Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1992 Nov;60(11):4898–4906. doi: 10.1128/iai.60.11.4898-4906.1992

Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labelling with biotin: comparison with other techniques.

M Casanova 1, J L Lopez-Ribot 1, J P Martinez 1, R Sentandreu 1
PMCID: PMC258246  PMID: 1383159

Abstract

Candida albicans ATCC 26555 blastoconidia and blastoconidia bearing germ tubes were metabolically labelled by incubating the cells with 14C-labelled protein hydrolysate and were subsequently tagged with biotin. Double-labelled (radioactive and biotinylated) cell wall proteins and glycoproteins were extracted from intact cells of both growth forms by treatment with 2-mercaptoethanol (beta ME) and with beta-glucanases (Zymolyase) after treatment with beta ME. The beta ME- and Zymolyase-extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotted (immunoblotted) to nitrocellulose paper. Polyacrylamide gels were stained with Coomassie blue and processed for fluorography. Western blot analysis was performed either with peroxidase conjugated-concanavalin A (ConA) or Extravidin. Blotted proteins were also reacted with polyclonal antibodies and monoclonal antibodies against mannoprotein components from mycelial cell walls of the ATCC 26555 strain. Labelling with biotin allowed identification of a complex array of cell wall protein and glycoprotein components within a very wide molecular mass range (from 650 to 13 kDa). These appeared to be genuine cell wall components. Biotinylated high-molecular-mass glycoproteins that were not stained with Coomassie blue or that appeared as poorly resolved polydisperse bands by indirect ConA-peroxidase staining of Western blots were detected as sharply defined bands following reaction with the Extravidin-peroxidase conjugate. Biotinylated molecules retained unaltered reactivities against ConA, polyclonal antibodies, and monoclonal antibodies.

Full text

PDF
4899

Images in this article

4901Image
on p.4901
4902Image
on p.4902
4902Image
on p.4902
4903Image
on p.4903
4904Image
on p.4904

