旋毛虫烯醇酶（TsEno）激活宿主纤溶系统协助幼虫侵入肠黏膜的机制

The critical process of the establishment of Trichinella infection is whether the Trichinella spiralis larvae invade intestinal mucosa to develop further. However, the mechanism of larval invasion is still unclear till now. In the process of invasion, parasites need to break through a variety of tissue barriers, and the targeted degradation induced by protease and fibrinolytic system is responsible. The fibrinolytic system not only exists in blood, but also in extravascular tissue fluids. The extracellular matrix (ECM) could be degraded after the activation of fibrinolytic system. Previous studies indicate that enolase can bind plasminogen (PLG) in extravascular fluids and activate the fibrinolytic system of host. Although the mechanism of the activation is not clear, the enhancement of fibrinolysis is surely helpful for the invasion of parasites. In order to figure out these unknown mechanisms, T. spiralis enolase (TsEno) gene will be cloned, expressed and purified. The antigenicity of recombinant TsEno protein (rTsEno) will be identified by ELISA and western blot, and the level of TsEno gene transcription and expression will be evaluated by real-time PCR and Western blot. The immunofluorescent test (IFT) will be used to locate the TsEno in the parasite tissues. The enzymatic activity of rTsEno and its specific binding capacity with PLG will be identified by biochemical methods, ELISA and Western blot. The mechanism of fibrinolytic system activation by rTsEno will be clarified by enzymatic reaction and competitive protein binding assay at molecular level. The scientific hypothesis that TsEno promotes larval invasion of intestinal mucosa by activating the host’s fibrinolytic system will be validated through ECM degration effect, the in vitro model of larval invading intestinal epithelial cells (IECs), animal experiments and RNAi at cellular and animal level, respectively. The scientific hypothesis will be tested at different point of view. The host’s contribution in the process of larval invasion is adequately considered when the experimental scheme is established. In order to mimic the real internal environment in normal host at the maximum level, the balanceable fibrinolytic system is added into the reaction in vitro. This project will elucidate the mechanism that TsEno promotes larval invasion of intestinal mucosa by activating host’s fibrinolytic system, and will provide some theoretical foundation for the study of interaction between parasites and hosts, and is helpful for development of new thrombolytic drugs.

旋毛虫幼虫侵入宿主肠黏膜是感染与致病的关键，但侵入机制尚不清楚。寄生虫突破宿主组织屏障需借助蛋白酶和纤溶系统的水解作用；纤溶系统不仅位于血管内，在组织液中也广泛分布，被激活后可降解细胞外基质，协助寄生虫侵入；烯醇酶可结合宿主组织液中的纤溶酶原，激活纤溶系统，但机制未明。我们前期研究发现，旋毛虫表面蛋白中含有旋毛虫烯醇酶（TsEno），且幼虫与肠上皮细胞孵育后TsEno表达量显著上调，提示TsEno可能与旋毛虫侵入肠黏膜有关。本项目将克隆表达TsEno，鉴定其酶活性及与纤溶酶原的特异性结合；通过酶促反应和竞争性结合实验，明确TsEno激活纤溶系统的具体方式；通过细胞外基质降解、旋毛虫体外侵入细胞模型、动物试验和RNAi，正反向验证TsEno通过激活纤溶系统协助幼虫侵入肠黏膜的科学假设。本项目对阐明宿主纤溶系统在旋毛虫侵入肠黏膜中的作用与机制具有重要的科学意义，并可为研发新型溶栓药物提供依据。

旋毛虫幼虫侵入宿主肠黏膜是感染与致病的关键，幼虫蛋白酶极可能介导了侵入过程。研究提示，旋毛虫烯醇酶（TsEno）可能通过与宿主纤溶酶原（PLG）的相互作用促进幼虫入侵，但机制未明。本项目应用生物信息学技术，分析了TsEno的基本特征并构建了3D模型，采用ZDOCK与已解析晶体结构的人PLG对接，VMD、LigPlot+（DIMPLOT）及PDBePISA等软件分析，确定关键位点及氨基酸残基间的互作方式。体外表达、纯化出了rTsEno和定点突变的M-rTsEno；应用qPCR、IFA观察了TsEno的转录表达及组织定位；应用Far-Western、ELISA、竞争性结合和PLG激活试验，验证了TsEno结合并促进PLG激活的作用。采用体外侵入模型、RNAi及动物试验，证实了TsEno在幼虫侵入宿主肠黏膜时的功能。结果表明，TsEno与PLG构象互补，TsEno的K90、K289、K291和K300在与PLG结合时发挥重要作用；PLG的活性决定位点W761与TsEno的E303之间存在较强的疏水相互作用。TsEno在旋毛虫各发育期均表达，集中分布在杆状体、皮层和胚胎。TsEno与PLG的结合具有特异性，肌幼虫ES包含结合PLG的52 kDa分子，rTsEno可特异结合小鼠肠黏膜组织。rTsEno和M-rTsEno均具催化活性；在t-PA存在时，rTsEno、M-rTsEno、肌幼虫可溶和ES均可促进PLG激活。rTsEno可促进幼虫侵入，抗rTsEno血清则具有抑制作用；以siRNA干扰TsEno表达后，可阻止49.82%的幼虫侵入IECs；rTsEno可诱导小鼠产生免疫保护，成虫和肌幼虫减虫率依次为31.79%和47.15%。提示，TsEno具有促进旋毛虫幼虫侵入的功能，是重要的侵入蛋白，可能是抗旋毛虫新药物或疫苗研发的候选靶标。