Molecular basis of antimalarial drug resistance in Plasmodium vivax

项目摘要 由间日疟原虫感染引起的疟疾是全世界巨大的公共卫生负担， 和严重发病的原因。抗疟药氯喹是治疗P。 间日疟原虫在大多数国家，并已被证明非常有效。然而，氯喹抗性（CQR）间日疟原虫 感染被广泛报道，严重阻碍了病例管理、疟疾控制工作， 消除方案。大多数关于抗药性机制的研究都集中在恶性疟原虫上，而恶性疟原虫的抗药性机制研究较少。 对间日疟原虫的研究仍然很少;然而，间日疟原虫对根除构成了主要障碍。分子 间日疟原虫CQR和对其他抗疟药耐药的决定因素尚不清楚，主要是由于缺乏 体外培养，排除了反向遗传学和强大的药物测定。几个候选药物转运蛋白基因 在间日疟原虫中鉴定出具有序列和表达水平多态性， CQR和/或对其他抗疟药耐药。这些基因包括pvmdr 1、pvcrt-o和pvmrp 1， 恶性疟原虫pfmdr 1，pfcrt和pfmrp 1基因，分别是恶性疟原虫pfmdr 1，pfcrt和pfmrp 1基因的关键决定因素， 抗疟药敏感性的研究间日疟原虫对氯喹的体外敏感性与特异性 P.间日疟原虫药物转运蛋白多态性在一些但不是所有的研究。目前尚不清楚这些差异是否 代表寄生虫多样性和药物暴露史的区域差异;或由技术 差异，因为迄今为止还没有基因验证。在这里，我们提出了一个全面的分析， 间日疟原虫的耐药性多态性，利用两种利用人畜共患猕猴的新方法 诺氏疟原虫（Plasmodium knowlesi）在遗传学上比恶性疟原虫更接近间日疟原虫， 具有较高转染效率的上级体外遗传学;并且重要的是， 抗疟药敏感性我们的目标是利用尖端的进化基因组和实验 前向遗传筛选，以确定间日疟原虫耐药性的假定决定因素。我们随后将使用 体外反向遗传方法在诺氏疟原虫中功能性评估优先间日疟原虫遗传的重要性 基因多态性介导抗疟药物耐药性。我们假设这些多态性， 单独或组合，已在世界特定地区通过抗间日疟原虫的抗疟用途而被选择。 鉴定介导对氯喹的不同敏感性水平的特异性多态性， 目前临床使用的其他抗疟化合物，包括青蒿素及其伙伴药物， 它们在监测、分子流行病学研究和设计延长艾滋病毒/艾滋病传播的战略方面的用途， 这些药物的效用。

Project Summary Malaria caused by infection with Plasmodium vivax is an enormous public health burden throughout the world, and the cause of significant morbidity. The antimalarial drug chloroquine is the first line of drug treatment for P. vivax in most countries, and has proven highly efficacious. However, chloroquine resistant (CQR) P. vivax infections have been widely reported, seriously hampering case management, malaria control efforts, and elimination programs. Most work on resistance mechanisms has focused on Plasmodium falciparum, while P. vivax remains poorly studied; nevertheless, P. vivax poses a major impediment to eradication. The molecular determinants of CQR and resistance to other antimalarials in P. vivax remain unclear, largely due to the lack of in vitro culture, precluding reverse genetics and robust drug assays. Several candidate drug transporter genes have been identified in P. vivax with polymorphism in sequence and expression level that could be associated with CQR and/or resistance to other antimalarials. These genes include pvmdr1, pvcrt-o and pvmrp1, orthologs of the Plasmodium falciparum pfmdr1, pfcrt and pfmrp1 genes, respectively, that are key determinants of antimalarial susceptibility in P. falciparum. Ex vivo P. vivax susceptibility to chloroquine is associated with specific P. vivax drug transporter polymorphisms in some but not all studies. It is not clear whether these discrepancies represent regional differences in parasite diversity and history of exposure to drugs; or result from technical differences, as no genetic validation has hitherto been possible. Here, we propose a comprehensive analysis of drug-resistance polymorphisms of P. vivax, utilizing two novel approaches leveraging the zoonotic macaque parasite Plasmodium knowlesi, which is much closer to P. vivax phylogenetically than is P. falciparum; possesses superior in vitro genetics with higher transfection efficiencies; and importantly, permits robust determinations of antimalarial drug susceptibility. We aim to utilize both cutting-edge evolutionary genomic and experimental forward genetic screening to identify putative determinants of P. vivax drug-resistance. We will subsequently use in vitro reverse genetic methods in P. knowlesi to functionally assess the importance of prioritized P. vivax genetic polymorphisms in mediating antimalarial drug-resistance. We hypothesize that these polymorphisms, either singly or in combination, have been selected in specific regions of the world by antimalarial use against P. vivax. The identification of specific polymorphisms that mediate different levels of susceptibility to chloroquine, and other antimalarial compounds in current clinical use, including artemisinin and its partner drugs, will be key to their use in surveillance, molecular epidemiological studies, and the design of strategies to prolong the usefulness of these drugs.