1887

Abstract

To elucidate the implications of secreted aspartyl proteinase (Sap)2p in the pathogenesis of infections, the gene was expressed in and overexpressed in . The coding region of , including its signal sequence and propeptide, was amplified by PCR and cloned downstream of the or promoter. Plasmid expression of in showed that the signal peptide was functional. Integrative transformation of and was accomplished by homologous recombination within the locus for and the locus for . Negative control transformants carried plasmids either without the insert or with mutated and transformants showed similar growth rates to their parental strains or negative controls, when grown in medium containing amino acids. However, in medium with BSA as sole nitrogen source, constitutive expression of enabled to grow and increased the growth rate of . In both media, only transformants harbouring secreted the enzyme, as confirmed by proteinase activity assays and immunoblotting. When C. albicans was grown in amino acids medium, the enzyme was detected exclusively in transformants constitutively expressing . However, in BSA medium these strains secreted enzyme earlier and secreted higher amounts of enzyme and total proteinase activity. In pathogenicity studies in intact mice, expression of Sap2p as a sole putative virulence factor did not cause to become virulent and constitutive overexpression of did not augment virulence of in experimental oral or systemic infection.

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1998-08-01
2021-04-22
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References

  1. Banerjee A., Ganesan K., Datta A. 1991; Induction of secretory acid proteinase in Candida albicans. . J Gen Microbiol 137:2455–2461
    [Google Scholar]
  2. Belhumeur P., Lee A., Tam R., Di Paolo T., Fortin N., Clark M.W. 1993; GSP1 and GSP2, genetic suppressors of the prp20-l mutant in Saccharomyces cerevisiae-. GTP-binding proteins involved in the maintenance of nuclear organization. . Mol Cell Biol 13:2152–2161
    [Google Scholar]
  3. Bertram G., Swoboda R.K., Gooday G.W., Gow N.A.R., Brown A.J.P. 1996; Structure and regulation of the Candida albicans ADH1 gene encoding an immunogenic alcohol dehydrogenase. . Yeast 12:115–127
    [Google Scholar]
  4. Borg M., Rüchel R. 1988; Expression of extracellular acid proteinase by proteolytic Candida spp. during experimental infection of oral mucosa. . Infect Immun 56:626–631
    [Google Scholar]
  5. Cassone A., De Bernardis F., Mondello F., Ceddia T., Agatensi L. 1987; Evidence for a correlation between proteinase secretion and vulvovaginal candidosis. . J Infect Dis 156:777–783
    [Google Scholar]
  6. Chakir J., Côté L., Coulombe C., Deslauriers N. 1994; Differential pattern of infection and immune response during experimental oral candidiasis in BALB/c and DBA/2 (H-2d) mice. . Oral Microbiol Immunol 9:88–94
    [Google Scholar]
  7. Clemons K.V., McCusker J.H., Davis R.W., Stevens D.A. 1994; Comparative pathogenesis of clinical and nonclinical isolates of Saccharomyces cerevisiae. . J Infect Dis 169:859–867
    [Google Scholar]
  8. Colina A.-R., Aumont F., Belhumeur P., de Repentigny L. 1996a; Development of a method to detect secretory mucinolytic activity from Candida albicans. . J Med Yet Mycol 34:401–406
    [Google Scholar]
  9. Colina A.-R., Aumont F., Deslauriers N., Belhumeur P., de Repentigny L. 1996b; Evidence for degradation of gastrointestinal mucin by Candida albicans secretory aspartyl proteinase. . Infect Immun 64:4514–4519
    [Google Scholar]
  10. Cormack B.P., Bertram G., Egerton M., Gow N.A.R., Falkow S., Brown A.J.P. 1997; Yeast enhanced-green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. . Microbiology 143:303–311
    [Google Scholar]
  11. Crandall M., Edwards J.E. JR 1987; Segregation of proteinasenegative mutants from heterozygous Candida albicans. . J Gen Microbiol 133:2817–2824
    [Google Scholar]
  12. Cutler J.E. 1991; Putative virulence factors of Candida albicans. . Annu Rev Microbiol 45:187–218
