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The apicomplexan parasite Toxoplasma gondii is the causative agent of life-threatening encephalitis in immunocompromised patients and in addition can cause a variety of birth defects if the infection is contracted congenitally. The pathology associated with disease originates in fast rounds of lytic intracellular replication cycles, causin extensive tissue damage. Since parasite replication only occurs within a cell from the mammalian host, the related processes of host cell invasion and egress mark essential steps in the pathogenesis. It has been firmly established that the signaling pathways underlying invasion and egress revolve around the cytoplasmic rise of Ca2+ concentration. Two small, Ca2+-binding proteins, calmodulin (CaM; 4 EF-hands) and the calcineurin phosphatase (the regulatory subunit has 4 EF hands), have been associated with activation of egress/invasion through pharmacological studies. Furthermore, a related group of small, 4-EF-hand containing proteins consisting of three centrins (Cen1-3) are critical components to the cell division machinery: Cen1 and Cen3 in centrosome duplication, required for mitosis and coordination of the unusual cell division process; Cen2 in contraction of the basal complex required for daughter parasite segregation.<p/> In essence, this family of EF-hand proteins plays pivotal roles in the defining steps of pathogenesis: invasion/egress and cell division. However this protein family has never been comprehensively studied, despite their critical roles in pathogenesis. To fill this void, the researchers identified a total of 16 such proteins in the genome, most of which have not been studied at all but likely execute additional functions in these processes. Preliminary sub-cellularlocalization dynamics of these proteins throughout the lytic cycle support this hypothesis and permits putative assignment to roles in cell division or invasion/egress.<p/> It is proposed to complete these preliminary studies for the whole family followed by conditional gene knock-outs of the ten prioritized candidates. To illustrate the power of this approach, these experiments willfor the first time directly address whether CaM or calcineurin have a role in egress/invasion, for which only indirect pharmacological data is currently available. Furthermore, using the Cen2 and CaM as a pilot, the direct dissection of the role of Ca2+-binding in the family members with critical roles in invasion or cell division will be pioneered. <p/>It is anticipated that these studieswill lift the veil on the enigmatic role of Cen2 in Toxoplasma cell division and/or cytoskeleton structure. In conclusion, this study of a poorly studied gene family is expected to provide key insights into two key aspects in apicomplexan parasite pathogenesis.
PUBLIC HEALTH RELEVANCE: Ca2+-mediated processes and proteins are critical in progression through two key events in Toxoplasma gondii pathogenesis: parasite cell division and invasion/egress from a host cell. The proposed work will examine the role and mechanism of small proteins with multiple EF-hands, domains that bind Ca2+ and translate intracellular Ca2+ concentration fluxes into action. Completion of the proposed work will enhance insights in key events in the parasite's life cycle and pathogenesis, which may ultimately lead to new treatment options.