PROJECT SUMMARY Precisely how anti-trypanosomatid drugs kill parasites remains largely unknown. Trypanosomatid infections, in the form of African trypanosomes, American trypanosomes, and Leishmania spp., directly contribute to disease and poverty of over 1 billion people. Thus, gaps in knowledge have a significant human impact. The well-established treatments nifurtimox, eflornithine, benznidazole, pentamidine, suramin, and melarsoprol all suffer from complex treatment regimens, host toxicity, and burgeoning drug resistance. The high host toxicity of melarsoprol (encephalopathy in ¼ of patents) made development of new drugs an imperative, which has been answered by NECT (nifurtimox-eflornithine combination, intravenous) and fexinidazole (oral) therapies. Despite this progress, mechanisms of cell death and drug resistance are unknown for fexinidazole and other significant drugs. The ORFeome-based Trypanosoma brucei Gain-of-Function Library is the state-of- the-art-tool for identification of both direct drug targets and mechanisms of drug resistance in trypanosomatids. Discoveries from a published melarsoprol Gain-of-Function screen identified novel aspects of resistance (including mitochondrial proteins). Unpublished data from a fexinidazole genetic screen demonstrated that drug resistant survivors arise from induced expression of a clearly identifiable set of genes, which remain to be elucidated. Multiple trypanocidal drugs converge on the same set of cytology phenotypes, suggesting shared pathways to cell death. Based on genetic screening data and cytology-based phenotypes, this proposal will test the central hypothesis that anti-trypanosomatid drugs share common mechanisms of cell killing and utilize pathways that can promote multi- and pan-resistance against widely used therapies. In AIM 1, all clinically relevant anti-trypanosomatid drugs (nifurtimox, eflornithine, benznidazole, pentamidine, suramin, and fexinidazole) will undergo GoF genetic screening and validation to identify a set of genes that promote multi- and pan-drug resistance. Multiple drugs converge on trypanosomatid redox and mitochondrial functions. AIM 2 will use genetically encoded fluorescent biosensors to test the working hypothesis that anti-trypanosomatid drug treatments perturb redox metabolism and ROS stress management in the cytosol and mitochondrion, filling a gap in our understanding of drug-induced redox stress. Drug cytotoxicity in T. brucei is associated with a set of established phenotypes, AIM 3 will determine how multi-resistance genes contribute to cell death phenotypes including: cell cycle, DNA damage, and loss of mitochondrial functions. The proposed research is of high significance because it will link trypanocidal phenotypes with their associated genes and genetic pathways for the first time. Genes whose expression promotes multidrug resistance will elucidate the pathways that lead to cell death in these parasites. Discoveries arising from these studies w...