    [Google Scholar]
  13. Dawson-Saunders B., Trapp R.G. 1990 Basic and Clinical Biostatistics. Norwalk, CT: Appleton & Large;
    [Google Scholar]
  14. De Bernardis F., Cassone A., Sturtevant J., Calderone R. 1995; Expression of Candida albicans SAP1 and SAP2 in experimental vaginitis. . Infect Immun 63:1887–1892
    [Google Scholar]
  15. De Bernardis F., Chiani P., Ciccozzi M., Pellegrini G., Ceddia T., D’Offizzi G., Quinti I., Sullivan P.A., Cassone A. 1996; Elevated aspartyl proteinase secretion and experimental pathogenicity of Candida albicans isolates from oral cavities of subjects infected with human immunodeficiency virus. . Infect Immun 64:466–471
    [Google Scholar]
  16. Fonzi W.A., Irwin M.Y. 1993; Isogenic strain construction and gene mapping in Candida albicans. . Genetics 134:717–728
    [Google Scholar]
  17. Gillum A.M., Tsay W.Y.H., Kirsch D.R. 1984; Isolation of the Candida albicans gene for orotidine-5ʹ-phosphate decarboxylase by complementation of the S. cerevisiae and E. coli pyrF mutations. . Mol Gen Genet 198:179–185
    [Google Scholar]
  18. Harlow E., Lane D. 1988 Antibodies: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  19. Hoffman C.S., Winston F. 1987; A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. . Gene 57:267–272
    [Google Scholar]
  20. Homma M., Kanbe T., Chibana H., Tanaka K. 1992; Detection of intracellular forms of secretory aspartic proteinase in Candida albicans. . J Gen Microbiol 138:627–633
    [Google Scholar]
  21. Hube B. 1996; Candida albicans secreted aspartyl proteinases. . Curr Top Med Mycol 7:55–69
    [Google Scholar]
  22. Hube B., Monod M., Schofield D.A., Brown A.J.P., Gow N.A.R. 1994; Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. . Mol Microbiol 14:87–99
    [Google Scholar]
  23. Hube B., Sanglard D., Odds F.C, Hess D., Monod M., Schäfer W., Brown A.J.P., Gow N.A.R. 1997; Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2 and SAP3 of Candida albicans attenuates virulence. . Infect Immun 65:3529–3538
    [Google Scholar]
  24. Kaplan E.L., Meier P. 1958; Nonparametric estimation from incomplete observations. . J Am Stat Assoc 53:457–481
    [Google Scholar]
  25. Kimberly F., Bausch K., Noonan J., Huguenel E., Tamburini P. 1997; Role of aspartic proteases in disseminated Candida albicans infection in mice. . Infect Immun 65:551–556
    [Google Scholar]
  26. Kwon-Chung K.J., Lehman D., Good C., Magee P.T. 1985; Genetic evidence for role of extracellular proteinase in virulence of Candida albicans. . Infect Immun 49:571–575
    [Google Scholar]
  27. Lacasse M., Fortier G, Trudel L., Collet A.J., Deslauriers N. 1990; Experimental oral candidosis in the mouse: microbiological and histological aspects. . J Oral Pathol Med 19:136–141
    [Google Scholar]
  28. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. . Nature 227:680–685
    [Google Scholar]
  29. Leberer E., Ziegelbauer K., Schmidt A., Harcus D., Dignard D., Ash J., Johnson L., Thomas D.Y. 1997; Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p. . Curr Biol 7:539–546
    [Google Scholar]
  30. Luna G.L. 1992 Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts. Downers Grove, IL: Johnson Printers;
    [Google Scholar]
  31. Lussier M., White A.-M., Sheraton J. 17 other authors 1997; Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. . Genetics 147:435–450
    [Google Scholar]
  32. Macdonald F., Odds F.C. 1983; Virulence for mice of a proteinase-secreting strain of Candida albicans and a proteinase- deficient mutant. . J Gen Microbiol 129:431–438
    [Google Scholar]
  33. Magee B.B., D’Souza T.M., Magee P.T. 1987; Strain and species identification by restriction fragment length poly-morphisms in the ribosomal DNA repeat of Candida species. . J Bacteriol 169:1639–1643
    [Google Scholar]
  34. Milewski S., Mignini F., Covelii I., Borowski E. 1994; Specific inhibition of acid proteinase secretion in Candida albicans by Lys-Nva-FMDP. . J Med Vet Mycol 32:1–11
    [Google Scholar]
