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. 1998 Feb 1;329(Pt 3):571–577. doi: 10.1042/bj3290571

A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers.

Y E Korchev 1, C L Bashford 1, C Pederzolli 1, C A Pasternak 1, P J Morgan 1, P W Andrew 1, T J Mitchell 1
PMCID: PMC1219078  PMID: 9445384

Abstract

Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined, Trp-433-->Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433-->Phe was least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1 nS) conductance steps. Small- and medium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433-->Phe formed large channels that were insensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433-->Phe. Electron microscopy revealed that Trp-433-->Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433-->Phe variant. We conclude that the change Trp-433-->Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels.

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Selected References

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

  1. Bashford C. L., Alder G. M., Graham J. M., Menestrina G., Pasternak C. A. Ion modulation of membrane permeability: effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents. J Membr Biol. 1988 Jul;103(1):79–94. doi: 10.1007/BF01871934. [DOI] [PubMed] [Google Scholar]
  2. Bashford C. L., Alder G. M., Menestrina G., Micklem K. J., Murphy J. J., Pasternak C. A. Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations. J Biol Chem. 1986 Jul 15;261(20):9300–9308. [PubMed] [Google Scholar]
  3. Bashford C. L., Alder G., Micklem K. J., Pasternak C. A. A novel method for measuring intracellular pH and potassium concentration. Biosci Rep. 1983 Jul;3(7):631–642. doi: 10.1007/BF01172873. [DOI] [PubMed] [Google Scholar]
  4. Bashford C. L., Menestrina G., Henkart P. A., Pasternak C. A. Cell damage by cytolysin. Spontaneous recovery and reversible inhibition by divalent cations. J Immunol. 1988 Dec 1;141(11):3965–3974. [PubMed] [Google Scholar]
  5. Bashford C. L., Pasternak C. A. Plasma membrane potential of Lettré cells does not depend on cation gradients but on pumps. J Membr Biol. 1984;79(3):275–284. doi: 10.1007/BF01871066. [DOI] [PubMed] [Google Scholar]
  6. Bashford C. L., Pasternak C. A. Plasma membrane potential of neutrophils generated by the Na+ pump. Biochim Biophys Acta. 1985 Jul 11;817(1):174–180. doi: 10.1016/0005-2736(85)90080-x. [DOI] [PubMed] [Google Scholar]
  7. Bhakdi S., Muhly M., Füssle R. Correlation between toxin binding and hemolytic activity in membrane damage by staphylococcal alpha-toxin. Infect Immun. 1984 Nov;46(2):318–323. doi: 10.1128/iai.46.2.318-323.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Blumenthal R., Habig W. H. Mechanism of tetanolysin-induced membrane damage: studies with black lipid membranes. J Bacteriol. 1984 Jan;157(1):321–323. doi: 10.1128/jb.157.1.321-323.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Boulnois G. J., Paton J. C., Mitchell T. J., Andrew P. W. Structure and function of pneumolysin, the multifunctional, thiol-activated toxin of Streptococcus pneumoniae. Mol Microbiol. 1991 Nov;5(11):2611–2616. doi: 10.1111/j.1365-2958.1991.tb01969.x. [DOI] [PubMed] [Google Scholar]
  10. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  11. Forda S. R., Gillies G., Kelly J. S., Micklem K. J., Pasternak C. A. Acute membrane responses to viral action. Neurosci Lett. 1982 Apr 26;29(3):237–242. doi: 10.1016/0304-3940(82)90323-8. [DOI] [PubMed] [Google Scholar]
  12. GREEN H., BARROW P., GOLDBERG B. Effect of antibody and complement on permeability control in ascites tumor cells and erythrocytes. J Exp Med. 1959 Nov 1;110:699–713. doi: 10.1084/jem.110.5.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Impraim C. C., Foster K. A., Micklem K. J., Pasternak C. A. Nature of virally mediated changes in membrane permeability to small molecules. Biochem J. 1980 Mar 15;186(3):847–860. doi: 10.1042/bj1860847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Korchev Y. E., Alder G. M., Bakhramov A., Bashford C. L., Joomun B. S., Sviderskaya E. V., Usherwood P. N., Pasternak C. A. Staphylococcus aureus alpha-toxin-induced pores: channel-like behavior in lipid bilayers and patch clamped cells. J Membr Biol. 1995 Jan;143(2):143–151. doi: 10.1007/BF00234660. [DOI] [PubMed] [Google Scholar]
