Functional analysis of erythrocyte determinants of malaria infection

描述（由申请人提供）：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环境中的人们，并且是儿童死亡的最重要原因之一。耐药性不断破坏抗疟方案的有效性。迫切需要开发新的治疗策略。恶性疟原虫利用多种配体-受体相互作用入侵人红细胞。许多工作都集中在寄生虫配体的特征上，目标是将它们开发为候选疫苗。主要由于去核人红细胞的遗传不稳定性，宿主人红细胞受体在配体-受体相互作用中的功能尚未得到全面评估。我们已经开发出一种方法，结合体外红细胞培养系统，支持恶性疟原虫的侵袭和生长，慢病毒转导产生转基因红细胞。恶性疟原虫中最具特征的配体-受体相互作用是配体EBA-175与其受体糖蛋白A的相互作用，EBA-175正被开发为候选疫苗。使用该系统，我们已经实现了糖蛋白A表达的敲低，遗传学证明它是有效的菌株特异性寄生虫入侵所必需的。我们假设靶向有限数量的红细胞受体将足以消除恶性疟原虫在所有恶性疟原虫寄生虫系中的侵袭。本项目将功能分析恶性疟原虫侵袭过程的红细胞决定因素。我们将使用1)功能缺失方法在假定的红细胞入侵受体之间建立层次结构，2)组合敲低方法来识别入侵所必需的红细胞表面决定因子的最小集合，3)基因筛选来识别作为入侵新决定因子的人类血型抗原。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-受体相互作用的功能理解，为疫苗开发和宿主靶向治疗的设计提供信息。

DESCRIPTION (provided by applicant): 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 erythrocytes. Much work has focused on the characterization of parasite ligands, with the goal of developing them as vaccine candidates. Largely due to the genetic intractabiliy of enucleated human erythrocytes, the function of host human erythrocyte receptors in ligand-receptor interactions has not been comprehensively assessed. We have developed an approach combining an in vitro erythrocyte culture system, which supports P. falciparum invasion and growth, with lentiviral transduction to generate genetically modified erythrocytes. The best characterized ligand-receptor interaction in P. falciparum is that of the ligand EBA-175 and its receptor glycophorin A, with EBA-175 being developed as a vaccine candidate. Using this system, we have achieved knockdown in expression of glycophorin A, genetically demonstrating that it is required for efficient strain-specific parasite invasion. We hypothesize that targeting a limited number of erythrocyte receptors will be sufficient to abrogate P. falciparum invasion in all P. falciparum parasite lines. This project will functionally analyze erythrocyte determinants of the invasion process of P. falciparum. We will use 1) a loss-of- function approach to establish a hierarchy amongst putative erythrocyte receptors for invasion, 2) a combinatorial knockdown approach to identify minimal sets of erythrocyte surface determinants that are essential for invasion and 3) a genetic screen to identify human blood group antigens that are novel determinants of invasion. 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.