  35. Monod M., Togni G., Hube B., Sanglard D. 1994; Multiplicity of genes encoding secreted aspartic proteases in Candida species. . Mol Microbiol 13:357–368
    [Google Scholar]
  36. Odds F.C. 1988 Candida and Candidosis, 2nd edn.. London: Ballière Tindall;
    [Google Scholar]
  37. Ollert M.W., Wende C., Görlich M., McMullan-Vogel C.G., Borg-vonZepelin M., Vogel C.-W., Korting H.C. 1995; Increased expression of Candida albicans secretory proteinase, a putative virulence factor, in isolates from human immunodeficiency virus-positive patients. . J Clin Microbiol 33:2543–2549
    [Google Scholar]
  38. Robinson J.S., Klionski D.J., Banta L.M., Emr S.D. 1988; Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. . Mol Cell Biol 8:4936–4948
    [Google Scholar]
  39. Rodrigue L., Marion D., Trudel L., Barthe C., Lavoie M.C. 1989; Comparison of methods for the evaluation of the oral microbiota of mice. . J Microbiol Methods 10:71–82
    [Google Scholar]
  40. Rose M.D, Winston F., Hieter P. 1990 Methods in Yeast Genetics-, a Laboratory Course Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  41. Ross I.K., De Bernardis F., Emerson G.W., Cassone A., Sullivan P.A. 1990; The secreted aspartate proteinase of Candida albicans: physiology of secretion and virulence of a proteinase-deficient mutant. . J Gen Microbiol 136:687–694
    [Google Scholar]
  42. Rothstein N. 1983; One step gene disruption in yeast. . Methods Enzymol 101:202–211
    [Google Scholar]
  43. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  44. Sanger F., Nicklen S., Coulson A.R. 1977; DNA sequencing with chain-terminating inhibitors. . Proc Natl Acad Sei USA 745463–5467
    [Google Scholar]
  45. Sanglard D., Togni G., de Viragh P.A., Monod M. 1992; Disruption of the gene encoding the secreted acid protease (ACP) in the yeast Candida tropicalis. . FEMS Microbiol Lett 95:149–156
    [Google Scholar]
  46. Sanglard D., Hube B., Monod M., Odds F.C., Gow N.A.R. 1997; A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5 and SAP6 of Candida albicans causes attenuated virulence. . Infect Immun 65:3539–3546
    [Google Scholar]
  47. Schiestl R.H., Manivasakam P., Woods R.A., Gietz R.D. 1993; Introducing DNA into yeast by transformation. . Methods Enzymol 5:79–85
    [Google Scholar]
  48. Sikorski R.S., Hieter P. 1989; A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. . Genetics 122:19–27
    [Google Scholar]
  49. Smolenski G., Sullivan P.A., Cutfield S.M., Cutfield J.F. 1997; Analysis of secreted aspartic proteinases from Candida albicans: purification and characterization of individual Sapl, Sap2 and Sap3 isoenzymes. . Microbiology 143:349–356
    [Google Scholar]
  50. Togni G., Sanglard D., Monod M. 1994; Acid proteinase secreted by Candida tropicalis: virulence in mice of a proteinase negative mutant. . J Med Met Mycol 32:257–265
    [Google Scholar]
  51. Togni G., Sanglard D., Quadroni M., Foundling S.I., Monod M. 1996; Acid proteinase secreted by Candida tropicalis: functional analysis of preproregion cleavages in C.tropicalis and Saccharomyces cerevisiae. . Microbiology 142:493–503
    [Google Scholar]
  52. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. . Proc Natl Acad Sei USA 764350–4354
    [Google Scholar]
  53. Vernet T., Dignard D., Thomas D.Y. 1987; A family of yeast expression vectors containing the phage fl intergenic region. . Gene 52:225–233
    [Google Scholar]
  54. White T., Agabian N. 1995; Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type and levels are determined by environmental factors. . J Bacteriol 177:5215–5221
    [Google Scholar]
  55. White T., Köhler G.A., Miyasaki S.H., Agabian N. 1995; Expression of virulence factors in Candida albicans. . Can J Bot 73: suppl. 1 S1054–S1058
    [Google Scholar]
  56. Wright R.J., Carne A., Hieber A.D., Lamont I.L., Emerson G.W., Sullivan P.A. 1992; A second gene for a secreted aspartate proteinase in Candida albicans. . J Bacteriol 174:7848–7853
    [Google Scholar]
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