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（T3 SS）的蛋白质输出对于细菌鞭毛的自组装以及许多革兰氏阴性病原体（包括肠道沙门氏菌）的毒力相关注射体的构建和功能是必不可少的。鞭毛是旋转装置，其促进细菌在表面上和通过液体环境的移动。相反，注射体通过针状附件分泌和注射多种细菌效应蛋白到真核宿主细胞中。核心T3 SS是一个复杂的蛋白分泌机器，由五种高度保守的蛋白质（FliPQR FlhBA / SpaPQRS InvA）组成，它们在鞭毛和注射体的基部形成实际的输出装置。我们最近可以证明，T3 SS输出装置的生产性组装对于这些纳米机器的构建是必不可少的，并且在鞭毛T3 SS的情况下涉及专用的膜集成组装分子伴侣FliO。基于我们先前的工作和额外的初步结果，我们假设输出装置的组装在多个水平上受到高度调节和协调，包括翻译起始、膜整合和亚复合物形成。本研究的目的是获得分子质量控制机制的机械理解，该机制允许细菌协调T3 SS输出装置的组装，鞭毛和注射体，这两个相关系统进行深入的比较分析，在肠道沙门氏菌。特别地，我们的目标是阐明（i）鞭毛特异性的、完整的膜伴侣FliO和FliP的信号肽如何促进核心鞭毛输出装置的复合物形成，（ii）鉴定高度保守的fliPQR/spaPQR基因顺序与输出装置组装的相关性，和（iii）剖析在共翻译膜靶向期间这些输出装置组分的基因的翻译偶联的作用，除了分子生物学、细菌遗传学和膜蛋白靶向的经典方法之外，我们的分析的中心方法是：半胱氨酸可及性筛选以分析输出装置蛋白的拓扑发生;评估翻译效率和T3 SS功能的基于内切酶的报告系统;以及体内光交联、交联-质谱和蓝色非变性PAGE来分析蛋白质-蛋白质相互作用和蛋白质复合物的组装，和选择性核糖体分析来研究翻译和共-在进化上相关的和实验上非常容易获得的T3 SS输出装置组件代表了极好的模型系统，以阐明质量管理的基本原理和使用这些技术的膜蛋白组装过程的协调。比较分析。

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.