Genetic manipulation of erythrocytes: Identification and characterization of essential host cell determinants for Plasmodium infection

疟疾是一种重要的媒介传播传染病，尤其影响撒哈拉以南非洲的儿童。疟疾的病原体是疟原虫属的专性细胞内寄生虫，控制这种疾病的尝试收效甚微。许多确定红细胞感染疟原虫的寄生虫已被详细研究过。相比之下，影响寄生虫入侵和生长的宿主细胞因素的特征很差。我们缺乏对红血球侵袭决定因素的了解，部分原因是红血球对基因操作的抵抗力。了解红细胞决定因素的一个主要进展是造血干细胞的体外培养和利用慢病毒转导进行基因操作的最新进展。因此，现在可以通过反向遗传学直接研究红血球表面蛋白在疟原虫入侵中的作用。利用这一遗传学方法，我们建议研究红细胞抗原的功能，重点是它们对疟原虫入侵红细胞的影响。首先，我们将重点介绍已知的疟原虫入侵的红细胞受体。在对它们对寄生虫入侵的贡献的个体深入表征的基础上，我们将使用双倍和三倍耗竭来定义对不同恶性疟原虫株的入侵所必需的最小一组红细胞受体。同时，我们建议确定迄今为止未知的疟原虫入侵受体；候选的是众所周知的血型抗原。这两项战略结合在一起，将能够对红细胞蛋白及其对疟疾的贡献进行直接基因检查。它们构成了一种检查基本宿主-寄生虫相互作用的新颖和创新的方法，并可以为几乎未被探索的以宿主为靶向的疟疾治疗方法确定基本靶点。

Malaria is an important vector-borne infectious disease that particularly affects children in sub-Saharan Africa. The etiological agents of malaria are obligate intracellular parasites of the genus Plasmodium and attempts to control the disease have been of limited success. Many parasite deter-minants of erythrocyte infection by Plasmodium have been studied in detail. In contrast, respective host cell factors that influence parasite invasion and growth are poorly characterized. This lack in our knowledge of erythrocyte determinants of invasion is in part due to the refractoriness of red blood cells to genetic manipulation. A major advance for understanding erythrocyte determinants are recent developments in the ex vivo culture of hematopoietic stem cells and their genetic manipulation employing lentiviral transduction. Hence, it is now possible to directly examine the role of red blood cell surface proteins in Plasmodium invasion by reverse genetics. Utilizing this genetic approach, we propose to study the function of erythrocyte antigens with a focus on their impact on invasion of the erythrocyte by Plasmodium parasites. Initially, we will focus on known erythrocyte receptors of Plasmodium invasion. Building on the individual in-depth characterization of their contribution to parasite invasion, we will use double and triple depletions to define a minimal set of erythrocyte receptors that are mandatory for invasion of diverse P. falciparum strains. In parallel, we propose to identify hitherto unknown receptors for Plasmodium invasion; candidates are the well-described blood group antigens. Together these two strategies will enable the direct genetic examination of erythrocytic proteins and their contribution to malaria. They constitute a novel and innovative approach for examining essential host-parasite interactions, and can identify essential targets for the barely explored avenue of host-targeted malaria therapeutics.