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

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 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.Candida albicans causes hematogenously disseminated and mucosal infections (7, 33). Despite current antifungal therapy, mortality and morbidity are unacceptably high (1,16,34). Therefore, new prophylactic and therapeutic strategies are urgently needed. The identification of virulence genes that can be targeted to alter pathogenicity is a necessary step in devising novel strategies to treat or prevent Candida infections.Virulence factors in C. albicans include proteins that mediate adherence to and invasion of host tissues (43), morphological change from yeast to hyphae (29,30), secretion of lytic enzymes (17,27,41), maintenance of cell wall integrity (55), and avoidance of the host immune response (39). Many of these virulence factors are glycosylphosphatidylinositol (GPI)-anchored proteins, which comprise 88% of all covalently linked cell wall proteins in C. albicans (23). Examples of GPI-anchored virulence factors are Phr1p and Phr2p, which mediate cell wall biogenesis and hyphal formation in response to changes in pH (15); Hwp1p, an epithelial adhesin and biofilm promoter (32) that is required for virulence during murine hematogenously disseminated candidiaisis (48); Als1p and Als3p, adhesins with broad substrate specificity (13,22,43); and Gpi7, an antivirulence factor that reduces candidal resistance to macrophages and virulence in mice (36). Numerous GPI proteins have been identified...

E1210, a New Broad-Spectrum Antifungal, Suppresses Candida albicans Hyphal Growth through Inhibition of Glycosylphosphatidylinositol Biosynthesis

“…The differences in the amino acid sequence of this region between fungi and mammals might confer the species specificity of Gwt1p to E1210. Virulence factors in C. albicans include proteins which mediate adherence to and invasion of host tissues, morphological changes from yeast to hyphae, secretion of lytic enzymes, maintenance of cell wall integrity, and avoidance of the host immune response (16). Some of these virulence factors are GPI-anchored proteins, which comprise 88% of all covalently linked cell wall proteins in C. albicans (25).…”

Candida albicans-epithelial interactions and pathogenicity mechanisms: scratching the surface

“…One of the more recently discovered adhesins is Iff4p. Overexpression of this cell surface protein increases adherence to oral epithelial cells, 34 while an iff4D/D null mutant shows reduced binding to plastic. 35 …”

Adhesins in Human Fungal Pathogens: Glue with Plenty of Stick

“…The molecular substrates of the Iff/Hyr family proteins have not yet been identified, but they are clearly of clinical relevance. Overexpression of IFF4 in C. albicans increased adherence to plastic and epithelial cells (79). In animal models, both overexpression and underexpression of IFF4 resulted in a reduction of virulence, indicating that a specific expression level is required for maximal virulence (108).…”