Abstract
Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigate how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.
Highlights
Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second
While FliP features a short, highly conserved loop of three methionines—for which we previously reported evidence that it acts as a gasket to close the T3SS channel6—FliR exposes instead a bulky domain that was hypothesized to act as a plug to seal the channel[5] (Fig. 1b, c)
The T3SSs of the flagellum and the injectisome are capable of translocating substrate proteins across the inner membrane with a remarkable speed of several thousand amino acids per second, which is several orders of magnitude faster than protein export in other pore-based protein channels[1,23]
Summary
Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. We recently demonstrated that secretion of flagellin via the fT3SS of Salmonella enterica occurs through an injection-diffusion mechanism with a remarkably fast initial injection speed—up to tens of thousands amino acids per second1—and similar results were reported in Vibrio alginolyticus[2]. This high rate of secretion, without equivalent in other pore-based protein channels, raises the question of how membrane gating is preserved. We demonstrate that gating of the EA is needed during active type-III protein secretion and that mutations compromising the membrane barrier are detrimental to bacterial fitness
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