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Vincent Deruelle

Identification of a new eukaryotic protein essential for the membrane localization of ExoU toxin from Pseudomonas aeruginosa

Published on 10 November 2020
Thesis presented November 10, 2020

Abstract:
Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium, responsible for different types of infection, in particular for nosocomial diseases. The acute infections caused by this pathogen are promoted by a broad panel of virulence factors; the most aggressive one being the Type III Secretion System. Among the four effectors injected by this system into the cytoplasm of target cells, ExoU is the most cytotoxic one. ExoU+ strains are associated with the most severe pathologies and to higher multiresistance to antibiotics. The toxin possesses a phospholipase activity inducing cell necrosis upon plasma membrane rupture. However, the trafficking of the toxin in host cells to reach the plasma membrane is unknown.
In this study, I aimed at identifying ExoU host partners, using a genome-wide screen employing the CRISPR-Cas9 system. Likewise, I identified the gene encoding a co-chaperone protein, named DNAJC5. This protein, located at the surface of late endosomes, is involved in an unconventional secretion pathway required for the release of misfolded cytosolic proteins. Using human cellular models, as well as Drosophila, we demonstrate that lack of DNAJC5 confers resistance to ExoU+ strain infection. In the host cytoplasm, the toxin localizes at the surface of DNAJC5+ vesicles. Mutations in DNAJC5 gene, preventing vesicle trafficking to the plasma membrane, also impede ExoU targeting to the plasma membrane and inhibit its cytotoxicity. We conclude that, once delivered into the host cell cytoplasm, ExoU uses DNAJC5+ vesicles to be transported to the plasma membrane, a process strictly required for ExoU-induced cell necrosis.

Keywords:
Pseudomonas aeruginosa, infection, T3SS, ExoU, DNAJC5 (CSPα)