Toxoplasma gondii and related parasites, exert great health and economic burden on society. Unfortunately, drugs against these parasites are limited, and treatments are often toxic and resistance is a serious challenge. Accordingly, the discovery of novel therapeutics is a priority. As these are obligate intracellular parasites, identifying novel therapeutic targets requires a thorough understanding of events and proteins that are unique to the parasite. Interestingly, Toxoplasma encodes for numerous plant-like proteins that are absent in mammalian cells. To exploit this unique feature, we have focused on the kelch domain containing protein phosphatase TgPPKL, which closest homolog is the plant phosphatase BSU1. We have shown that TgPPKL is in the cytoplasm of the parasite and that it associates with nascent cytoskeletal structures during parasite division. We have shown that PPKL is essential for parasite survival and conditional knockdown results in aberrant parasite division. PPKL’s plant homolog, BSU1, is central to one of the best characterized plant signaling pathways, the brassinosteroid cascade. Brassinosteroid activates a cascade that includes activation of BSU1, which in turns dephosphorylates the kinase BIN2 at a highly conserved tyrosine, inactivating it. When phosphorylated, BIN2 inactivates transcription factors through direct phosphorylation. Interestingly, Toxoplasma also has a close homologue of BIN2 that is phosphorylated at the conserved tyrosine. We have shown that TgBIN2 is in the nucleus of non-dividing parasites, but accumulates in the cytoplasm during division. Importantly, we have shown that TgBIN2 interacts with a complex of transcription regulators including two plant-like AP2 transcription factors, suggesting that, as its plant homologue, it is involved in regulating gene expression. In total, our preliminary data show that expression and localization of both Toxoplasma PPKL and BIN2 change according to the division cycle, knockdown of PPKL affects parasite division, and BIN2 interacts with transcription factors. Accordingly, we hypothesize that TgPPKL and TgBIN2 act in concert as part of a novel signaling pathway to regulate parasite division and structure. Our first aim will be to analyze the effect of TgPPKL disruption at the ultrastructural level, test for a physical and functional interaction between TgPPKL and TgBIN2, and identify signaling proteins upstream of TgPPKL. In a second aim we will focus on TgBIN2’s function by generating and characterizing a knockdown strain and identifying TgBIN2 substrates. These studies will combine state of the art cell biology, proteomic and molecular genetic approaches to elucidate the function of these two unique and essential signaling proteins. In conjunction, our experiments will elucidate a unique signaling pathway in Toxoplasma that is driven by homologs of the plant brassinosteroid pathway. This work will undoubtedly uncover proteins that can be exploited as drug targets and will shed light on the regulation of parasite division.
