背景:子孢子侵入肝细胞是疟原虫生命周期中必不可少的步骤,并且具有相似性,在细胞水平上,裂殖子侵入红细胞。在疟原虫血液阶段,鉴定宿主-病原体蛋白质-蛋白质相互作用的努力产生了重要的见解,包括候选疫苗。在子孢子-肝细胞侵袭的情况下,所涉及的宿主-病原体蛋白质-蛋白质相互作用了解甚少。
方法:为了更好地了解子孢子配体和宿主受体之间的蛋白质-蛋白质相互作用,进行了系统的筛选。先前的恶性疟原虫和人类表面蛋白胞外域文库被大量扩展,导致产生包含88个恶性疟原虫子孢子蛋白编码序列和182个编码人肝细胞表面蛋白的序列的新文库。从这些序列表达重组蛋白,使用了基于平板的测定法,能够检测重组蛋白之间的低亲和力相互作用,修改以提高吞吐量,筛选蛋白质的相互作用。在筛选中鉴定出的新的相互作用在生物化学上进行了表征,使用抗体和基因操纵的疟原虫寄生虫进一步阐明了它们在寄生虫入侵中的重要作用。
结果:在能够检测至少1.2µMKD的相互作用的条件下测试了总共7540个子孢子-肝细胞蛋白对。本文鉴定并报道了人成纤维细胞生长因子受体4(FGFR4)与恶性疟原虫蛋白Pf34之间的相互作用,表征其亲和力并证明试剂对相互作用的阻断,包括单克隆抗体.此外,Pf34和第二个恶性疟原虫头颈蛋白之间的进一步相互作用,鉴定了PfRON6和人低密度脂蛋白受体(LDLR)与恶性疟原虫蛋白PIESP15之间的PfRON6。条件遗传缺失证实了PfRON6在血液阶段的重要性,与这种蛋白质在寄生虫生命周期中的重要作用一致。Pf34难以尝试进行遗传修饰。Pf34的抗体消除了相互作用,对子孢子侵入原代人肝细胞的影响不大。
结论:Pf34和PfRON6可能是功能上重要的侵袭复合体的成员,可作为未来干预的目标。改进的相互作用筛选试验,本文报道的蛋白质表达文库和恶性疟原虫突变寄生虫可能是蛋白质相互作用发现和抗原候选物筛选的有用工具,这对科学界可能具有更广泛的价值。
BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of
sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood.
METHODS: To gain a better understanding of the protein-protein interaction between the
sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum
sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites.
RESULTS: A total of 7540
sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes.
CONCLUSIONS: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.