ROLE OF MITOCHONDRIAL DNA REPAIR ENZYME IN DRUG RESISTANCE & DVL'T IN T GONDII

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 寄生虫弓形虫是温血动物中最广泛和最成功的原生动物寄生虫之一，并且可以在人类中致病。我们最近分离了一个T。弓形虫突变体，对包括抗球虫莫能菌素和盐霉素在内的几种药物以及烷化剂N-甲基-N-亚硝基脲表现出强烈的抗性。我们已经确定，这种耐药突变体在一种新的基因TgMSH-1中被破坏，该基因编码与错配修复酶MutS同源的蛋白质。 在野生型T.弓形虫菌株重现了莫能菌素耐药表型，并且具有TgMSH-1功能拷贝的原始突变体的互补恢复了药物敏感性，这表明TgMSH-1的破坏直接导致了弓形虫的耐药性。刚地。 我们还表明，TgMSH-1定位于寄生虫的胞体。有趣的是，在其他生物体中，MutS同源物（MSH）被认为参与指导细胞凋亡和细胞周期停滞以响应某些压力，缺乏MSH的癌细胞对DNA损伤药物具有抗性。 对不同压力的反应是细胞内寄生虫（如T。刚地。这一点在某些应激因素，如pH变化、免疫原性反应和线粒体抑制，诱导T。弓形虫转化为包囊形式，以逃避免疫系统和其他应激诱导条件。 因此，我们假设TgMSH-1是弓形虫线粒体应激反应信号传导的中心，并且在寄生虫发育和发病机制中起作用。 目的1：鉴定TgMHS 1在药物应答中的信号伙伴。 我们证明了T.弓形虫以MSH依赖性方式对莫能菌素敏感。 我们的目标是通过鉴定其功能伴侣和研究该途径激活的特定细胞效应来了解TgMHS 1在该药物应答过程中的作用。具体而言，我们将： + 鉴定和表征与TgMSH-1直接相互作用的蛋白质。 + 分析野生型和TgMSH 1敲除寄生虫中参与线粒体应激途径的信号分子对药物治疗的响应的表达和激活。 + 研究细胞凋亡和细胞周期在TgMSH-1依赖性药物应答中的作用。 目的2：确定TgMSH 1在寄生虫发育和发病机制中的作用。 在感染的动物中，T.弓形虫从快速分裂的速殖子分化为成囊的潜伏的缓殖子以逃避免疫应答。 虽然分化是这种寄生虫的传播和发病机制的关键，但很少有人知道触发发育变化的信号传导机制。 在组织培养中，T.弓形虫将响应于低pH和线粒体抑制剂而分化为缓殖子形式。 鉴于TgMSH-1的定位及其在应激信号传导中的潜在作用，我们将研究TgMSH-1在组织培养和体内缓殖子发育中的作用。 +敲除T中的TgMSH-1。适合发育和毒力研究的弓形虫菌株。 +测定突变株在组织培养中转化为缓殖子阶段的能力。 +测定用敲除菌株感染的小鼠体内包囊形成、毒力和寄生虫分布。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The parasite Toxoplasma gondii is one of the most widespread and successful protozoan parasites of warm-blooded animals and can be pathogenic in humans. We have recently isolated a T. gondii mutant that exhibits strong resistance to several drugs including the anti-coccidians monensin and salinomycin, and the alkylating agent N-methyl-N-nitrosourea. We have determined that this drug resistance mutant is disrupted in a novel gene, TgMSH-1, which encodes a protein homologous to the mismatch repair enzyme, MutS. A directed knock-out of this gene in a wild-type T. gondii strain recapitulates the monensin-resistant phenotype, and complementation of the original mutant with a functional copy of TgMSH-1 restores drug sensitivity, indicating that the disruption of TgMSH-1 is directly responsible for conferring drug resistance in T. gondii. We have also shown that TgMSH-1 localizes to the mitochondrion of the parasite. Interestingly, in other organisms, MutS Homologs (MSHs) are believed to be involved in directing the cell to apoptosis and cell cycle arrest in response to certain streses, and cancer cells lacking MSHs are resistant to DNA damaging drugs. Responding to different stresses is key to the survival of an intracellular parasite such as T. gondii. This is most evident in the fact that certain stressors, such as pH changes, immunogenic response and mitochondrial inhibition, induce T. gondii to convert to an encysted form as to escape the immune system and other stress inducing conditions. Thus, it is our hypothesis that TgMSH-1 is central in mitochondrial stress response signaling in Toxoplasma and that it plays a role in parasite development and pathogenesis. Aim 1: Identify signaling partners of TgMHS1 in drug response. We have shown that T. gondii is sensitive to monensin in a MSH dependent manner. It is our goal to understand the role of TgMHS1 in this drug response process by identifying its functional partners and by studying the specific cellular effects of activation of this pathway. Specifically we will: + Identify and characterize proteins that directly interact with TgMSH-1. + Analyze expression and activation of signaling molecules involved in the mitochondrial stress pathway in wild type and TgMSH1 knock out parasite in response to drug treatment. + Study the role of apoptosis and cell cycle in the TgMSH-1 dependent response to drugs. Aim 2: Determine role of TgMSH1 in parasite development and pathogenesis. In an infected animal, T. gondii differentiates from the rapidly dividing tachyzoite to the encysted latent bradyzoite as to evade the immune response. While differentiation is key to the transmission and pathogenesis of this parasite, very little is known of the signaling mechanisms involved in triggering developmental changes. In tissue culture, T. gondii will differentiate to the bradyzoite form in response to low pH and mitochondrial inhibitors. Given the localization of TgMSH-1 and its potential role in stress signaling we will investigate the role of TgMSH-1 in bradyzoite development in tissue culture and in vivo. +Knock out TgMSH-1 in a T. gondii strain suitable for developmental and virulence studies. +Determine ability of mutant strain to convert to the bradyzoite stage in tissue culture. +Determine in vivo cyst formation, virulence and parasite distribution in mice infected with the knock-out strain.