No abstract

14‐3‐3 proteins are phosphoserine/phosphothreonine‐recognizing adapter proteins that regulate the activity of a vast array of targets. There are also examples of 14‐3‐3 proteins binding their targets via unphosphorylated motifs. Here we present a structural and biological investigation of the phosphorylation‐independent interaction between 14‐3‐3 and exoenzyme S (ExoS), an ADP‐ribosyltransferase toxin of Pseudomonas aeruginosa. ExoS binds to 14‐3‐3 in a novel binding mode mostly relying on hydrophobic contacts. The 1.5 Å crystal structure is supported by cytotoxicity analysis, which reveals that substitution of the corresponding hydrophobic residues significantly weakens the ability of ExoS to modify the endogenous targets RAS/RAP1 and to induce cell death. Furthermore, mutation of key residues within the ExoS binding site for 14‐3‐3 impairs virulence in a mouse pneumonia model. In conclusion, we show that ExoS binds 14‐3‐3 in a novel reversed orientation that is primarily dependent on hydrophobic residues. This interaction is phosphorylation independent and is required for the function of ExoS.

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram‐negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersiniaouter protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two‐hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non‐secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N‐terminal domain and a C‐terminal amphipathic α‐helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic α‐helix abolished the YopD–LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild‐type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram‐negative pathogens secrete and subsequently translocate antihost effector proteins into target eukaryotic cells by a common type III secretion system (TTSS). In this process, YopD (Yersiniaouter protein D) is essential to establish regulatory control of Yop synthesis and the ensuing translocation process. YopD function depends upon the non‐secreted TTSS chaperone LcrH (low‐calcium response H), which is required for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A lcrH null mutant constitutively produced Yops, even when this strain was engineered to produce wild‐type levels of YopD. Furthermore, the YopD–LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced in trans into a lcrH null mutant to assess their impact on yop regulation and the subsequent translocation of YopE, a Rho‐GTPase activating protein, across the plasma membrane of eukaryotic cells. Two mutants, LcrHK20E, E30G, I31V, M99V, D136G and LcrHE30G lost all regulatory control, even though YopD binding and secretion and the subsequent translocation of YopE was indistinguishable from wild type. Moreover, these regulatory deficient mutants showed a reduced ability to bind YscY in the two‐hybrid assay. Collectively, these findings confirm that LcrH plays an active role in yop regulation that might be mediated via an interaction with the Ysc secretion apparatus. This chaperone–substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in stabilization and secretion of YopD or a regulatory protein responsible for co‐ordinating synthesis of Yersinia virulence determinants. We suggest that LcrH can exhibit both of these activities.

14‐3‐3 proteins play an important role in a multitude of signalling pathways. The interactions between 14‐3‐3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho‐specific manner. Recently, a phosphorylation‐independent interaction has been reported to occur between 14‐3‐3 and several proteins, for example 5‐phosphatase, p75NTR‐associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP‐ribosyltransferase from Pseudomonas aeruginosa. In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14‐3‐3. Furthermore, we show that a peptide derived from ExoS, containing the 14‐3‐3 interaction site, effectively competes out the interaction between ExoS and 14‐3‐3. In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay. We show that the ExoS protein interacts with all isoforms of the 14‐3‐3 family tested. Moreover, in vivo an ExoS protein lacking the 14‐3‐3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g. Ras, and shows a reduced capacity to change the morphology of infected cells.