ROLE OF COMPLEMENT RECEPTOR 1 IN ERYTHROCYTE INVASION BY PLASMODIUM FALCIPARUM IN

描述（由申请人提供）：疟原虫入侵的主要途径是由红细胞表面糖蛋白的唾液酸（SA）残基和疟原虫蛋白的红细胞结合抗原（EBA）家族介导的。然而，一些恶性疟原虫菌株能够侵入缺乏SA的经神经氨酸酶处理的红细胞，这表明存在其他不依赖SA的侵袭机制。在我们最近鉴定出补体受体1 （CR1）是恶性疟原虫实验室菌株使用的主要sa独立侵袭受体之前，介导sa独立侵袭的红细胞受体一直是未知的。随后的研究表明，恶性疟原虫蛋白网状细胞结合样同源物4 （Rh4）是CR1的配体。该应用程序将我们的研究扩展到加纳的恶性疟原虫临床分离株，以检查野外寄生虫入侵半免疫个体红细胞的分子机制，并确定允许寄生虫逃避适应性免疫反应的入侵表型的变化。从加纳三个地理上不同的地区收集的具有不同传播强度的寄生虫分离物将通过测试它们入侵经神经氨酸酶、胰蛋白酶或凝乳胰蛋白酶处理的红细胞的能力来检查其红细胞入侵机制，这些酶选择性地破坏特定的红细胞受体。此外，我们将通过测定抗CR1抗体和可溶性CR1抑制红细胞侵袭的能力来研究CR1作为受体的作用。半免疫个体的红细胞CR1表达也将通过流式细胞术测量，并将研究与外周血寄生虫密度的关系。此外，针对主要sa依赖性配体（包括EBA-140、EBA-175和EBA-181）以及CR1配体Rh4的抗体滴度将通过ELISA测定，并使用多元统计模型检验它们对CR1与寄生虫病之间关系的影响。对于已确定入侵表型的寄生虫分离物，将通过定量实时RT-PCR确定已知sa独立的寄生虫配体Rh2a， Rh2b和Rh4的基因表达，以及我们正在进行的CR1配体研究中将确定的候选配体。红细胞受体使用、抗eba抗体滴度和配体基因表达之间的关系将被检查，以明确显示抗eba滴度的增加是否与CR1配体基因表达的增加相关，并转换为CR1依赖的入侵表型。这些拟议研究的成功完成将大大增加我们对加纳疟疾寄生虫入侵机制的理解，这对设计潜在的血液阶段干预措施具有广泛的意义。

DESCRIPTION (provided by applicant): A major pathway used by the malaria parasite for invasion is mediated by sialic acid (SA) residues of glycophorins present on the erythrocyte surface and the erythrocyte binding antigen (EBA) family of parasite proteins. However, some P. falciparum strains have the ability to invade neuraminidase-treated erythrocytes which lack SA, demonstrating the existence of alternative SA-independent invasion mechanisms. The erythrocyte receptors that mediate SA-independent invasion had previously been unknown until we recently identified complement receptor 1 (CR1) as the major SA-independent invasion receptor used by laboratory strains of P. falciparum. Subsequent studies have implicated the P. falciparum protein reticulocyte-binding like homologue 4 (Rh4) as the ligand for CR1. This application will extend our investigations to clinical isolates of P. falciparum in Ghana to examine the molecular mechanisms used by field parasites for invasion of erythrocytes in semi-immune individuals and to determine the changes in invasion phenotypes that allow the parasites to evade the adaptive immune response. Parasite isolates collected from three geographically distinct zones of Ghana with varying transmission intensities will be examined for their erythrocyte invasion mechanisms by testing their ability to invade erythrocytes treated with neuraminidase, trypsin, or chymotrypsin, which selectively disrupt specific erythrocyte receptors. In addition, the contribution of CR1 as a receptor will be investigated by measuring the ability of anti-CR1 antibodies and soluble CR1 to inhibit erythrocyte invasion. Erythrocyte CR1 expression in semi-immune individuals will also be measured by flow cytometry and the association with peripheral parasite density will be investigated. In addition, titers of antibodies against the major SA-dependent ligands including, EBA-140, EBA-175, and EBA-181, as well as the CR1 ligand Rh4, will be determined by ELISA and their impact on the relationship between CR1 and parasitemia will be examined using a multivariate statistical model. For parasite isolates whose invasion phenotypes are determined, gene expression of the known SA-independent parasite ligands Rh2a, Rh2b, and Rh4, as well as candidate ligands that will be identified in our ongoing investigations of CR1 ligands, will be determined by quantitative real time RT-PCR. The relationship between erythrocyte receptor usage, anti-EBA antibody titers and ligand gene expression will then be examined to definitively show if increased anti-EBA titers are associated with an increased CR1 ligand gene expression and switch to CR1-dependent invasion phenotype. Successful completion of these proposed studies will substantially increase our understanding of the invasion mechanisms that are used by malaria parasites in Ghana which has broad implications on the design of potential blood- stage interventions. PUBLIC HEALTH RELEVANCE: Improving health for populations around the globe has become an important international priority. Perhaps one of the greatest challenges in infectious disease research has been decreasing the morbidity and mortality associated with malaria. Clinical symptoms of malaria are caused by the erythrocytic cycle of the parasite; therefore, interventions targeting this stage could potentially be an effective strategy for preventing disease. However, the development of a blood stage vaccine is hampered by an inadequate understanding of the molecular mechanisms through which the parasite invades erythrocytes. We recently discovered that the red cell surface protein complement receptor 1 (CR1) is a major mediator of malaria parasite invasion. This proposal aims to extend our studies to the field to examine the role of CR1 in mediating red cell invasion in semi-immune individuals. Successful completion of these proposed studies will substantially increase our understanding of the invasion mechanisms that are used by malaria parasites in Ghana which has broad implications on the design of potential blood-stage interventions. In addition, these investigations will significantly enhance the career of the PI and contribute towards capacity-building at the University of Ghana, both in infrastructure and technology for malaria research.