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Structural and functional characterization of components of bacterial type III secretion systems

University of British Columbia

Many Gram-negative pathogens use a type III secretion system (T3SS) to inject effector proteins into the host cytoplasm, where they manipulate host processes to the advantage of the bacterium. The T3SS is composed of a cytoplasmic export apparatus, a membrane-spanning basal body with a central channel formed by the inner rod, an extra-cellular needle filament and a translocon complex that inserts in the host membrane. In this thesis, proteins involved in T3SS assembly, as well as a T3SS effector protein were structurally and functionally characterized. The structure of EtgA, a T3SS-associated peptidoglycan (PG)-cleaving enzyme from enteropathogenic Escherichia coli (EPEC) was solved. The EtgA active site has features in common with lytic transglycosylases (LTs) and hen egg-white lysozyme (HEWL). EtgA contains an aspartate that aligns with lysozyme Asp52 (a residue critical for catalysis), a conservation not observed in LT families to which the conserved T3SS enzymes were presumed to belong. Mutation of the EtgA catalytic glutamate conserved across LTs and HEWL, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in T3SS assembly. EtgA forms a complex with the T3SS inner rod component, which enhances PG-lytic activity of EtgA in vitro, providing localization and regulation of the lytic activity to prevent cell lysis. After assembly of the basal body and needle, the gatekeeper protein ensures the translocon assembles at the needle tip prior to secretion of effector proteins. The gatekeeper from EPEC (SepL) was crystallized and it was shown that it has three X-bundle domains, which likely mediate protein-protein interactions to control translocon and effector secretion. Comparison of SepL to structurally characterized orthologs revealed several conserved residues, which may be required to regulate secretion of translocators or effectors. Finally, SopB, a Salmonella effector protein, in complex with host Cdc42, an Rho GTPase that regulates critical events in eukaryotic cytoskeleton organization and membrane trafficking was structurally characterized. Structural and biochemical analysis of the SopB/ Cdc42 complex shows that SopB structurally and functionally mimics a host guanine nucleotide dissociation inhibitor (GDI) by contacting key residues in the regulatory switch regions of Cdc42 and slowing Cdc42 nucleotide exchange.

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2015-07-27

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/54176

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/