PROJECT SUMMARY Candida albicans and Candida parapsilosis are major cause of infectious morbidity and mortality in premature neonates, in patients with hematologic malignancies, and in organ transplant recipients. Our pilot studies reveal that the GI tract harbors C. albicans and C. parapsilosis strains that expand and dominate the microbiota prior to translocation into the bloodstream of allogeneic hematopoietic cell transplant recipients, despite receipt of antifungal prophylaxis. Although the GI tract represents a major source of fungal bloodstream infections, the molecular and cellular constituents of the intestinal immune system required to maintain colonization resistance against Candida remain poorly defined. Our recent finding that specific C. albicans strains can colonize the GI tract to intermediate levels in the absence of antibiotics represents an important advance to investigate this question, because it enables us to explore the impact of host cellular and molecular constituents in the context of an intact bacterial microbiota. Thus, we can compare colonization phenotypes and susceptibility to invasive disease in experimental models with defined molecular and cellular lesions in host immune function, but with an intact microbiota. To define essential immune-mediated mechanisms that limit Candida colonization, we conducted in vivo screens of pattern recognition receptor pathways and host cellular requirements required for this process. We found an essential requirement for TLR2/4, IL-1 receptor, and MyD88 signaling and for innate lymphoid/NK cells to maintain Candida colonization resistance in otherwise unperturbed mice. In addition, we have described a HIF-1/anti-microbial peptide circuit in intestinal epithelial cells that can regulate Candida colonization in the murine intestinal tract. Based on these findings, we will explore the central hypothesis that MyD88 and HIF-1 cooperate to couple recognition of the bacterial microbiota and tissue hypoxia to innate lymphoid-natural killer cell and epithelial effector programs that limit Candida colonization and risk of invasive disease. The aims will define (1) the precise role and essential cellular sources of TLR2/4, IL-1 receptor and MyD88 signaling required for Ca and Cp colonization resistance, (2) characterize the functional requirements for intercellular crosstalk among immune and epithelial cells, and (3) harness complementary immune activation strategies to restrict Candida colonization, reduce the reduce the risk of systemic dissemination with immune injury, and provide an experimental platform to interrogate fungal strain dynamics in the intestine. These studies will incorporate C. albicans and C. parapsilosis mutants in morphogenesis and virulence factor expression in collaboration with Project 1, will generate metabolomics datasets to inform studies in Project 3, and will work closely with the Gnotobiotic Core to dissect the role of individual molecular and cellular determina...