Project Summary: The fungus Candida albicans is a frequent commensal of the gastrointestinal (GI) tract but is also an important cause of both mucosal and systemic disease. The GI population is particularly relevant to human health as cells can translocate out of this niche to cause disseminated infections. C. albicans cells in the gut also play key roles in regulating local and systemic immune responses that can be either beneficial or detrimental to the host. There is therefore a pressing need to understand how C. albicans colonizes the GI tract and to define how changes to the commensal environment impact fungal behavior. Attention has focused on the ability of C. albicans to transition between yeast and filamentous states, with yeast-locked cells shown to exhibit higher fitness in the GI tract than wildtype cells. However, these studies have extensively relied on models that require antibiotic supplementation for stable fungal colonization. In preliminary studies, we examined C. albicans fitness in colonization models without antibiotic treatment. Surprisingly, we show that yeast-locked cells are defective for gut colonization in hosts containing high bacterial loads, including those colonized with defined bacterial consortia. Furthermore, we demonstrate that Candidalysin, the first toxin identified in a human fungal pathogen, is critical for gut colonization in hosts carrying high bacteria loads but not in those given antibiotics, indicating that this factor supports fungal commensalism by enabling competition with the bacterial microbiota. To build on these observations, we will examine how C. albicans morphology (and co-regulated genes) determine GI colonization fitness. Experiments will utilize a variety of murine GI models carrying native or defined bacterial populations to determine how interkingdom interactions influence fungal gut commensalism (Aim 1). We are particularly interested in determining how Candidalysin regulates fungal fitness in the GI niche, and whether this toxin acts intrinsically on fungal cells, to inhibit bacterial cells, or via its impact on host epithelial cells (Aim 2). Experiments will also perform fitness selection assays to identify novel factors that determine gut colonization fitness in hosts carrying different gut bacterial populations (Aim 3). Together, these experiments will provide novel insights into the fundamental mechanisms used by C. albicans to colonize the gut, including the role of Candidalysin toxin in increasing the fitness of fungal cells in the competitive GI niche. Given the importance of gut colonization to fungal-host interactions, these experiments are critical for understanding how C. albicans operates as a human pathobiont.