Project Summary/Abstract Microsporidia are unicellular, fungal parasites with a wide host-range, from insects to humans. They are emerging pathogens, classified as NIAID Category B opportunistic pathogens, and cause microsporidiosis in immunocompromised patients. To gain entry into a target cell, microsporidia employ a remarkably unique and specialized harpoon-like invasion machinery called the polar tube, which is conserved among microsporidial species. While initially coiled neatly within the spore of the parasite, infection of a new cell begins with the rapid extrusion of the polar tube from the spore on a fast timescale (< 2s), which anchors the spore to the host cell. After it has been fired, the polar tube is thought to act as a conduit for the transfer of the infectious “sporoplasm” into the target cell, where replication can begin. Early work has yielded global insights into this process, and the molecular and structural underpinnings of the invasion process are ripe for exploration with modern techniques, such as cryo electron microscopy. This work aims to address fundamental questions and paradoxes in our understanding of the microsporidial polar tube machinery and how it drives invasion into host cells. We will use a combined bottom-up (structural biology, biochemistry and other in vitro techniques on purified proteins) and top-down (in vivo light microscopy, electron tomography) approach; the intersection of these approaches will allow us to unravel the mechanistic biology of this unique invasion process. Here we focus on three human pathogens: Anncaliia algerae, Encephalitozoon cuniculi and Encephalitozoon hellem. The specific aims are 1) To characterize the dynamics of polar tube firing and movement of sporoplasm through the tube using high-speed optical microscopy, and to comprehensively define the composition of the polar tube using mass spectrometry; 2) To biochemically and structurally characterize the individual protein components of the polar tube organelle using X-ray crystallography, single particle cryo electron microscopy and protein-protein interaction assays; 3) To elucidate the overall architecture and packing of the polar tube in the spore using structural cell biology techniques such as serial block face scanning electron microscopy (SBFSEM) and cryo focused ion beam scanning electron microscopy (cryo FIB-SEM) followed by cryo electron tomography (cryo ET).