Development of an Agrobacterium-Mediated Transformation System for Candida glabrata

Objectives: Candida species cause the most opportunistic fungal infections in humans with Candida albicans being the most prevalent species. However, Candida glabrata has emerged as the second most important member of this genus and is responsible for various mucosal and systemic infections. Caspofungin, one of three licensed echinocandin antifungals, is currently being used to treat Candida infections. However, recent evidence has shown various clinical cases of resistant C. glabrata. Thus far, little is known about the genes that may contribute to caspofungin resistance in C. glabrata. Methods: In order to identify these genes, we have developed a novel insertional mutagenesis strategy for C. glabrata using an Agrobacterium-mediated transformation system. Agrobacterium tumafaciens is a natural bacterial pathogen of plants that has been adapted for DNA transfer into a variety of organisms. Results: In this study we designed a recombinant Agrobacterium tumafaciens plasmid containing the NAT1 gene (a dominant selectable marker for candida species) for Agrobacterium uptake and use in transforming C. glabrata. We have also developed adequate transformation and selection conditions, which yielded 96% transformation stability. Conclusions: Our work is the first such study for C. glabrata and demonstrates the development and utilization of a natural DNA transformation system that will be useful for identifying genes that affect susceptibility and resistance to caspofungin in C. glabrata.