A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins
A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins
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DOI：
10.1074/mcp.o113.028357
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发表时间：
2013-12-01
影响因子：
7
通讯作者：
Wright, Gavin J.
中科院分区：
文献类型：
--
作者：
Crosnier, Cecile;Wanaguru, Madushi;Wright, Gavin J.
疟疾是一种由疟原虫属寄生虫引起的传染病，是世界上主要的公共卫生问题之一，每年导致多达一百万人死亡，其中大部分是由于恶性疟原虫感染。所有的临床症状都与疾病的血液阶段有关，这是寄生虫生命周期的必然部分，此时一种称为裂殖子的寄生虫识别并侵入宿主红细胞。在红细胞入侵过程中，裂殖子直接暴露于宿主体液免疫系统，使寄生虫的血液阶段成为概念上有吸引力的治疗目标。然而，恶性疟原虫裂殖子蛋白的功能和分子表征方面的进展受到以生化活性重组形式表达这些蛋白相关的技术挑战的阻碍。对于细胞外蛋白来说，这一挑战尤其严峻，细胞外蛋白可能是宿主抗体反应的目标，因为它们包含一些重组表达系统未添加的结构上关键的翻译后修饰。在这里，我们报告了一种方法的开发，该方法使用哺乳动物表达系统来编译包含 42 种恶性疟原虫裂殖子分泌的完整胞外域和细胞表面蛋白的蛋白质资源，其中许多蛋白质以前尚未被表征。重要的是，我们能够通过证明重组 MSP1-MSP7 和 P12-P41 直接相互作用，以及重组 EBA175 和 EBA140 都能以唾液酸依赖性方式结合人红细胞，来概括已知的生化活性。最后，我们使用来自疟疾暴露的免疫成人的血清来分析蛋白质的相对免疫反应性，并表明大多数抗原含有构象（热不稳定）表位。我们预计，这种重组蛋白资源将为恶性疟原虫感染的血液阶段的分子理解做出宝贵的贡献，并促进作为血液阶段候选疫苗的抗原的比较筛选。
Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.