reduced activity on erythrocytes and polymorphonuclear leukocytes, being 6 and 20 times less active than the wild type, respectively. However, all the modified toxins formed oligomers in erythrocyte membranes to the same extent as the wild-type recombinant toxin. This suggests that the cysteine residue at position 428 is involved in neither the binding of toxin to membranes nor its insertion into the membrane, and also that the formation of oligomers is not by itself sufficient for toxin activity.

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Selected References

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

  1. Bhakdi S., Tranum-Jensen J. Damage to cell membranes by pore-forming bacterial cytolysins. Prog Allergy. 1988;40:1–43. [PubMed] [Google Scholar]
  2. 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]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  5. Dourmashkin R. R., Rosse W. F. Morphologic changes in the membranes of red blood cells undergoing hemolysis. Am J Med. 1966 Nov;41(5):699–710. doi: 10.1016/0002-9343(66)90031-3. [DOI] [PubMed] [Google Scholar]
  6. Geoffroy C., Alouf J. E. Interaction of alveolysin A sulfhydryl-activated bacterial cytolytic toxin with thiol group reagents and cholesterol. Toxicon. 1982;20(1):239–241. doi: 10.1016/0041-0101(82)90208-2. [DOI] [PubMed] [Google Scholar]
  7. Jackett P. S., Andrew P. W., Aber V. R., Lowrie D. B. Hydrogen peroxide and superoxide release by alveolar macrophages from normal and BCG-vaccinated guinea-pigs after intravenous challenge with Mycobacterium tuberculosis. Br J Exp Pathol. 1981 Aug;62(4):419–428. [PMC free article] [PubMed] [Google Scholar]
  8. Johnson M. K., Geoffroy C., Alouf J. E. Binding of cholesterol by sulfhydryl-activated cytolysins. Infect Immun. 1980 Jan;27(1):97–101. doi: 10.1128/iai.27.1.97-101.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kehoe M. A., Miller L., Walker J. A., Boulnois G. J. Nucleotide sequence of the streptolysin O (SLO) gene: structural homologies between SLO and other membrane-damaging, thiol-activated toxins. Infect Immun. 1987 Dec;55(12):3228–3232. doi: 10.1128/iai.55.12.3228-3232.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Mengaud J., Chenevert J., Geoffroy C., Gaillard J. L., Cossart P. Identification of the structural gene encoding the SH-activated hemolysin of Listeria monocytogenes: listeriolysin O is homologous to streptolysin O and pneumolysin. Infect Immun. 1987 Dec;55(12):3225–3227. doi: 10.1128/iai.55.12.3225-3227.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mitchell T. J., Walker J. A., Saunders F. K., Andrew P. W., Boulnois G. J. Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties. Biochim Biophys Acta. 1989 Jan 23;1007(1):67–72. doi: 10.1016/0167-4781(89)90131-0. [DOI] [PubMed] [Google Scholar]
  15. Nakane P. K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem. 1974 Dec;22(12):1084–1091. doi: 10.1177/22.12.1084. [DOI] [PubMed] [Google Scholar]
  16. Paton J. C., Ferrante A. Inhibition of human polymorphonuclear leukocyte respiratory burst, bactericidal activity, and migration by pneumolysin. Infect Immun. 1983 Sep;41(3):1212–1216. doi: 10.1128/iai.41.3.1212-1216.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Paton J. C., Rowan-Kelly B., Ferrante A. Activation of human complement by the pneumococcal toxin pneumolysin. Infect Immun. 1984 Mar;43(3):1085–1087. doi: 10.1128/iai.43.3.1085-1087.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pinkney M., Beachey E., Kehoe M. The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity. Infect Immun. 1989 Aug;57(8):2553–2558. doi: 10.1128/iai.57.8.2553-2558.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shumway C. N., Klebanoff S. J. Purification of pneumolysin. Infect Immun. 1971 Oct;4(4):388–392. doi: 10.1128/iai.4.4.388-392.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tweten R. K. Nucleotide sequence of the gene for perfringolysin O (theta-toxin) from Clostridium perfringens: significant homology with the genes for streptolysin O and pneumolysin. Infect Immun. 1988 Dec;56(12):3235–3240. doi: 10.1128/iai.56.12.3235-3240.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Walker J. A., Allen R. L., Falmagne P., Johnson M. K., Boulnois G. J. Molecular cloning, characterization, and complete nucleotide sequence of the gene for pneumolysin, the sulfhydryl-activated toxin of Streptococcus pneumoniae. Infect Immun. 1987 May;55(5):1184–1189. doi: 10.1128/iai.55.5.1184-1189.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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