Functional analysis of Plasmodium vivax drug resistance polymorphisms

项目摘要 疟疾的抗药性是治疗、控制和消灭疟疾的最大障碍之一。推定 在间日疟原虫中发现了耐药性转运蛋白的多态性， 所有导致人类疟疾的物种的基因。然而，这些多态性赋予氯喹的能力 耐药性或由这些突变赋予的耐药性的程度仍然未知。我们之间的差距 知识主要是由于缺乏体外培养系统，强大的药物测定和反向遗传学 用于间日疟原虫的研究 在这个建议中，我们试图了解多态性在假定的耐药转运蛋白中的影响， 在间日疟原虫的分子流行病学研究中发现的。我们将利用 猕猴寄生虫诺氏疟原虫用于分析天然存在的间日疟原虫多态性，由于 它与间日疟原虫亲缘关系密切，而且它作为反向遗传学和药物系统的多功能性 敏感性测定我们将直接在诺氏毕赤酵母中利用基于CRISPR/Cas9的方法进行功能性重组。 间日疟原虫药物转运蛋白基因及其多态性分析。我们最近对一个P. 诺氏寄生虫株在人类红细胞中生长，这非常适合我们的研究。 间日疟原虫特异性药物转运蛋白多态性的反向遗传分析对我们的研究至关重要。 了解它们与抗疟药耐药性的相关性，制定战略， 耐药性扩散或逆转，以及它们作为群体水平耐药性标志物的验证 研究和监测控制和消除措施。我们的工作也将建立P. knowlesi作为一个 强大的异源平台，用于研究与间日疟原虫相关的许多过程中涉及的关键基因。

Project Summary Drug resistance in malaria is one of the most formidable barriers to treatment, control and elimination. Putative polymorphisms in drug resistance transporters have been identified in Plasmodium vivax, the most widespread of all of the species causing human malaria. However, the ability of these polymorphisms to confer chloroquine resistance, or the magnitude of the resistance conferred by these mutations remains unknown. This gap in our knowledge is largely due to the absence of an in vitro culture system, robust drug assays, and reverse genetics for the study of P. vivax. In this proposal, we seek to understand the impact of polymorphisms in putative drug resistance transporters that have been identified in molecular epidemiological studies of P. vivax. We will take advantage of the macaque parasite Plasmodium knowlesi for the analysis of naturally occurring P. vivax polymorphisms, due to its close phylogenetic relatedness to P. vivax, and its versatility as a system for reverse genetics and drug susceptibility assays. We will utilize CRISPR/Cas9-based approaches directly in P. knowlesi for the functional analysis of the P. vivax drug transporter genes and their polymorphisms. We recently culture-adapted a P. knowlesi parasite line to grow in human red blood cells, which is ideally suited for our studies. Reverse genetic analyses of specific drug transporter polymorphisms of P. vivax will be critical to our understanding of their relevance to antimalarial resistance, the development of strategies for limiting the spread or reversal of drug resistance, and their validation as drug resistance markers in population level studies and surveillance for control and elimination measures. Our work will also establish P. knowlesi as a powerful heterologous platform for the study of critical genes involved in many processes relevant to P. vivax.