Phosphatidylinositol 4,5-Bisphosphate-Dependent Oligomerization of the Pseudomonas aeruginosa Cytotoxin ExoU

Abstract

The type III secretion system delivers effector proteins directly into target cells, allowing the bacterium to modulate host cell functions. ExoU is the most cytotoxic of the known effector proteins and has been associated with more severe infections in humans. ExoU is a patatin-like A phospholipase requiring the cellular host factors phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] and ubiquitin for its activation We demonstrated that PI(4,5)P also induces the oligomerization of ExoU and that this PI(4,5)P-mediated oligomerization does not require ubiquitin. Single amino acid substitutions in the C-terminal membrane localization domain of ExoU reduced both its activity and its ability to form higher-order complexes in transfected cells and Combining inactive truncated ExoU proteins partially restored phospholipase activity and cytotoxicity, indicating that ExoU oligomerization may have functional significance. Our results indicate that PI(4,5)P induces the oligomerization of ExoU, which may be a mechanism by which this coactivator enhances the phospholipase activity of ExoU.

Pseudomonas aeruginosa Toxin ExoU as a Therapeutic Target in the Treatment of Bacterial Infections

“…The C-terminal MLD of ExoU, containing the four-helical bundle, has a high binding affinity for PIP 2 [68,92]. Models suggest that binding to PIP 2 causes conformation changes in the structure of ExoU, including the conformational rearrangement of the four-helical bundle of ExoU, allowing it to insert into the lipid membrane [84,93]. PIP 2 binding has also been demonstrated to promote ExoU multimerisation [93].…”

“…Models suggest that binding to PIP 2 causes conformation changes in the structure of ExoU, including the conformational rearrangement of the four-helical bundle of ExoU, allowing it to insert into the lipid membrane [84,93]. PIP 2 binding has also been demonstrated to promote ExoU multimerisation [93]. SEC-MALS analysis and phospholipase assays indicate that, in vitro, in the presence PIP 2 , ExoU can form multimers that have greatly enhanced catalytic activity, in the presence of ubiquitin, when compared to ExoU and ubiquitin alone [93].…”

“…PIP 2 binding has also been demonstrated to promote ExoU multimerisation [93]. SEC-MALS analysis and phospholipase assays indicate that, in vitro, in the presence PIP 2 , ExoU can form multimers that have greatly enhanced catalytic activity, in the presence of ubiquitin, when compared to ExoU and ubiquitin alone [93].…”

“…Although the mechanisms of ExoU regulation are not fully understood, evidence for ubiquitin-binding as an activator and recruitment to the plasma membrane and oligomerisation followed by phospholipase A2 activity have been described in ExoU associated pathology [48,64]. These result in multiple dynamic conformational changes that could be targeted by small molecules to attenuate ExoU activity in clinical infections (Figure 1) [84,85,93,99].…”

Evaluation of ExoU inhibitors in aPseudomonas aeruginosascratch infection assay

“…There is the potential that ExoU is rapidly digested by bacterial proteases shortly after induction of expression. Thus, previous purification procedures have adopted a short 3 hour induction of ExoU expression followed by immediate purification from BL21(DE3)pLysS E. coli [21,28,29,35]. Alternate tagged variants, including glutathione-S-transferase (GST) and maltose binding protein (MBP) ExoU fusion proteins, were also quickly degraded, which was apparent from a large abundance of GST/MBP proteins with ExoU cleaved away after respective pull-downs during purification (data not shown).…”