Structure and mechanism of the polar tube invasion apparatus from microsporidian parasites

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 microsporidian species. While initially coiled neatly within the parasite spore (pre-firing state), infection of a new cell begins with the rapid firing of the long polar tube from the spore on a fast timescale (~300 ms), which anchors the spore to the host cell. After it has been fired, the long, linear polar tube (post-firing state) is thought to act as a conduit for the transfer of the infectious “sporoplasm” into the target cell, where parasite 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 in our understanding of the microsporidian 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 the human pathogen Encephalitozoon intestinalis, one of the main human-infecting species. The specific aims are 1) To study the architecture of the polar tube in its post-fired state, and capture intermediates of the firing process, including cargo transport, using cryo-electron tomography; 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 define the mechanism of host cell invasion by the polar tube using live-cell optical microscopy.