Candidiasis, the most prevalent fungal disease, affects millions annually in the U.S. alone. Patients with cancer, AIDS and diabetes, as well as premature infants and individuals requiring intensive care, are among those at risk. Dissemination of primary epithelial Candida infection is associated with a high rate of crude and attributable mortality. Widespread resistance of Candida to the limited number of available antifungal agents demonstrates the urgent need for therapeutic alternatives. Photodynamic therapy (PDT) is a promising strategy for treatment of organisms resistant to conventional chemotherapy. Optimizing approaches for antimicrobial PDT will facilitate its translation to the clinic. Mitochondrial respiratory-deficient mutants of Candida are significantly more sensitive to oxidative stress induced by PDT than parental strains. The central hypothesis of this proposal is that specific targeting of mitochondrial respiratory activity in Candida increases its sensitivity to PDT. Our long-range goal is to develop a two-step therapeutic strategy by which fungal respiration is first selectively inhibited, resulting in an increased susceptibility to oxidative stress induced by PDT leading to cell death. The immediate objective of the proposed research is to identify relevant targets for inhibiting respiration in Candida. Using defined genetic mutants, we have identified three major mitochondrial metabolic pathways that can be targeted to increase the sensitivity of Candida to PDT. Importantly, each of the three fungal pathways utilizes proteins with no known ortholog in mammals;we will test mutants in each pathway for sensitivity to PDT both in vitro and in vivo. Targeting a broad range of potential molecular targets increases the likelihood of identifying compounds to enhance the sensitivity of Candida to PDT. We propose three Specific Aims. Respiratory competence in yeast requires mitochondrial mRNA processing and maturation. Specific Aim 1 will determine whether blocking the mitochondrial mRNA processing pathway increases sensitivity to PDT. Proteins known to participate in translational activation and assembly of cytochrome c oxidase in Candida are absent in mammals. Specific Aim 2 will evaluate whether targeting translational activation and assembly of the cytochrome c oxidase complex increases sensitivity to PDT. The sulfur containing amino acids cysteine and methionine are primary targets of oxidative damage in proteins. The third critical pathway to be targeted is linked to maintenance of mitochondrial redox balance by thioredoxins, glutaredoxins and glutathione transferases. Specific Aim 3 will determine whether Candida mutants in mitochondrial redox pathways display increased sensitivity to PDT. The sensitivity of selected Candida mutants in each pathway to PDT will be evaluated in vivo using a mouse model of infection. In this way, we will link the immediate objective of identifying relevant targets for inhibiting respiration in Candida with the development of improved PDT strategies for treating infections caused by this important fungal pathogen.