Steps for Shigella Gatekeeper MxiC Function in Hierarchical Type III Secretion Regulation

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. While 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, next, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gate-keeper protein, MxiC in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation: 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. INTRODUCTION Type III secretion systems (T3SSs) are central devices in the virulence of many major Gram-negative bacterial pathogens of humans, animals and plants. They translocate virulence proteins into the membranes and cytoplasm of eukaryotic host cells to manipulate them during infection. T3SSs are key to the virulence of enteric pathogens such as E. coli, Salmonella and Shigella species. Shigella species are the etiological agent of bacillary dysentery in humans. 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). Physical contact with eukaryotic host cells activates the secretion system, which initiates secretion and leads to creation of a pore, formed by the bacterial proteins IpaB and IpaC, in hostcell membranes (3). The effectors are translocated through the needle (4) and pore channels, to facilitate host cell invasion (3). The needle tip complex (TC), which contains IpaD and IpaB, is the host cell sensor and transforms itself into the translocation pore (5) via addition of IpaC upon secretion activation (6,7). IpaD is hydrophilic and required for tip recruitment of the other two proteins, which are hydrophobic, and hence chaperoned by IpgC intrabacterially (8). The three proteins are collectively called the translocators. T3SSs are assembled, using a broadly conserved morphogenesis pathway (9), following Genetic & biophysical study of Shigella T3SS component MxiC 2 detection of environmental cues indicating entry into the host. In addition, virulence effectors acting late in the host cell manipulation cascade are only expressed once the presynthesised early effectors have been secreted at host cell contact. Most components and/or molecular mechanisms of these regulatory pathways diverge from one T3SS-carrying organism to another (10). Yet, one regulatory cascade is conserved, a process allowing hierarchical secretion of substrates, although the stages it covers vary: needle vs. translocator components in plant pathogens or translocators and then early effectors in animal ones (11). We focus here on how this cascade functions in animal pathogens. After T3SS assembly, effector secretion is prevented through the concerted action of surface TC proteins and regulators that control secretion from within the bacterial cytoplasm. The TC may prevent premature effector secretion by allosterically constraining the T3SS in a secretion “off” conformation without blocking the secretion channel (12-14). Upon physical contact of the TC with host cells, a signal, termed Signal 1, is transmitted via the TC (15) and needle (12,16) to the cytoplasm where it triggers secretion. Next, translocators are secreted to form the pore in the host cell membrane (3). Successful pore formation at the needle tip generates Signal 2, also transmitted via the needle, that allows inactivation or T3S-mediated removal of a conserved cytoplasmic regulatory protein, MxiC in Shigella (12,16). Third, early effector proteins are secreted and translocated into the host cell and late effector expression is activated (17). MxiC belongs to a class of “gate-keeper” proteins that is conserved among different type III secretion systems (18). They repress effector secretion in the absence of a secretion signal, but have different roles in translocator secretion, impairing it in a ΔmxiC mutant (12) while stimulating it in a Yersinia ΔyopN mutant (19). While gate-keepers are clearly involved in the cytoplasmic steps controlling T3SS secretion hierarchy upon activation, their mechanism of action remains unclear. The gate-keepers have conserved structures (20,21): after an N-terminal secretion signal and putative chaperone-binding domain (CBD), three α-helical X-bundles (domains 1-3; supplemental Fig. S1A-C) form a flat, elongated structure (21) typical for “hub proteins” regulating processes via interaction with multiple partners. In some species, gate-keepers are composed of two proteins where the second polypeptide covers the C-terminal X-bundle (domain 3; supplemental Fig. S1D-E (20)). MxiC is secreted by the type III secretion system (22). Its N-terminal 30 residues contain the secretion signal (23). Immediately thereafter is a domain similar to the chaperone-binding domain of Yersinia YopN (20,21). This domain is partially conserved (18) even though not every MxiC homologue has an identified chaperone. While this area is enriched in hydrophobic residues that mediate interactions with the chaperones, fewer hydrophobic residues are found in MxiC. Many type III secreted proteins are bound by a chaperone inside the bacterium. These chaperones have various roles, including stabilisation of their binding partners, aiding their secretion and mediation of secretion hierarchy (24). Several MxiC homologues bind to specific heterodimeric chaperones. For instance, Yersinia YopN binds to the SycN/YscB heterodimer (20,25). It wraps around its heterodimeric chaperone in a conformation similar to other effector/chaperone complexes (20). This domain is disordered in the absence of the chaperones. Interestingly, the first ~75 residues of MxiC are likely also disordered (21). Yet, so far no chaperone has been identified for MxiC (23). MxiC’s helix 9 of is a straight helix, while the structurally equivalent helix in YopN/TyeA is kinked into two smaller helices. The structure of the EPEC MxiC homolog, SepL, is also bent at an equivalent location (26). Thus, one face of the molecule is flat in MxiC, while it is concave in YopN/TyeA and SepL (21,26). Interestingly, this surface contains a negatively charged patch (E201, E276, E293; (21)) we showed is important for MxiC functions that involve IpaD (15). Furthermore, the Chlamydia hydrophobic translocator chaperone Scc3 binds to its gatekeeper at the flat interface between domain 2 and 3 (27), which the kink in the YopN/TyeA renders convex. Deane et al. (2008) already suggested this Genetic & biophysical study of Shigella T3SS component MxiC 3 structural difference between MxiC and YopN/TyeA could be a “conformational switch” and these new findings suggest it might allow the switch from hydrophilic to hydrophobic translocator secretion. To dissect MxiC’s interconnected functions we used site-directed mutagenesis. Mutant design was guided by the description of MxiC structure by Deane et al. (2008) and the sequence alignment of MxiC homologues by Pallen et al. (2005) (18). Our mutations (Fig. 1) focussed on the N-terminal non-crystallised region and domains 2 and 3 of the crystal structure (21). We identified mutants predominantly affecting all known features of MxiC regulation: secretion of translocators, MxiC and effectors. Using molecular genetics to map at which point in the regulatory cascade the mutants were affected we further dissected MxiC’s role. Analysis of some of these mutants led us to electron paramagnetic resonance (EPR) experiments that, together with phenotypic analysis of the mutants, provide evidence that MxiC’s conformation is regulated via a direct interaction with IpaD.