1887

Microbiology Society logo

Volume 148, Issue 12

The variant undecapeptide sequence of the haemolysin, pyolysin, is required for full cytolytic activity Free

Abstract

The cholesterol-dependent cytolysins (CDCs) are characterized by an undecapeptide sequence (ECTGLAWEWWR) that is located near the C terminus and within domain 4 of these proteins. Pyolysin (PLO), the CDC of , has a variant undecapeptide sequence (ETGLAWWW). Site-directed mutants were constructed in undecapeptide residues in a recombinant PLO molecule containing a hexahistidine tag (His-PLO). Mutations in each of the three undecapeptide tryptophan residues resulted in low haemolytic activity, confirming the importance of these residues in the protein. Deletion of a proline residue (P), inserted in PLO, or substitution of this residue with either phenylalanine or glycine resulted in mutant proteins with undetectable or low haemolytic activities, indicating that P is essential for His-PLO haemolytic activity. Substitution of the PLO undecapeptide sequence with a consensus undecapeptide resulted in a His-PLO protein with only 01% activity, confirming that the variant PLO undecapeptide is required for the full cytolytic activity of this toxin. The presence of the conserved undecapeptide cysteine residue either alone (His-PLO.C) or in a consensus sequence resulted in His-PLO molecules which were activated in the presence of reducing compounds, confirming the importance of this residue in the thiol-activated nature of many CDC toxins. The ability of His-PLO mutant proteins to bind cholesterol mimicked haemolytic activity, with the exception of His-PLO.C, which, despite having reduced haemolytic activity, showed an increased ability to bind cholesterol compared to His-PLO. Despite reductions in haemolytic activity and cholesterol-binding, all mutant proteins were still able to bind to erythrocyte membranes, suggesting that other regions of PLO may recognize host-cell membranes, through receptors other than cholesterol.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): amino acid substitution , BME, β-mercaptoethanol , CDC, cholesterol-dependent cytolysin , cholesterol-dependent cytolysin , ILY, intermedilysin , LLO, listeriolysin O , PFO, perfringolysin O , PLO, pyolysin , PLY, pneumolysin , SRBC, sheep red blood cell and virulence factor
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-12-3947
2002-12-01
2024-12-09
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/12/1483947a.html?itemId=/content/journal/micro/10.1099/00221287-148-12-3947&mimeType=html&fmt=ahah

