PROJECT SUMMARY The major goal of this project is to elucidate the mechanisms by which the gut microbiota and/or microbiota- derived metabolites modulate Candida colonization in the mammalian gut. While it has long been appreciated that intestinal bacteria and more recently bacterial-derived metabolites likely play a role in Candida colonization resistance (CCR), there is a key knowledge gap in understanding the mechanisms by which specific gut microbiota and/or microbiota-derived metabolites impact CCR. In preliminary data presented in this proposal, we show that unbiased in vitro and in vivo screening approaches led to the identification of both short-chain fatty acids, SCFA, and medium chain fatty acids, MCFAs, as being significantly associated with CCR in mice. We further validated that specific SCFAs inhibit Candida growth in vitro and inhibited Candida albicans (Ca) GI colonization in mice. Select SCFAs inhibit Ca glucose uptake and downregulate genes important for glucose metabolism. Pilot microbiome and metabolomic profiling data generated from fecal samples collected from human allogeneic hematopoietic cell transplant (allo-HCT) patients demonstrated a reduction in fecal SCFA abundance and linked this reduction to C. parapsilosis (Cp) intestinal domination in the clinic. Thus, our central hypothesis is that gut microbiota-derived metabolites (i.e., SCFAs/MCFAs) impede Candida growth directly by inhibition of fungal glucose metabolism and restrict GI colonization indirectly via the induction of host immune effectors (including antimicrobial peptides) that target fungal cells. In Aim 1, we will determine the mechanisms by which SCFA/MCFA directly inhibit Candida growth by using functional metabolomic profiling studies to examine levels of glucose-derived metabolites, glycolytic intermediates, and TCA cycle intermediates, and complementary unbiased proteomics and metabolomic studies will be utilized to assess other metabolite- based mechanisms of direct inhibition. In Aim 2, we will assess the effect of SCFAs/MCFAs on induction of immune effectors in intestinal epithelial cells and innate immune cells. In Aim 3, we will define the role of the microbiome/metabolome in modulating Cp GI colonization by perform unbiased microbiome and metabolomic profiling of our human allo-HCT fecal specimen library to identify associations between candidate taxa, metabolite mediators and Cp colonization phenotypes. We will also utilize complementary preclinical models in parallel to address molecular mechanisms of CCR.