FOLATE AND DRUG METABOLISM AND RESISTANCE IN TOXOPLASMA

原生动物寄生虫弓形虫是一种普遍存在的人类病原体 它已经成为一种主要的机会性感染， 艾滋病弓形虫病的临床管理传统上依赖于 抗叶酸剂，但需要与慢性治疗相关的并发症 对于免疫缺陷患者， 这种毁灭性的疾病。该提案旨在利用新开发的 研究药物敏感性的遗传和药理学工具， 弓形虫的耐药性，特别关注叶酸代谢， 寄生虫的双功能二氢叶酸还原酶-胸苷酸合酶 DHFR-TS酶。这项研究的目标包括开发 改善急性弓形虫病的治疗策略。 序列和探针来源于T.弓形虫DHFR-TS基因已被 用于检查野生型中酶的预测结构 寄生虫，并开发功能性载体，用于瞬时和稳定的 寄生虫的分子转化抗叶酸剂的临床病例- 抗弓形虫病和类似的突变体在实验室分离将是 筛选了对组合的敏感性差异， 乙胺嘧啶/磺胺治疗（除了对 乙胺嘧啶或磺胺单独），并检查可能的DHFR-TS突变 或改变基因表达。不依赖DHFR的耐药机制将 通过遗传学手段识别。将评估DHFR-TS酶功能 转基因寄生虫携带的突变来自四个来源： 自然发生的等位基因变异，从药物中鉴定的突变， 耐药实验室菌株和临床分离株， 霸王弓形虫酶和点突变被认为与 相关疟原虫野外分离株的抗叶酸剂抗性 恶性疟原虫（疟疾）。这项研究的结果将与 重组DHFR-TS酶的结构/功能研究，以协助 设计具有改善的抗肿瘤活性的新型抗叶酸剂， 寄生虫 由于基因技术的巨大力量， 药物靶点的鉴定和分析，有效转化 方案将被修改，以允许：（1）靶向基因破坏， 置换，在此背景下用于检查DHFR-TS的突变 （2）插入诱变和标记拯救，用于克隆 治疗干预的潜在靶点和阴性选择 用于基因置换研究的标记物;（3） 互补，重点是DHFR非依赖性抗叶酸剂抗性基因 这似乎是特别重要的乙胺嘧啶耐药性， 弓形虫;和（4）重组体的最佳表达和过表达 蛋白除了对叶酸代谢的分析有价值外， 和耐药性，这些工具应该广泛适用于其他 研究弓形虫的生物学和生物化学。

The protozoan parasite Toxoplasma gondii is a ubiquitous human pathogen which has emerged as a leading opportunistic infection associated with AIDS. Clinical management of toxoplasmosis has traditionally relied on antifolates, but complications associated with the chronic therapy needed for immunodeficient patients have left us with no adequate treatment for this devastating disease. This proposal seeks to employ newly developed genetic and pharmacological tools to investigate drug sensitivity and resistance in Toxoplasma, focusing particularly on folate metabolism and the parasite's bifunctional dihydrofolate reductase-thymidylate synthase enzyme (DHFR-TS). Goals of this research include the development of improved treatment strategies for acute toxoplasmosis. Sequences and probes derived from the T. gondii DHFR-TS gene have been employed to examine the predicted structure of the enzyme in wild-type parasites, and to develop functional vectors for transient and stable molecular transformation of the parasite. Clinical cases of antifolate- resistant toxoplasmosis and similar mutants isolated in the lab will be screened for differences in sensitivity to combined pyrimethamine/sulfonamide treatment (in addition to sensitivity to pyrimethamine or sulfa alone), and examined for possible DHFR-TS mutations or altered gene expression. Mechanisms of DHFR-independent resistance will be identified by genetic means. DHFR-TS enzyme function will be assessed in transgenic parasites bearing mutations derived from four sources: naturally occurring allelic variation, mutations identified from drug- resistant laboratory strains and clinical isolates, modeling studies on the T. gondii enzyme, and point mutations thought to be associated with antifolate resistance in field isolates of the related parasite Plasmodium falciparum (malaria). Findings from this research will be combined with structure/function studies on the recombinant DHFR-TS enzyme, to assist in the design of novel antifolates with improved activity against the parasite. Because of the tremendous power of genetic techniques for the identification and analysis of drug targets, available transformation schemes will be modified to permit: (1) Targeted gene disruption and replacement, used in this context to examine mutations at the DHFR-TS locus; (2) Insertional mutagenesis and marker rescue, used to clone potential targets for therapeutic intervention and negative selectable markers for gene replacement studies; (3) Molecular cloning by complementation, focusing on DHFR-independent antifolate resistance genes which appear to be particularly important for pyrimethamine resistance in Toxoplasma; and (4) Optimal expression and overexpression of recombinant protein. In addition to their value for the analysis of folate metabolism and drug resistance, these tools should be broadly applicable to other studies on the biology and biochemistry of Toxoplasma.