Backbone Interactions Between Transcriptional Activator ExsA and Anti-Activator ExsD Facilitate Regulation of the Type III Secretion System in \u3cem\u3ePseudomonas aeruginosa\u3c/em\u3e

The type III secretion system (T3SS) is a pivotal virulence mechanism of many Gram-negative bacteria. During infection, the syringe-like T3SS injects cytotoxic proteins directly into the eukaryotic host cell cytoplasm. In Pseudomonas aeruginosa, expression of the T3SS is regulated by a signaling cascade involving the proteins ExsA, ExsC, ExsD, and ExsE. The AraC-type transcription factor ExsA activates transcription of all T3SS-associated genes. Prior to host cell contact, ExsA is inhibited through direct binding of the anti-activator protein ExsD. Host cell contact triggers secretion of ExsE and sequestration of ExsD by ExsC to cause the release of ExsA. ExsA does not bind ExsD through the canonical ligand binding pocket of AraC-type proteins. Using site-directed mutagenesis and a specific in vitro transcription assay, we have now discovered that backbone interactions between the amino terminus of ExsD and the ExsA beta barrel constitute a pivotal part of the ExsD-ExsA interface. Follow-up bacterial two-hybrid experiments suggest additional contacts create an even larger protein–protein interface. The discovered role of the amino terminus of ExsD in ExsA binding explains how ExsC might relieve the ExsD-mediated inhibition of T3SS gene expression, because the same region of ExsD interacts with ExsC following host cell contact

Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus▿

Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE∼P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD∼P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD∼P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD∼P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE∼P

Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion

Secretion of Yop effector proteins by the Yersinia pestis plasmid pCD1-encoded type III secretion system (T3SS) is regulated in response to specific environmental signals. Yop secretion is activated by contact with a eukaryotic cell or by growth at 37 degrees C in the absence of calcium. The secreted YopN protein, the SycN/YscB chaperone and TyeA form a cytosolic YopN/SycN/YscB/TyeA complex that is required to prevent Yop secretion in the presence of calcium and prior to contact with a eukaryotic cell. The mechanism by which these proteins prevent secretion and the subcellular location where the block in secretion occurs are not known. To further investigate both the mechanism and location of the YopN-dependent block, we isolated and characterized several YopN mutants that constitutively block Yop secretion. All the identified amino-acid substitutions that resulted in a constitutive block in Yop secretion mapped to a central domain of YopN that is not directly involved in the interaction with the SycN/YscB chaperone or TyeA. The YopN mutants required an intact TyeA-binding domain and TyeA to block secretion, but did not require an N-terminal secretion signal, an intact chaperone-binding domain or the SycN/YscB chaperone. These results suggest that a C-terminal domain of YopN complexed with TyeA blocks Yop secretion from a cytosolic, not an extracellular, location. A hypothetical model for how the YopN/SycN/YscB/TyeA complex regulates Yop secretion is presented

Cartoon model of a full-length dimeric ExsA-DNA complex.

<p>This model was generated by first overlaying the structure of the ExsA-NTD A/A’ dimer and a homology model of ExsA-CTD (based on the MarA-DNA crystal structure) onto the structure of ToxT. Subsequently, crystallographic two-fold axis was applied to create a model of the full-length protein with a dimer interface corresponding to A/A’ dimer observed in the crystal.</p

Crystal structure of the ExsA-NTD.

<p><b>(A)</b> Model of a monomer encompassing amino acids 2–166 which produced clearly defined electron density. Blue to red rainbow coloring traces the backbone from the N to the C-terminus. Secondary structure elements are numbered. <b>(B)</b> Packing contacts in the crystal suggest the possible structure of the biological dimer. Chains A and B constitute the asymmetric unit of the crystal. Application of two crystallographic two-fold axes produces two additional pairs of chains labeled with a prime and a double-prime, respectively. Contacts between either chains A and A’ or between chains B and B” are proposed to mediate ExsA dimerization <i>in vivo</i>. (C) Shown in gray are the overlaid backbones traces of chains A and B. Also displayed are the symmetry-related molecules A’ and B” to highlight similarities and differences between the two possible quaternary structures. The B” molecule is rotated by approximately 23° around helix α-3. The rotation is visualized by marking the angle between the P20 residues of A’ and B” in the figure.</p

Mapping of the ExsA dimer interface.

<p>(A) The shown A/A’ ExsA-NTD interface suggests involvement of helix α-2 in ExsA dimerization. Previously identified interface residues are indicated in the same color as the protein backbone. G124 and L117 are colored violet and yellow in the respective molecules. (B) Shown is a sample gel of measurements testing the impact of the L117R and G124R mutations on the ability of ExsA to activate transcription <i>in vitro</i>. Three concentrations of each protein were tested to ensure that the experiments were conducted in a sensitive range. (C) Graphical representation of the <i>in vitro</i> transcription assays from triplicate experiments. Going from left to right: wtExsA, ExsAG124R, and ExsAL117R.</p