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

描述（由申请方提供）：疟疾寄生虫用于入侵的主要途径是由红细胞表面上存在的血型糖蛋白的唾液酸（SA）残基和寄生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具有侵入神经氨酸酶处理的缺乏SA的红细胞的能力，这表明存在替代的SA非依赖性侵入机制。介导SA非依赖性侵袭的红细胞受体以前一直是未知的，直到我们最近确定补体受体1（CR 1）作为恶性疟原虫实验室菌株使用的主要SA非依赖性侵袭受体。随后的研究表明恶性疟原虫蛋白质网织红细胞结合样同源物4（Rh 4）是CR 1的配体。本申请将我们的调查扩展到加纳的恶性疟原虫临床分离株，以研究田间寄生虫入侵半免疫个体红细胞的分子机制，并确定入侵表型的变化，使寄生虫逃避适应性免疫反应。从加纳三个地理上不同的区域收集的寄生虫分离株具有不同的传输强度，将检查其红细胞侵入机制，通过测试他们的能力，侵入红细胞神经氨酸酶，胰蛋白酶，胰凝乳蛋白酶，选择性地破坏特定的红细胞受体处理。此外，将通过测量抗CR 1抗体和可溶性CR 1抑制红细胞侵袭的能力来研究CR 1作为受体的贡献。还将通过流式细胞术测量半免疫个体中的红细胞CR 1表达，并研究其与外周寄生虫密度的相关性。此外，将通过ELISA测定针对主要SA依赖性配体（包括EBA-140、EBA-175和EBA-181）以及CR 1配体Rh 4的抗体滴度，并使用多变量统计模型检查其对CR 1和寄生虫血症之间关系的影响。对于确定其侵袭表型的寄生虫分离株，将通过定量真实的时间RT-PCR确定已知SA非依赖性寄生虫配体Rh 2a、Rh 2b和Rh 4以及将在我们正在进行的CR 1配体研究中鉴定的候选配体的基因表达。然后将检查红细胞受体使用、抗EBA抗体滴度和配体基因表达之间的关系，以明确显示抗EBA滴度增加是否与CR 1配体基因表达增加相关，并转换为CR 1依赖性侵袭表型。成功完成这些拟议的研究将大大增加我们对加纳疟疾寄生虫所使用的入侵机制的理解，这对潜在的血液阶段干预措施的设计具有广泛的影响。 公共卫生相关性：改善地球仪各地人口的健康状况已成为一项重要的国际优先事项。也许传染病研究的最大挑战之一是降低与疟疾有关的发病率和死亡率。疟疾的临床症状是由寄生虫的红细胞周期引起的;因此，针对这一阶段的干预措施可能是预防疾病的有效战略。然而，血液阶段疫苗的开发受到寄生虫侵入红细胞的分子机制的不充分理解的阻碍。我们最近发现，红细胞表面蛋白补体受体1（CR 1）是疟原虫入侵的主要介质。该提案旨在将我们的研究扩展到该领域，以研究CR 1在介导半免疫个体红细胞侵袭中的作用。成功完成这些拟议的研究将大大增加我们对加纳疟疾寄生虫所使用的入侵机制的理解，这对潜在的血液阶段干预措施的设计具有广泛的影响。此外，这些调查将大大提高PI的职业生涯，并有助于加纳大学在疟疾研究基础设施和技术方面的能力建设。

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.