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Assembly and substrate switching of the T3S protein export apparatus in enteropathogenic E. coli
Book - Dissertation
The Type III protein secretion (T3S) pathway is common in Gram-negative pathogens, like enteropathogenic E. coli(EPEC). EPEC can use the T3S machinery, also called injectisome, to deliver virulence factors into the host cell. The injectisome comprises the basal body with a cytoplasm-facing export apparatus channel and a surface-exposed needle. The export apparatus comprises a conical SctR5S4T1export channel, decorated by SctU, and enveloped by SctV. The large cytoplasmic domain (C-domain) of SctV binds T3S chaperone/substrate complexes and forms a putative ante-chamber leading to the membrane translocase.The aim of this study is to dissect the assembly of the export apparatus and the mechanism of Ca2+mediated substrate switching.Dissection of EPEC T3SS and determination of its structure are hindered by the low numbers of the injectisomes per cell. Firstly, we report that the plasmid encoded regulator Per and the master regulator Ler can improve the production of the injectisomes by 2-3 fold. That suggests that it's possible to maximize the production of the injectisomefor further biochemical and biophysical analysis of the T3SS in vitro. SctW is critical in controlling substrate switching from translocators to effectors as well as Ca2+. In this study, we would like to figure out the connection between Ca2+andSctW related substrate switching in EPEC. The optimized M9 medium isestablished and used because of its less complexity and easier manipulation.No coincidence between SctW secretion and effectors secretion was seen, which negates the hypothesis that secretion of SctW initiates effector secretion. Besides, Ca2+did not change SctW membrane assocication, which indicates that SctW triggers effector secretion without releasing from membrane. To probe the mechanism of assembly and function of the T3S export apparatus, we first used live cell imaging. SctV assembles in oligomeric clusters at cell periphery of both EPEC and a non-T3SS E.colistrain. Non-ionic detergents extracted SctV homo-nonamers from membranes of both strains. The reconstituted His-SctV9-PR peptidiscs show an elongated, tripartite particle of ~22 nm with a membrane domain and a narrow linker connecting to a C-domain. The C-domain assembles in a hollow asymmetric ring with a 5-6 nm inner opening. SctV9is necessary and sufficient to act as a receptor for chaperone/substrate complexes via its C-domain. These findings mechanistically uncouple chaperone/substrate targeting from the succeeding translocation step and advance structural understanding of injectisome assembly.
Number of pages: 133