Identifaction of the genetic basis of primaquine resistance in malaria parasites

描述（由申请人提供）：2009年报告了大约2.25亿例疟疾病例，导致781，000人死亡，其中大多数是5岁以下的儿童。在引起疟疾的5种疟原虫中，恶性疟原虫的病死率最高，因此也是研究最多的疟原虫。这导致恶性疟原虫的流行率下降，但在许多已根除恶性疟原虫疟疾的国家，间日疟仍然是地方病。间日疟原虫是地理分布最广的疟原虫属，超过三分之一的世界人口面临感染的风险。间日疟原虫与恶性疟原虫的独特不同之处在于，间日疟原虫能够在肝脏中持续处于休眠、无症状阶段，其可以在初始感染后数月至数年重新出现以引起临床疾病。这些复发事件有助于在流行地区维持传播。伯氨喹是唯一获得许可的药物，将消除休眠的肝脏阶段间日疟原虫寄生虫，从而防止复发。抗药性的发展是控制疟疾和伯氨喹抗药性的一个长期障碍。尽管伯氨喹已经使用了50多年，但其作用机制尚不清楚。这项研究金申请的目的是确定疟原虫对伯氨喹耐药性的分子决定因素。确定这些决定因素将为开发针对肝脏期间日疟原虫的新抗疟药物提供信息。为了实现这一目标，我将使用一个体内模型来发展伯喹疟原虫的耐药性。间日疟原虫不能在体外培养，因此我将使用啮齿动物疟疾模型伯氏疟原虫。首先，我将开发一种全基因组测序方法来检测伯氏疟原虫的遗传多态性。然后，我将通过在用递增浓度的伯氨喹处理的小鼠中连续传代伯氏疟原虫的血液阶段来进化寄生虫对伯氨喹的抗性。然后可以将起始进化菌株的全基因组序列与抗性进化菌株进行比较，以确定与伯氨喹抗性相关的遗传修饰。然后可以测试该抗性品系，以查看其在植物中是否也是抗性的。 通过让感染了耐药品系的蚊子以用伯氨喹治疗过的小鼠为食，随后观察是否会出现血液阶段的感染，来观察肝脏阶段的感染。伯氨喹耐药性的分子决定因素的鉴定，然后可以用来建立分子标记，可以检测耐药的现场分离。这些分子决定因素也将为药物发现工作提供信息，以确定其他肝脏阶段的抗疟药物。这两项研究结果的应用将有助于消灭间日疟。

DESCRIPTION (provided by applicant): In 2009 approximately 225 million cases of malaria were reported resulting in 781,000 deaths, mostly in children under 5 years old. Of the 5 Plasmodium species that cause malaria, P. falciparum has the highest case fatality rate and is subsequently the most studied. This has resulted in a decrease in the prevalence of P. falciparum, yet in many of the countries where falciparum malaria has been eradicated, vivax malaria remains endemic. P. vivax is the most geographically widespread of the Plasmodium spp., and over one-third of the world's population is at risk of infection. P. vivax is uniquely different from P. falciparum in that P. vivax is able to persist in a dormant, asymptomatic stage in the liver that can reemerge months to years after initial infection to cause clinical disease. These relapse episodes help to sustain transmission in endemic regions. Primaquine is the only licensed drug that will eliminate dormant liver-stage P. vivax parasites and thus prevent relapse. The development of resistance is a consistent hurdle faced when trying to control malaria and primaquine resistance has been reported. Despite being in use for over 50 years, the mechanism of action of primaquine is unknown. The objective of this fellowship application is to identify the molecular determinants of primaquine resistance in Plasmodium spp. Identification of these determinants will inform the development of new antimalarials that target liver-stage P. vivax. To accomplish this objective, I will use an in vivo model to evolve primaquine resistance in Plasmodium parasites. P. vivax cannot be cultured in vitro, therefore I will use a rodent model of malaria, P. berghei. First I will develop a whole-genome sequencing approach to detect genetic polymorphisms in P. berghei. I will then evolve parasite resistance to primaquine by serial passage of blood stages of P. berghei in mice treated with increasing concentrations of primaquine. The whole-genome sequence of the starting evolution strain can then be compared with the resistance-evolved strain to determine genetic modifications that are associated with primaquine resistance. This resistant line can then be tested to see if it is also resistant in the liver stage by allowing mosquitoes infected with the resistant line to feed on mice treated with primaquine and subsequently see if a blood-stage infection develops. Identification of the molecular determinants of primaquine resistance can then be used to establish molecular markers that can detect resistance in field isolates. These molecular determinants will also inform drug discovery efforts to identify additional liver-stage acting antimalarials. Both applications of the proposed results will aide in the eradication of vivax malaria.