Gene Overexpression/Suppression Analysis of Candidate Virulence Factors of Candida albicans

Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502; Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90024

^* To whom correspondence should be addressed. Email: yfu{at}labiomed.org.

	Abstract

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 (TR) promoter in front of the gene of interest. To validate the strategy, a library of genes encoding glycosyl phosphatidylinositol (GPI)-anchored surface proteins was screened for virulence phenotypes in vitro. During screening, overexpression of IFF4 was found to increase adherence of C. albicans to plastic and human epithelial cells, but not endothelial cells. Consistent with the in vitro results, IFF4 overexpression modestly increased tissue fungal burden during murine vaginal candidiasis. In additional 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 twelve other GPI-proteins did not affect normal GPI-protein cell surface accumulation, demonstrating that the overexpression strategy did not affect capacity utilization of 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.