Project Summary Unraveling the biology of human pathogens is fundamental toward understanding mechanisms of pathogenesis and identifying genes essential for survival in the host. This application focuses on the protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa. There are no vaccines, and therapeutic drugs have serious side effects and decreasing efficacy. Thus, there is a pressing need for research to better understand the biology of these human pathogens and the mechanisms they use to survive within their hosts. T. brucei undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector, which among others involves changes in cell morphology, surface coat composition, metabolism, signaling pathways and gene expression. Consequently, these parasites have evolved adaptations to allow for their survival in both the gut and salivary glands of the tsetse fly, as well as in the bloodstream of their mammalian host. By overexpressing a single RNA-binding protein (RBP6) in non-infectious trypanosomes, we recapitulated in vitro the events leading to acquisition of infectivity in the insect vector, including the expression of metacyclic variant surface glycoproteins (mVSGs). The overall goal of the proposed work here is to identify the mechanism by which RBP6 activates developmental progression to infectious metacyclics and to characterize the RNA binding protein network that regulates the developmental program leading to epimastigotes and infectious metacyclics. To accomplish these goals, we will build on our major findings in the previous funding period, namely the identification of initial targets of RBP6 function, the depiction of the molecular characteristics of metacyclics, and the identification of a large compendium of molecules linked to the developmental program leading to infective metacyclic parasites. We will use innovative approaches involving a screen for RBP6 function with deep mutational scanning by combining metacyclic selection and high-throughput DNA sequencing and analyze gene expression at the single cell level with RNA-Seq. Taken together our research plan provides unique opportunities to illuminate the developmental program leading from non-infective procyclics to infectious metacyclics, a crucial process in the T. brucei life cycle.