Functional analysis of erythrocyte determinants of malaria infection

描述(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匮乏环境中的人，是导致儿童死亡的最重要原因之一。抗药性不断削弱抗疟疾方案的有效性。迫切需要开发新的治疗策略。恶性疟原虫利用多种配体-受体相互作用侵袭人类红细胞。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。很大程度上由于去核人红细胞的遗传难治性，宿主人红细胞受体在配体-受体相互作用中的功能还没有得到全面的评估。我们开发了一种将支持恶性疟原虫侵袭和生长的体外红细胞培养系统与慢病毒转导相结合的方法，以生成转基因红细胞。恶性疟原虫中最具特征性的配体-受体相互作用是配体EBA-175及其受体血型糖蛋白A的相互作用，EBA-175正被开发为候选疫苗。利用这个系统，我们已经实现了血糖素A的表达下调，从遗传学上证明了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灭所有恶性疟原虫品系中的恶性疟原虫入侵。本项目将从功能上分析恶性疟原虫入侵过程中的红细胞决定因素。我们将使用1)功能丧失的方法来建立可能的侵袭红细胞受体之间的层次结构，2)组合敲除方法来确定对侵袭至关重要的最小的红细胞表面决定因素集，以及3)基因筛查来识别作为新的侵袭决定因素的人类血型抗原。从长远来看，我们希望我们的研究将为了解恶性疟原虫侵袭红细胞的关键配体-受体相互作用提供功能上的理解，从而为疫苗的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.