Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump

Kristensen, R.; Andersen, J. B.; Levin Rybtke, M.; Jansen, C. U.; Fritz, B. G.; Kiilerich, R.; Uhd, J.; Bjarnsholt, T.; Qvortrup, K.; Tolker-Nielsen, T.; Givskov, M.; Jakobsen, T. H.

doi:10.1101/2023.10.27.564458

bioRxiv · 10.1101/2023.10.27.564458

Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump

Kristensen, R.·Andersen, J. B.·Levin Rybtke, M.·Jansen, C. U.·Fritz, B. G.·Kiilerich, R.·Uhd, J.·Bjarnsholt, T.·Qvortrup, K.·Tolker-Nielsen, T.·Givskov, M.·Jakobsen, T. H.

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Abstract

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa a chemical library consisting of 3280 small compounds from LifeArc was screened. A series of ten conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were resynthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing revealing a strong inhibitor of QS-regulated genes and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump thereby inhibiting the auto-generated positive feedback and its enhanced signal-molecule synthesis.

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Kristensen, R., Andersen, J. B., Levin Rybtke, M., Jansen, C. U., Fritz, B. G., Kiilerich, R., Uhd, J., Bjarnsholt, T., Qvortrup, K., Tolker-Nielsen, T., Givskov, M., Jakobsen, T. H.. 2023-10-28. Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump. https://doi.org/10.1101/2023.10.27.564458

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