Collaboration : Yohan Couté, Edyp
Bacterial toxins target key cellular functions to promote infections. Among the most potent bacterial toxins are phospholipases, which damage the plasma membrane and lead to necrosis and tissue lesions. P. aeruginosa produces ExoU, a phospholipase that is exported and injected into the host cytoplasm by the Type III Secretion System (T3SS), or injectisome. We previously made two major discoveries : one concerning the structure of ExoU in complex with its chaperon [1] (Figure 1), and another concerning its intracellular trafficking inside DNAJC5-positive vesicles [2] (Figure 2).
Figure 1 : Structure cristallographique de ExoU en complexe avec sa chaperonne SpcU (adapted from Gendrin et al. 2012)
More recently, we used proximity labeling (PL) tools and integrated cellular approaches to better understand how ExoU highjacks the host’s trafficking machinery. We fused and expressed the C-terminus of ExoU to the biotin ligase UltraID. The secretion, cytotoxicity and requirement of DNAJC5 of the fusion protein were confirmed. In the presence of exogenous biotin, UltraID biotinylates the proteins in close proximity to ExoU. Streptavidin-affinity captured proteins underwent tryptic digestion followed by LC-MS/MS. MS analysis revealed 8 significantly enriched human proteins in the UltraID condition compared to the control, among them RAB27B, SNAP23 and SLC3A2 which we prioritized due to their known roles in a variety of trafficking pathways.
We found, using KO cells for each of the targets that there is a fine equilibrium between ExoU toxicity and host cell defenses.
Figure 2 : ExoU injection and trafficking within cells (created in https://BioRender.com)