Effectors of Brucella intracellular replication

项目摘要 细胞内细菌病原体引起大量具有公共卫生重要性的疾病。他们的 细胞内循环是毒力的关键，并在一个专门用于它们的生态位的生物发生中达到高潮。 存活、增殖或持久性，这是通过它们颠覆宿主细胞功能而实现的壮举。 确定用于创建这些小生境的机制对于理解其发病机制至关重要， 为微生物共有的致病主题提供了广泛的见解。细菌对宿主细胞的颠覆 功能调用传递效应蛋白，其作用模式的关键，他们的致病性 机制等布氏杆菌属细菌是世界性人畜共患病的病原体 布鲁氏菌病，产生一个复制允许的细胞器，含布鲁氏菌的空泡（rBCV）， 其发病机制是必需的，来源于宿主内质网（ER）。rBCV生物发生 需要布鲁氏菌VirB IV型分泌系统（T4SS）和宿主分泌运输，这表明 由调节特定分泌功能的T4SS递送效应蛋白控制。然而 rBCV生物发生的细菌效应子和宿主因子大多是未知的。我们最近发现 靶向宿主分泌途径的布鲁氏菌VirB T4SS效应子（BspA、BspB和BspF） rBCV生物发生或rBCV内的细菌生长，并与各种宿主蛋白相互作用 功能上与高尔基体的ER相关降解（ERAD）或分泌运输相关 设备.这些调查结果及时提供了机会，以解决有关 rBCV生物发生的分子机制。在这里，我们建议阐明的机制， 新鉴定的T4SS效应蛋白BspA、BspB和BspF调节不同的宿主分泌功能 促进rBCV生物合成和细菌增殖。我们将使用分子和细胞的方法 首先剖析BspB在调节高尔基体相关分泌运输中的作用模式， 与COG复合物的相互作用，并定义它如何有助于rBCV生物发生。二是 确定视神经磷酸酶（OPTN）介导的BspF靶向功能是否在高尔基体后囊泡中起作用， 运输介导其在布鲁氏菌细胞内复制中的作用。最后，我们将研究BspA是否 通过靶向ERAD E3泛素连接酶MARCH 6调节ER相关降解（ERAD） 以促进rBCV生物合成和布鲁氏菌增殖。该项目的成功完成将 提供了通过靶向宿主参与rBCV生物发生的布鲁氏菌效应子的首次表征 分泌功能通过鉴定参与布鲁氏菌细胞内循环的新宿主功能， 拟议的研究将确定病原体利用宿主分泌途径的新范例 适用于靶向该细胞区室的许多细胞内微生物，因此具有 对微生物发病机理、细胞生物学和先天免疫等研究领域产生了广泛的影响。

Project Summary Intracellular bacterial pathogens cause a large number of diseases of public health importance. Their intracellular cycle is key to virulence and culminates in the biogenesis of a niche dedicated to their survival, proliferation or persistence, a feat achieved via their subversion of host cell functions. Determining the mechanisms used to create these niches is critical to understanding their pathogenesis, providing broad insights into pathogenic themes shared by microbes. Bacterial subversion of host cell functions invokes delivery of effector proteins, whose modes of action hold keys to their pathogenic mechanisms. Bacteria of the genus Brucella, the causative agents of the world-widespread zoonosis brucellosis, generate a replication-permissive organelle, the Brucella-containing vacuole (rBCV), which is essential to their pathogenesis and derived from the host endoplasmic reticulum (ER). rBCV biogenesis requires the Brucella VirB Type IV secretion system (T4SS) and host secretory trafficking, suggesting it is controlled by T4SS-delivered effector proteins that modulate specific secretory functions. Yet, the bacterial effectors and host factors of rBCV biogenesis are mostly unknown. We recently discovered Brucella VirB T4SS effectors (BspA, BspB and BspF) that target the host secretory pathway, contribute to either rBCV biogenesis or bacterial growth within rBCVs, and interact with various host proteins functionally associated with either ER-associated degradation (ERAD) or secretory trafficking at the Golgi apparatus. These findings provide timely opportunities to address outstanding questions about the molecular mechanisms of rBCV biogenesis. Here we propose to elucidate the mechanisms by which the newly identified T4SS effector proteins BspA, BspB and BspF modulate distinct host secretory functions to promote rBCV biogenesis and bacterial proliferation. We will use molecular and cellular approaches to first dissect the mode of action of BspB in modulating Golgi-associated secretory trafficking via its interaction with the COG complex, and define how it contributes to rBCV biogenesis. Second, we will determine whether BspF targeting of optineurin (OPTN)-mediated functions in post-Golgi vesicular trafficking mediates its role in Brucella intracellular replication. Last, we will examine whether BspA modulates ER-associated degradation (ERAD) via its targeting of the ERAD E3 ubiquitin ligase MARCH6 to facilitate rBCV biogenesis and Brucella proliferation. The successful completion of this project will provide the first characterizations of Brucella effectors involved in rBCV biogenesis via targeting of host secretory functions. By identifying novel host functions involved in the Brucella intracellular cycle, the proposed research will define new paradigms of pathogenic exploitation of the host secretory pathway applicable to the many intracellular microbes that target this cellular compartment, therefore having a broad impact on the research areas of microbial pathogenesis, cell biology and innate immunity.