Defining the role of the Trichomonas vaginalis cytoskeleton during infection - Project Summary Nearly 200 million people globally are infected with Trichomonas vaginalis each year. This single-cell, extracellular parasite infects its host by colonizing the urogenital tract. It represents the most common non-viral sexually transmitted disease worldwide and accounts for more than 5 million annual infections in the United States. In addition to this high incidence, infection with T. vaginalis increases the risk of other sexually transmitted diseases, such as HIV, and raises the chances of premature birth and low birth weight. Despite this large burden of disease, only one class of drugs is available to treat infections and it is estimated that reinfections occur in nearly 20% of patients. Given these alarming statistics, identifying new therapeutic targets is critically needed. One promising target is the evolutionarily unique cytoskeletal network of T. vaginalis, which is made up of three major components: two large microtubule-based structures, called the pelta and axostyle, which run along the length of the parasite and the costa, a striated filament located adjacent to these assemblies. Because of their size and position in the cell, these structures likely play essential roles in Trichomonas biology and have been implicated in critical cellular processes, including motility, cell division, and adherence to the host. This proposal aims to define the functions of the major cytoskeletal components of T. vaginalis using a multidisciplinary approach that combines proteomics, structural biology, functional analyses, and in-vivo imaging. In Aim 1, I will use mass spectrometry and single-particle electron cryomicroscopy (cryo-EM) to define the composition and structure of each cytoskeletal component. Aim 2 will use CRISPR-Cas9 knockout studies to dissect the roles of proteins that specifically localize to the pelta, axostyle, or costa in cell division, motility, and adherence to host epithelium. This functional analysis will be done in two steps: first, to characterize six currently known proteins that localize to the pelta, axostyle, and costa and second, to investigate the proteins identified in Aim 1. In Aim 3 I will use live-cell fluorescence microscopy to track the cytoskeletal changes that occur during adherence. Concurrent focused ion beam (FIB)-milling enabled electron cryotomography (cryo- ET) will be used to visualize the native cytoskeleton and determine the molecular basis of the host-pathogen interface. This work has the potential to transform the field of T. vaginalis research by characterizing individual components of its divergent cytoskeleton and probing the host-pathogen interface. My exceptional committee of mentors will provide me with invaluable training in multiple disciplines, foster collaborations, and support my pathway toward an independent scientific career.
