Leishmania donovani Utilize Sialic Acids for Binding and Phagocytosis in the Macrophages through Selective Utilization of Siglecs and Impair the Innate Immune Arm

BackgroundLeishmania donovani, belonging to a unicellular protozoan parasite, display the differential level of linkage-specific sialic acids on their surface. Sialic acids binding immunoglobulin-like lectins (siglecs) are a class of membrane-bound receptors present in the haematopoetic cell lineages interact with the linkage-specific sialic acids. Here we aimed to explore the utilization of sialic acids by Leishmania donovani for siglec-mediated binding, phagocytosis, modulation of innate immune response and signaling pathways for establishment of successful infection in the host.Methodology/Principle FindingsWe have found enhanced binding of high sialic acids containing virulent strains (AG83+Sias) with siglec-1 and siglec-5 present on macrophages compared to sialidase treated AG83+Sias (AG83-Sias) and low sialic acids-containing avirulent strain (UR6) by flow cytometry. This specific receptor-ligand interaction between sialic acids and siglecs were further confirmed by confocal microscopy. Sialic acids-siglec-1-mediated interaction of AG83+Sias with macrophages induced enhanced phagocytosis. Additionally, sialic acids-siglec-5 interaction demonstrated reduced ROS, NO generation and Th2 dominant cytokine response upon infection with AG83+Sias in contrast to AG83-Sias and UR6. Sialic acids-siglecs binding also facilitated multiplication of intracellular amastigotes. Moreover, AG83+Sias induced sialic acids-siglec-5-mediated upregulation of host phosphatase SHP-1. Such sialic acids-siglec interaction was responsible for further downregulation of MAPKs (p38, ERK and JNK) and PI3K/Akt pathways followed by the reduced translocation of p65 subunit of NF-κβ to the nucleus from cytosol in the downstream signaling pathways. This sequence of events was reversed in AG83-Sias and UR6-infected macrophages. Besides, siglec-knockdown macrophages also showed the reversal of AG83+Sias infection-induced effector functions and downstream signaling events.Conclusions/SignificancesTaken together, this study demonstrated that virulent parasite (AG83+Sias) establish a unique sialic acids-mediated binding and subsequent phagocytosis in the host cell through the selective exploitation of siglec-1. Additionally, sialic acids-siglec-5 interaction altered the downstream signaling pathways which contributed impairment of immune effector functions of macrophages. To the best of our knowledge, this is a comprehensive report describing sialic acids-siglec interactions and their role in facilitating uptake of the virulent parasite within the host.背景 杜氏利什曼原虫（Leishmania donovani）是一种单细胞原生动物寄生虫，其表面表达具有不同连接特异性的唾液酸（sialic acids）。唾液酸结合免疫球蛋白样凝集素（sialic acid-binding immunoglobulin-like lectins，简称siglecs）是一类表达于造血细胞谱系的膜结合受体，可与具有连接特异性的唾液酸结合。本研究旨在探究杜氏利什曼原虫如何利用唾液酸，通过siglec介导的黏附、吞噬作用、先天免疫应答调控及信号通路，以在宿主体内成功建立感染。 研究方法与主要结果 我们通过流式细胞术检测发现，与经唾液酸酶处理的AG83+Sias组（AG83-Sias）以及低唾液酸含量的无毒株UR6组相比，高唾液酸含量的有毒株AG83+Sias与巨噬细胞表面的siglec-1、siglec-5的结合能力显著增强。上述唾液酸与siglecs之间的特异性受体-配体相互作用，进一步通过共聚焦显微镜得到验证。AG83+Sias通过唾液酸-siglec-1介导的与巨噬细胞的相互作用，可诱导吞噬作用增强。此外，与AG83-Sias组及UR6组相比，AG83+Sias感染的巨噬细胞中，唾液酸-siglec-5相互作用可导致活性氧（Reactive Oxygen Species, ROS）、一氧化氮（Nitric Oxide, NO）生成减少，并引发以Th2型为主的细胞因子应答。唾液酸-siglec结合还可促进细胞内无鞭毛体（amastigotes）的增殖。此外，AG83+Sias可通过唾液酸-siglec-5介导，诱导宿主磷酸酶SHP-1的表达上调。此类唾液酸-siglec相互作用可进一步下调丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinases, MAPKs，包括p38、ERK及JNK）与PI3K/Akt通路，进而导致下游信号通路中核因子κB（Nuclear Factor κB, NF-κB）的p65亚基从细胞质向细胞核的转位减少。在AG83-Sias及UR6感染的巨噬细胞中，上述一系列事件均被逆转。此外，siglec敲低的巨噬细胞中，AG83+Sias感染诱导的效应功能及下游信号事件也得到了逆转。 结论与意义 综上，本研究表明，有毒株AG83+Sias可通过选择性利用siglec-1，在宿主细胞中建立独特的唾液酸介导的黏附及后续吞噬过程。此外，唾液酸-siglec-5相互作用可改变下游信号通路，进而削弱巨噬细胞的免疫效应功能。据我们所知，本研究是首个全面阐述唾液酸-siglec相互作用及其在促进宿主体内有毒株摄取过程中作用的报道。