1887

Abstract

The ALS (gglutinin-ike equence) gene family encodes eight large cell-surface glycoproteins. The work presented here focuses on Als2p and Als4p, and is part of a larger effort to deduce the function of each Als protein. Both alleles were deleted from the genome and the phenotype of the mutant strain (Δ/Δ; named 2034) studied. Loss of Als4p slowed germ tube formation of cells grown in RPMI 1640 medium and resulted in decreased adhesion of to vascular endothelial cells. Loss of Als4p did not affect adhesion to buccal epithelial cells, biofilm formation in a catheter model, or adhesion to or destruction of oral reconstituted human epithelium (RHE). Although deletion of one allele was achieved readily, a strain lacking the second allele was not identified despite screening thousands of transformants. The remaining allele was placed under control of the promoter to create an Δ/ strain (named 2342). Real-time RT-PCR analysis of strain 2342 grown in glucose-containing medium (non-inducing conditions) showed that although transcript levels were greatly reduced compared to wild-type cells, some transcript remained. The decreased expression levels were sufficient to slow germ tube formation in RPMI 1640 and Lee medium, reduce adhesion to vascular endothelial cells and to RHE, decrease RHE destruction, and impair biofilm formation. Growth of strain 2342 in maltose-containing medium (inducing conditions) restored the wild-type phenotype in all assays. Real-time RT-PCR analysis demonstrated that in maltose-containing medium, strain 2342 overexpressed compared to wild-type cells; however no overexpression phenotype was apparent. Microarray analysis revealed little transcriptional response to deletion, but showed twofold up-regulation of orf19.4765 in the glucose-medium-grown Δ/ strain. orf19.4765 encodes a protein with features of a glycosylated cell wall protein with similarity to Ccw12p, although initial analysis suggested functional differences between the two proteins. Real-time RT-PCR measurement of and transcript copy number showed a 2·8-fold increase in expression in the Δ/Δ strain and a 3·2-fold increase in expression in the Δ/ strain, suggesting the potential for compensatory function between these related proteins.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27763-0
2005-05-01
2019-09-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/5/mic1511619.html?itemId=/content/journal/micro/10.1099/mic.0.27763-0&mimeType=html&fmt=ahah

