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Investigating the assembly and function of the bacterial injectisome by hybrid structural methods Majewski, Dorothy Diana

Abstract

The bacterial injectisome is an essential virulence factor for many Gram-negative pathogens. Resembling a 50-100 nm long syringe, the injectisome creates a continuous channel between the bacterial and host cytosols through which the bacterium secretes effector proteins to modulate host signalling. The aim of the following work is to contribute to the structural characterization of the injectisome, focusing on proteins involved in its assembly and substrate selection. The outer membrane pore of the injectisome, termed the secretin, relies on a pilotin protein for its localization. The X-ray crystallographic structure of the Salmonella enterica SPI-1 pilotin InvH reveals an α-helical dimer, confirmed to exist in solution through biophysical experiments. The pilotin-secretin interface, characterized by X-ray crystallography and NMR spectroscopy, is mutually exclusive with the InvH dimer and results in a 1:1 complex. The inner membrane pore protein has a vital role in injectisomal secretion hierarchy. The cryo-EM structure of the IM pore EscV from enteropathogenic Escherichia coli (EPEC) demonstrates that the protein forms a nonameric ring in solution. Of its four subdomains, two contribute to ring formation while the remaining two have some rotational freedom. The cytosolic ATPase complex is essential to separating effector proteins from their cognate chaperones prior to secretion. The cryo-EM analysis of the EPEC ATPase-stalk complex, EscN-EscO, yielded an asymmetric homohexamer structure resembling F1- and V-ATPases. Kinetic studies show that oligomerization and the presence of the stalk are required for efficient ATP hydrolysis. The similarity to rotary ATPases supports the hypothesis that the injectisomal ATPase complex is a rotary motor, with the stalk acting as the rotor. How this rotation contributes to secretion is yet to be discovered.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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