Giardiasis, the disease caused by the eukaryotic parasite Giardia lamblia, is the most common intestinal parasitic disease in the U.S. and a major cause of morbidity in children throughout the world; estimates indicate 280 million cases of Giardia annually. Newly emerging drug-resistant strains are proving difficult to treat, and the front-line treatment, metronidazole, has a high incidence of side effects. Therefore, there is a critical need for anti-Giardia drugs that target novel molecular pathways. Giardia belongs to one of the earliest diverging groups of eukaryotes and is therefore quite divergent from metazoans. Opportunities for developing targeted therapeutics exist within this divergent biology. This proposal explores the biology of Rho family GTPases in Giardia. These proteins act as molecular switches that control essential cellular processes. Giardia contains a single Rho family GTPase homolog, gRac, previously demonstrated to play a conserved role in regulating polarity, membrane trafficking, and the cytoskeleton, all of which are essential to viability and pathogenesis. Functional assays have demonstrated that gRac plays a role in cyst formation. This process requires pulsed production, processing, and secretion of cyst wall protein (CWP) to make environmentally resistant cysts and is the only known regulated secretory pathway in Giardia. Rho GTPases are known to regulate ER-Golgi transport and secretory events in plants and animals. How CWP secretion is triggered in Giardia remains enigmatic, yet gRac is a likely candidate for regulating this process.
Aim 1 of this proposal identifies the specific role of gRac n infectious cyst formation by following cyst wall markers through the secretory process. Also unknown is how gRac signaling is linked to downstream processes, previously unidentified downstream effectors (Giardia-specific or potentially ancient and undiscovered) are hypothesized to link gRac signaling to the cytoskeleton and membrane trafficking. Using affinity purification and mass spectroscopy, 199 putative gRac interactors were identified.
Aim 2 proposes to validate four of these gRac interactors as downstream effectors of membrane trafficking and/or the cytoskeleton through microscopy and knockdown studies. Upstream of gRac is a set of six gRac modulators that act to activate or inhibit gRac signaling.
Aim 3, proposes to determine their contribution to gRac signaling and identify specific biological roles for each modulator using a combination of depletion studies and assays to measure cell proliferation, parasite attachment, and the ability to form infectious cysts. The proposed experiments are expected to define the central role of gRac in Giardia biology and pave the way toward discovery of novel therapeutic targets. Furthermore, because the Giardia genome contains only a single Rho GTPase and a handful of upstream modulators, the system is highly tractable and may uncover fundamental biology that has been obscured by the more complex signaling systems found in model eukaryotes.
Giardia is the causative agent of giardiasis, a prevalent gastrointestinal illness that results in diarrhea and malabsorption in 280 million people each year. This proposal investigates the function of Giardia Rac, a protein we have shown to be critical for cellular processes in the parasite. Importantly, Giardia Rac and the downstream effectors controlling these essential functions are divergent from their human homologs; therefore, these proteins represent potential therapeutic targets.
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