Selected References

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

  1. Bouchara J. P., Tronchin G., Annaix V., Robert R., Senet J. M. Laminin receptors on Candida albicans germ tubes. Infect Immun. 1990 Jan;58(1):48–54. doi: 10.1128/iai.58.1.48-54.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brawner D. L., Cutler J. E., Beatty W. L. Caveats in the investigation of form-specific molecules of Candida albicans. Infect Immun. 1990 Feb;58(2):378–383. doi: 10.1128/iai.58.2.378-383.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  4. Calderone R. A., Braun P. C. Adherence and receptor relationships of Candida albicans. Microbiol Rev. 1991 Mar;55(1):1–20. doi: 10.1128/mr.55.1.1-20.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Calderone R. A., Linehan L., Wadsworth E., Sandberg A. L. Identification of C3d receptors on Candida albicans. Infect Immun. 1988 Jan;56(1):252–258. doi: 10.1128/iai.56.1.252-258.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casanova M., Chaffin W. L. Cell wall glycoproteins of Candida albicans as released by different methods. J Gen Microbiol. 1991 May;137(5):1045–1051. doi: 10.1099/00221287-137-5-1045. [DOI] [PubMed] [Google Scholar]
  7. Casanova M., Chaffin W. L. Phosphate-containing proteins and glycoproteins of the cell wall of Candida albicans. Infect Immun. 1991 Mar;59(3):808–813. doi: 10.1128/iai.59.3.808-813.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Casanova M., Gil M. L., Cardeñoso L., Martinez J. P., Sentandreu R. Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans. Infect Immun. 1989 Jan;57(1):262–271. doi: 10.1128/iai.57.1.262-271.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Casanova M., Martìnez J. P., Chaffin W. L. Identification of germ tube cell wall antigens of Candida albicans. J Med Vet Mycol. 1991;29(4):269–272. doi: 10.1080/02681219180000391. [DOI] [PubMed] [Google Scholar]
  10. Casanova M., Martínez J. P., Chaffin W. L. Fab fragments from a monoclonal antibody against a germ tube mannoprotein block the yeast-to-mycelium transition in Candida albicans. Infect Immun. 1990 Nov;58(11):3810–3812. doi: 10.1128/iai.58.11.3810-3812.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cassone A. Cell wall of Candida albicans: its functions and its impact on the host. Curr Top Med Mycol. 1989;3:248–314. doi: 10.1007/978-1-4612-3624-5_10. [DOI] [PubMed] [Google Scholar]
  12. Chaffin W. L., Stocco D. M. Cell wall proteins of Candida albicans. Can J Microbiol. 1983 Oct;29(10):1438–1444. doi: 10.1139/m83-220. [DOI] [PubMed] [Google Scholar]
  13. Douglas L. J. Adhesion of Candida species to epithelial surfaces. Crit Rev Microbiol. 1987;15(1):27–43. doi: 10.3109/10408418709104446. [DOI] [PubMed] [Google Scholar]
  14. Elorza M. V., Marcilla A., Sentandreu R. Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties. J Gen Microbiol. 1988 Aug;134(8):2393–2403. doi: 10.1099/00221287-134-8-2393. [DOI] [PubMed] [Google Scholar]
  15. Elorza M. V., Murgui A., Sentandreu R. Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cell walls. J Gen Microbiol. 1985 Sep;131(9):2209–2216. doi: 10.1099/00221287-131-9-2209. [DOI] [PubMed] [Google Scholar]
  16. Elorza V., Mormeneo S., Garcia de la Cruz F., Gimeno C., Sentandreu R. Evidence for the formation of covalent bonds between macromolecules in the domain of the wall of Candida albicans mycelial cells. Biochem Biophys Res Commun. 1989 Aug 15;162(3):1118–1125. doi: 10.1016/0006-291x(89)90789-4. [DOI] [PubMed] [Google Scholar]
  17. Fukayama M., Calderone R. A. Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants. Infect Immun. 1991 Apr;59(4):1341–1345. doi: 10.1128/iai.59.4.1341-1345.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gil M. L., Casanova M., Martínez J. P., Sentandreu R. Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species. J Gen Microbiol. 1991 May;137(5):1053–1061. doi: 10.1099/00221287-137-5-1053. [DOI] [PubMed] [Google Scholar]
  19. Hawkes R. Identification of concanavalin A-binding proteins after sodium dodecyl sulfate--gel electrophoresis and protein blotting. Anal Biochem. 1982 Jun;123(1):143–146. doi: 10.1016/0003-2697(82)90634-0. [DOI] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. 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]
  22. Lee K. L., Buckley H. R., Campbell C. C. An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida Albicans. Sabouraudia. 1975 Jul;13(2):148–153. doi: 10.1080/00362177585190271. [DOI] [PubMed] [Google Scholar]
  23. Linehan L., Wadsworth E., Calderone R. Candida albicans C3d receptor, isolated by using a monoclonal antibody. Infect Immun. 1988 Aug;56(8):1981–1986. doi: 10.1128/iai.56.8.1981-1986.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lopez-Ribot J. L., Casanova M., Martinez J. P., Sentandreu R. Characterization of cell wall proteins of yeast and hydrophobic mycelial cells of Candida albicans. Infect Immun. 1991 Jul;59(7):2324–2332. doi: 10.1128/iai.59.7.2324-2332.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marcilla A., Elorza M. V., Mormeneo S., Rico H., Sentandreu R. Candida albicans mycelial wall structure: supramolecular complexes released by zymolyase, chitinase and beta-mercaptoethanol. Arch Microbiol. 1991;155(4):312–319. doi: 10.1007/BF00243448. [DOI] [PubMed] [Google Scholar]
  26. Martinez J. P., Gil M. L., Casanova M., Lopez-Ribot J. L., Garcia De Lomas J., Sentandreu R. Wall mannoproteins in cells from colonial phenotypic variants of Candida albicans. J Gen Microbiol. 1990 Dec;136(12):2421–2432. doi: 10.1099/00221287-136-12-2421. [DOI] [PubMed] [Google Scholar]
  27. Martinez J. P., Lopez-Ribot J. L., Gil M. L., Sentandreu R., Ruiz-Herrera J. Inhibition of the dimorphic transition of Candida albicans by the ornithine decarboxylase inhibitor 1,4-diaminobutanone: alterations in the glycoprotein composition of the cell wall. J Gen Microbiol. 1990 Oct;136(10):1937–1943. doi: 10.1099/00221287-136-10-1937. [DOI] [PubMed] [Google Scholar]
  28. Millette C. F., Scott B. K. Identification of spermatogenic cell plasma membrane glycoproteins by two-dimensional electrophoresis and lectin blotting. J Cell Sci. 1984 Jan;65:233–248. doi: 10.1242/jcs.65.1.233. [DOI] [PubMed] [Google Scholar]
  29. Ponton J., Jones J. M. Identification of two germ-tube-specific cell wall antigens of Candida albicans. Infect Immun. 1986 Dec;54(3):864–868. doi: 10.1128/iai.54.3.864-868.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pytela R., Pierschbacher M. D., Argraves S., Suzuki S., Ruoslahti E. Arginine-glycine-aspartic acid adhesion receptors. Methods Enzymol. 1987;144:475–489. doi: 10.1016/0076-6879(87)44196-7. [DOI] [PubMed] [Google Scholar]
  31. Saxena A., Calderone R. Purification and characterization of the extracellular C3d-binding protein of Candida albicans. Infect Immun. 1990 Feb;58(2):309–314. doi: 10.1128/iai.58.2.309-314.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shepherd M. G., Poulter R. T., Sullivan P. A. Candida albicans: biology, genetics, and pathogenicity. Annu Rev Microbiol. 1985;39:579–614. doi: 10.1146/annurev.mi.39.100185.003051. [DOI] [PubMed] [Google Scholar]
  33. Sundstrom P. M., Kenny G. E. Enzymatic release of germ tube-specific antigens from cell walls of Candida albicans. Infect Immun. 1985 Sep;49(3):609–614. doi: 10.1128/iai.49.3.609-614.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Tronchin G., Bouchara J. P., Robert R., Senet J. M. Adherence of Candida albicans germ tubes to plastic: ultrastructural and molecular studies of fibrillar adhesins. Infect Immun. 1988 Aug;56(8):1987–1993. doi: 10.1128/iai.56.8.1987-1993.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilchek M., Bayer E. A. The avidin-biotin complex in bioanalytical applications. Anal Biochem. 1988 May 15;171(1):1–32. doi: 10.1016/0003-2697(88)90120-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES