Quality management in assembly of bacterial type III secretion systems – a comparative analysis of the construction of flagella and injectisomes

通过III型分泌系统（T3SS）输出蛋白质对于细菌鞭毛的自组装以及包括肠沙门氏菌在内的许多革兰氏阴性病原体的毒力相关注射体的构建和功能至关重要。鞭毛是一种旋转装置，有助于细菌在表面和液体环境中移动。相比之下，注射体通过针状阑尾分泌并注入多种细菌效应蛋白到真核宿主细胞中。核心T3SS是一个复杂的蛋白质分泌机器，由五个高度保守的蛋白质（FliPQR FlhBA / SpaPQRS InvA）组成，它们在鞭毛和注射体的底部形成实际的出口装置。我们最近可以证明，T3SS出口设备的生产组装对于这些纳米机器的构建至关重要，并且涉及到鞭毛T3SS的专用膜集成组装伴侣FliO。基于我们之前的工作和额外的初步结果，我们假设出口装置的组装在多个层面上受到高度调节和协调，包括翻译起始、膜整合和亚复合物的形成。因此，本研究的目的是通过对肠道沙门氏菌中鞭毛和注射体这两个相关系统进行深入的比较分析，以获得细菌协调T3SS输出装置组装的分子质量控制机制的机制理解。特别是，我们的目标是阐明(i)鞭毛特异性的完整膜伴侣FliO和FliP的信号肽如何促进核心鞭毛输出装置的复杂形成，（ii）确定高度保守的fliPQR/spaPQR基因顺序与输出装置组装的相关性，以及（iii）解剖这些输出装置组件的基因在共翻译膜靶向过程中的翻译偶联作用。插入和Fli/SpaPQR复合体组装。除了分子生物学、细菌遗传学和膜蛋白靶向的经典方法外，我们的分析中心方法是：半胱氨酸可及性筛选来分析输出装置蛋白的拓扑发生；基于荧光素酶的报告系统评估t3ss的翻译效率和功能；在体内光交联、交联质谱和蓝色原生PAGE分析蛋白质-蛋白质相互作用和蛋白质复合物的组装，以及选择性核糖体分析研究输出装置蛋白的翻译和共翻译靶向。在比较分析中，进化相关和实验上非常容易获得的T3SS出口设备组件代表了很好的模型系统，可以阐明使用这些技术进行膜蛋白组装过程的质量管理和协调的基本原理。

Protein export via type III secretion systems (T3SS) is essential for the self-assembly of bacterial flagella as well as for construction and function of virulence-associated injectisomes of many Gram-negative pathogens, including Salmonella enterica. Flagella are rotary devices that facilitate movement of bacteria on surfaces and through liquid environments. In contrast, injectisomes secrete and inject a great diversity of bacterial effector proteins through a needle-like appendix into eukaryotic host cells.The core T3SS is a sophisticated protein secretion machine and is made of five highly conserved proteins (FliPQR FlhBA / SpaPQRS InvA) that form the actual export apparatus at the base of flagella and injectisomes. We could recently show that productive assembly of the T3SS export apparatus is essential for the construction of these nanomachines and involves in case of the flagellar T3SS a dedicated membrane-integrated assembly chaperone, FliO. Based on our previous work and additional preliminary results, we hypothesize that assembly of the export apparatus is highly regulated and coordinated on multiple levels, including translation initiation, membrane integration, and sub-complex formation.Accordingly, the goal of this study is to obtain a mechanistic understanding of the molecular quality control mechanisms that allow bacteria to coordinate the assembly of the T3SS export apparatus of both the flagellum and injectisome by performing an in-depth comparative analysis of these two related systems in Salmonella enterica. In particular, we aim to elucidate (i) how the flagellum-specific, integral membrane chaperone FliO and signal peptide of FliP facilitate complex formation of the core flagellar export apparatus, (ii) to identify the relevance of the highly conserved gene order of fliPQR/spaPQR for assembly of the export apparatus, and (iii) to dissect the role of translational coupling of the genes of these export apparatus components during co-translational membrane targeting, insertion and Fli/SpaPQR complex assembly.Central methods of our analysis are besides classical approaches of molecular biology, bacterial genetics, and membrane protein targeting: cysteine accessibility screens to analyze the topogenesis of export apparatus proteins; luciferase-based reporter systems to assess the efficiency of translation and the function of T3SSs; and in vivo photocrosslinking, crosslinking-mass spectrometry, and blue native PAGE to analyze protein-protein interactions and assembly of protein complexes, and selective ribosome profiling to investigate translation and co-translational targeting of export apparatus proteins.The evolutionarily related and experimentally very accessible T3SS export apparatus components represent excellent model systems to elucidate basic principles of quality management and coordination of membrane protein assembly processes using these techniques in a comparative analysis.