Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / JOB Viviana
Contact person(s) related to this article / JOB Viviana
Contact person(s) related to this article / JOB Viviana

ExoU effect

The majority of clinical P. aeruginosa strains translocates ExoU or ExoS, ExoT and ExoY toxins in the cytoplasm of target cells.
We have investigated the effects of these three exotoxins in human endothelial cells and described their role in cell signaling disruption [1-4].

https://youtu.be/Kg5dgp1863Y

Fig. 1 : Live-imaging quantification of PA14 (ExoU+) cell infection by fluorescence intensity measurement of propidium Iodide incorporation.

Eukaryotic DNAJC5 is need for ExoU toxicity

We used a genome-wide screen using CRISPR-Cas9 technology to discover that the main P. aeruginosa toxin ExoU requires DNAJC5, a host chaperone, for its necrotic activity. DNAJC5 is known to participate in an unconventional secretory pathway for misfolded proteins called anterograde vesicular traffic. We show that DNAJC5-deficient human epitelial cells or Drosophila flies knocked-down for the DNAJC5 orthologue, are largely resistant to ExoU-dependent cell-lyses and vurulence. ExoU colocalizes with DNAJC5-positive vesicles in the host cytoplasm. DNAJC5 mutations preventing vesicle trafficking inhibit ExoU-dependent cell lysis. Our results suggest that, once injected into the host cytoplasm, ExoU docks to DNAJC5-positive secretory vesicles to reach the plasma membrane, where it can exert its phospholipase activity.

Fig. 2 Colocalization (in yellow) de ExoU (red) et DNAJC5-FLAG (green). Epithelial cells A549 were infected by Pseudomonas aeruginosa expressing an inactive ExoU. Cells were processed for immunofluorescence and visualized by Apotome microscope. One z-section is shown

Fig. 3 Proposed model of ExoU trafficking in host cells. Upon delivery into the host cytoplasm by the T3SS, the toxin uses probably an endocytic pathway toreach the perinuclear region, as suggested by ExoU/EEA1 colabeling (not shown). Then, ExoU binds to the late endosome’s limiting membrane (decorated byLamp2 and DNAJC5), thanks to the interaction of ExoU with an as yet unidentified specific receptor at the vesicle’s surface. ExoU remains at the external side of the vesicle’s membrane and co-opts the DNAJC5-dependent MAPS pathway to achieve anterograde transport toward the cellular periphery (where vesicles loose Lamp2), and eventually the plasma membrane (PM). Fusion of vesicles with the PM brings ExoU close to PM’s inner leaflet, where its membrane localization domain binds to PI(4,5)P2. PI(4,5)P2 binding triggers conformational changes in ExoU, leading to toxin oligomerization and activation of its phospholipase activity, which eventually induces PM rupture.

ExoS – ExoT effect


Fig. 4 : Live-imaging quantification of PAO1 (ExoS+) cell infection by fluorescence intensity measurement of Hoechst-labelled nuclei. Wallez Y. et al (2018) Scientific Reports

References
[1] Huber et al (2014) Cell Mol Life Sci 71(10):1927-1941.,
[2] Bouillot et al (2015) Infection and immunity 83(5):1820-1829.,
[3] Walley et al (2018) Scientific reports 8(1):5. doi : 10.1038/s41598-017-18501-9.
[4] Deruelle et al (2021) Nat Commun, doi.org/10.1038/s41467-021-24337-9