Effectors of Brucella intracellular replication

项目摘要 细胞内的细菌病原体导致了大量对公共卫生具有重要意义的疾病。他们的 细胞内循环是毒力的关键，并最终导致致力于它们的生态位的生物发生 生存、增殖或持久，这是一项通过颠覆宿主细胞功能实现的壮举。 确定用于创建这些生态位的机制对于了解它们的发病机制至关重要， 对微生物共有的致病主题提供了广泛的见解。宿主细胞的细菌颠覆 功能激活效应蛋白的传递，其作用模式是致病的关键 机械装置。布鲁氏菌属细菌，世界范围内广泛流行的人畜共患病的病原体 布鲁氏菌病，产生一种允许复制的细胞器，即含有布鲁氏菌的液泡(RBCV)，它是 在其发病机制中是必不可少的，来源于宿主内质网(ER)。RBCV的生物发生 需要布鲁氏菌VIRB IV型分泌系统(T4SS)和宿主分泌运输，提示它 是由T4SS传递的调节特定分泌功能的效应蛋白控制的。然而， RBCV生物发生的细菌效应因子和宿主因子大多尚不清楚。我们最近发现 针对宿主分泌途径的布鲁氏菌VIRB T4SS效应物(BspA、BSPB和BspF)起作用 RBCV的生物发生或细菌生长，并与各种宿主蛋白相互作用 在功能上与内质网相关的退化(ERAD)或高尔基体的分泌物贩运有关 仪器。这些调查结果提供了及时解决有关 RBCV生物发生的分子机制。在这里，我们建议阐明 新发现的T4SS效应蛋白BspA、BSPB和BspF调节不同的宿主分泌功能 促进rBCV的生物生成和细菌增殖。我们将使用分子和细胞方法 首先剖析BSPB通过ITS调控高尔基体相关分泌物运输的作用模式 与COG复合体的相互作用，并定义它对rBCV生物发生的贡献。第二，我们将 确定BspF靶向视神经磷酸酶(OPTN)在高尔基体后小泡中的作用 运输调节其在布鲁氏菌细胞内复制中的作用。最后，我们将检查BSPA是否 通过靶向ERAD E3泛素连接酶MARCH6调控ER相关降解(ERAD 以促进rBCV的生物发生和布鲁氏菌的增殖。这一项目的成功完成将 首次通过靶向宿主提供了参与rBCV生物发生的布鲁氏菌效应子的特征 分泌功能。通过识别参与布鲁氏菌细胞内循环的新宿主功能， 拟议的研究将定义病原性利用宿主分泌途径的新范式 适用于许多以这个细胞隔间为目标的细胞内微生物，因此具有 对微生物发病机制、细胞生物学和先天免疫等研究领域产生广泛影响。

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.