References

  1. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1994 Current Protocols in Molecular Biology New York: Greene Publishing Associates and Wiley;
     [Google Scholar]
  2. Baumann C. D., Davidson W. R., Roscoe D. E., Beheler-Amass K. 2001; Intracranial abscessation in white-tailed deer of North America. J Wildl Dis 37:661–670 [CrossRef]
     [Google Scholar]
  3. Billington S. J., Jost B. H., Cuevas W. A., Bright K. R., Songer J. G. 1997; The Arcanobacterium ( Actinomyces ) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family. J Bacteriol 179:6100–6106
     [Google Scholar]
  4. Billington S. J., Jost B. H., Songer J. G. 2000; Thiol-activated cytolysins: structure, function and role in pathogenesis. FEMS Microbiol Lett 182:197–205 [CrossRef]
     [Google Scholar]
  5. Boulnois G. J., Paton J. C., Mitchell T. J., Andrew P. W. 1991; Structure and function of pneumolysin, the multifunctional, thiol-activated toxin of Streptococcus pneumoniae . Mol Microbiol 5:2611–2616 [CrossRef]
     [Google Scholar]
  6. Carter G. R., Chengappa M. M. 1991 Essentials of Veterinary Bacteriology and Mycology, 4th edn. Philadelphia, PA: Lea & Febiger;
     [Google Scholar]
  7. de los Toyos J. R., Mendez F. J., Aparicio J. F. 7 other authors 1996; Functional analysis of pneumolysin by use of monoclonal antibodies. Infect Immun 64:480–484
     [Google Scholar]
  8. Ding H., Lämmler C. 1996; Purification and further characterization of a haemolysin of Actinomyces pyogenes . Zentralbl Veterinarmed (B) 43:179–188
     [Google Scholar]
  9. Dubail I., Autret N., Beretti J.-L., Kayal S., Berche P., Charbit A. 2001; Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes . Microbiology 147:2679–2688
     [Google Scholar]
  10. Funk P. G., Staats J. J., Howe M., Nagaraja T. G., Chengappa M. M. 1996; Identification and partial characterization of an Actinomyces pyogenes hemolysin. Vet Microbiol 50:129–142 [CrossRef]
     [Google Scholar]
  11. Gilbert R. J. C., Jimenez J. L., Chen S., Tickle I. J., Rossjohn J., Parker M., Andrew P. W., Saibil H. R. 1999; Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae . Cell 97:647–655 [CrossRef]
     [Google Scholar]
  12. Heuck A. P., Hotze E. M., Tweten R. K., Johnson A. E. 2000; Mechanism of membrane insertion of a multimeric β-barrel protein: perfringolysin O creates a pore using ordered and coupled conformational changes. Mol Cell 6:1233–1242 [CrossRef]
     [Google Scholar]
  13. Hotze E. M., Heuck A. P., Czajkowsky D. M., Shao Z., Johnson A. E., Tweten R. K. 2002; Monomer–monomer interactions drive the prepore to pore conversion of a β-barrel-forming cholesterol-dependent cytolysin. J Biol Chem 277:11597–11605 [CrossRef]
     [Google Scholar]
  14. Jacobs T., Cima-Cabal M. D., Darji A. 7 other authors 1999; The conserved undecapeptide shared by thiol-activated cytolysins is involved in membrane binding. FEBS Lett 459:463–466 [CrossRef]
     [Google Scholar]
  15. Jonsson P., Olsson S.-O., Olofson A.-S., Fälth C., Holmberg O., Funke H. 1991; Bacteriological investigations of clinical mastitis in heifers in Sweden. J Dairy Res 58:179–185 [CrossRef]
     [Google Scholar]
  16. Jost B. H., Songer J. G., Billington S. J. 1999; An Arcanobacterium ( Actinomyces ) pyogenes mutant deficient in production of the pore-forming cytolysin pyolysin has reduced virulence. Infect Immun 67:1723–1728
     [Google Scholar]
  17. Jost B. H., Post K. W., Songer J. G., Billington S. J. 2002; Isolation of Arcanobacterium pyogenes from the porcine gastric mucosa. Vet Res Commun 26:419–425 [CrossRef]
     [Google Scholar]
  18. Korchev Y. E., Bashford C. L., Pederzolli C., Pasternak C. A., Morgan P. J., Andrew P. W., Mitchell T. J. 1998; A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers. Biochem J 329:571–577
     [Google Scholar]
  19. Lechtenberg K. F., Nagaraja T. G., Leipold H. W., Chengappa M. M. 1988; Bacteriologic and histologic studies of hepatic abscesses in cattle. Am J Vet Res 49:58–62
     [Google Scholar]
  20. Michel E., Reich K. A., Favier R., Berche P., Cossart P. 1990; Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitution in listeriolysin O. Mol Microbiol 4:2167–2178 [CrossRef]
     [Google Scholar]
  21. Nagamune H. 1997; Streptococcal cytolysins. Seikagaku 69:343–348
     [Google Scholar]
  22. Nagamune H., Ohnishi C., Katsuura A., Fushitani K., Whiley R. A., Tsuji A., Matsuda Y. 1996; Intermedilysin, a novel cytotoxin specific for human cells, secreted by Streptococcus intermedius UNS46 isolated from a human liver abscess. Infect Immun 64:3093–3100
     [Google Scholar]
  23. Nagaraja T. G., Laudert S. B., Parrott J. C. 1996; Liver abscesses in feedlot cattle. Part I. Causes, pathogenesis, pathology, and diagnosis. Comp Cont Edu Pract Vet 18:S230–S241 S256
     [Google Scholar]
  24. Nakamura M., Sekino-Suzuki N., Mitsui K.-I., Ohno-Iwashita Y. 1998; Contribution of tryptophan residues to the structural changes in perfringolysin O during interaction with liposomal membranes. J Biochem 123:1145–1155 [CrossRef]
     [Google Scholar]
  25. Narayanan S., Nagaraja T. G., Wallace N., Staats J., Chengappa M. M., Oberst R. D. 1998; Biochemical and ribotypic comparison of Actinomyces pyogenes and A. pyogenes -like organisms from liver abscesses, ruminal wall, and ruminal contents of cattle. Am J Vet Res 59:271–276
     [Google Scholar]
  26. Owen R. H. G., Boulnois G. J., Andrew P. W., Mitchell T. J. 1994; A role in the cell-binding for the C-terminus of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae . FEMS Microbiol Lett 121:217–221 [CrossRef]
     [Google Scholar]
  27. Pinkney M., Beachey E., Kehoe M. 1989; The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity. Infect Immun 57:2553–2558
     [Google Scholar]
  28. Rossjohn J., Feil S. C., McKinstry W. J., Tweten R. K., Parker M. W. 1997; Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form. Cell 89:685–692 [CrossRef]
     [Google Scholar]
  29. Rossjohn J., Gilbert R. J. C., Crane D. 7 other authors 1998; The molecular mechanism of pneumolysin, a virulence factor from Streptococcus pneumoniae . J Mol Biol 284:449–461 [CrossRef]
     [Google Scholar]
  30. Saunders F. K., Mitchell T. J., Walker J. A., Andrew P. W., Boulnois G. J. 1989; Pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae , does not require a thiol group for in vitro activity. Infect Immun 57:2547–2552
     [Google Scholar]
  31. Sekino-Suzuki N., Nakamura M., Mitsui K.-I., Ohno-Iwashita Y. 1996; Contribution of individual tryptophan residues to the structure and activity of θ-toxin (perfringolysin O), a cholesterol-binding cytolysin. Eur J Biochem 241:941–947 [CrossRef]
     [Google Scholar]
  32. Shatursky O., Heuck A. P., Shepard L. A., Rossjohn J., Parker M. W., Johnson A. E., Tweten R. K. 1999; The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins. Cell 99:293–299 [CrossRef]
     [Google Scholar]
  33. Shepard L. A., Heuck A. P., Hamman B. D., Rossjohn J., Parker M. W., Ryan K. R., Johnson A. E., Tweten R. K. 1998; Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an α-helical to β-sheet transition identified by fluorescence spectroscopy. Biochemistry 37:14563–14574 [CrossRef]
     [Google Scholar]
  34. Shepard L. A., Shatursky O., Johnson A. E., Tweten R. K. 2000; The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane β-hairpins. Biochemistry 39:10284–10293 [CrossRef]
     [Google Scholar]
  35. Shimada Y., Nakamura M., Naito Y., Nomura K., Ohno-Iwashita Y. 1999; C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin. J Biol Chem 274:18536–18542 [CrossRef]
     [Google Scholar]
  36. Tweten R. K., Parker M. W., Johnson A. E. 2001; The cholesterol-dependent cytolysins. Curr Top Microbiol Immunol 257:15–33
     [Google Scholar]
/content/journal/micro/10.1099/00221287-148-12-3947
Loading
The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity
Microbiology 148, 3947 (2002); https://doi.org/10.1099/00221287-148-12-3947
/content/journal/micro/10.1099/00221287-148-12-3947
/content/journal/micro/10.1099/00221287-148-12-3947
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error