弓形虫顶质体中丙酮酸的代谢机制研究

Apicoplast is a unique organelle in most apicomplexa parasites including Toxoplasma gondii. Type 2 fatty acid synthesis (FAS II) and methylerythritol phosphate (MEP) pathways are the most important metabolic pathways in apicoplast and both of which require pyruvate as substrate. However, the sources and the metabolic processes of apicoplast-localized pyruvate in T.gondii are pooly understood. Pyruvate kinase 2 (PYK2) and pyruvate dehydrogenase (PDH) are known to localize to the apicoplast, meanwhile Toxoplasma genome encodes potential pyruvate carrier proteins (MPC) and an intact MEP pathway. Therefore we propose that pyruvate in T. gondii apicoplast is derived from the catalysis of PYK2 and/or imported by MPC, subsequently it is used by 1- deoxy -D- wood ketone -5- phosphate synthetase (DXS) to enter the MEP pathway, or by PDH to join the FAS II pathway. This study will focus on the four genes PYK2, MPC, PDH and DXS, using genetic approaches to modify these genes and analyze their impact on parasite growth, development and differentiation both in vitro and in vivo. Then changes in pyruvate and other metabolites will be examined by mass spectrometry. This study will systematically analyze the metabolic mechanisms of pyruvate in apicoplast of T. gondii, which will offer significant insights into the understanding of the mechanisms governing parasite growth and development, and help the design of new anti-toxoplasmic vaccines and drugs.

顶质体是顶复门寄生虫特有的细胞器，II型脂肪酸合成（FASⅡ）与类异戊二烯合成途径（MEP）是顶质体中最重要的代谢途径，而它们均需要丙酮酸作为底物，但弓形虫顶质体中丙酮酸的来源以及代谢机制都不清楚。现已证实弓形虫丙酮酸激酶2（PYK2）和丙酮酸脱氢酶（PDH）定位于顶质体，基因组学分析发现弓形虫存在潜在丙酮酸转运载体（MPC）和完整的MEP途径，为此我们假设：弓形虫顶质体中丙酮酸来源于PYK2的催化或/和MPC的转运，然后由1-脱氧-D-木酮糖-5-磷酸合成酶（DXS）催化进入MEP途径，或经PDH催化进入FASⅡ途径。本项目拟对PYK2、MPC、PDH、DXS这四个基因进行遗传学改造，在细胞和动物水平观察突变虫株生长发育以及分化的变化，通过质谱学方法检测丙酮酸及其相关物质的含量变化，解析弓形虫顶质体中丙酮酸的代谢机制，为阐明弓形虫生长发育机制、开发新型基因工程疫苗和药物提供理论依据。

顶质体是顶复门寄生虫特有的细胞器，II型脂肪酸合成（FASⅡ）与类异戊二烯合成途径（MEP） 是顶质体中最重要的代谢途径，而它们均需要丙酮酸作为底物，但弓形虫顶质体中丙酮酸的来源以及代谢机制都不清楚。为此本研究对弓形虫顶质体中的丙酮酸来源和糖酵解途径以及丙酮酸脱氢酶（PDH）对于FASII脂肪酸合成途径的关键作用及对顶质体代谢的意义进行研究。研究结果表明：定位于顶质体的TgPDH复合物起源于蓝藻，TgPDH-E1α对寄生虫生长重要，但不是生存所必需的，Δpdh-e1α突变体的脂肪酸合成受损，PDH对于弓形虫的毒力没有影响，PDH突变体的顶质体丢失与其生长表型相关。顶质体中的PYK2和PGK2对寄生虫的生长是非必须的，胞质的TPI1和顶质体中的TPI2对弓形虫的裂解循环都是必需的，定位于顶质体的TPI2失活后导致通过MEP途径的类异戊二烯前体合成减少，GAPDH2是顶质体代谢和寄生虫生长所必需的。上述结果为弓形虫病的预防和治疗以及药物设计、新型基因工程疫苗开发提供理论依据和思路。