Combining structural biology and genetics to understand the function of a multi-gene family expanded in neglected human malaria parasites

全世界三分之一以上的人口面临感染疟疾的风险，每年有2亿多病例，导致近50万人死亡。疟疾是由多种疟原虫寄生虫引起的，它们通过按蚊在人与人之间传播。大多数疟疾研究都集中在恶性疟原虫上，恶性疟原虫是非洲的主要物种，也是造成大多数疟疾死亡的原因。然而，恶性疟原虫与非洲以外引起大多数疟疾病例的物种只有相对较远的关系。这些属于不同的进化组，其中包括间日疟原虫，全球疟疾的第二大主要原因，以及诺氏疟原虫，一种主要感染东南亚猴子的寄生虫，但也可以传播给人类，在那里它可能导致严重的疾病和死亡。这组寄生虫的生物学，统称为疟原虫亚属，是显着的探索不足，部分原因是只有P.诺氏可以在实验室环境中常规生长和实验操作。在这个项目中，我们将集中在一组蛋白质，色氨酸丰富的抗原（TRAgs），发现在所有疟原虫寄生虫，但在疟原虫亚属内的物种中存在显着较高的数字。间日疟原虫基因组包含38个编码TRAg的基因，而诺氏疟原虫包含29个TRAg-相比之下，主要的非洲疟疾寄生虫恶性疟原虫只包含3个。这种蛋白质家族的功能尚不清楚，但它们在疟原虫亚属中扩展的事实表明它们在这些被忽视的人类疟疾寄生虫物种中特别重要。我们最近解决了一种间日疟原虫TRAG的三维蛋白质结构，揭示了它与其他生物体中的蛋白质之间的相似性，这些蛋白质与构成所有细胞膜的脂质结合，包括疟原虫寄生虫在其中生长的红细胞。我们随后证实，间日疟原虫和诺氏疟原虫TRAg都可以直接与脂质结合，并且在一种诺氏疟原虫TRAg的情况下，这种相互作用参与了这些寄生虫识别和侵入人类红细胞的过程。这是第一次提出了一个明确的假设TRAg功能-他们结合到脂质和参与操纵人类红细胞膜，无论是在过程中或之后的invasion.We将探讨这一假设进行了大量的TRAgs在疟原虫亚属寄生虫物种的第一次系统性研究。我们将专注于P. knowlesi，它可以在实验室中生长并进行遗传操作，这使得探测单个TRAg的位置和功能成为可能。我们将扩展我们的结构研究，探索不同的TRAg是否对不同的脂质具有特异性，并解决与人类红细胞膜中发现的脂质复合的TRAg蛋白的结构。我们将确定哪些TRAg基因在诺氏毕赤酵母实验室菌株中表达，并使用先进的显微镜建立最丰富的TRAg蛋白的特异性定位。我们还将利用最新的技术进步来创建诺氏疟原虫株系，其中这些高表达的TRAg基因已使用CRISPR-Cas9基因组编辑删除，并监测对寄生虫生长和入侵的影响。总的来说，这项研究将提供关于被忽视的疟疾寄生虫物种中未充分研究的蛋白质家族功能的关键信息。

More than a third of the world's population is at risk of contracting malaria and there are more than 200 million cases each year, leading to nearly half a million deaths. Malaria is caused by multiple species of Plasmodium parasite, which are spread from person to person by Anopheles mosquitoes. Most malaria research has focussed on Plasmodium falciparum, the dominant species in Africa and the one that causes the majority of malaria deaths. However, P. falciparum is only relatively distantly related to the species that cause most malaria cases outside Africa. These fall into a different evolutionary group which includes both Plasmodium vivax, the second most significant cause of malaria globally, and Plasmodium knowlesi, a parasite that predominantly infects monkeys in Southeast Asia but can also be transmitted to humans where it can cause severe disease and death. The biology of this group of parasites, collectively referred to as the Plasmodium subgenus, is significantly underexplored, in part because only P. knowlesi can be routinely grown and experimentally manipulated in a lab setting.In this project we will focus on a group of proteins, Tryptophan-rich antigens (TRAgs) that are found in all Plasmodium parasites but are present in significantly higher numbers in species within the Plasmodium subgenus. The P. vivax genome contains 38 genes that encode TRAgs, while P. knowlesi contains 29 TRAgs - by contrast the major African malaria parasite P. falciparum contains only 3. The function of this protein family is unknown, but the fact that they are expanded in the Plasmodium subgenus suggests that they are particularly important in these neglected human malaria parasite species. We have recently solved the three-dimensional protein structure of one Plasmodium vivax TRAG, which revealed similarities between it and proteins in other organisms that bind to the lipids that make up the membranes of all cells, including the red blood cells that Plasmodium parasites grow inside. We subsequently confirmed that both P. vivax and P. knowlesi TRAgs can bind directly to lipids, and in the case of one P. knowlesi TRAg, that this interaction is involved in the process by which these parasites recognise and invade human red blood cells. This for the first time raises a clear hypothesis for TRAg function - that they bind to lipids and are involved in manipulating human red blood cell membranes, either during or after the process of invasion.We will explore this hypothesis by carrying out the first systematic study of a large number of TRAgs in a Plasmodium subgenus parasite species. We will focus on P. knowlesi, which can be grown in the lab and genetically manipulated, which makes it possible to probe the location and function of individual TRAgs. We will extend our structural studies, exploring whether different TRAgs have specificity for different lipids, and solving the structure of TRAg proteins in complex with lipids found in the membrane of human red blood cells. We will identify which TRAg genes are expressed in the lab strain of P. knowlesi and establish the specific localisation of the most abundant TRAg proteins using advanced microscopy. We will also leverage recent technical advances to create P. knowlesi parasite lines in which these highly expressed TRAg genes have been deleted using CRISPR-Cas9 genome editing and monitor the effect on parasite growth and invasion. Overall, this research will provide critical information about the function of an understudied protein family in a neglected malaria parasite species.