T. GONDII CHLOROQUINE RESISTANCE TRANSPORTER AND REDOX

 描述(申请人提供)：弓形虫、隐孢子虫和疟疾寄生虫通过它们的系统发育关系和它们对艾滋病毒/艾滋病患者的威胁生命的影响而统一。疟疾对氯喹的抗性是由氯喹抗性转运体(PfCRT)的突变授予的，PfCRT是一种完整的膜蛋白，能够将氯喹从疟疾消化液泡(DV)中穿梭出来，在那里它抑制血红素的解毒。我们最近发现，弓形虫表达PfCRT的同源基因，称为TgCRT，并且这个假定的转运蛋白与弓形虫的消化细胞器有关，称为空泡室或VAC。TgCRT的靶向缺失会导致VAC肿胀，影响寄生虫的复制，减弱寄生虫的毒力，并导致慢性感染期间神经组织囊肿减少90%。在疟疾中，CRT被认为与谷胱甘肽的运输和氧化还原电位的调节有关，但这些研究是用PfCRT的氯喹抗性突变体完成的。无法去除PfCRT也阻碍了了解这种重要蛋白质的天然功能的努力。几乎对中国的氧化还原法规一无所知。 尽管弓形虫在抵抗感染期间的氧化应激方面起到了作用。在这里，我们建议利用弓形虫的遗传易感性来剖析TgCRT的功能，方法是首次在弓形虫体内安装能够测量相关隔室中氧化还原状态的生物传感器，并测试TgCRT消融对氧化还原电位和氧化应激敏感性的调节的影响。我们还将确定与TgCRT和/或 作为一种确定其与其他可能参与氧化还原调节的蛋白质的关系的手段。证明TgCRT对氧化应激具有抵抗力将为评估氧化应激在控制弓形虫感染中的重要性创造新的途径。表明弓形虫VAC有助于氧化还原调节，将加强该细胞器与疟疾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产生了影响，这些途径影响了艾滋病毒/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.