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Toxoplasma gondii , an obligate intracellular parasite, is capable of invading virtually any nucleated cell. Active invasion by the parasite is a precise process and intracellular parasites reside and replicate inside a parasitophorous vacuole (PV). The membrane of the PV functions as a molecular sieve allowing for its ionic milieu to be in equilibrium with the host cytosol. The parasite would thus be exposed to the ionic fluctuations of the host cytoplasm, such as increases in host cytosolic Ca 2+ during Ca 2+ signaling events. Ca 2+ is a universal signaling molecule and both the host cell and T. gondii will utilize Ca 2+ signaling to stimulate diverse cellular functions. Using T. gondii tachyzoites and host cells expressing genetically encoded Ca 2+ indicators, we demonstrate that host Ca 2+ signaling impacts intracellular Ca 2+ levels of the parasite. We propose that Ca 2+ influx derived from host Ca 2+ signaling into the parasite is utilized to maintain the intracellular Ca 2+ stores replenished as the parasite replicates within the low Ca 2+ environment of the host cell. Intracellular Ca 2+ store(s) release is needed to reach the initiation spike in Ca 2+ that meets the threshold of Ca 2+ needed to initiate the tightly coordinated process of egress. Post activation of the signal and subsequent microneme secretion, would cause breakdown of the host cell and allow for extracellular Ca 2+ influx, causing a second, larger spike in Ca 2+ and activation of the glideosome, the main motility machinery. To directly deliver precise concentrations of ions needed for egress, we used patch-clamped infected host cells and monitored parasite egress. In doing so, we discovered an alternative role for K + in timing parasite egress. This is the first study using whole-cell patch clamp to study the role of ions such as K + and Ca 2+ in T. gondii egress. Author Summary Toxoplasma gondii is an intracellular parasite that causes disease by engaging in multiple rounds of a lytic cycle which consists of invasion, replication inside a parasitophorous vacuole (PV) and egress, resulting in lysis of the host cell. In intracellular parasites, the release of Ca 2+ from intracellular stores forms part of a series of signaling events that culminate in stimulation of motility and egress. This means that intracellular parasites must be able to uptake Ca 2+ while replicating to keep their intracellular stores filled to be used during egress. This is not an insignificant task considering that they are inside a PV surrounded by the host cytosol which contains Ca 2+ below 100 nM. We present data showing that the host Ca 2+ increases are able to reach the parasite cytosol through a Ca 2+ influx mechanism sensitive to nifedipine. This influx causes oscillations that are the result of Ca 2+ increase and re-uptake by intracellular stores. A Ca 2+ threshold was needed for egress, extracellular Ca 2+ influx was relevant for egress and potassium had a modulatory effect on egress. We used infected patched host cells, a novel approach that leaves the host cell intact while allowing changes of PV surrounding buffers.

Toxoplasma gondii is the causative agent responsible for toxoplasmosis and serves as one of the most common parasites in the world. For a successful lytic cycle, T. gondii must traverse biological barriers in order to invade host cells, and as such, motility is critical for its virulence. Calcium signaling, governed by fluctuations in cytosolic calcium (Ca2+) concentrations, is utilized universally across life and regulates many cellular processes, including the stimulation of T. gondii virulence factors such as motility. Therefore, increases in cytosolic calcium, called calcium oscillations, serve as a means to link and quantify the intracellular signaling processes that lead to T. gondii motility and invasion. Here, we describe our work extracting, quantifying and modeling motility patterns of T. gondii before and after the addition of pharmacological drugs and/or extracellular calcium. We demonstrate a computational pipeline including a robust tracking system using optical flow and dense trajectory features to extract T. gondii motility patterns. Using this pipeline, we were able to track changes in T.gondii motility in response to cytosolic Ca2+ fluxes in extracellular parasites. This allows us to study how Ca2+ signaling via release from intracellular Ca2+ stores and/or from extracellular Ca2+ entry relates to motility patterns, a crucial first step in developing countermeasures for T. gondii virulence., 7 pages, 13 figures, KDDBigDas Workshop