Small Molecule Approaches to Studying T. gondii Invasion

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DESCRIPTION (provided by applicant): Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in the congenitally-infected fetus and in immunocompromised persons. T. gondii uses a unique form of gliding motility to invade host cells and disseminate through the body during infection, and motility is essential to parasite virulence. The machinery that powers gliding motility is well conserved in apicomplexan parasites, but very little is known about how the unconventional myosin motor protein at the heart of the motility machinery is regulated. A recent high-throughput screen identified 24 novel small-molecule inhibitors and six enhancers of host cell invasion by T. gondii. Strikingly, 21 of the 24 small molecules that inhibit invasion were found to inhibit parasite motility, and all six of the invasion enhancers enhance parasite motility. This proposal is focused on the detailed mechanism of action of one of the most promising of the motility inhibitors, compound 115556. When compound 115556 is added to T. gondii, it causes the single myosin light chain (TgMLC1) associated with the myosin motor that drives motility (TgMyoA) to undergo a pronounced electrophoretic mobility shift, due to an as yet uncharacterized posttranslational modification. Isolated T. gondii motor complexes containing the modified TgMLC1 show decreased motor activity in an in vitro motility assay. In the course of this work, TgMLC1 was also shown to be phosphorylated and dimethylated. The central hypothesis of this project is that posttranslational modifications of TgMLC1, including the modification induced by 115556, regulate the function of the TgMyoA motor and parasite motility. The Specific Aims of the project are to: (1) Determine the nature of the 115556-induced modification to TgMLC1 and its effect on TgMyoA function and directly test the hypothesis that the 115556-induced modification of TgMLC1 is responsible for the inhibition of parasite motility by the compound; (2) Identify the target of 115556. The preliminary data suggest that 115556 does not bind to and act directly on TgMLC1. The direct target of 115556 will be identified, and the pathway connecting this target to the modification of TgMLC1 elucidated; (3) Determine the functional consequences of TgMLC1 phosphorylation and methylation on TgMyoA activity and parasite motility, and establish whether any of the other motility inhibitors or enhancers identified in the high-throughput screen act through phosphorylation or methylation of TgMLC1. This work will provide new insights into the proteins and pathways involved in the motility and invasion of host cells by T. gondii. A greater understanding of how motility is regulated in T. gondii and other apicomplexan parasites is likely to lead to new chemotherapeutic approaches to treating the devastating diseases these parasites cause.