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

剖析宿主受体背景和细胞骨架破坏在疟疾寄生虫入侵中的作用

• 批准号: MR/V010506/1
• 负责人: Ashley Toye
• 金额: $ 74.57万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FV010506%2F1
• 关键词: Dissecting; role; host; receptor; context

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.

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岁以下的儿童。引起疟疾的寄生虫通过附着在循环红细胞的表面然后穿透循环红细胞而存活，然后在循环红细胞中繁殖。红细胞（RBC）具有高度专业化的膜结构，这是由质膜内的蛋白质与称为细胞骨架的蛋白质细丝的柔性底层网络之间的复杂相互作用引起的，该蛋白质细丝允许细胞挤压通过毛细血管。为了穿透坚固的RBC膜，寄生虫附着在细胞表面的蛋白质上，并诱导这种膜-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RBC在毛细血管中挤压所需的短暂破坏有一些相似之处。尽管已经显示出几种关键蛋白质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质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，阻止了通常用于操纵其他细胞类型中蛋白质表达的遗传技术的应用。红细胞发育（红细胞生成）领域的最新进展改变了这一点。现在可以培养年轻的红细胞（网织红细胞），容易受到寄生虫的入侵，导致严重的疟疾，从一个永生细胞系，允许红细胞的前体（成红细胞）可以无限生长或安全储存。令人兴奋的是，我们已经表明，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是可能的，以引入在细胞分裂后维持的变化。细胞失去细胞核变成红细胞。该技术可用于防止已知参与入侵的特定RBC蛋白质被表达，并且还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RBC打开了大门，这些特征可用于探索哪些蛋白质对疟疾寄生虫附着或入侵很重要，它们的膜环境和特性的重要性，以及这些宿主细胞蛋白质在寄生虫成功入侵事件中如何参与或被寄生虫操纵。本项目将使用这种方法产生的具有新特性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侵入红细胞的机制。使用疟疾寄生虫入侵检测，生物化学和成像技术的组合，它将揭示红细胞蛋白如何在入侵中发挥关键但知之甚少的作用。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RBC挤压和成功侵入的机制的性质和程度是共享的（由寄生虫增选）或独特的，我们将努力确定在不损害体内RBC正常功能的情况下靶向抑制侵入的方法。

项目成果

Generation of red blood cells from stem cells: Achievements, opportunities and perspectives for malaria research.

• DOI: 10.3389/fcimb.2022.1039520
• 发表时间: 2022
• 期刊: FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY
• 影响因子: 5.7
• 作者: Satchwell, Timothy J.

Missense mutations in PIEZO1, which encodes the Piezo1 mechanosensor protein, define Er red blood cell antigens.

编码压电1机械传感器蛋白的压电1中的错义突变定义了红细胞抗原。

• DOI: 10.1182/blood.2022016504
• 发表时间: 2023-01-12
• 期刊: BLOOD
• 影响因子: 20.3
• 作者: Crew, Vanja Karamatic;Tilley, Louise A.;Satchwell, Timothy J.;AlSubhi, Samah A.;Jones, Benjamin;Spring, Frances A.;Walser, Piers J.;Freire, Catarina Martins;Murciano, Nicoletta;Rotordam, Maria Giustina;Woestmann, Svenja J.;Hamed, Marwa;Alradwan, Reem;AlKhrousey, Mouza;Skidmore, Ian;Lewis, Sarah;Hussain, Shimon;Jackson, Jane;Latham, Tom;Kiloy, Mark D.;Lester, William;Becker, Nadine;Rapedius, Markus;Toye, Ashley M.;Thornton, Nicole M.
• 通讯作者: Thornton, Nicole M.