PROJECT SUMMARY The etiological agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite that infects an estimated 10 million people in the Americas, with an at-risk population of 70 million. Despite its recognition as the highest impact parasitic infection of the Americas, Chagas disease remains underreported, understudied and underfunded. Basic research into the biology of T. cruzi has previously been hindered by a lack of efficient genetic tools, but the advent of CRISPR/Cas9 gene editing technology has cleared the way for more in-depth molecular analyses. Among the human infecting parasitic trypanosomatids, T. cruzi is extremely unique for a number of reasons: it develops into its infectious form in the hindgut of insects, is transmitted to its mammalian host via the feces of its insect vector, lives directly in the cytosol of its mammalian host cell and utilizes an ancient feeding organelle to endocytose extracellular material in a manner much like its bacterivorous free- living relatives. This endocytic structure is composed of a long tubular invagination (cytopharynx) starting at a surface plasma membrane pore (cytostome) which we refer to here as the cytostome/cytopharynx complex or SPC. The SPC is a highly dynamic organelle that is present and functional only in the replicating forms of the parasite and disassembles during the transition to its infectious stages. Until recently the SPC had only been examined using electron tomography techniques and had resisted molecular analysis as the protein components comprising it remained elusive. Our initial published work described the first proteins known to be targeted to the SPC and was followed by the identification of a family of SPC targeted myosin motors that we show contribute directly to the endocytic process. Recently, however, we have identified an essential myosin regulatory component of the SPC (MyAP1), a knockout of which resulted in parasites that are completely devoid of measurable endocytosis and who are defective in their ability to scavenge host lipids. Although viable in culture, we have also demonstrated through infections of the insect vector (R. prolixus) that endocytosis is critical for the parasite to be able to establish a robust infection and colonize the insect hindgut, a necessary step for effective transmission to its mammalian host. Completion of this study will allow us to continue to elucidate the molecular machinery responsible for the formation and function of the SPC as well as give insight into why it is that T. cruzi retained this ancient mode of nutrient uptake. Our endocytic-null mutant also gives us the unique opportunity to finally examine the “endocytome” of this parasite. Although not limited to lipids, we are excited by preliminary data allowing us for the first time to now distinguish which lipids are actively being scavenged through endocytosis from those which the parasite is able to endogenously produce for itself. The goal...