TLR5及NAIP5/NLRC4炎症小体介导梅毒螺旋体鞭毛蛋白致炎反应的分子机制研究

Syphilis is a chronic multistage sexually transmitted disease (STDs) caused by the spirochaete Treponema pallidum. In China, syphilis has become one of the top five most reported infectious diseases and the most frequently reported STDs. During early infection, T. pallidum is able to elicit a vigorous inflammation and the ensuing adaptive immune response. It is probable that inflammation and the ensuing adaptive immune cooperate in damaging the tissues. However, which proteins in triggering inflammation and orchestrating adaptive immune response have not been identified. Flagellin is a potent immunogen that activates the innate immune system via TLR5 and Naip5, and generates strong inflammation responses.Furthermore, whether the flagellin of T. pallidum is able to induce inflammation response and the interactions with innate immune system is unclear. In this study, we will explore the role of flagellin in the pathogenesis of T. pallidum and the TLR5 signaling pathway in flagellin induced inflammation response, through gene cloning and expression, confocal immunofluorescence, flow cytometry, Western Blot, Real Time-PCR and other methods. To study the mechanism of flagellin with the NAIP5/NLRC4 inflammasome, LFn-flagellin will be developed by fusing recombinant flagellin carboxy-terminal to the amino-terminal domain of anthrax lethal factor. LFn-flagellin together with protective antigen (PA) of Bacillus anthracis, can efficiently translocate heterologous fusion proteins into mammalian cytosol through endocytosis-mediated entry. We further investigate the conserved domains of flagellin of T. pallidum sensed by TLR5 and NAIP5 receptors. Characterization of TLR5 and NAIP5/NLRC4 inflammasome response to the flagellin of T.pallidum will be useful for understanding the pathogenesis of T. pallidum,and be helpful for the prevention and treatment of syphilis.

梅毒是由梅毒螺旋体（T.pallidum，Tp）引起的严重危害人类健康的性传播疾病，其发病率居各类性传播疾病之首。目前，Tp致病机制仍不清楚,研究认为Tp感染诱导的炎症反应是造成免疫病理损伤的主要因素，细菌鞭毛蛋白在诱导炎症反应中发挥重要作用，但与细菌鞭毛蛋白高度同源的Tp鞭毛蛋白是否为导致梅毒炎症反应的重要因子及作用机制并不明确。本项目拟采用基因克隆与表达、免疫荧光共聚焦、流式细胞术、Western Blot、实时荧光定量PCR、基因敲除和负显性技术等方法，从分子、细胞及动物不同层面，探索Tp鞭毛蛋白在梅毒感染过程中的致炎作用，研究Tp鞭毛蛋白与TLR5及其胞内NAIP5/NLRC4炎症小体的相互作用及关键位点，明确Tp鞭毛蛋白导致炎症反应过程中的关键因子及信号通路，最终阐明Tp鞭毛蛋白在梅毒炎症反应及免疫病理损伤中的作用与机制，为Tp致病机制、预防与治疗以及疫苗研发提供新的思路与依据。

梅毒是由梅毒螺旋体（Treponema pallidum, Tp）引起的严重危害人类健康的性传播疾病，并可明显增加感染和传播艾滋病的危险性。近年来我国梅毒发病率呈逐年上升趋势。目前，Tp的致病物质和致病机制仍不清楚，研究认为Tp感染诱导的炎症反应是造成免疫病理损伤的主要因素。细菌鞭毛蛋白作为一种经典病原相关分子模式，在促进病原体侵袭和诱导宿主炎症反应中发挥重要作用。本项目通过体外基因克隆与表达了Tp鞭毛蛋白FlaA2、FlaB2和FlaB3。并采用间接免疫荧光、RT-PCR、Western Blot、RNA干扰、蛋白芯片、基因突变和免疫共沉淀等技术探究了Tp鞭毛蛋白在梅毒感染过程中的致炎作用及分子机制。结果显示：Tp鞭毛鞘蛋白FlaA2可经TLR2途径诱导单核细胞产生炎症因子IL-6；Tp鞭毛蛋白FlaB1、FlaB2和FlaB3可通过TLR5诱导皮肤角质形成细胞表达基质金属蛋白酶，进而降解细胞外基质，引起皮肤免疫炎症反应并促进Tp侵袭；此外，Tp鞭毛蛋白B3可通过胞内NLRC4炎症小体诱导人巨噬细胞表达IL-1β 和IL-18；同时Tp鞭毛蛋白FlaB1、FlaB2和FlaB3还可通过其ND1区的R89、L93和E113三个位点氨基酸与单核细胞胞外TLR5相互作用产生炎症因子。本项目的开展阐明了Tp鞭毛蛋白在梅毒炎症反应及免疫病理损伤中的作用与机制，为梅毒防控及疫苗研发提供新的思路与依据。