Functional analysis of red blood cell determinants of Plasmodium invasion

项目摘要 疟疾仍然是一种主要的全球传染病，主要影响生活在资源贫乏地区的人们。 环境污染是造成儿童死亡的最重要原因之一。抗药性不断地 破坏了抗疟疗法的有效性。迫切需要开发新的 治疗策略恶性疟原虫寄生虫利用多种配体-受体相互作用， 入侵人体红细胞，其中许多是多余的。不同的寄生虫菌株依赖于替代的 配体相互作用被称为入侵途径，用于入侵，可能用于免疫逃避和入侵 多态性红细胞 我们最近开发了红细胞基因的体外遗传分析方法， 造血干细胞我们对两种红细胞蛋白BSG和CD 55进行了功能表征， 已经表明，它们是所有恶性疟原虫株入侵所必需的，这表明它们是株- 超越，优先考虑它们的相互作用作为治疗开发的靶点，而不是菌株特异性 交互.在评估RBC中发现的所有膜蛋白的正向遗传敲除筛选中， 蛋白质组，我们已经确定了一个短名单的高优先级红细胞决定因素，我们假设是 参与宿主红细胞和疟疾寄生虫之间的应变超越必要的相互作用。 在这项提议中，我们将验证这些基因作为应变的红细胞决定因素-超越或应变- 具体入侵。为此，我们还将利用CRISPR/Cas9介导的基因编辑的发展， 红系细胞系支持恶性疟原虫入侵，并促进RBC蛋白质的敲除。的 将鉴定由每个红细胞决定簇介导的恶性疟原虫侵袭的精确步骤。我们 将使用我们产生的RBC突变体来识别特定的寄生虫配体， 无偏见的筛选方法。总之，这些研究将有助于改变范式， 替代冗余入侵途径的功能分析的必要应变超越主机- 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一个功能性的理解， 恶性疟原虫侵入红细胞的关键配体-受体相互作用为疫苗开发提供信息 和设计针对宿主的治疗方法。

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.