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

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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.