Pseudomonas aeruginosa (Pa) is a multi-drug resistant and a major human Gram-negative opportunistic pathogen able to cause a variety of infections. It assembles on its surface an arsenal of virulence factors including the potent Type 3 Secretion System (T3SS) which injects four toxins into eukaryotic cells. Among them, the most aggressive toxin, the phospholipase ExoU targets the host plasma membrane (PM) leading to a rapid necrotic cell death. Recently, our team identified the host chaperone DNAJC5, critical for the Misfolding-Associated Protein Secretion (MAPS) pathway, as essential for targeting ExoU to the PM. However, other host factors governing the intracellular journey of ExoU remain unknown.
​ In this study, we employed proximity labeling (PL) and integrated cellular approaches, to better understand the crosstalk between ExoU and the host’s trafficking machinery. To do so, we used the biotin ligase, UltraID as PL tool. We expressed ExoU fused to UltraID in Pa and performed PL assays during infection. In the presence of exogenous biotin, UltraID mediates the biotinylation of proteins in close proximity to ExoU. The resulting proteins are then captured by streptavidin and undergo tryptic digestion followed by LC-MS/MS. Analysis revealed nine significantly enriched human proteins when ExoU-UltraID is injected, among them SNAP23 and SLC3A2, which we prioritized due to their known roles in trafficking and/or DNAJC5-dependent pathways. Cells that lack either of these two proteins showed increased sensitivity to an infection with ExoU+ strain. The mutants and their parental cell lines were similarly susceptible to other T3SS (ExoS) and PM-damaging (ExlA) toxins indicating an ExoU specific phenotype. In addition, immunofluorescence revealed a partial colocalization of ExoU with SLC3A2 and CD63, marker of extracellular vesicles (EVs). Given that SNAP23 mediates the exocytosis of CD63-positive vesicles, we hypothesized that ExoU is detoxified via this pathway. Consistent with this, ExoU was detected in isolated EVs from the supernatant of infected cells. For the first time, we applied PL to a T3SS injected toxin of an extracellular pathogen. We identified host factors taking part in the host cell response to ExoU, underlining a complex host-ExoU interplay
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