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