Functional analysis of red blood cell determinants of Plasmodium invasion

项目总结 疟疾仍然是一种主要的全球传染病，主要影响生活在资源匮乏地区的人们。 这是造成儿童死亡的最重要原因之一。抗药性不断地 削弱了抗疟疾疗法的有效性。迫切需要发展新的 治疗策略。恶性疟原虫利用多个配体-受体相互作用 入侵人类红细胞，其中许多是多余的。不同的寄生虫菌株依赖于替代 配体相互作用称为侵袭途径，用于侵袭，推测用于免疫逃避和侵袭 多态的红细胞。 我们最近开发了用于红细胞基因的体外遗传分析的方法 造血干细胞。我们已经鉴定了两种红细胞蛋白，BSG和CD55，以及 已经表明它们对所有恶性疟原虫株的入侵是必不可少的，这表明它们是株- 超凡的，将它们的相互作用作为治疗开发的目标，而不是特定的菌株 互动。在正向基因敲除筛查中，评估在红细胞中发现的所有膜蛋白 蛋白质组，我们已经确定了我们假设的高优先级红细胞决定因素的简短列表 参与宿主红细胞和疟疾寄生虫之间的菌株超越的基本相互作用。 在这项建议中，我们将验证这些基因是否为菌株-超越或菌株-的红细胞决定因素。 特定的入侵。为此，我们还将利用我们开发的CRISPR/Cas9介导的基因编辑在 支持恶性疟原虫入侵并促进RBC蛋白敲除的红系细胞系。这个 恶性疟原虫入侵的确切步骤将被确定，它是由每个红细胞决定因素介导的。我们 将使用我们生成的RBC突变体来识别特定的寄生虫配体，使用候选和 不带偏见的筛选方法。总而言之，这些研究将有助于将范式从关注 替代冗余入侵途径用于基本品系超越性寄主的功能分析 寄生虫的相互作用。从长远来看，我们希望我们的研究将提供对 恶性疟原虫侵袭红细胞的关键配体-受体相互作用为疫苗开发提供信息 以及针对宿主的治疗方法的设计。

PROJECT SUMMARY Malaria remains a major global infectious disease, largely affecting people living in resource poor environments, and is one of the most important causes of childhood mortality. Drug-resistance is constantly undermining the usefulness of antimalarial regimens. There is an urgent need for the development of new therapeutic strategies. Plasmodium falciparum parasites utilize multiple ligand-receptor interactions for the invasion of human red blood cells, many of which are redundant. Different parasite strains rely on alternative ligand-interactions known as invasion pathways, for invasion, presumably for immune evasion and to invade polymorphic red blood cells. We have recently developed methods for the in vitro genetic analysis of red blood cell genes using hematopoietic stem cells. We have functionally characterized two red blood cell proteins, BSG and CD55, and have shown that they are essential for invasion by all P. falciparum strains, suggesting that they are strain- transcendent, prioritizing their interactions as targets for therapeutic development over strain-specific interactions. In forward genetic knockdown screens that assess all of membrane proteins found in the RBC proteome, we have identified a short list of high priority red blood cell determinants that we hypothesize are involved in strain-transcendent essential interactions between the host red blood cell and malaria parasite. In this proposal we will validate these genes as red blood cell determinants of strain- transcendent or strain- specific invasion. For this we will also leverage our development of CRISPR/Cas9-mediated gene editing in an erythroid cell-line that supports P. falciparum invasion, and facilitates the knockout of RBC proteins. The precise step of P. falciparum invasion that is mediated by each red blood cell determinant will be identified. We will use the RBC mutants that we generate to identify specific parasite ligands using both candidate and unbiased screening approaches. Together, these studies will serve to shift the paradigm from a focus on alternative redundant invasion pathways to the functional analysis of essential strain-transcendent host- parasite interactions. In the long-term we hope that our studies will provide a functional understanding of critical ligand-receptor interactions for P. falciparum invasion of erythrocytes to inform vaccine development and the design of host-targeted therapeutics.