Luman在布鲁氏菌调节非折叠蛋白反应和细胞凋亡抑制中的作用及其机制研究

The unfolded protein response (UPR) activated by Brucella infection is crucial for its colonization and persistent infection due to the apoptosis inhibition of the infected cell. It is verified that IRE1/PERK expression can be activated in Brucella infecton. However, the exact regulation mechanism is still unclear. Luman is a novel transcription factor involved in the unfolded protein response (UPR), and plays a critical role in the pathway of ER stress-induced apoptosis. Furthermore, our previous study proved its participation regulating the Brucella infection. In this study, the goat immortalized trophoblastic cells and Luman gene knockout/overexpression trophoblastic cells were used to detect the functions of Luman in responses to the infection of different Brucella strains or key virulence factors (VceC) by Western Blot, quantitative PCR, FCM and RNAi techniques. ①The function of Luman in BCV's formation, migration and proliferation in Brucella infected trophoblastic cells. ②The influence of Luman on trophoblastic cells' secretion activity in Brucella infected GTCs. ③The regulating effect of Luman on UPR activation infected with Brucella/VceC. ④The effects of Luman on the expressions of IRE1, IRE1/XBP1 and HERP/EDEM, and analysing its pathway in Brucella infection. The study will illustrates the effects and regulation mechanisms of Luman on apoptosis inhibition of infected host cells and proliferation of Brucella from a new perspective. The results may contribute to experimental bases and theoretical basis for further investigation of the response between Brucella and host, exploring new prevention and control targets. Meanwhile, Our results would also provide the theoretical basis for understanding the mechanisms involved in infertility and abortions caused by Brucella infection.

布鲁氏菌激活细胞的UPR是其抑制细胞凋亡并在细胞内存活和感染的关键。研究显示，布鲁氏菌可激活内质网膜上IRE1/PERK的表达，但其机制尚不清楚。Luman是调节UPR的关键性宿主因子，前期研究证明其参与了布鲁氏菌感染的调节。本项目以山羊滋养层细胞为模型，通过RNAi、qPCR、western blot、免疫荧光、电镜及流式细胞术等方法，测定布鲁氏菌/VceC感染细胞后，Luman ①对布鲁氏菌在滋养层细胞内BCV形成、迁移与增殖的影响；②对布鲁氏菌损伤滋养层细胞分泌活性的影响，③对布鲁氏菌/VceC激活滋养层细胞UPR的调节作用；④对IRE1表达、IRE1/XBP1信号通路以及Herp/EDEM表达的影响，分析其作用途径。揭示Luman调节布鲁氏菌细胞内增殖与诱导细胞凋亡抑制的作用及其机制。为深入研究布鲁氏菌-宿主相互作用、导致不育和流产的机制，探寻布病防控新靶点提供实验依据和理论基础。

布鲁氏菌（Brucella）等胞内感染菌引起的人兽共患传染病至今依然是世界范围内严重威胁人类健康和动物健康养殖的重大疫病，极大地影响动物产品的对外贸易，国际上将其列为B类生物恐怖战剂和农业生物恐怖战剂(Skendros et al. 2011)，对该病的研究具有重大的实际应用价值和理论意义。近年来，我国布病新患病人数快速上升，动物疫区扩散，已经严重的危害人民健康和影响畜牧业发展。面对如此大的经济压力和扑灭难度，探寻新的控制、预防和治疗措施成为该领域面临的重大挑战。充分揭示布鲁氏菌的病原特点、全面了解布鲁氏菌与宿主相互作用的分子机制是探寻布鲁氏菌病防控的关键，也是该领域研究的核心。本项目以布鲁氏菌感染的主要靶细胞—奶山羊胎盘滋养层细胞为模型，揭示Luman在布鲁氏菌，尤其是VceC在诱导感染细胞凋亡抑制中的作用及其机制。同时，结合近年该领域的研究进展和布鲁氏菌病防控的实际需求，以B. suis S2为基础，针对参与布鲁氏菌细胞周期、细胞膜稳态维持和调节宿主免疫的基因，构建基因敲除株，检测其生物学特性和免疫活性的变化。证明布鲁氏菌通过调节宿主细胞内质网应激反应增强其在体内的生存和增殖，同时证明：Brucella BI-1 缺失导致B. suis S2生长抑制、分裂受阻、细胞生存力降低、应激敏感性增强和细胞膜性质改变。DnaA缺失菌株胞内增殖减少以及形成rBCV受阻, 毒力显著降低，且炎性细胞因子IL-6的表达升高; Brucella BI-1的缺失显著降低了B. suis S2 在小鼠模型中的毒力，但能够显著促进B. suis S2 介导的巨噬细胞的炎症反应。上述成果为进一步完善布鲁氏菌感染的调控机理、探寻布鲁氏菌防控的新靶点提供新的理论依据，同时，为新型安全基因缺失疫苗的研发提供了材料。