T. GONDII CHLOROQUINE RESISTANCE TRANSPORTER AND REDOX

 描述（由申请方提供）：弓形虫、隐孢子虫属和疟疾寄生虫因其致病关系和对HIV/AIDS患者的生命威胁而统一。疟疾对氯喹的抗性是由氯喹抗性转运体（PfCRT）的突变赋予的，PfCRT是一种整合的膜蛋白，能够将氯喹穿梭出疟疾消化泡（DV），在那里它抑制血红素解毒。我们最近发现T.弓形虫表达PfCRT的直系同源物，称为TgCRT，并且这种推定的转运蛋白与T.弓形虫消化细胞器称为空泡室或VAC。TgCRT的靶向缺失导致VAC肿胀，影响寄生虫复制，减弱寄生虫毒力，并导致慢性感染期间神经组织囊肿减少90%。在疟疾中，CRT与谷胱甘肽的转运和氧化还原电位的调节有关，但这些研究是用PfCRT的氯喹抗性突变体进行的。无法消融PfCRT也阻碍了理解这种重要蛋白质的天然功能的努力。事实上，人们对生物体内的氧化还原调节机制一无所知， T.弓形虫，尽管它的作用是对抗感染过程中的氧化应激。在此，我们建议利用T. gondii，首次在弓形虫体内实现了TgCRT的功能。弓形虫氧化还原生物传感器，能够测量相关隔室中的氧化还原状态，并测试TgCRT消融对氧化还原电位调节和对氧化应激敏感性的影响。我们还将鉴定与TgCRT和/或 作为定义其与其他潜在参与氧化还原调节的蛋白质的关系的手段。证明TgCRT能抵抗氧化应激将为评估氧化应激在控制T细胞中的重要性创造新的途径。弓形虫感染表明T.弓形虫VAC有助于氧化还原调节，将加强这种细胞器和疟疾DV之间的进化联系。总之，这些发现对VAC和氧化还原途径的未来拮抗作用具有影响，以影响艾滋病毒/艾滋病相关疾病的结果，包括弓形虫病，隐孢子虫病和疟疾。

 DESCRIPTION (provided by applicant): Toxoplasma gondii, Cryptosporidium spp and malaria parasites are unified by their phylogenic relationships and by their life threatening impact on individuals with HIV/AIDS. Malaria resistance to chloroquine is conferred by mutations in chloroquine resistance transporter (PfCRT), an integral membrane protein capable of shuttling chloroquine out of the malaria digestive vacuole (DV) where it inhibits heme detoxification. We recently discovered that T. gondii expresses an ortholog of PfCRT termed TgCRT, and that this putative transporter is associated with a T. gondii digestive organelle termed the vacuolar compartment or VAC. Targeted deletion of TgCRT causes bloating of the VAC, affects parasite replication, attenuates parasite virulence and results in a 90% decrease in neural tissue cysts during chronic infection. In malaria, CRT has been implicated it in the transport of glutathione and the regulation of redox potential, but these studies were done with chloroquine resistant mutants of PfCRT. The inability to ablate PfCRT has also impeded efforts to understand the native function of this important protein. Virtually nothing is known about the redox regulation in T. gondii despite its role in combatting oxidative stress during infection. Herein we propose to exploit the genetic tractability of T. gondii to dissect the function of TgCRT by implementing for the first time in T. gondii redox biosensors capable of measuring redox status in relevant compartments and testing the impact of TgCRT ablation on the regulation of redox potential and susceptibility to oxidative stress. We will also identify proteins that associate with TgCRT and/or are proximal to it as a means of defining its relationships with other proteins potentially involve in redox regulation. Demonstrating that TgCRT confers resistance to oxidative stress will create new avenues to assessing the importance of oxidative stress in controlling T. gondii infection. Showing that the T. gondii VAC contributes to redox regulation will strengthen the evolutionary link between this organelle and the malaria DV. Together the findings have implications for future antagonism of the VAC and redox pathways to affect the outcome of HIV/AIDS associated diseases including toxoplasmosis, cryptosporidiosis and malaria.