1887

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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-12-3947
2002-12-01
2019-12-08
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.
  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.
  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]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-12-3947
Loading
/content/journal/micro/10.1099/00221287-148-12-3947
Loading

Data & Media loading...

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