Identifaction of the genetic basis of primaquine resistance in malaria parasites

描述（由申请人提供）：2009年，据报告约有2.25亿疟疾病例，造成781 000人死亡，其中大多数是5岁以下儿童。在引起疟疾的5种疟原虫中，恶性疟原虫的致死率最高，因此研究最多。这导致恶性疟原虫的流行率下降，但在许多已经根除恶性疟疾的国家，间日疟疾仍然流行。间日疟原虫是疟原虫在地理上分布最广的一种，世界上三分之一以上的人口面临感染的危险。间日疟原虫与恶性疟原虫的独特之处在于，间日疟原虫能够在肝脏中持续处于休眠、无症状阶段，在初次感染后数月至数年再次出现，导致临床疾病。这些复发事件有助于维持流行地区的传播。伯氨喹是唯一获得许可的药物，可以消除潜伏的肝期间日疟原虫，从而防止复发。在努力控制疟疾时，耐药性的发展一直是面临的障碍，有报告称对伯氨喹产生了耐药性。尽管使用了50多年，但伯氨喹的作用机制尚不清楚。本研究申请的目的是确定疟原虫对伯氨喹耐药的分子决定因素，这些决定因素的确定将为开发针对肝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，因此我将使用一种啮齿动物的疟疾模型，伯氏疟原虫。首先，我将开发一种全基因组测序方法来检测柏氏假体的遗传多态性。然后，我将通过在增加伯氨喹浓度的小鼠中连续传递伯氏疟原虫的血液阶段来进化寄生虫对伯氨喹的抗性。然后可以将开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。然后可以测试这条抗性线，看看它是否也具有抗性

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.