Membrane and host cytoskeleton reorganization during malaria parasite egress from erythrocytes

疟疾是主要的全球杀手，对发展中国家的儿童来说是最致命的。在感染人类的五种物种中，恶性疟原虫是最致命的。虽然目前有有效的抗疟疾药物，但恶性疟原虫耐药株的出现和传播构成了越来越大的威胁。这种寄生虫有一个复杂的生命周期，在蚊子和人类宿主中有几个不同的阶段，但人类的临床症状源于无性血液阶段寄生虫的一波又一波释放，在这个阶段，寄生虫入侵红细胞，并在称为液泡的内膜室中繁殖。不断增长的寄生虫劫持它们的宿主血细胞，消耗它们的血红蛋白，并重新引导细胞活动，以造福于寄生虫。尤其重要的是，这种寄生虫输出自己的一些蛋白质，以便在血细胞表面建立新的结构。恶性疟原虫一个独特的致命方面是它在血细胞上产生表面突起(称为旋钮)，使其附着在血管系统的衬里上。这可以防止被感染的细胞被脾捕获和破坏，但也可以阻止脑血管毛细血管，这是疟疾感染的主要死亡原因。一旦成熟，16-24个子寄生虫就会穿过周围的液泡膜和红细胞膜(这一过程统称为出口)进入血液，在那里它们立即入侵新一轮的血细胞。侵袭、黏附和外泄的过程是通过一系列酶反应以及结构成分的表达和运输来调节的。其中一些成分是疟疾独有的，使它们成为未来药物开发的潜在目标。目前，大多数寄生虫的特性都很差。在这个项目中，我们重点研究成熟寄生虫穿过两个结合膜进行出口的步骤。一系列高度受监管的步骤会导致寄生虫从血细胞中爆炸性地释放。为了详细检查这些膜破裂事件，我们使用电子显微镜以纳米级分辨率成像寄生虫发育后期感染细胞的三维结构。这一成像结合了寄生虫突变体、类药物分子和酶抑制剂的使用，以在不同的出口步骤捕获寄生虫。这种方法已经让我们在出口中发现了一个以前没有被检测到的新的、最初的步骤。我们现在知道，这一过程始于寄生虫导致其液泡周围的膜变得泄漏。随后，这一膜被完全破坏，使寄生虫能够在血细胞内自由移动。在那之后不久，血细胞膜本身就会渗漏，最后细胞膜及其下面的细胞骨架破裂，使寄生虫得以逃逸并入侵新的宿主血细胞。随着基因编辑技术的最新进展，现在已经有可能有条件地修改恶性疟原虫的基因表达，我们将利用这项强大的技术来探索疟疾关键成分的分子性质、功能和亚细胞定位，这些成分参与了恶性疟原虫出口过程中的一系列步骤。这些研究的结果可能为未来新疗法的开发奠定基础。

Malaria is a major global killer, most deadly for children in the developing world. Of the five species that infect humans, Plasmodium falciparum is the most lethal. Although there are currently effective anti malarial drugs, the appearance and spread of resistant strains of P falciparum pose an increasing threat. The parasite has a complex life cycle with several different stages in its mosquito and human hosts, but the clinical symptoms in humans arise from waves of parasite release during the asexual blood stages, in which parasites invade red blood cells and multiply within an internal membrane compartment called a vacuole. The growing parasites hijack their host blood cells, consume their haemoglobin, and redirect the cell activity for the benefit of the parasite. Of particular importance, the parasite exports some of its own proteins to build new structures on the surface of the blood cell. A uniquely lethal aspect of P. falciparum is that it creates surface protrusions (called knobs) on the blood cell that make it adhere to the lining of blood vasculature. This prevents the infected cells from being captured and destroyed by the spleen, but can also block brain blood capillaries, the main cause of death in malaria infection. Once they have matured, 16-24 daughter parasites break through their surrounding vacuole membrane as well as the red blood cell membrane (a process collectively called egress) to enter the bloodstream, where they immediately invade a fresh round of blood cells. The processes of invasion, adhesion and egress are regulated through a cascade of enzyme reactions, as well as expression and transport of structural components. Some of these components are unique to malaria, making them potential targets for future drug development. Currently, most of them are poorly characterised.In this project, we focus on the steps by which the mature parasites break through the two bounding membranes to undergo egress. A highly regulated series of steps leads to the explosive release of parasites from the blood cell. To examine these membrane breakage events in detail, we use electron microscopy to image at nanoscale resolution the three-dimensional structures of the infected cells during the late stages of parasite development. This imaging is combined with use of parasite mutants and drug-like molecules and enzyme inhibitors to trap the parasites at different steps of egress. This approach has already led us to discover a new, initial step in egress that had not been previously detected. We now know that the process begins with the parasites causing the membrane surrounding their vacuole to become leaky. Subsequently, this membrane is completely disrupted, allowing the parasites to move freely inside the blood cell. Shortly after that, the blood cell membrane itself becomes leaky and then finally the cell membrane and its underlying cytoskeleton rupture to allow the parasites to escape and invade new host blood cells. With recent advances in gene editing technology, it has now become possible to conditionally modify gene expression in P. falciparum, and we will use this powerful technology to probe the molecular nature, functions and subcellular localisations of key malaria components involved in the sequence of steps during egress. The results of these studies could form the basis for future development of novel therapeutics.