Eukaryotic Cell
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Eukaryotic Cell, March 2008, p. 483-492, Vol. 7, No. 3
1535-9778/08/$08.00+0     doi:10.1128/EC.00445-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Gene Overexpression/Suppression Analysis of Candidate Virulence Factors of Candida albicans{triangledown}

Yue Fu,1,2* Guanpingsheng Luo,1 Brad J. Spellberg,1,2 John E. Edwards Jr.,1,2 and Ashraf S. Ibrahim1,2

Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California 90502,1 Department of Medicine, David Geffen School of Medicine at University of California—Los Angeles, Los Angeles, California 900242

Received 7 December 2007/ Accepted 18 December 2007

We developed a conditional overexpression/suppression genetic strategy in Candida albicans to enable simultaneous testing of gain or loss of function in order to identify new virulence factors. The strategy involved insertion of a strong, tetracycline-regulated promoter in front of the gene of interest. To validate the strategy, a library of genes encoding glycosylphosphatidylinositol (GPI)-anchored surface proteins was screened for virulence phenotypes in vitro. During the screening, overexpression of IFF4 was found to increase the adherence of C. albicans to plastic and to human epithelial cells, but not endothelial cells. Consistent with the in vitro results, IFF4 overexpression modestly increased the tissue fungal burden during murine vaginal candidiasis. In addition to the in vitro screening tests, IFF4 overexpression was found to increase C. albicans susceptibility to neutrophil-mediated killing. Furthermore, IFF4 overexpression decreased the severity of hematogenously disseminated candidiasis in normal mice, but not in neutropenic mice, again consistent with the in vitro phenotype. Overexpression of 12 other GPI proteins did not affect normal GPI protein cell surface accumulation, demonstrating that the overexpression strategy did not affect the cell capacity for making such proteins. These data indicate that the same gene can increase or decrease candidal virulence in distinct models of infection, emphasizing the importance of studying virulence genes in different anatomical contexts. Finally, these data validate the use of a conditional overexpression/suppression genetic strategy to identify candidal virulence factors.

* Corresponding author. Mailing address: Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, 1124 W. Carson St., Torrance, CA 90502. Phone: (310) 222-6425. Fax: (310) 782-2016. E-mail: yfu{at}labiomed.org

{triangledown} Published ahead of print on 4 January 2008.

Eukaryotic Cell, March 2008, p. 483-492, Vol. 7, No. 3
1535-9778/08/$08.00+0     doi:10.1128/EC.00445-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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