References

  1. Boeke, J. D., Lacroute, F. & Fink, G. R. ( 1984; ). A positive selection for mutants lacking orotidine 5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197, 345–346.[CrossRef]
    [Google Scholar]
  2. Brown, D. H., Jr, Slobodkin, I. V. & Kumamoto, C. A. ( 1996; ). Stable transformation and regulated expression of an inducible construct in Candida albicans using restriction enzyme-mediated integration. Mol Gen Genet 251, 75–80.
    [Google Scholar]
  3. Calderone, R. A. & Braun, P. C. ( 1991; ). Adherence and receptor relationships of Candida albicans. Microbiol Rev 55, 1–20.
    [Google Scholar]
  4. Chu, W.-S., Magee, B. B. & Magee, P. T. ( 1993; ). Construction of an SfiI macrorestriction map of the Candida albicans genome. J Bacteriol 175, 6637–6651.
    [Google Scholar]
  5. 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 F. M. Ausubel. New York: Wiley.
  6. Dudoit, S., Yang, Y. H., Callow, M. J. & Speed, T. P. ( 2002; ). Statistical methods for identifying genes with differential expression in replicated cDNA microarray experiments. Stat Sin 12, 111–139.
    [Google Scholar]
  7. Fonzi, W. A. & Irwin, M. Y. ( 1993; ). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728.
    [Google Scholar]
  8. Frieman, M. B., McCaffery, J. M. & Cormack, B. P. ( 2002; ). Modular domain structure in the Candida glabrata adhesin Epa1p, a β1,6 glucan-cross-linked cell wall protein. Mol Microbiol 46, 479–492.[CrossRef]
    [Google Scholar]
  9. 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]
  10. 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]
  11. Green, C. B., Cheng, G., Chandra, J., Mukherjee, P., Ghannoum, M. A. & Hoyer, L. L. ( 2004; ). RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms. Microbiology 150, 267–275.[CrossRef]
    [Google Scholar]
  12. Green, C. B., Zhao, X., Yeater, K. M. & Hoyer, L. L. ( 2005; ). Construction and real-time RT-PCR validation of Candida albicans PALS-GFP reporter strains and their use in flow cytometry analysis of ALS gene expression in budding and filamenting cells. Microbiology 151, 1051–1060.[CrossRef]
    [Google Scholar]
  13. Hagen, I., Ecker, M., Lagorce, A. & 8 other authors ( 2004; ). Sed1p and Srl1p are required to compensate for cell wall instability in Saccharomyces cerevisiae mutants defective in multiple GPI-anchored mannoproteins. Mol Microbiol 52, 1413–1425.[CrossRef]
    [Google Scholar]
  14. Heid, C. A., Stevens, J., Livak, K. J. & Williams, P. M. ( 1996; ). Real-time quantitative PCR. Genome Res 6, 986–994.[CrossRef]
    [Google Scholar]
  15. Hoyer, L. L. ( 2001; ). The ALS gene family of Candida albicans. Trends Microbiol 9, 176–180.[CrossRef]
    [Google Scholar]
  16. Hoyer, L. L., Payne, T. L. & Hecht, J. E. ( 1998; ). Identification of Candida albicans ALS2 and ALS4 and localization of Als proteins to the fungal cell surface. J Bacteriol 180, 5334–5343.
    [Google Scholar]
  17. Kapteyn, J. C., Hoyer, L. L., Hecht, J. E., Muller, W. H., Andel, A., Verkleij, A. J., Makarow, M., Van Den Ende, H. & Klis, F. M. ( 2000; ). The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants. Mol Microbiol 35, 601–611.
    [Google Scholar]
  18. 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]
  19. Lee, K. L., Buckley, H. R. & Campbell, C. C. ( 1975; ). An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida albicans. Sabouraudia 13, 148–153.[CrossRef]
    [Google Scholar]
  20. Littel, R. C., Milliken, G. A., Stroup, W. W. & Wolfinger, E. D. ( 1996; ). SAS System for Mixed Models. Cary, NC: SAS Institute.
  21. Livak, K. J. & Schmittgen, T. D. ( 2001; ). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔC T method. Methods 25, 402–408.[CrossRef]
    [Google Scholar]
  22. Loza, L., Fu, Y., Ibrahim, A. S., Sheppard, D. C., Filler, S. G. & Edwards, J. E., Jr ( 2004; ). Functional analysis of the Candida albicans ALS1 gene product. Yeast 21, 473–482.[CrossRef]
    [Google Scholar]
  23. MacKenzie, D. J., McLean, M. A., Mukerji, S. & Green, M. ( 1997; ). Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction. Plant Dis 81, 222–226.[CrossRef]
    [Google Scholar]
  24. Mrsa, V., Ecker, M., Strahl-Bolsinger, S., Nimtz, M., Lehle, L. & Tanner, W. ( 1999; ). Deletion of new covalently linked cell wall glycoproteins alters the electrophoretic mobility of phosphorylated wall components of Saccharomyces cerevisiae. J Bacteriol 181, 3076–3086.
    [Google Scholar]
  25. Odds, F. C. ( 1988; ). Candida and Candidosis, 2nd edn. London: Baillière Tindall.
  26. Oh, S.-H., Cheng, G., Nuessen, J. A., Jajko, R., Yeater, K. M., Zhao, X., Pujol, C., Soll, D. R. & Hoyer, L. L. ( 2005; ). Functional specificity of Candida albicans Als3p proteins and clade specificity of ALS3 alleles discriminated by the number of copies of the tandem repeat sequence in the central domain. Microbiology 151, 673–681.[CrossRef]
    [Google Scholar]
  27. Porta, A., Ramon, A. M. & Fonzi, W. A. ( 1999; ). PRR1, a homolog of Aspergillus nidulans palF, controls pH-dependent gene expression and filamentation in Candida albicans. J Bacteriol 181, 7516–7523.
    [Google Scholar]
  28. Sheppard, D. C., Yeaman, M. R., Welch, W. H. & 8 other authors ( 2004; ). Functional and structural diversity in the Als protein family of Candida albicans. J Biol Chem 279, 30480–30489.[CrossRef]
    [Google Scholar]
  29. Soll, D. R. & Pujol, C. ( 2003; ). Candida albicans clades. FEMS Immunol Med Microbiol 39, 1–7.[CrossRef]
    [Google Scholar]
  30. Wilson, R. B., Davis, D., Enloe, B. M. & Mitchell, A. P. ( 2000; ). A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruption. Yeast 16, 65–70.[CrossRef]
    [Google Scholar]
  31. Wolfinger, R. D., Gibson, G., Wolfinger, E. D., Bennett, L., Hamadeh, H., Bushel, P., Afshari, C. & Paules, R. S. ( 2001; ). Assessing gene significance from cDNA microarray expression data via mixed models. J Comput Biol 8, 625–637.[CrossRef]
    [Google Scholar]
  32. Zhao, X., Oh, S.-H., Cheng, G., Green, C. B., Nuessen, J. A., Yeater, K., Leng, R. P., Brown, A. J. P. & Hoyer, L. L. ( 2004; ). ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin; functional comparison between Als3p and Als1p. Microbiology 150, 2415–2428.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27763-0
Loading
/content/journal/micro/10.1099/mic.0.27763-0
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