Identifaction of the genetic basis of primaquine resistance in malaria parasites

说明(申请人提供)：2009年，报告的疟疾病例约为2.25亿例，导致781,000人死亡，其中大部分是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。这导致恶性疟的流行率下降，但在许多已根除恶性疟疾的国家，间日疟仍是地方病。间日疟原虫是间日疟原虫中地理分布最广的一种，世界上超过三分之一的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持续处于休眠、无症状的阶段，在最初感染后几个月至几年内重新出现，导致临床疾病。这些复发病例有助于维持流行地区的传播。伯氨喹是唯一一种获得许可的药物，可以消除休眠的肝期间日疟原虫，从而防止复发。抗药性的发展是试图控制疟疾时面临的一个一贯障碍，据报道，伯喹抗药性也是如此。尽管伯喹已经使用了50多年，但其作用机制尚不清楚。本次联谊会申请的目的是鉴定疟原虫对伯喹耐药性的分子决定因素。这些决定因素的识别将为针对肝期间日疟原虫的新抗疟药的开发提供信息。为了实现这一目标，我将使用体内模型来进化疟原虫对伯氨喹的耐药性。间日疟原虫不能在体外培养，因此我将使用疟疾的啮齿动物模型--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拟青霉的遗传多态。然后，我将通过伯氏疟原虫血液阶段的一系列传代来进化寄生虫对伯克喹的抵抗力，用浓度增加的伯克喹治疗的小鼠。然后，可以将开始进化的菌株的全基因组序列与抗药性进化菌株进行比较，以确定与伯喹耐药性相关的遗传修饰。然后可以测试这个抗性品系，看看它是否也在 通过允许感染了抗药性品系的蚊子以伯喹处理过的小鼠为食，然后观察是否发生血液期感染，可以使蚊子进入肝期。对伯喹抗性分子决定因素的鉴定可用于建立能够检测田间菌株抗药性的分子标记。这些分子决定因素也将为药物发现工作提供信息，以识别更多的肝期作用抗疟疾药物。这两项拟议成果的应用都将有助于根除间日疟。

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.