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

结合结构生物学和遗传学来了解在被忽视的人类疟疾寄生虫中扩展的多基因家族的功能

• 批准号: MR/Y012895/1
• 负责人: Julian Rayner
• 金额: $ 116.09万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FY012895%2F1
• 关键词: Combining; structural; biology; genetics; understand

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.

世界上超过三分之一的人口面临感染疟疾的风险，每年有超过 2 亿病例，导致近 50 万人死亡。疟疾是由多种疟原虫寄生虫引起的，这些寄生虫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，它是非洲的主要物种，也是导致大多数疟疾死亡的原因。然而，恶性疟原虫与导致非洲以外大多数疟疾病例的物种关系较远。它们属于不同的进化组，其中包括间日疟原虫（全球疟疾的第二大原因）和诺氏疟原虫（一种主要感染东南亚猴子的寄生虫​​，但也可以传播给人类，导致严重疾病和死亡） 。这组寄生虫（统称为疟原虫亚属）的生物学尚未得到充分研究，部分原因是只有诺氏疟原虫可以在实验室环境中常规生长和实验操作。在这个项目中，我们将重点关注一组蛋白质富含色氨酸的抗原 (TRAgs)，存在于所有疟原虫寄生虫中，但在疟原虫亚属物种中的数量明显更高。间日疟原虫基因组包含 38 个编码 TRAgs 的基因，而诺氏疟原虫包含 29 个 TRAgs - 相比之下，主要的非洲疟疾寄生虫恶性疟原虫仅包含 3 个。该蛋白质家族的功能尚不清楚，但事实上它们已扩展疟原虫亚属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。我们最近解析了一种间日疟原虫 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内部生长的红细胞。我们随后证实，间日疟原虫和诺氏疟原虫 TRAgs 都可以直接与脂质结合，并且就一种诺氏疟原虫 TRAg 而言，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。这首次提出了 TRAg 功能的明确假设 - 它们与脂质结合并参与在入侵过程中或之后操纵人类红细胞膜。我们将通过进行第一个系统研究来探索这一假设疟原虫亚属寄生虫物种中的大量 TRAgs。我们将重点关注诺氏假单胞菌，它可以在实验室中生长并进行基因操作，这使得探测单个 TRAgs 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TRAg蛋白与人红细胞膜中发现的脂质复合物的结构。我们将鉴定哪些 TRAg 基因在诺氏疟原虫实验室菌株中表达，并使用先进的显微镜确定最丰富的 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寄生虫系，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。总体而言，这项研究将提供有关被忽视的疟疾寄生虫物种中待研究的蛋白质家族功能的关键信息。