Ago-associated proteins in macrophages.

The intracellular protozoan parasite Leishmania causes leishmaniasis in humans, leading to serious illness and death in tropical and subtropical areas worldwide. Unfortunately, due to the unavailability of approved vaccines for humans and the limited efficacy of available drugs, leishmaniasis is on the rise. A comprehensive understanding of host-pathogen interactions at the molecular level could pave the way to counter leishmaniasis. There is growing evidence that several intracellular pathogens target RNA interference (RNAi) pathways in host cells to facilitate their persistence. The core elements of the RNAi system are complexes of Argonaute (Ago) proteins with small non-coding RNAs, also known as RNA-induced silencing complexes (RISCs). Recently, we have shown that Leishmania modulates Ago1 protein of host macrophages for its survival. In this study, we biochemically characterize the Ago proteins’ interactome in Leishmania-infected macrophages compared to non-infected cells. For this, a quantitative proteomic approach using stable isotope labelling by amino acids in cell culture (SILAC) was employed, followed by purification of host Ago-complexes using a short TNRC6 protein-derived peptide fused to glutathione S-transferase beads as an affinity matrix. Proteomic-based detailed biochemical analysis revealed Leishmania modulated host macrophage RISC composition during infection. This analysis identified 51 Ago-interacting proteins with a broad range of biological activities. Strikingly, Leishmania proteins were detected as part of host Ago-containing complexes in infected cells. Our results present the first report of comprehensive quantitative proteomics of Ago-containing complexes isolated from Leishmania-infected macrophages and suggest targeting the effector complex of host RNAi machinery. Additionally, these results expand knowledge of RISC in the context of host-pathogen interactions in parasitology in general.细胞内寄生原生动物利什曼原虫（Leishmania）可引发人类利什曼病，在全球热带及亚热带地区造成严重疾病甚至死亡。遗憾的是，由于尚无获批的人用疫苗，且现有药物疗效有限，利什曼病的发病率正逐年攀升。从分子层面全面解析宿主-病原体互作机制，或将为对抗利什曼病开辟新的路径。越来越多的研究证据表明，多种细胞内病原体可靶向宿主细胞的RNA干扰（RNA interference, RNAi）通路，以促进自身在宿主体内的持续定植。RNAi系统的核心组分是Argonaute（Ago）蛋白与小型非编码RNA形成的复合物，即RNA诱导沉默复合物（RNA-induced silencing complexes, RISCs）。此前本团队已证实，利什曼原虫可通过调控宿主巨噬细胞的Ago1蛋白实现自身存活。本研究通过对比感染利什曼原虫与未感染的巨噬细胞，从生化层面解析了Ago蛋白的互作组。为此，本研究采用基于细胞培养氨基酸稳定同位素标记（stable isotope labelling by amino acids in cell culture, SILAC）的定量蛋白质组学方法，并使用融合谷胱甘肽S-转移酶（glutathione S-transferase）的短TNRC6蛋白衍生肽作为亲和基质，对宿主Ago复合物进行纯化。基于蛋白质组学的详细生化分析显示，利什曼原虫感染期间会重塑宿主巨噬细胞的RISC组成。本次分析共鉴定出51种与Ago存在互作的蛋白，其生物学功能覆盖广泛的生理活动范畴。尤为引人注目的是，在感染细胞的宿主Ago复合物中检测到了利什曼原虫自身的蛋白组分。本研究结果首次报道了从利什曼原虫感染的巨噬细胞中分离得到的Ago复合物的全面定量蛋白质组学分析，并提示可靶向宿主RNAi机器的效应复合物。此外，本研究结果还拓宽了寄生虫学领域中宿主-病原体互作背景下关于RISC的认知。