Project Summary Toxoplasma gondii is a protozoan parasite that infects between 30-50% of the world’s population. The parasite causes toxoplasmosis, which is a potentially life-threatening disease in the developing fetus and immunocompromised individuals. Currently available drugs to treat toxoplasmosis often cause adverse side effects that lead to discontinuation of treatment, highlighting the need for new and improved treatments. One potential target for drug development is the motile system of T. gondii, as motility is essential for virulence. T. gondii uses its unique form of cellular motility to invade host cells, traverse numerous biological barriers, and disseminate throughout the host organism. An unconventional myosin motor protein, TgMyoA, drives motility, and this project will test the hypothesis that motor function and parasite motility are regulated by calcium- mediated phosphorylation of TgMyoA. Aim 1 seeks to investigate the role in motility of a calcium-dependent protein kinase (TgCDPK3) that was recently shown to phosphorylate TgMyoA. A recently developed 3D motility assay will be used to determine the specific effect(s) that disruption of TgCDPK3 has on motility, and recombinant TgMyoA expression to establish how phosphorylation affects motor function. As TgCDPK3 phosphorylates many parasite proteins in addition to TgMyoA, Aim 1 will also determine whether expressing TgMyoA with phosphomimetic amino acids at the sites normally phosphorylated by TgCDPK3 is sufficient to overcome the motility defects seen in parasites lacking functional TgCDPK3. Aim 2 will determine if the oscillations in parasite intracellular calcium levels observed during motility play a role in regulating the regular oscillations in parasite velocity seen in 3D. Using calcium indicator-expressing parasites, the calcium oscillations will be altered pharmacologically (e.g., with caffeine, which lengthens the calcium oscillations) to determine whether the velocity oscillations are correlation and/or causative. Combined, the two Aims will provide novel insights into the role of calcium and TgMyoA phosphorylation in motility regulation. Understanding the regulation of TgMyoA will be critical to determining how to most effectively target motility for drug development.