Effects of antifungal drug treatment and immune dysfunction on the evolutionary dynamics of gut-colonizing Candida glabrata

Invasive fungal infections are estimated to cause ~2.5 million deaths per year worldwide, constituting an enormous health and economic burden. Many invasive fungal infections are opportunistic: fungi harmlessly colonize mucosal surfaces, such as the gastrointestinal (GI) tract, in healthy individuals, but can cause life- threatening systemic infections in those with immune dysfunction. Such infections are usually caused by the individual’s own commensal strains and can be preceded by fungal expansions in the GI tract (e.g., due to the use of antibiotics). The GI tract is also a key reservoir where drug-sensitive commensal populations produce drug-resistant isolates, for which very few therapeutic options are available due to our limited antifungal drug armamentarium. Thus, the host GI tract is a key fungal commensal niche whose population dynamics can have dire clinical consequences. However, these dynamics are almost entirely unexplored either under normal conditions, during antifungal drug treatment, or when gut mucosal immunity is impaired. Candida glabrata is a major opportunistic fungal pathogen and a common human gut commensal fungus. C. glabrata infections are challenging to cure, in part due to its widely documented propensity to rapidly evolve antifungal drug resistance. We have used a mouse model of C. glabrata gut colonization to track the evolution of resistance to caspofungin (CSF), one of the echinocandin class antifungal drugs. Using this model and amplicon and whole genome sequencing, we showed that the evolution of CSF resistance is unexpectedly complex, with multiple small-effect drug susceptibility-reducing mutations occurring rapidly and fluctuating until the emergence of a large-effect mutation in the drug’s target gene (FKS2) conferring clinical resistance. This limited analysis using standard genomic tools suggests that during antifungal drug treatment, gut-colonizing C. glabrata undergoes complex population dynamics with clonal interference, which underlie the evolution of antifungal drug resistance. We propose to perform an in-depth analysis of C. glabrata evolutionary dynamics in the host GI tract by using barcode-enabled lineage tracking, a technique developed in the model fungus Saccharomyces cerevisiae (closely related to C. glabrata) that has yielded many insights about its evolution in culture under different stresses. We now have access to a collection of C. glabrata strains carrying hundreds of thousands of unique DNA barcodes, which has been developed and validated in the lab of Dr. Gavin Sherlock, a pioneer of lineage tracking technology. We propose to use this unique resource in our mouse gut colonization model to elucidate the population and evolutionary dynamics of gut-colonizing C. glabrata during treatment with two different echinocandins (CSF and micafungin, Aim 1) and under clinically-relevant conditions of immunodeficiency (neutropenia, p47phox-/-, and CARD9-/-, Aim 2). The results will elucidate how antifungal drugs or immune dysfunction influence the adaptation and evolution of gut-colonizing C. glabrata and may help develop improved strategies of fungal control in the immunocompromised and reduce the emergence of drug-resistant mutants.