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

描述(申请人提供)：疟疾寄生虫用于入侵的一个主要途径是由存在于红细胞表面的血糖蛋白的唾液酸(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，一些恶性疟原虫株具有侵袭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不依赖的侵袭机制。在我们最近确定补体受体1(CR1)是恶性疟原虫实验室株使用的主要SA非依赖性侵袭受体之前，介导SA非依赖性侵袭的红细胞受体一直不为人所知。随后的研究表明恶性疟原虫蛋白网织红细胞结合样同源4(Rh4)是CR1的配体。这一应用将把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，并确定使寄生虫逃避适应性免疫反应的入侵表型的变化。从加纳三个不同地理位置、不同传播强度的地区收集的寄生虫分离株将通过测试它们入侵神经氨酸酶、胰蛋白酶或凝乳酶处理的红细胞的能力来检查它们的红细胞入侵机制，神经氨酸酶、胰蛋白酶或凝乳酶处理的红细胞选择性地破坏特定的红细胞受体。此外，将通过检测抗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仪进行检测，并将调查其与外周寄生虫密度的关系。此外，针对主要SA依赖配体包括EBA-140、EBA-175和EBA-181以及CR1配体Rh4的抗体效价将用ELISA法测定，它们对CR1和寄生虫血症之间关系的影响将用多元统计模型来检验。对于侵袭表型已确定的寄生虫分离株，已知的SA非依赖性寄生虫配体Rh2a、Rh2b和Rh4以及将在我们正在进行的CR1配体研究中确定的候选配体的基因表达将通过实时定量RT-PCR来确定。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CR1配体基因表达增加相关，并转换为CR1依赖的侵袭表型。这些拟议研究的成功完成将大大提高我们对加纳疟疾寄生虫入侵机制的了解，这对设计潜在的血液阶段干预措施具有广泛的影响。 公共卫生相关性：改善全球人口的健康已成为一项重要的国际优先事项。也许传染病研究中最大的挑战之一是降低与疟疾相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循环引起的；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，由于对寄生虫入侵红细胞的分子机制了解不足，血液期疫苗的开发受到阻碍。我们最近发现，红细胞表面蛋白补体受体1(CR1)是疟疾原虫侵袭的主要介体。这项建议旨在将我们的研究扩展到该领域，以检查CR1在半免疫个体中介导红细胞侵袭的作用。这些拟议研究的成功完成将大大提高我们对加纳疟疾寄生虫入侵机制的了解，这对设计潜在的血液阶段干预措施具有广泛的影响。此外，这些调查将极大地促进和平协会的事业，并有助于加纳大学在疟疾研究的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.