Remodeling of intracellular membrane traffic by Brucella effectors

项目摘要 大多数具有公共卫生意义的细菌性疾病是由具有细胞内生命周期的病原体引起的 这是其毒性的组成部分。这些微生物利用多种宿主细胞功能来确保 它们的细胞内存活和增殖，通过递送调节特异性免疫应答的效应蛋白或毒素， 宿主过程，包括液泡病原体的细胞内膜运输。高尔基体 在宿主细胞成分的分泌运输中起核心作用，并被许多细菌靶向， 液泡生物发生和维持或细胞内营养获取的目的。我们所知甚少， 然而，关于利用这种基本细胞器的细菌效应器，也不是病原体如何破坏其 用于致病目的。布鲁氏杆菌属细菌，引起全球人畜共患病 布鲁氏菌病，产生一个复制许可的细胞器，来源于宿主内质网（ER）， 布鲁氏菌空泡（rBCV），这是必不可少的，他们的发病机制。rBCV生物发生需要 VirB IV型分泌系统（T4 SS）介导的调节特异性宿主的效应蛋白的递送 分泌功能我们鉴定了其中几种效应子，其中BspB靶向保守的 寡聚高尔基体（COG）复合物，高尔基体相关囊泡运输的主要调节因子，以促进 rBCV生物发生和细菌复制。另外的T4 SS效应子（BspD和BspF）调节高尔基体- 相关的运输功能，进一步支持了高尔基体在 布氏杆菌细胞内循环。我们的研究结果还表明，T4 SS效应子（BspB/RicA和BspF/BspD） 在调节膜转运途径方面功能性合作，认为布鲁氏菌 高尔基体相关过程的调节由多种效应子功能的整合引起。这里 我们将检验布鲁氏菌传递一系列T4 SS效应蛋白的假设， 膜转运在高尔基体界面起作用以协调地促进细菌增殖。我们将 使用遗传，细胞和生物化学方法，首先表征高尔基体运输途径， 有助于布鲁氏菌增殖并确定Golgi靶向T4 SS的靶点和功能网络 效应器其次，我们将确定高尔基体靶向T4 SS效应物的分子作用模式 BspB、BspD和BspF。最后，我们将定义i）BspB和RicA如何协调调节ER-高尔基体囊泡 ii）BspF和BspD如何共同调节TGN相关的转运 促进布鲁氏菌复制。该项目的成功完成将揭示细菌效应子- 驱动机制的调节高尔基体相关的膜运输功能，并定义新的 细菌病原体中效应子功能和协调的范例，广泛影响了研究 微生物发病机理、细胞生物学和先天免疫领域。

Project Summary Most bacterial diseases of public health significance are caused by pathogens with an intracellular lifecycle that is integral to their virulence. These microorganisms exploit a variety of host cell functions to ensure their intracellular survival and proliferation, via delivery of effector proteins or toxins that modulate specific host processes, including intracellular membrane transport by vacuolar pathogens. The Golgi apparatus plays a central role in the host secretory traffic of cellular components and is targeted by many bacteria for the purpose of vacuole biogenesis and maintenance or intracellular nutrient acquisition. Little is known, however, about the bacterial effectors that exploit this essential organelle, nor how pathogens subvert its functions for pathogenic purposes. Bacteria of the genus Brucella, which cause the global zoonosis brucellosis, generate a replication-permissive organelle derived from the host endoplasmic reticulum (ER), the Brucella-containing vacuole (rBCV), which is essential to their pathogenesis. rBCV biogenesis requires VirB Type IV secretion system (T4SS)-mediated delivery of effector proteins that modulate specific host secretory functions. We identified several of these effectors, among which BspB targets the conserved oligomeric Golgi (COG) complex, a master regulator of Golgi-associated vesicular trafficking, to promote rBCV biogenesis and bacterial replication. Additional T4SS effectors (BspD and BspF) modulate Golgi- associated trafficking functions, further supporting a previously unsuspected role of the Golgi apparatus in the Brucella intracellular cycle. Our findings also suggest that T4SS effectors (BspB/RicA and BspF/BspD) functionally cooperate in their modulation of membrane transport pathways, arguing that Brucella modulation of Golgi-associated processes results from the integration of multiple effector functions. Here we will test the hypothesis that Brucella delivers an array of T4SS effector proteins that modulate membrane transport functions at the Golgi interface to coordinately promote bacterial proliferation. We will use genetic, cellular and biochemical approaches to first characterize the Golgi transport pathways that contribute to Brucella proliferation and define the targets and functional network of Golgi-targeting T4SS effectors. Second, we will determine the molecular modes of action of the Golgi-targeting T4SS effectors BspB, BspD and BspF. Last, we will define i) how BspB and RicA coordinately modulate ER-Golgi vesicular transport to promote rBCV biogenesis and ii) how BspF and BspD co-modulate TGN-associated transport to promote Brucella replication. The successful completion of this project will uncover bacterial effector- driven mechanisms of modulation of Golgi-associated membrane transport functions and define new paradigms of effector functions and coordination in bacterial pathogens, broadly impacting the research areas of microbial pathogenesis, cell biology and innate immunity.