FOLATE METABOLISM AND DRUG RESISTANCE IN TOXOPLASMA

拟议的项目旨在研究分子，遗传和 细胞内叶酸代谢的药理学方面 原生动物寄生虫弓形虫，一种常见的传染因子 这是一种特别阴险的机会致病菌， 免疫缺陷患者。 急性弓形虫病也是一种主要的 胎儿感染导致的神经异常的原因。 除了它的临床重要性，弓形虫是一个有吸引力的 供实验室研究的微生物：寄生虫易于生长 在体外，并有助于自己的遗传分析更容易比 做其他原生动物寄生虫。 因为T.弓形虫不能 胸苷补救，叶酸途径是一个关键的目标， 临床治疗，并可能引入可选择的 在实验室进行遗传学研究的标记。 这项提案的具体目的是研究药理学 阻断叶酸代谢不同阶段的药物，分离 各种类型的突变体在它们对这些试剂的反应中发生改变， 并克隆和表征编码寄生虫的融合基因 二氢叶酸还原酶/胸苷酸合成酶。 的 药理学研究利用体外试验 开发用于评估药物给药时观察到的协同作用程度 影响叶酸途径中的不同步骤， 组合. 这些分析将有助于表征药物- 耐药突变体，建立药物敏感性谱 在实验室和临床分离物中对T.弓形虫，并评估 新的治疗药物的前景 弓形虫病 从临床病例分离的突变寄生虫或 在实验室中选择将进行研究，以确定 弓形虫耐药性的分子基础，并完善 治疗策略，以尽量减少并发症所引起的 出现耐药菌株。 cDNA克隆的序列分析 （已经分离的）编码寄生虫DHFR/TS酶将被 分析突变菌株所必需的，以及组合 基因组克隆将为工程载体提供基础 for the development发展of transfection转染protocols协议. DHFR/TS序列 也将被认为是有价值的，在阐明进化 寄生原生动物的关系和这种起源 有趣的融合酶。 针对DHFR/TS制备的抗体 蛋白质和合成多肽将用于纯化蛋白质 对于未来的结构和酶的研究，这将是有价值的， 在合理设计新型杀寄生虫药物方面。 除了协助设计新的治疗策略外， 这项研究启动了PI的全面努力， 进一步发展弓形虫作为一种有用的范例， 寄生虫细胞遗传分析和分子生物学。 在 特别是，最近证明的能力， 操纵寄生虫卵囊应该有助于分离 后代形成遗传杂交，并提供了一种新的方法， 寄生虫转染系统的寄生虫的开发。

The proposed project seeks to investigate molecular, genetic, and pharmacological aspects of folate metabolism in the intracellular protozoan parasite Toxoplasma gondii, a common infectious agent which is a particularly insidious opportunistic pathogen of immunodeficient patients. Acute toxoplasmosis is also a leading cause of neurological abnormalities resulting from fetal infection. Aside from its clinical importance, toxoplasma is an appealing organisms for laboratory study: the parasite is convenient to grow in vitro, and lends itself to genetic analysis more readily than do other protozoan parasites. Because T. gondii is incapable of thymidine salvage, the folate pathway is a key target for both clinical therapy and, potentially, the introduction of selectable markers for genetic studies in the laboratory. The specific aims of this proposal are to study the pharmacology of drugs which block different stages of folate metabolism, isolate various types of mutants altered in their response to these agents, and to clone and characterize the fused gene encoding the parasite dihydrofolate reductase/thymidylate synthase enzyme. The pharmacological studies take advantage of an in vitro assay developed to assess the extent of synergism observed when drugs affecting different steps in the folate pathway are used in combination. These assays will be useful in characterizing drug- resistant mutants, establishing the spectrum of drug sensitivity in laboratory and clinical isolates to T. gondii, and assessing the promise of new therapeutic agents for the treatment of toxoplasmosis. Mutant parasites isolated from clinical cases or selected in the laboratory will be studied to determine the molecular basis of drug resistance in Toxoplasma, and to refine treatment strategies to minimize complications arising from the emergence of resistant strains. Sequence analysis of cDNA clones (already isolated) encoding the parasite DHFR/TS enzyme will be necessary for the analysis of mutant strains, and in combination with genomic clones will provide the basis for engineering vectors for the development of transfection protocols. DHFR/TS sequences will also be of consider able value in elucidating the evolutionary relationship of the parasitic protozoa and the origins of this interesting fused enzyme. Antibodies prepared to the DHFR/TS protein and synthetic polypeptides will be used to purify protein for future structural and enzymatic studies, which will be valuable in the rational design of new parasiticidal drugs. In addition to assisting in the design of new treatment strategies, this study initiates a comprehensive effort on the part of the PI to further the development of Toxoplasma as a useful paradigm for the genetic analysis of parasite cell and molecular biology. In particular, the recently demonstrated ability to microscopically manipulate the parasite oocyst should facilitate the isolation of progeny form genetic crosses, and offers a novel approach to the development of a parasite of a parasite transfection system.