Backbone Interactions Between Transcriptional Activator ExsA and Anti-Activator ExsD Facilitate Regulation of the Type III Secretion System in Pseudomonas aeruginosa

Abstract

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.

Figure 1

Conserved surface residues of ExsA are not critical for ExsD binding. (a) Structure of the ExsA-NTD wherein the targeted surface residues are highlighted. The figure was generated using Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). (b) Representative autoradiograms of three independent in vitro transcription experiments analyzing the susceptibility of ExsA variants containing substitutions in conserved surface residues to regulation by ExsD. All variants were sensitive to the presence of ExsD in the assay. Quantitative analysis of the autoradiograms was performed with ImageQTL. The bar diagrams show the averages obtained from three independent experiments with the error bars representing the standard deviations. . Gel image shown is representative of the results we obtained from three replicates.

Figure 2

Backbone contacts between ExD and ExsA are critical for binding. (a) The Gly₅∆7ExsA variant was insensitive to the presence of ExsD in the assay. However, ∆7ExsA protein behaved like wtExsA. Point substitutions in the periphery of the ExsA N-terminus showed no effect on ExsD regulation in our in vitro transcription assay. The bar diagrams show the averages obtained from three independent experiments with the error bars representing the standard deviations. The gel images shown are representative of the results we obtained from three replicates. (b) Residues in β-strands β-1 and β-2 of ExsA were replaced by stretches of alanines to determine if the side chains are important for ExsA binding. Both variants still respond to ExsD, demonstrating that the side chains of the substituted residues are not critical. However, proline substitutions of strand β-2 residues Y24 and the V26 impact ExsD dependent regulation. The bar diagrams show the averages obtained from three independent experiments with the error bars representing the standard deviations. The gel images shown are representative of the results we obtained from three replicates. (c) Results from a BACTH test demonstrate that, although ExsAY24P and ExsAV26P are insensitive to the regulation by ExsD, the two ExsA mutant still interact with ExsD, suggesting the presence of a larger ExsA-ExsD interface. Image shown is representative of the results we obtained from three biological replicates.

Figure 3

The Amino-terminus of ExsD is essential for the inhibition of ExsA-dependent transcription. (a) Results of an in vitro transcription assay comparing the concentration-dependent inhibition of in vitro transcription by three different ExsD constructs demonstrating that the ExsD N-terminus is essential but alone not sufficient for ExsD function. The bar diagrams show the averages obtained from three independent experiments with the error bars representing the standard deviations. Gel image shown is representative of the results we obtained from three replicates.. (b) Model of an ExsA-promoter (AraC-domain in marine, NTD in cyan, DNA shown as gray surface) complex with the ExsD N-terminal peptide (shown in red). The figure was generated using Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).