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Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A.

ABSTRACT: Pseudomonas aeruginosa produces the virulence factor, ETA (exotoxin A), which catalyses an ADP-ribosyltransferase reaction of its target protein, eEF2 (eukaryotic elongation factor-2). Currently, this protein-protein interaction is poorly characterized and this study was aimed at identifying the contact sites between eEF2 and the catalytic domain of ETA (PE24H, an ETA from P. aeruginosa, a 24 kDa C-terminal fragment containing a His6 tag). Single-cysteine residues were introduced into the toxin at 21 defined surface-exposed sites and labelled with the fluorophore, IAEDANS [5-(2-iodoacetylaminoethylamino)-1-napthalenesulphonic acid]. Fluorescence quenching studies using acrylamide, and fluorescence lifetime and wavelength emission maxima analyses were conducted in the presence and absence of eEF2. Large changes in the microenvironment of the AEDANS [5-(2-aminoethylamino)-1-naphthalenesulphonic acid] probe after eEF2 binding were not observed as dictated by both fluorescence lifetime and wavelength emission maxima values. This supported the proposed minimal contact model, which suggests that only small, discrete contacts occur between these proteins. As dictated by the bimolecular quenching constant (k(q)) for acrylamide, binding of eEF2 with toxin caused the greatest change in acrylamide accessibility (>50%) when the fluorescence label was near the active site or was located within a known catalytic loop. All mutant proteins showed a decrease in accessibility to acrylamide once eEF2 bound, although the relative change varied for each labelled protein. From these data, a low-resolution model of the toxin-eEF2 complex was constructed based on the minimal contact model with the intention of enhancing our knowledge on the mode of inactivation of the ribosome translocase by the Pseudomonas toxin.

SUBMITTER: Yates SP

PROVIDER: S-EPMC1224111 | biostudies-other | 2004 May

REPOSITORIES: biostudies-other

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Project description:The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

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Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A.

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