New Insights into Organization, Assembly and Function of the Export Apparatus of Bacterial Type III Secretion Systems

Abstract

Bacterial type III secretion systems (T3SSs) are big multi-protein complexes which span both membranes of Gram-negative bacteria and are used to translocate effector proteins directly into the cytoplasm of target host cells. The export apparatus, a substructure centrally located in the inner membrane inside of these systems is essential for their proper assembly and functionality. In the Salmonella T3SS1, encoded on the Salmonella pathogenicity island 1 (SPI-1), the export apparatus is composed of the five membrane proteins SpaPQRS and InvA. This substructure is also present in the closely related flagella system, which bacteria like Salmonella use as a motility device. In the Salmonella flagella system the homologs of the export apparatus proteins are FliPQR, FlhB and FlhA. In this work the structural organization and assembly of the core components of this subcomplex from both systems were investigated. It was found that in both systems the homologs SpaP/FliP form a stable complex with SpaR/FliR independently of all other components. SpaP/FliP was identified as T3SS assembly nucleation point, which needs immediate stabilization by SpaR/FliR. This solid subcomplex acts as a platform onto which subsequently SpaQ/FliQ and later on SpaS/FlhB as well as InvA/FlhA get recruited. For the injectisome, the core component SpaP was identified as the pore forming unit in the inner membrane. This protein forms a pentamer with a donut-like shape which allows the passage of molecules with a size of 500 Da, indicating a pore diameter of about 15 Å. For its homolog FliP in the flagella system it was found that this protein forms a hexamer, built by a trimer of dimers. The hexamer is organized, as its respective injectisome homolog, in a donut-like shape, suggesting a similar function. Furthermore, the function of an additional flagella-associated protein FliO was investigated. This bitopic membrane protein is missing in the injectisome and has been reported to be connected to FliP functionality. In this work, it was shown that FliO acts as a chaperone for FliP, preventing its degradation via the Lon pathway and stabilizing its multimerization until FliR gets recruited. FliO itself builds multimers and is organized in a ring structure with flexible clamp like side arms to which FliP is able to bind.