SUMMARY Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, while many of the calcium-dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation we have determined that a nonsense mutation in a previously uncharacterized protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localizes to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network (TVN). Our findings support a connection between the TVN and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.

Calcium ions regulate a diversity of cellular functions in all eukaryotes. The cytosolic Ca concentration is tightly regulated at the physiological cytosolic concentration of 50-100 nm. The Toxoplasma gondii genome predicts the presence of several genes encoding potential Ca channels, pumps, and transporters. Many of these genes are weakly expressed and likely tightly regulated due to their potential impact to the physiology of the cell. Endogenous tagging has been widely used to localize proteins in T. gondii but low level of expression of many of them makes visualization of tags difficult and sometimes impossible. The use of high-performance tags for labeling proteins expressed at low level is ideal for investigating the localization of these gene products. We designed a Carboxy-terminus tagging plasmid containing the previously characterized "spaghetti monster-HA" (smHA) or "spaghetti monster-MYC" (smMYC) tags. These tags consist of 10 copies of a single epitope (HA or MYC) inserted into a darkened green fluorescence protein scaffold. We localized six proteins of various levels of expression. Clonal lines were isolated and validated by PCR, western blot, and immunofluorescence analyses. Some gene products were only visible when tagged with smHA and in one case the smHA revealed a novel localization previously undetected.

Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca2+) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca2+ signaling is essential for the cellular and developmental changes that support T. gondii parasitism. The characterization of the molecular players and pathways directly activated by Ca2+ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca2+ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca2+ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.

Transient Receptor Potential (TRP) channels participate in calcium ion (Ca2+) influx and intracellular Ca2+ release. TRP channels have not been studied in Toxoplasma gondii or any other apicomplexan parasite. In this work we characterize TgGT1_310560, a protein predicted to possess a TRP domain (TgTRPPL-2) and determined its role in Ca2+ signaling in T. gondii, the causative agent of toxoplasmosis. TgTRPPL-2 localizes to the plasma membrane and the endoplasmic reticulum (ER) of T. gondii. The ΔTgTRPPL-2 mutant was defective in growth and cytosolic Ca2+ influx from both extracellular and intracellular sources. Heterologous expression of TgTRPPL-2 in HEK-3KO cells allowed its functional characterization. Patching of ER-nuclear membranes demonstrates that TgTRPPL-2 is a non-selective cation channel that conducts Ca2+. Pharmacological blockers of TgTRPPL-2 inhibit Ca2+ influx and parasite growth. This is the first report of an apicomplexan ion channel that conducts Ca2+ and may initiate a Ca2+ signaling cascade that leads to the stimulation of motility, invasion and egress. TgTRPPL-2 is a potential target for combating Toxoplasmosis.