Abstract
Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.
Highlights
Shigella species are the etiological agent of bacillary dysentery in humans (1)
The Shigella T3SS consists of a cytoplasmic portion and a transmembrane region traversing both bacterial membranes, into which a hollow needle, made of MxiH, is embedded protruding from the bacterial surface (2)
After T3SS assembly, effector secretion is prevented through the concerted action of surface tip complex (TC) proteins and regulators that control secretion from within the bacterial cytoplasm
Summary
MxiC’s Secretion Signal Is Not Required for Promoting Inducible Translocator Secretion—A non-secretable form of MxiC lacking residues 2–30 is unable to prevent effector secretion (23). The resulting mxiC⌬Cterm mutant was stably expressed but unable to complement ⌬mxiC (supplemental Fig. S4); translocators were only weakly induced, and effector proteins were leaked. Under the straight mutant mxiC(I251A,T253A,S254A,D255E), induced secretion of both IpaB and IpaC was reduced, whereas that of IpaD was as efficient as in the complemented strain or even increased (Fig. 5D) This mutant did not affect secretion of MxiC or effector proteins. Its own inducible secretion was reduced and that of all translocators slightly affected (Fig. 5D) These results suggest that a “straightened” MxiC favors the earliest step in the induction hierarchy, i.e. IpaD secretion, whereas a bent MxiC cannot prevent or favors, independently of its own secretion, the final one, i.e. effector release. We could only analyze the “wildtype” His-MxiC(Cys)
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