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。

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.