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Abstract
T. gondii is a parasite that forms part of the apicomplexan phylum. Members of this phylum can cause a wide range of devastating diseases to humans and livestock. T. gondii is an excellent model organism as it can be easily cultured and is a genetically tractable organism. The hallmark of T. gondii pathogenesis is the lytic cycle. This cycle causes rapid lysis and ultimately destruction of the host cell. Our lab established that calcium (Ca2+) stimulates all of the steps of the lytic cycle. The ability to manipulate the genome has allowed us to utilize reverse and forward genetic techniques to learn more about Ca2+ signaling within the parasite. This work shows a new member of the Ca2+ signaling toolkit, a calcium/proton exchanger TgCAXL1. TgCAXL1 has proven to be critical for parasite invasion of the host cell and regulated Ca2+ signaling and pH homeostasis at the Golgi apparatus and the endoplasmic reticulum (ER). We found that pH is critical for proper protein function, specifically the T. gondii sarco/endoplasmic Ca- ATPase, TgSERCA, a pump on the ER essential for sequestration of Ca2+. To perform Ca2+ measurements in a high-throughput manner, we developed a dual fluorescent parasite to produce ratiometric Ca2+ measurements. We were able to measure cytosolic Ca2+ measurements in a large number of clones. Lastly, we investigated the top hit froma CRISPR-genome wide screen, known as the ClpP protease, performed in the presence of a voltage-gated Ca2+ channel inhibitor, cilnidipine. To conclude, our work revealed various genes and techniques that have allowed us to learn about Ca2+ regulation in T. gondii.