ExoU is the most potent type III-secreted toxin synthesized by P. aeruginosa
(Lee et al., 2005
), but its mechanism of action remained unclear due to limited homology with other proteins and its potent cytotoxicity in mammalian cells (Sato and Frank, 2004
). The use of yeast as a model system revealed a phenotype of vacuolar fragmentation suggesting membrane destruction or remodeling (Sato et al., 2003
). These data, combined with the inhibition of cytotoxicity in both mammalian and yeast cells by phospholipase A2
inhibitors, allowed alignment with phospholipases and the identification of a catalytic dyad, S142 and D344 with similarity to the patatin protein family (Phillips et al., 2003
, Sato et al., 2003
). In vitro
phospholipase activity was detectable only in the presence of other cellular materials indicating the requirement for a eukaryotic activator (Sato et al., 2003
). Affinity methods for identifying this activator generally failed as ExoU appeared to interact with multiple proteins and the inclusion of detergents or other agents that decrease nonspecific protein-protein interactions inhibited enzyme activity (Sato et al., 2005
). It was unclear whether this inhibition was due to the inability of ExoU to associate with phospholipid substrates or activator or both. Proteomic approaches and the use of enzymatic activity as a screen resulted in the discovery of eukaryotic SOD1 as an activator (Sato et al., 2006
). Caveats pertaining to SOD1, however, were that it had poor specific activity as a cofactor, saturable kinetics for ExoU could not be obtained in vitro
and only particular commercial preparations of the protein displayed activation capabilities (Sato et al., 2006
, Benson et al., 2010
). The goal of this study was to determine the specific properties of yeast and bovine SOD1 that mediated the activation of ExoU. Our results indicate that ExoU is a unique toxin in that it specifically associates with ubiquitin and/or ubiquitylated proteins to activate the enzyme. The mechanism of activation coupled with type III delivery ensures that eukaryotic cells are specifically and potently targeted and that the bacterium is protected from its own enzyme.
In addition to using ubiquitin as an activator, ExoU is itself ubiquitylated (Stirling et al., 2006
). Ubiquitylation of proteins delivered by type III and type IV secretion systems has been shown to play critical roles in effector stability and trafficking (Angot et al., 2007
; Kubori an Galan, 2003
; Patel et al., 2009
; Schnupf et al., 2006
). For ExoU, two monoubiquitin molecules are added to K178 predominantly via a K63 linkage (Stirling et al., 2006
). Modification appears to have no significant impact on the half-life of the toxin. In terms of intracellular localization, ubiquitylated, catalytically inactive ExoU (ExoU-S142A) as well as ExoUS142A that cannot be ubiquitylated at K178 (K178R) traffic to the plasma membrane suggesting that ubiquitin modification is not involved with plasma membrane localization (Stirling et al., 2006
). The observation of cytotoxicity in the prokaryotic dual expression system also suggests that eukaryotic proteins, other than monoubiquitin, are not required for ExoU to traffic to membrane substrates, or compromise membrane integrity.
The discovery of ubiquitin as an activator and the fact that ExoU is modified by ubiquitin in cells suggests the hypothesis that ubiquitylated ExoU may self-activate. In this model, the injection of ExoU would lead to ubiquitlyation at K178, followed by a conformational change of the molecule. This conformational change might be mediated by the intramolecular recognition of attached diubiquitin by another domain within ExoU. Structure-function analyses of ExoU have implicated the importance of C-terminal residues for phospholipase activity (Finck-Barbançon and Frank, 2001
; Sato et al., 2003
; Schmalzer et al., 2010
; Benson et al., 2010
). It is also clear from EPR analyses that ExoU N- and C-terminal residues change conformation in the presence of ubiquitin (bSOD1, Benson et al., 2011
; data not shown). To account for these observations, we considered whether the C-terminus of ExoU might encode an ubiquitin-binding domain. Using a variety of bioinformatic approaches, no known ubiquitin binding motifs or domains were identified (data not shown) in the C-terminus or other regions of ExoU. Importantly, Stirling et al. showed that ExoU K178R retained full toxicity, implying that ubiquitylation of ExoU is not required for phospholipase activity. Finally, rExoU is produced in bacteria and the in vitro
enzyme activity assay used to measure phospholipase activity lacks ATP and other enzymes required for ubiquitylation. Whether intracellular ubiquitylation of ExoU serves to accelerate activation is unclear and will require direct testing of ubiquitin-modified ExoU derivatives.
The activation of ExoU is highly specific to ubiquitin as ubiquitin-like proteins SUMO-1, ISG15, FAT10 and NEDD8 (10 μM) do not activate ExoU (data not shown and Figure S2C
). Although the specificity of activation relates to ubiquitin, different chain lengths, types of linkages or conjugation to other proteins all function to activate ExoU. Longer chains of ubiquitin, however, have a greater ability to activate phospholipase activity in vitro
. We postulate that the interaction of ExoU with ubiquitin may involve multiple sites accounting for the apparent high affinities measured in the solid phase binding assays and the absence of an identifiable motif. Polyubiquitin may act as the best scaffold on which ExoU folds to produce an active enzyme. Alternatively, cofactor interaction may serve a bi-functional role in this toxin’s activation, facilitating both a global conformational change (Benson et al., 2011
) as well as contributing to catalysis. Our kinetic data suggests that a single ubiquitin molecule may not be able to efficiently accomplish both tasks, as high concentrations of monoubiquitin are required to reach saturable kinetics relative to chain-linked counterparts ( and Table S2
). These data indicate that the ExoU-ubiquitin interaction may define a novel type of binding or unique motifs.
In summary, we have demonstrated that the P. aeruginosa phospholipase toxin ExoU is activated by several ubiquitin isoforms, as well as by ubiquitylated proteins. This is, to our knowledge, the first report of ubiquitin serving as an activator for a bacterial toxin. The exact role of ubiquitin in the activation process is unknown but is postulated to facilitate a conformational change in ExoU to allow catalysis. Polyubiquitin molecules associate with and activate ExoU with the greatest efficiency in vitro suggesting that either the size of the cofactor or multiple interaction sites within ExoU are important for phospholipase activity. The association of ExoU with ubiquitin apparently involves novel structural contacts, as no recognizable ubiquitin binding motifs were identified within the ExoU sequence. The toxicity and membrane degradation observed in an E. coli dual expression system for ExoU and ubiquitin reinforces the importance and absolute requirement of a eukaryotic cofactor in regulating the activity of this potent type III effector.