Genetically Encoded Calcium Indicators in Toxoplasma gondii

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DESCRIPTION (provided by applicant): Fluctuations of the cytosolic free calcium (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. However, if Ca2+ elevates over the physiological level it becomes damaging to the cell. Cells contain a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in these Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, as for example heart failure or neuronal death. Apicomplexan parasites include a number of pathogens of medical and veterinary relevance. The role of Ca2+ regulation and homeostasis in the infection cycle of Toxoplasma gondii has been very well documented. There is also some information on the role of Ca2+ in the life cycle of Plasmodium spp. In Toxoplasma, Ca2+ signaling is involved in the stimulation of microneme secretion, gliding motility, conoid extrusion and invasion. However, the information on how this happens mechanistically and the molecules involved is fragmented or missing. In addition, the vast majority of the known Ca2+-related genes remain uncharacterized. An interesting case is that the presence of experimental evidence for an inositol 1,4,5- trisphosphate (IP3)-dependent Ca2+ response in Plasmodium spp. and T. gondii, but no IP3 (IP3R) or ryanodine (RyR) receptor orthologs have been identified in either of those genomes. Our own preliminary data supports the presence of a highly regulated mechanism for Ca2+ entry but the candidate genes for these regulatory molecules are not known. Several potential Ca2+ channels are present in T. gondii but not in Plasmodium spp. or Cryptosporidium spp. genomes. The available information on Ca2+ storage and function in Apicomplexans, although fragmented, point toward the presence of unique Ca2+-mediated pathways in these parasites. The information on the role of Ca2+ in the lytic cycle of T. gondii has been obtained using indirect methods such as labeling of extracellular parasites with fluorescent dyes or using intracellular Ca2+ chelators and/or ionophores. Our tools will allow performing direct real-time observations of Ca2+ changes during T. gondii gliding motility, conoid extrusion, microneme secretion and host-cell invasion and egress. We propose to generate T. gondii tachyzoites expressing Genetically Encoded Ca2+ Indicators (GECI) targeted to different cellular compartments to reveal the dynamics of Ca2+ in live parasites and explore the requirements for Ca2+ signaling in host-cell invasion and egress. Toxoplasma is the ideal system because of its genetic tractability, the easiness of isolating clonal lines and its clear-cut response to Ca2+ making validation of these tools feasible. These cells will allow performing direct measurements and will make feasible studies in intracellular tachyzoites. We will be able to study the role of Ca2+ in host-cell egress and physiological Ca2+ fluxes between different Ca2+ stores.