Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacteria, including animal and plant pathogens. They inject ‘effector’ proteins through a ‘needle’ protruding from the bacterial surface directly into eukaryotic cells after assembly of a ‘translocator’ pore in the host plasma membrane. Secretion is a tightly regulated process, which is blocked until physical contact with a host cell takes place. Host cell sensing occurs through a distal needle ‘tip complex’ and translocators are secreted before effectors. MxiC, a Shigella T3SS substrate, prevents premature effector secretion. Here, we examine how the different parts of T3SSs work together to allow orderly secretion. We show that T3SS assembly and needle tip composition are not altered in an mxiC mutant. We find that MxiC not only represses effector secretion but that it is also required for translocator release. We provide genetic evidence that MxiC acts downstream of the tip complex and then the needle during secretion activation. Finally, we show that the needle controls MxiC release. Therefore, for the first time, our data allow us to propose a model of secretion activation that goes from the tip complex to cytoplasmic MxiC via the needle.

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Type III secretion systems (T3SSs) are widely distributed virulence determinants of Gram-negative bacteria. They translocate bacterial proteins into host cells to manipulate them during infection. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region, and a hollow needle protruding from the bacterial surface. The distal tip of mature, quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. However, its mode of needle association is unknown. Here, we show that deletion of 3 or 9 residues at the C terminus of IpaB leads to fast constitutive secretion of late effectors, as observed in a ⌬ipaB strain. Like the ⌬ipaB mutant, mutants with C-terminal mutations also display hyperadhesion. However, unlike the ⌬ipaB mutant, they are still invasive and able to lyse the internalization vacuole with nearly wild-type efficiency. Finally, the mutant proteins show decreased association with needles and increased recruitment of IpaC. Taken together, these data support the notion that the state of the tip complex regulates secretion. We propose a model where the quiescent needle tip has an "off" conformation that turns "on" upon host cell contact. Our mutants may adopt a partially "on" conformation that activates secretion and is capable of recruiting some IpaC to insert pores into host cell membranes and allow invasion.

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.

SummaryThe NifA protein from Azotobacter vinelandii belongs to a family of enhancer binding proteins (EBPs) that activate transcription by RNA polymerase containing the sigma factor s s s s 54 . These proteins have conserved AAA+ domains that catalyse ATP hydrolysis to drive conformational changes necessary for open complex formation by s s s s 54 -RNA polymerase. The activity of the NifA protein is highly regulated in response to redox and fixed nitrogen through interaction with the antiactivator protein NifL. Binding of NifL to NifA inhibits the ATPase activity of NifA, and this interaction is controlled by the amino-terminal GAF domain of NifA that binds 2-oxoglutarate. Mutations conferring resistance to NifL are located in both the GAF and the AAA+ domains of NifA. To investigate the mechanism by which the GAF domain regulates the activity of the AAA+ domain, we screened for second-site mutations that suppress the NifL-resistant phenotype of mutations in the AAA+ domain. One suppressor mutation, F119S, in the GAF domain restores inhibition by NifL to an AAA+ domain mutation, E356K, in response to fixed nitrogen but not in response to oxygen. The biochemical properties of this mutant protein are consistent with the in vivo phenotype and demonstrate that interdomain suppression results in sensitivity to inhibition by NifL in the presence of the signal transduction protein GlnK, but not to the oxidized form of NifL. In the absence of an AAA+ domain mutation, the F119S mutation confers hypersensitivity to repression by NifL. Isothermal titration calorimetry demonstrates that this mutation prevents binding of 2-oxoglutarate to the GAF domain. Our data support a model in which the GAF domain plays an essential role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. These observations are of general significance in considering how the activities of EBPs are controlled in response to environmental signals.