Homologs of brassinosteroid signaling proteins in Toxoplasma gondii regulate parasite division

刚地弓形虫及其相关寄生虫对社会健康和经济造成巨大负担。不幸的是，针对这些寄生虫的药物是有限的，而且治疗往往是有毒的，耐药性是一个严重的挑战。因此，发现新的治疗方法是当务之急。由于这些是专性细胞内寄生虫，确定新的治疗靶点需要彻底了解寄生虫特有的事件和蛋白质。有趣的是，弓形虫编码许多哺乳动物细胞中不存在的植物样蛋白质。为了利用这一独特的特征，我们重点研究了含有蛋白磷酸酶TgPPKL的kelch结构域，其最接近的同源物是植物磷酸酶BSU1。我们已经证明TgPPKL存在于寄生虫的细胞质中，并且在寄生虫分裂过程中与新生细胞骨架结构相关。我们已经证明PPKL对寄生虫的生存至关重要，条件敲除会导致寄生虫的异常分裂。PPKL的植物同源物BSU1在油菜素内酯级联这一最具特征的植物信号通路中起着核心作用。油菜素内酯激活了包括BSU1激活在内的级联反应，BSU1依次使高度保守的酪氨酸激酶BIN2去磷酸化，使其失活。当被磷酸化时，BIN2通过直接磷酸化使转录因子失活。有趣的是，弓形虫也有一个与BIN2相近的同源物，在保守的酪氨酸位点被磷酸化。我们已经证明，TgBIN2存在于非分裂寄生虫的细胞核中，但在分裂期间在细胞质中积累。重要的是，我们已经证明TgBIN2与包括两种植物样AP2转录因子在内的转录调节剂复合物相互作用，这表明，作为其植物同源物，它参与调节基因表达。总之，我们的初步数据表明弓形虫PPKL和BIN2的表达和定位都根据分裂周期而变化，PPKL的敲低影响寄生虫的分裂，BIN2与转录因子相互作用。因此，我们假设TgPPKL和TgBIN2作为一种新的信号通路的一部分协同作用，调节寄生虫的分裂和结构。我们的第一个目标将是在超微结构水平上分析TgPPKL破坏的影响，测试TgPPKL和TgBIN2之间的物理和功能相互作用，并确定TgPPKL上游的信号蛋白。在第二个目标中，我们将通过产生和表征敲低菌株和鉴定TgBIN2底物来关注TgBIN2的功能。这些研究将结合先进的细胞生物学、蛋白质组学和分子遗传学方法来阐明这两种独特而重要的信号蛋白的功能。同时，我们的实验将阐明弓形虫中由植物油菜素内酯途径的同源物驱动的独特信号通路。这项工作无疑将发现可以作为药物靶点的蛋白质，并将阐明寄生虫分裂的调节。

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.