TY - JOUR

T1 - Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump

AU - Kristensen, Rasmus

AU - Andersen, Jens Bo

AU - Rybtke, Morten

AU - Jansen, Charlotte Uldahl

AU - Fritz, Blaine Gabriel

AU - Kiilerich, Rikke Overgaard

AU - Uhd, Jesper

AU - Bjarnsholt, Thomas

AU - Qvortrup, Katrine

AU - Tolker-Nielsen, Tim

AU - Givskov, Michael

AU - Jakobsen, Tim Holm

N1 - Publisher Copyright: © 2024 American Society for Microbiology. All rights reserved.

PY - 2024

Y1 - 2024

N2 - 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 3,280 small compounds from LifeArc was screened. A series of 10 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 re-synthesized 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 autogenerated positive feedback and its enhanced signal-molecule synthesis.

AB - 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 3,280 small compounds from LifeArc was screened. A series of 10 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 re-synthesized 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 autogenerated positive feedback and its enhanced signal-molecule synthesis.

KW - MexEF-oprN

KW - Pseudomonas aeruginosa

KW - quorum sensing

KW - quorum-sensing inhibitor

KW - thiol-reactive compound

KW - virulence factors

U2 - 10.1128/aac.01387-23

DO - 10.1128/aac.01387-23

M3 - Journal article

C2 - 38189278

AN - SCOPUS:85184518010

VL - 68

JO - Antimicrobial Agents and Chemotherapy

JF - Antimicrobial Agents and Chemotherapy

SN - 0066-4804

IS - 2

ER -