Project Summary During chronic fungal infections, heterogeneous subpopulations can arise. While these diversified populations can pose significant challenges for treatment, they also provide an opportunity to identify pathways under selection in vivo. This proposal focuses on the study of unique longitudinally-collected sets of Candida (Clavispora) lusitaniae isolates from three individuals with cystic fibrosis (CF). In each case, the C. lusitaniae infections replaced prior chronic bacterial infections, were associated with high levels of airway inflammation, and were resistant to treatment. C. lusitaniae is an emerging agent of candidiasis known to develop resistance to antifungal drugs and is a close relative of Candida auris, a multidrug resistant pathogen that has repeatedly caused hospital associated outbreaks with high mortality. Through the genomic and phenotypic analysis of variable traits in these chronic infection populations, via a productive collaboration between the Hogan and Stajich Labs, we found striking heterogeneity in two genes: MRR1, which encodes a transcription factor known for its ability to confer resistance to azoles, bacterial toxins, and host antimicrobial peptides, and MRS4, a mitochondrial iron transporter that affects metabolism and metal uptake. We propose that these genes strongly impact host interactions and fungal physiology in vivo. Our studies revealed that Mrr1 controls a large regulon of resistance, metabolic and metal acquisition genes, and we discovered the first endogenous inducer of Mrr1, methylglyoxal (MG), which spontaneously forms from intermediates in glycolysis. Further, we found that repeated loss-of-function mutations in a second gene, MRS4, biases cells towards a glycolytic metabolism, increased MG production, and increased Mrr1 signaling. We propose that these changes promote survival in an inflammatory environment. Specifically, we propose to test the hypotheses that (Aim 1) endogenous MG directly stimulates inducible Mrr1 variants, (Aim 2) that MRS4 loss-of-function mutations induce MG Mrr1 signaling through increased glycolysis, and (Aim 3) that increased glycolysis decreases ROS accumulation in co-culture with activated neutrophils. As we show in published and preliminary data, the pathways and mechanisms studied here are conserved broadly across diverse Candida pathogens including C. auris. Through this work, we aim to further develop our understanding of C. auris, and ways in which C. lusitaniae can be used as a highly tractable, parallel system with an expanded tool kit including genetic, genomic, transcriptomic, and metabolomic resources. We propose that the studies will reveal new broadly relevant mechanisms by which fungi adapt to the host environment which can inform new treatment strategies.