Elements of the Ca2+ signal transduction pathway of Toxoplasma gondii

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Toxoplasma gondii is an obligate intracellular parasite that replicates inside host cells. T. gondii belongs to the Apicomplexan phylum which also includes a number of pathogens of medical and veterinary relevance. The clinical manifestations of these diseases are a direct result of the growth of parasites within host cells. Replication and dissemination within the host are essential mechanisms by which T. gondii causes disease. T. gondii engages in multiple rounds of a lytic cycle, which consists of attachment and secretion of unique adhesins, invasion of host cells, replication, egress and search of another host cell to invade. Almost all of these biological functions are triggered by an increase in cytosolic free calcium (Ca2+), followed by stimulation of signaling cascades that are poorly characterized. Many of the transducing elements downstream to Ca2+ are either not known or have not been characterized or their interaction with other elements in the signaling cascade is not clear. Discovery and characterization of new signaling elements is highly significant because Ca2+ signaling forms part of the signaling mechanisms by which T. gondii and other related pathogens, cause disease. In addition, essential parasite calcium signaling players can be developed as targets for anti-parasitic chemotherapy.Fluctuations of the cytosolic Ca2+ concentration regulate a variety of cellular functions in all eukaryotes. Ca2+ signaling starts by an increase in cytosolic Ca2+ that results from influx from the extracellular milieu or release from intracellular stores. The information encoded in transient Ca2+ signals is deciphered by various intracellular Ca2+ binding proteins (CBPs) that convert the signals into a wide variety of biochemical changes. CBPs bind Ca2+ through specific domains like the EF-hand domains composed of EF-hands. Calmodulin (CaM), with four EF hands plays a central role in Ca2+ signaling and it is the main mechanism by which Ca2+ signals are amplified to the scale of proteins and is transduced into biological responses. Binding of Ca2+ triggers a dramatic change in CaM shape favoring its interaction with target proteins resulting in diverse effects like relieve of autoinhibition, changes in domains structures, remodeling of active sites and also protein dimerization. In this proposal we aim at discovering new Ca2+ signaling players by exploring T. gondii CaM (TgCaM) binding sensors. Almost nothing is known about TgCaM and its downstream sensors, which most likely play essential roles in T. gondii by transducing information from Ca2+ signals. It is likely that some of the targets/sensors have been identified but the mechanistic basis for their activation, potentially by binding to TgCaM has not been shown. We believe that our work will lead to the discovery of novel bridging elements in the Ca2+ signaling cascade offering potentially novel chemotherapeutic targets. Additionally, the discovery of new protein players within established signaling pathways has the potential to generate novel insight into the early origins of complex signaling networks.