  16. Korchev Y. E., Bashford C. L., Pasternak C. A. Differential sensitivity of pneumolysin-induced channels to gating by divalent cations. J Membr Biol. 1992 May;127(3):195–203. doi: 10.1007/BF00231507. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Menestrina G., Bashford C. L., Pasternak C. A. Pore-forming toxins: experiments with S. aureus alpha-toxin, C. perfringens theta-toxin and E. coli haemolysin in lipid bilayers, liposomes and intact cells. Toxicon. 1990;28(5):477–491. doi: 10.1016/0041-0101(90)90292-f. [DOI] [PubMed] [Google Scholar]
  20. Michel E., Reich K. A., Favier R., Berche P., Cossart P. Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O. Mol Microbiol. 1990 Dec;4(12):2167–2178. doi: 10.1111/j.1365-2958.1990.tb00578.x. [DOI] [PubMed] [Google Scholar]
  21. Micklem K. J., Nyaruwe A., Pasternak C. A. Permeability changes resulting from virus-cell fusion: temperature-dependence of the contributing processes. Mol Cell Biochem. 1985 Mar;66(2):163–173. doi: 10.1007/BF00220784. [DOI] [PubMed] [Google Scholar]
  22. Mitchell T. J., Andrew P. W., Saunders F. K., Smith A. N., Boulnois G. J. Complement activation and antibody binding by pneumolysin via a region of the toxin homologous to a human acute-phase protein. Mol Microbiol. 1991 Aug;5(8):1883–1888. doi: 10.1111/j.1365-2958.1991.tb00812.x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Morgan P. J., Hyman S. C., Byron O., Andrew P. W., Mitchell T. J., Rowe A. J. Modeling the bacterial protein toxin, pneumolysin, in its monomeric and oligomeric form. J Biol Chem. 1994 Oct 14;269(41):25315–25320. [PubMed] [Google Scholar]
  25. Morgan P. J., Hyman S. C., Rowe A. J., Mitchell T. J., Andrew P. W., Saibil H. R. Subunit organisation and symmetry of pore-forming, oligomeric pneumolysin. FEBS Lett. 1995 Aug 28;371(1):77–80. doi: 10.1016/0014-5793(95)00887-f. [DOI] [PubMed] [Google Scholar]
  26. Paton J. C., Andrew P. W., Boulnois G. J., Mitchell T. J. Molecular analysis of the pathogenicity of Streptococcus pneumoniae: the role of pneumococcal proteins. Annu Rev Microbiol. 1993;47:89–115. doi: 10.1146/annurev.mi.47.100193.000513. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. SEARS D. A., WEED R. I., SWISHER S. N. DIFFERENCES IN THE MECHANISM OF IN VITRO IMMUNE HEMOLYSIS RELATED TO ANTIBODY SPECIFICITY. J Clin Invest. 1964 May;43:975–985. doi: 10.1172/JCI104983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Saunders F. K., Mitchell T. J., Walker J. A., Andrew P. W., Boulnois G. J. Pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae, does not require a thiol group for in vitro activity. Infect Immun. 1989 Aug;57(8):2547–2552. doi: 10.1128/iai.57.8.2547-2552.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thelestam M., Möllby R. Interaction of streptolysin O from Streptococcus pyogenes and theta-toxin from Clostridium perfringens with human fibroblasts. Infect Immun. 1980 Sep;29(3):863–872. doi: 10.1128/iai.29.3.863-872.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Vodyanoy I., Bezrukov S. M., Parsegian V. A. Probing alamethicin channels with water-soluble polymers. Size-modulated osmotic action. Biophys J. 1993 Nov;65(5):2097–2105. doi: 10.1016/S0006-3495(93)81245-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zimmerberg J., Parsegian V. A. Polymer inaccessible volume changes during opening and closing of a voltage-dependent ionic channel. Nature. 1986 Sep 4;323(6083):36–39. doi: 10.1038/323036a0. [DOI] [PubMed] [Google Scholar]
  35. de Kruijff B. Cholesterol as a target for toxins. Biosci Rep. 1990 Apr;10(2):127–130. doi: 10.1007/BF01116571. [DOI] [PubMed] [Google Scholar]

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