ANTIFUNGAL RESISTANCE GENE TARGETING IN CANDIDA GLABRATA BY TARGET CAPTURE

Most of what we know about antifungal drug research is derived from studies of non-pathogenic model fungi,such as Saccharomyces cerevisiae, which can easily be inserted into high throughput screening platforms,but are unsuitable as pan-fungal models. The rapid completion of genome sequences, and continuallyevolving bioinformatic manipulation of this vast and growing amount of data, has enabled new approaches toantifungal investigation. Presently, clinical mycology is faced with a growing problem of drug resistant andmulti drug resistant organisms, which is compounded by the lack of approved antifungals and difficulty ofdiscovering and bringing new ones to the clinic. These issues combine to limit antifungal choices forclinicians. To address this issue, we will develop a method that rapidly interrogates the fungal genome toreveal genes and pathways that are potential antifungal targets. The major objective of this study will be todevelop a way to rapidly and inexpensively identify these targets. To accomplish this goal we will work withan increasingly important yeast pathogen, Candida glabrata, and an insertional mutagenesis system basedon the bacterial pathogen, Agrobacterium tumefaciens. The first aim will be to improve the existingAgrobacterium tumefaciens transformation efficiency to yield enough transformants to produce a saturatedinsertional mutagenesis map. We will next develop a capture-probe based enrichment method for recoveringinsertion site fragments from the predominating non-junctional genomic DNA background. Finally, we willapply deep sequencing to these enriched fragments to identify each insertion site and its neighboring flankinggenomic DNA, and ultimately assemble a high density insertion map that will be used to identify genes thatare essential for survival, and therefore, potential antifungal targets. Three maps will be prepared, one will bea control map from the wild type genomic strain, and two will be maps prepared from cells exposed toFluconazole and Caspofungin, which will be compared to the wild type map to identify drug-specific genesand genes that are responsive to both drugs. The long term goals that will be possible after this study will beto rapidly identify the genes and pathways that are the targets of any antifungal lead candidate.