Signal perception and transduction regulating Giardia cyst formation

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PROJECT SUMMARY/ABSTRACT Giardia lamblia, the causative agent of giardiasis, leads to gastrointestinal disorders, long-term growth defects and even death. Estimates place worldwide incidence at over 200 million symptomatic cases per year. Of concern, up to 20% of giardiasis cases are resistant to front-line clinical treatments, and resistance to all major antigiardial drugs has been reported. In addition to their increasing lack of effectiveness, front-line and second- line nitro drugs act intracellularly via non-specific free radical generation, and therefore have a high incidence of negative side effects. There is a critical need to better understand the basic biology of the parasite in order to ultimately design improved intervention and treatment strategies. Flagellated trophozoites proliferate to colonize the intestine where cues including cholesterol starvation and increased pH at the end of the intestinal tract promote terminal differentiation into cysts that detach for transmission. While current treatments target the flagellated trophozoites, encystation could be exploited to clear Giardia infections. The regulation of encystation is poorly understood across the diversity of encysting parasites; thus, studies focused on the encystation pathway are of fundamental cell and developmental biology interest, as well as profound clinical relevance. The aim of this proposal is to understand how encystation cues are perceived and transduced to initiate the encystation program. Our preliminary studies indicate that elevated cAMP is necessary to activate encystation. Our preliminary studies identified a role for Giardia’s sole Rho family GTPase, GlRac, in the regulation of cAMP. Protein-protein interaction studies with GlRac identified a previously uncharacterized seven- transmembrane PQ-loop protein that belongs to the TOG (transporter/opsin/GPCR) superfamily. Knockdown of this protein results in increased encystation indicating that it negatively regulates encystation. We named this protein EncystR for its role in encystation. EncystR localizes to the plasma membrane in actively growing cells, but exposure to encystation stimuli (high pH and cholesterol depletion), causes rapid internalization. EncystR internalization requires GlRac, supporting a functional relationship between these proteins. EncystR negatively regulates encystation through control of cAMP levels, but the mechanism remains to be determined. Related PQ loop proteins are multi-function amino acid transporter-receptors that are known to be pH responsive and can regulate development. EncystR is an exciting new entry point into uncovering the regulation of Giardia’s developmental program. The overall goal of this project is to delineate the roles of EncystR and GlRac in regulating cAMP and the role of cAMP in eliciting the encystation program. This work will establish a framework for understanding the regulation of encystation from signal detection to encystation. Ultimately, we aim to identify the means to short circuit the normal encystation program such that cell cycle arrest and cytoskeletal disassembly can be activated without producing a protective cyst wall. This would clear infections without the risk of transmission.