Genetic approaches to probe virulence and drug resistance in Candida auris

Abstract Fungal infections have risen in frequency and severity over the last several decades, as the number of patients with increased susceptibility have also risen. Today, these infections are a serious clinical problem, and new species emerge to add to these challenges. Candida auris is one such species, having been first identified in a superficial infection in 2009. Subsequently, C. auris has been identified in four geographic foci on three continents, and spread throughout the world. A primary concern with C. auris is its resistance to antifungal drugs, with nearly all isolate resistance to azoles, and many to the polyenes and echinocandins. The rapidity with which this has happened is startling and has far outpaced research advances in this species. One advantage is that C. auris is a haploid species and this opens the door to powerful forward genetic approaches. To take advantage of this, we propose here to develop an insertional mutant library with thousands of mutants, which we will validate using two highly relevant genetic screens. To generate these mutants, we will use the bacterium Agrobacterium tumifaciens, which transfers an integrating vector (T-DNA) to a promiscuous range of host cells, including C. auris. We have adapted tools with which we propose to isolate ~5,000 mutants and array them in 96 well plates, then sequence each to identify the insertion site. Our primary goal is to develop this resource and make it available to the community. In doing so, we will validate this library with two screens that are particularly relevant to C. auris. In the first, we have developed an animal infection assay using the nematode model Caenorhabditis elegans that allows us to identify avirulent mutants. In other Candida species, this assay tracks murine virulence closely, and we have adapted it for C. auris. Our host strain, chosen for its virulence and transformation efficiency, is also highly fluconazole resistant and we will screen for insertions that increase susceptibility. At the conclusion of this project we expect to have (a) built a large mutant library of random insertional mutants which we will make available to the community, (b) generated two large datasets on virulence and drug resistance, (c) primed future mechanistic studies that will make substantial progress in improving the outcomes of patients, and (d) determined the applicability of the nematode model to a wide array of C. auris isolates..