Masters Theses

Date of Award

5-2009

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Todd Reynolds

Abstract

The pathogenic yeast Candida glabrata is the second-most common cause of candidiasis in humans after Candida albicans. Interestingly, C. glabrata is phylogenetically closer to Saccharomyces cerevisiae than C. albicans. One important virulence factor in C. glabrata is its inherent resistance to the azole class of antifungals, necessitating the continued discovery of novel antifungal agents. Many antifungals target ergosterol or ergosterol biosynthesis. In an attempt to look for new potential drug targets in C. glabrata, homologues of the genes in S. cerevisiae that regulate the transcription of phospholipid biosythesis (the inositol regulon) were examined. The S. cerevisiae inositol regulon consists of a heterodimeric transcriptional activator encoded by the genes INO2 and INO4 and a repressor encoded by OPI1, none of which are essential.The most well studied target of these genes is INO1, whose protein product converts glucose-6-phosphate to inositol-1-phosphate for the synthesis of phosphatidylinositol in the absence of inositol. Disruption of INO2 or INO4 blocks transcription of several phospholipid biosynthetic genes including INO1, resulting in inositol auxotrophy. Disruption of OPI1 causes overproduction of INO1 and other genes. Surprisingly, it was found that CgOPI1 is essential for viability in C. glabrata. This was found to be true for strains in both the BG2 and ATCC2001 backgrounds indicating that this is not just a strain-specific effect. This is very different from S. cerevisiae, where the Scopi1[Delta] mutant grows robustly. These results led to the hypothesis that the CgOPI1 gene is necessary for viability because it causes overexpression of a target of the inositol regulon transcriptional activator CgIno2p-CgIno4p. Experimental evidence suggests that this hypothesis is true.Disruption of CgINO2 or CgINO4 leads inositol auxotrophy due to the inability to transcribe regulon targets such as CgINO1. The Cgopi1[Delta] mutant's viability defect can be rescued by disruption of the Cgino2[Delta] gene. The Cgopi1[Delta] Cgino2[Delta] double mutant is viable in the absence of CgOPI1 on a plasmid. These results indicate that blocking the expression of a gene that is activated by the inositol regulon can rescue the Cgopi1[Delta] mutant's viability defect. Identification of this target will help elucidate the phenotype.

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