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Non-conventional regulation of essential apicomplexan guanylate cyclases

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Project Summary Most protists are free-living, but some infectious forms reproduce exclusively from within host cells. One group of obligate-intracellular protists constitute Apicomplexa, a phylum of globally-distributed parasites notorious for inflicting severe disease and death in humans and livestock. Since pathogenesis stems from their lytic lifecycles, I am interested in signaling pathways that all apicomplexans utilize to transmit from one cell to the next. This project centers around cyclic GMP (cGMP), which acts as a molecular switch for apicomplexan motility, facilitating cell to cell transmission. Although dozens of studies have focused on kinases activated by cGMP (i.e. PKGs), little is known about how cGMP is produced in these organisms. In other eukaryotes, guanylate cyclases (GCs) catalyze the formation of cGMP from GTP and are regulated by domains that sense nitric oxide or peptide hormones. However, apicomplexan GCs lack known regulatory domains, instead possess an N-terminal P-type ATPase domain of unknown function. To investigate the function of these unique enzymes, I chose Toxoplasma gondii as a model due to its clinical importance and excellent experimental tractability. I found that T. gondii GC localizes to the apex of the parasite where it functions as a gatekeeper for apical protein exocytosis, the initiating and rate-limiting step of parasite motility. Conditional loss of TgGC paralyzes parasites preventing lytic growth and spreading, phenocopying loss of TgPKG. Though TgGC possesses dual functional domains, cell-permeable cGMP rescues TgGC depletion, further indicating that its primary role is to synthesize cGMP. Through genetic complementation, I found that both domains of TgGC were required, suggesting that the P-type ATPase domain may function as a regulatory domain. These P-type ATPase domains most closely resemble P4-ATPases, which utilize energy from ATP to flip phospholipids across membranes to maintain the lipid asymmetry needed for a variety of cellular processes. Since P4- ATPases typically function as an ?/? heterodimer with a Cdc50 chaperone, apicomplexan GCs are likely to function in a complex with one or more other proteins. Recently I identified 13 candidate interactors of TgGC using BioID proximity labeling, including a Cdc50 protein that is predicted to be essential based on a prior genome-wide CRISPR screen. The goal of this K22 proposal is to understand how apicomplexan GCs are regulated, both intramolecularly and through interacting partners. This goal will be accomplished by two independent Specific Aims that will define the biochemical function of the P4-ATPase domain (Specific Aim 1) as well as the contributions of TgCdc50 and other interacting proteins (Specific Aim 2) with respect to TgGC function. This research will reveal the mechanism by which essential cGMP signaling is initiated in apicomplexans and will serve as a springboard for my independent career as a principle investigator at an academic research institution. My long-term plan is to use these protein function studies to develop inhibitors of cGMP signaling as potential therapeutic agents for treating apicomplexan infections.
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