Divergent Calcium Channels of the Apicomplexan parasite Toxoplasma gondii

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PROJECT SUMMARY/ABSTRACT Ca2+ ions impact almost every aspect of cellular life. Ca2+ signaling begins with the opening of Ca2+ channels in either the plasma membrane (PM) or the endoplasmic reticulum (ER) and results in a dramatic increase in the physiologically low (<100 nM) cytosolic Ca2+ levels. The temporal and spatial Ca2+ levels are exquisitely regulated and enable the precise and specific activation of critical biological processes like changes in gene expression, cell differentiation, muscle contraction, fertilization, or secretion of neurotransmitters to name a few. Ca2+ signaling regulates pathogenic pathways of apicomplexan parasites like Toxoplasma gondii which infects approximately one third of the world’s population. T. gondii is an opportunistic pathogen of immunocompromised patients like HIV-infected individuals, fetuses, and organ transplant recipients. As an obligate intracellular pathogen, T. gondii replicates inside cells and the clinical manifestations of toxoplasmosis are a direct result of its growth within cells and its dissemination. T. gondii relies on Ca2+ signals for the stimulation of specific features of its infection cycle and several Ca2+ signaling elements play essential roles in its parasitic cycle. However, the fundamental elements that initiate Ca2+ signals in T. gondii are largely unknown yet are likely essential for its viability and virulence. Discovery and characterization of the molecules that initiate Ca2+ signaling in T. gondii are hence central for the understanding of its pathogenesis. Active egress of T. gondii from host cells is critical for dissemination of the infection and our prior work has provided conclusive evidence that there is a cytosolic Ca2+ peak preceding egress. This parasitic cytosolic increase arises from release from intracellular stores, likely the endoplasmic reticulum. It is puzzling, however, that intracellular parasites replicate surrounded by the low host cytosolic Ca2+ but still store sufficient Ca2+ in their ER to trigger egress. Upon host cell rupture, extracellular Ca2+ influx across the PM contributes to a second Ca2+ peak enhancing motility of parasites, which then exit and seek another host cell to invade. Our hypothesis is that PM Ca2+ entry is essential for refilling of intracellular Ca2+ stores, and both intra and extracellular sources are necessary for triggering the cascade of molecular events that lead to the stimulation of parasitic functions like motility, secretion of adhesins, invasion of host cells, egress and dissemination. In this proposal we aim to characterize the proteins that enable PM Ca2+ influx. There is almost no information about the functional characteristics and roles of Ca2+ channels in T. gondii. This lack of knowledge could be due to lack of appropriate tools, techniques, and training in electrophysiology within the molecular parasitology field. We address this void with a collaboration with a mammalian electrophysiologist and a modeler. Channels are critical for the successful unicellular life of parasites, and they could be targeted by many therapeutically useful agents. Ion channels remain significantly under-exploited as therapeutic targets, even more so as antiparasitic agents.