PROJECT SUMMARY Chagas' disease (CD) is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Over 70 million people in South and Central America, as well as immigrant populations, are at risk of CD. There is currently no effective vaccine, and treatment options are limited. The parasite is transmitted by the feeding activity of triatomine insects. Once in the blood stream of the mammalian host, the parasite invades nucleated cells and proliferates until the cell ruptures, restarting the lytic infection cycle. T. cruzi has a unique morphology with a single, anchored flagellum enclosed by the flagellar pocket (FP), and undergoes several morphological changes during its life cycle that allow the parasite to adapt to the different environments within its mammalian and insect hosts. The location of the FP and the flagellum along the cell body change during these transitions. Of particular interest is the phase of the life cycle known as metacyclogenesis where insect forms of the parasite (epimastigotes) differentiate into metacyclic trypomastigotes which are infectious to the mammalian host cell. My goal is to identify novel molecular markers and describe the morphological changes that occur during metacyclogenesis using transcriptomics and microscopy. In the first year of this fellowship, I will focus on aim 1, where we will use single cell RNA sequencing to identify the gene cascades underlying metacyclogenesis. From this, we will be able to identify molecular markers unique to either epimastigotes or trypomastigotes, and be able to describe the gene regulatory cascades that underly both the epimastigote cell cycle and metacyclogenesis. It is currently unknown if the transition from epimastigote to trypomastigote requires a cell division, a question we will answer using live cell imaging. This will be facilitated by using fluorescently tagged genes identified in the single cell transcriptomes to allow for real time imaging of transitioning parasites to be validated by an expressed specific stage marker. At the end of the first year, and in the second and third year, I will focus on aim 2 where we will map the localization of FP proteins during metacyclogenesis and [validate their role in serum survival and host cell invasion efficiency]. In the former approach, we will use CRISPR/Cas9 to endogenously tag several FP components and view transitioning cells using a combination of conventional epifluorescence and ultrastructure expansion microscopy to generate high resolution structural information. [Next, we will take FP mutants and expose them to complement active serum and measure lysis, as well as allow them to invade host cell monolayers.] The findings of the proposed work will generate novel and critical resources for the field of CD research, and reveal unique and essential biological pathways that may be amenable [to transmission blocking] to combat CD. This work will be facilitated by the excellent research environme...