Dissecting the role of host receptor context and cytoskeletal disruption in malaria parasite invasion

每年，全世界有2亿多人感染疟疾，50多万人死于这种疾病，其中大多数是5岁以下儿童。导致疟疾的寄生虫通过附着在循环红细胞的表面并穿透红细胞而存活，然后在红细胞中繁殖。红细胞（rbc）具有高度特化的膜结构，这是由于质膜内蛋白质之间复杂的相互作用，以及被称为细胞骨架的灵活的蛋白质丝底层网络，使细胞能够通过毛细血管。为了穿透坚固的红细胞膜，寄生虫附着在细胞表面的蛋白质上，诱导这种膜-细胞骨架结构的协调和局部破坏，以促进入侵，这一过程也与使红细胞挤压毛细血管所需的短暂破坏有一些相似之处。尽管一些关键蛋白已经被证明参与或需要成功的入侵，但在大多数情况下，对这些宿主细胞蛋白在入侵过程中实际发挥的作用的了解严重或完全缺乏。从宿主红细胞的角度研究寄生虫入侵机制的最大障碍之一是无法直接操纵这些细胞中的蛋白质表达。与大多数细胞不同，红细胞不含DNA，这阻碍了基因技术在其他细胞类型中用于操纵蛋白质表达的应用。最近在红细胞发育（红细胞生成）领域取得的进展改变了这一点。现在有可能从一种不朽的细胞系中培养出易受引起严重疟疾的寄生虫侵袭的年轻红细胞（网织红细胞），这种细胞系允许红细胞前体（红细胞）无限期生长或安全储存。令人兴奋的是，我们已经证明，可以使用慢病毒和基因编辑技术来操纵这些有核细胞中的蛋白质表达，从而在细胞失去细胞核成为红细胞后维持这些变化。这项技术可用于防止已知参与入侵的特定RBC蛋白的表达，也允许它们被改变或替换为突变版本，其中膜内的定位，与其他蛋白质的相互作用或蛋白质本身的特性已经改变。这项技术为产生具有罕见甚至独特特征的红细胞打开了大门，这些红细胞可用于探索哪些蛋白质对疟疾寄生虫的附着或入侵很重要，它们的膜背景和性质的重要性，以及这些宿主细胞蛋白质如何参与或在成功的入侵事件中被寄生虫操纵。本项目将利用这种方法产生的具有新特征的红细胞与正常供体红细胞一起，从独特的宿主细胞角度研究疟原虫附着和入侵红细胞的机制。结合使用疟疾寄生虫入侵检测、生化和成像技术，它将揭示红细胞蛋白在入侵中发挥的关键作用如何参与这一过程。由于红细胞受体的附着或刺激也会诱导膜-细胞骨架蛋白相互作用的重构或破坏，我们还将研究介导膜和细胞骨架蛋白之间连接的关键细胞骨架衔接蛋白的参与和修饰。通过确定促进毛细血管中红细胞挤压和成功入侵的机制的性质和程度，我们将努力确定在不损害体内红细胞正常功能的情况下抑制入侵的方法。

Every year, across the world more than 200 million people contract malaria, and more than half a million people die, the majority of them children under the age of five, as a result of this disease. The parasites that cause malaria survive by attaching to the surface of and then penetrating circulating red blood cells in which they then multiply. Red blood cells (RBCs) have a highly specialised membrane structure that results from complex interactions between proteins within the plasma membrane and a flexible underlying meshwork of protein filaments called the cytoskeleton that allow the cell to squeeze through capillaries. To penetrate the robust RBC membrane, the parasite attaches to proteins at the cell surface and induces a coordinated and localised disruption of this membrane-cytoskeletal architecture to facilitate invasion, a process that also shares some similarities with the transient disruptions required to enable RBC squeezing in the capillaries. Although several key proteins have been shown to be involved in or required for successful invasion, in the majority of cases insight into the role that these host cell proteins actually play in the invasion process is severely or completely lacking. One of the biggest obstacles to investigating the mechanism of parasite invasion from the perspective of the host RBC is the inability to directly manipulate protein expression in these cells. Unlike most cells, RBCs contain no DNA, preventing the application of genetic techniques commonly used to manipulate protein expression in other cell types. Recent developments made in the field of RBC development (erythropoiesis) have changed this. It is now possible to culture young RBCs (reticulocytes) that are susceptible to invasion by the parasite that causes severe malaria, from an immortal cell line that allows the precursors of RBCs (erythroblasts) to be grown indefinitely or safely stored.Excitingly, we have shown it is possible to manipulate protein expression in these nucleated cells using lentivirus and gene editing techniques to introduce changes which are maintained after the cells lose their nucleus to become RBCs. This technology can be used to prevent specific RBC proteins that are known to be involved in invasion from being expressed and also allows them to altered or replaced with mutated versions in which the localisation within the membrane, interactions with other proteins or properties of the protein itself have been changed. This technology has opened the door to the generation of RBCs with rare and even unique characteristics that can be used to explore which proteins are important for malaria parasite attachment or invasion, the importance of their membrane context and properties and how these host cell proteins participate in or are manipulated by the parasite during a successful invasion event.This project will use RBCs with novel characteristics generated using this approach together with normal donor RBCs to investigate the mechanism of malaria parasite attachment and invasion of RBCs from a unique host cell perspective. Using a combination of malaria parasite invasion assays, biochemical and imaging techniques it will uncover how RBC proteins with crucial but poorly understood roles in invasion participate in this process. Since attachment to or stimulus of RBC receptors also induces reconfiguration or disruption of membrane-cytoskeletal protein interactions we will also investigate the involvement and modification of key cytoskeletal adaptor proteins that mediate connections between both membrane and cytoskeletal proteins. By determining the nature of and degree to which mechanisms that facilitate RBC squeezing in the capillaries and successful invasion are shared (co-opted by the parasite) or unique we will strive to identify ways in which invasion may be targeted for inhibition without impairing the normal function of the RBC within the body.