1887

Abstract

An RT-PCR assay was developed to analyse expression patterns of genes in the ALS (gglutinin-ike equence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from -inoculated buccal RHE showed that , , , , and were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from , and particularly from , was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between and its host.

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2004-02-01
2024-03-29
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References

  1. Bernhardt J., Herman D., Sheridan M., Calderone R. 2001; Adherence and invasion studies of Candida albicans strains, using in vitro models of esophageal candidiasis. J Infect Dis 184:1170–1175 [CrossRef]
    [Google Scholar]
  2. Chandra J., Mukherjee P. K., Leidich S. D., Faddoul F. F., Hoyer L. L., Douglas L. J., Ghannoum M. A. 2001; Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res 80:903–908 [CrossRef]
    [Google Scholar]
  3. Collart M. A., Oliviero S. 1993; Preparation of yeast RNA. In Current Protocols in Molecular Biology vol. 2 pp 13.12.1–13.12.5 Edited by Ausubel F. M. New York: Wiley;
    [Google Scholar]
  4. De Bernardis F., Sullivan P. A., Cassone A. 2001; Aspartyl proteinases of Candida albicans and their role in pathogenicity. Med Mycol 39:303–313 [CrossRef]
    [Google Scholar]
  5. Fu Y., Ibrahim A. S., Sheppard D. C., Chen Y. C., French S. W., Cutler J. E., Filler S. G., Edwards J. E. Jr 2002; Candida albicans Als1p: an adhesin that is a downstream effector of the EFG1 filamentation pathway. Mol Microbiol 44:61–72 [CrossRef]
    [Google Scholar]
  6. Gaur N. K., Klotz S. A. 1997; Expression, cloning, and characterization of a Candida albicans gene, ALA1 , that confers adherence properties upon Saccharomyces cerevisiae for extracellular matrix proteins. Infect Immun 65:5289–5294
    [Google Scholar]
  7. Gillum A. M., Tsay E. Y. H., Kirsch D. R. 1984; Isolation of the Candida albicans genes for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198:179–182 [CrossRef]
    [Google Scholar]
  8. Hoyer L. L. 2001; The ALS gene family of Candida albicans . Trends Microbiol 9:176–180 [CrossRef]
    [Google Scholar]
  9. Hoyer L. L., Hecht J. E. 2001; The ALS5 gene of Candida albicans and analysis of the Als5p N-terminal domain. Yeast 18:49–60 [CrossRef]
    [Google Scholar]
  10. Hoyer L. L., Scherer S., Shatzman A. R., Livi G. P. 1995; Candida albicans ALS1 : domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif. Mol Microbiol 15:39–54 [CrossRef]
    [Google Scholar]
  11. Hoyer L. L., Payne T. L., Bell M., Myers A. M., Scherer S. 1998a; Candida albicans ALS3 and insights into the nature of the ALS gene family. Curr Genet 33:451–459 [CrossRef]
    [Google Scholar]
  12. Hoyer L. L., Payne T. L., Hecht J. E. 1998b; Identification of Candida albicans ALS2 and ALS4 and localization of Als proteins to the fungal cell surface. J Bacteriol 180:5334–5343
    [Google Scholar]
  13. Hube B., Naglik J. 2001; Candida albicans proteinases: resolving the mystery of a gene family. Microbiology 147:1997–2005
    [Google Scholar]
  14. Hube B., Stehr F., Bossenz M., Mazur A., Kretschmar M., Schafer W. 2000; Secreted lipases of Candida albicans : cloning, characterisation and expression analysis of a new gene family with at least ten members. Arch Microbiol 174:362–374 [CrossRef]
    [Google Scholar]
  15. Kuhn D. M., Chandra J., Mukherjee P. K., Ghannoum M. A. 2002; Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect Immun 70:878–888 [CrossRef]
    [Google Scholar]
  16. Monod M., Borg-von Zepelin M. 2002; Secreted aspartic proteases as virulence factors of Candida species. Biol Chem 383:1087–1093
    [Google Scholar]
  17. Naglik J. R., Newport G., White T. C., Fernandes-Naglik L. L., Greenspan J. S., Greenspan D., Sweet S., Challacombe S. J., Agabian N. 1999; In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis. Infect Immun 67:2482–2490
    [Google Scholar]
  18. Naglik J. R., Rodgers C. A., Shirlaw P. J., Dobbie J. L., Fernandes-Naglik L. L., Greenspan D., Agabian N., Challacombe S. J. 2003; Differential expression of Candida albicans secreted aspartyl proteinase and phospholipase B genes in humans correlates with active oral and vaginal infections. J Infect Dis 188:469–479 [CrossRef]
    [Google Scholar]
  19. Odds F. C. 1988 Candida and Candidosis. A Review and Bibliography , 2nd edn. London: Baillière Tindall;
    [Google Scholar]
  20. Ripeau J.-S., Fiorillo M., Aumont F., Belhumeur P, de Repentigny L. 2002; Evidence for differential expression of Candida albicans virulence genes during oral infection in intact and human immunodeficiency virus type 1-transgenic mice. J Infect Dis 185:1094–1102 [CrossRef]
    [Google Scholar]
  21. Schaller M., Schafer W., Korting H. C., Hube B. 1998; Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity. Mol Microbiol 29:605–615 [CrossRef]
    [Google Scholar]
  22. Schaller M., Korting H. C., Schafer W., Bastert J., Chen W.-C., Hube B. 1999; Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis. Mol Microbiol 34:169–180 [CrossRef]
    [Google Scholar]
  23. Schaller M., Bein M., Korting H. C., Baur S., Hamm G., Monod M., Beinhauer S., Hube B. 2003; The secreted aspartyl proteinases Sap1 and Sap2 cause tissue damage in an in vitro model of vaginal candidiasis based on reconstituted human vaginal epithelium. Infect Immun 71:3227–3234 [CrossRef]
    [Google Scholar]
  24. Schofield D. A., Westwater C., Warner T., Nicholas P. J., Paulling E. E., Balish E. 2003; Hydrolytic gene expression during oroesophageal and gastric candidiasis in immunocompetent and immunodeficient gnotobiotic mice. J Infect Dis 188:591–599 [CrossRef]
    [Google Scholar]
  25. Zhang N., Harrex A. L., Holland B. R., Fenton L. E., Cannon R., Schmid J. 2003; Sixty alleles of the ALS7 open reading frame in Candida albicans : ALS7 is a hypermutable contingency locus. Genome Res 13:2005–2017 [CrossRef]
    [Google Scholar]
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