Assembly and Function of the Core Type-III Protein Export Apparatus of the Bacterial Flagellum


Many bacteria, including Salmonella Typhimurium, use rotation of a helical organelle, the flagellum, for directed movement in various environments. The flagellum is composed of three main parts: a basal body, a flexible, curved adapter structure, and a long external filament. The bacterial flagellum is functionally and structurally related to virulence-associated injectisome systems of pathogenic bacteria. Both nanomachines utilize a type-III secretion system (T3SS) to export proteins across the inner membrane in a proton motive force-dependent manner. Type-III protein secretion is essential for the assembly of the flagellum, the injectisome, as well as for secretion of effector proteins. The core protein export apparatus consists of a cytoplasmic ATPase-cargo delivery complex and six integral membrane proteins that are responsible for the proton motive force-driven export of substrate proteins. However, a detailed understanding of the protein export function on a molecular level is crucially missing. In case of the flagellum, a complex of the integral membrane components FliOPQR is presumed to form the secretion channel of the export apparatus. The core complex proteins FliOPQR are required for function of the T3SS and assembly of the flagellum, but little is known about the stoichiometry of the complex, orientation of the transmembrane domains, inter- and intrasubunit interactions, and molecular functions during protein export. FliP is the most conserved component of the core T3SS. Based on preliminary data and bioinformatical analyses, we hypothesize that oligomers of FliP form the protein-conducting channel of the flagellar T3SS. The goal of this project is to elucidate the molecular function of FliP using a synergistic combination of bioinformatics, genetics and biochemical approaches. We will first determine the orientation of secondary structure elements of FliP using site-specific accessibility of cysteine residues, which forms the basis to generate a structural model of the FliP export channel. We will next combine crosslinking strategies and high-resolution electron cryotomography to elucidate inter- and intra-molecular interactions and pinpoint sub-cellular localization of protein domains within the T3SS core complex. Various mutational approaches will corroborate the biochemical and microscopic analyses to study FliP function and pore complex formation. This information will be used to rationalize and iteratively improve a FliP structural model with the goal to generate a pseudo-atomic model of the T3SS core complex. In conclusion, the insights gained in this project will facilitate our mechanistic understanding of the function of bacterial type-III secretion systems and assembly of complex multi-protein machineries in general.


Principal Investigators
Erhardt, Marc Prof. Dr. (Details) (Bacterial Physiology)

Duration of Project
Start date: 11/2017
End date: 06/2020

Research Areas
Metabolism, Biochemistry and Genetics of Microorganisms, Parasitology and Biology of Tropical Infectious Disease Pathogens

Publications
1) Fabiani, F. D. et al. A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum. PLoS Biol 15, e2002267 (2017).

2) Ward, E. et al. Type-III secretion pore formed by flagellar protein FliP. Mol Microbiol 107, 94-103 (2018).

Last updated on 2021-15-09 at 10:42