具有GAP功能的BspI效应蛋白调节布鲁氏菌胞内生存的分子机制

The formation of rBCV (replicative Brucella-containing vacuole) is crucial for survival and replication of Brucella. The previous studies demonstrated that the activity of the small GTPase Rab2 plays an essential role in mediating the formation of rBCV. However, the mechanism by which Brucella regulates the activity of Rab2 remains unknown until now. Our preliminary data showed that the Brucella effector BspI, which contains GAP domain, can act as the GAP（GTPase-Activating Protein）of Rab2. Moreover, compared with wild type Brucella infection, the intracellular growth of BspI deficient Brucella was suppressed significantly. Based on the above observations, we propose that BspI regulates the formation of rBCV via its GAP of Rab2, which promotes Brucella intracellular survival. To test the hypothesis, the following experiments will performed: (i) the methods of cell biology and molecular bacteriology will be utilized to confirm that BspI is a specific Rab2 GAP; (ii) B.abortus 2308 strains, in which BspI gene is deleted or the relative BspI mutation is expressed, will be constructed; (iii) the roles of BspI in fusion with ER(Endoplamic Reticulum) or lysosome will be clarified via confocal microscopy; (iv) the roles of BspI in Brucella survival within macrophages will be determined through CFU( Colony-Forming Unit ) assay. . If the proposal is funded, our studies will not only help elucidate how BspI regulates the formation of rBCV and show a novel interpretation of Brucella survival, but will potentially give an insight in how to manipulate the bacterial effector to achieve more efficient elimination of intracellular Brucella. This is a topic of significance in the development of drugs to prevent and treat Brucellosis.

布鲁氏菌（布菌）在宿主细胞内形成复制性菌泡是其在胞内生存和增殖的关键。研究发现布菌菌泡上的小G蛋白Rab2活性在复制性菌泡形成中发挥重要作用，但是目前尚不清楚Rab2的活性是如何被调控的。我们的预实验显示：含有GTP酶活化蛋白结构域的效应蛋白BspI是Rab2的GAP，且缺失BspI的布菌在宿主细胞内的生长显著减少。据此，我们推测BspI可能通过其Rab2的GAP功能调节复制性菌泡的形成，从而促进布菌的胞内生存。为验证该假说，本课题拟用细胞生物学手段明确BspI是Rab2的特异性GAP；通过对BspI的GAP结构域分析构建BspI突变体的布菌菌株；利用该菌株感染巨噬细胞，通过检测布菌中期菌泡与溶酶体或内质网融合情况来探究BspI的GAP活性在复制性菌泡形成及促进布菌胞内生存中的作用。本课题首次从GAP分子调节复制性菌泡形成的角度研究布菌胞内生存机制，这将有助于我们深入理解布菌的感染机制。

项目背景：目前学界普遍认为布菌的IV型分泌系统（T4SS）可以通过其分泌的效应蛋白调节布菌胞内寄生，但是是哪一种效应蛋白起这种调节作用，以及其调节机制都未知。该假设的证明有助于我们深入了解布菌的胞内寄生机制，对未来设计靶向药物治疗布病将打下坚实的实验和理论基础。.主要研究内容：1）本项目利用生物信息学的相关知识分析已知的T4SS所有效应蛋白，锁定BspI作为研究对象；2）通过细胞生物学及分子细菌学手段证明BspI在调节布菌胞内生存中的功能；3）进一步证明其在调节布菌感染引起的炎症反应中的作用。.重要结果：1）具有GAP结构域的BspI效应蛋白通过T4SS的分泌进入宿主细胞调节布菌的胞内寄生；2）BspI通过其GAP结构域抑制布菌感染引起的炎症反应，促进布菌胞内寄生;3)在前两个结果的基础上，我们进一步研究T4SS在调节流产中的作用，结果表明：T4SS的存在是布菌感染导致流产的关键因素。.关键数据：1）证明具有GAP结构域的BspI效应蛋白是小G蛋白Rab2 的GAP分子；2）BspI 可以通过其GAP活性调节布菌胞内寄生；3）BspI 还可以通过其GAP活性负向调节布菌感染的巨噬细胞的炎症反应，同时我们还发现T4SS调节布菌感染引起的流产。.科学意义：1）促进了我们对布菌感染机制的理解；2）在该研究的基础上我们还扩展研究了T4SS与流产的关系，这对我们进一步探讨和研发相关治疗流产靶向药物提供了科学依据。