Dissecting the Red Blood Cell Invasion Pathways of the Malaria Parasite Plasmodium knowlesi

疟疾是人类最重要的传染病之一，世界上一半以上的人口有感染疟疾的危险，每年造成50多万人死亡。这种疾病是由疟原虫引起的，它是一种单细胞寄生虫，可以通过被感染的蚊子叮咬传染给人类。这种疾病的症状是由于寄生虫首先进入红细胞，在其中生长和复制，然后在一个连续的循环中爆发并入侵其他红细胞，从而破坏红细胞。寄生虫产生一系列粘附蛋白，使它们能够与红细胞表面的特定蛋白结合，并建立红细胞入侵过程。由于它们在入侵过程中的关键作用，这些寄生虫蛋白是重要的疫苗靶点。它们还决定了寄生虫复制的效率，从而影响疾病的严重程度，以及确定哪些宿主容易感染疟疾。在这个项目中，我将使用一种被称为诺氏疟原虫的疟疾寄生虫来研究这些蛋白质在入侵过程中的作用。这种寄生虫自然感染东南亚的猕猴，最近发现它是造成严重和致命人类感染的一个重要原因。在最近的工作中，我首次开发了用人类红细胞培养这种寄生虫的方法，并建立了高效的技术来对寄生虫进行基因改造。我将用这些技术来制造寄生虫，其中我删除了编码粘附蛋白的基因。这将使我能够确定哪些是入侵所必需的，哪些可以删除而不会对入侵过程产生任何影响。使用类似的技术，我还将为靶基因编码的每个蛋白质添加荧光“标签”，这样我就可以确定粘附蛋白在细胞中的位置以及它们在入侵过程中移动的位置。这些“标签”还将使我能够识别与粘附蛋白相互作用的寄生虫蛋白质，以及它们在宿主红细胞表面特异性结合的蛋白质。我将使用包括电子断层扫描和超分辨率显微镜在内的尖端成像技术分析基因缺失和“标记”的寄生虫系，这些技术以前从未被用于可视化这种寄生虫物种的入侵。这将为了解所有疟疾寄生虫的入侵机制提供关键的见解，并准确确定诺氏疟原虫入侵人类红细胞需要哪些寄生虫蛋白和宿主蛋白。后者尤其重要，因为它可以解释猕猴疟疾寄生虫如何能够传播感染人类，并确定寄生虫出现人际传播的可能性。虽然目前没有针对疟疾的疫苗，但人们对疟疾最常见和最严重的病因恶性疟原虫非常感兴趣，并且正在开发几种候选疫苗。然而，间日疟原虫是疟疾的第二大常见病因，它的疫苗开发由于无法在实验室中培养而受到阻碍。这意味着测试新疫苗将涉及让人类或非人类灵长类动物感染间日疟原虫。间日疟原虫的主要候选疫苗是疟原虫的粘附蛋白之一。诺氏疟原虫与间日疟原虫密切相关，也使用这种黏附蛋白的类似版本。通过对诺氏疟原虫进行基因修饰，将编码粘附蛋白的基因替换为间日疟原虫的基因，就有可能确定一种疫苗是否能在培养物中诱导出杀死寄生虫的抗体，然后再进行人体试验。因此，我将利用诺氏疟原虫独特的生物学特性以及该模型的技术优势，不仅研究其入侵过程，而且还将产生重要的工具，以加快至关重要的疟疾疫苗的开发。

Malaria is one of the most important infectious diseases of man with more than half of the world's population living at risk of the disease, and resulting in more than half a million deaths per year. The disease is caused by Plasmodium species, single-celled parasites that can be transferred to humans by the bite of an infected mosquito. Symptoms of the disease result from the parasite destroying red blood cells by first entering them, growing and replicating inside them, before bursting out and invading other red blood cells in a continuous cycle. The parasites produce a range of adhesive proteins enabling them to bind to specific proteins on the surface of red blood cells and establish the process of red blood cell invasion. Because of their crucial role in the invasion process these parasite proteins are important vaccine targets. They also determine how effectively parasites can replicate and so can affect disease severity as well as determining which hosts are susceptible to malaria.In this project I will investigate the role of these proteins during the invasion process using a malaria parasite known as Plasmodium knowlesi. This parasite naturally infects macaque monkeys in South-East Asia, and was recently found to be a significant cause of severe and fatal human infections. In recent work I have developed methods to grow this parasite in culture with human red blood cells for the first time, and established highly efficient techniques to genetically modify the parasite. I will use these techniques to generate parasites in which I have deleted genes encoding the adhesive proteins. This will enable me to determine which are essential for invasion and which can be deleted without any effect on the invasion process. Using similar techniques I will also add fluorescent "tags" to each of the proteins coded by the target genes, so that I can determine where the adhesive proteins are in the cell and where they move during the invasion process. The "tags" will also allow me to identify parasite proteins that interact with the adhesive proteins as well as what they specifically bind to on the host red blood cell surface. I will analyse both the gene deletion and "tagged" parasite lines using cutting edge imaging technologies including electron tomography and super resolution microscopy, which have never before been used to visualise invasion of this parasite species. This will provide critical insight into the mechanism of invasion of all malaria parasites, as well as identifying precisely which parasite proteins and host proteins are required for P. knowlesi to invade human red blood cells. The latter is of particular importance as it may explain how a macaque malaria parasite is able to spread to infect humans and determine the potential for emergence of human-to-human transmission of the parasite.Whilst there is currently no vaccine for malaria, there is great interest and several vaccine candidates under development for the most common and serious cause of malaria P. falciparum. However, vaccine development for the second most common cause of malaria P. vivax, is hampered by the fact that it cannot be grown in the laboratory. This means that testing new vaccines would involve infecting people or non-human primates with P. vivax. The primary vaccine candidate for P. vivax is one of the parasite's adhesive proteins. P. knowlesi is closely related to P. vivax and also uses a similar version of this adhesive protein. By genetically modifying P. knowlesi to replace the gene encoding the adhesive protein with the version from P. vivax, it will be possible to determine whether a vaccine can induce antibodies that kill parasites in culture, before it is necessary to test it in people. Thus I will use the unique biology of P. knowlesi along with the technical advantages of the model to not only study the process of invasion but also generate important tools to expedite the development of vital malaria vaccines.