Abstract
Quorum sensing (QS), a bacterial communication strategy, has been recognized as one of the control mechanisms of virulence in bacteria. Thus, targeting QS offers an interesting opportunity to impair bacterial pathogenicity and develop antivirulence agents. Aiming to enhance the discovery of QS inhibitors, we developed a bioreporter Escherichia coli JW5505 pET-Plsrlux and set up a cell-based assay for identifying inhibitors of autoinducer-2 (AI-2)-mediated QS. A comparative study on the performance of target- versus cell-based assays was performed, and 91 compounds selected with the potential to target the ATP binding pocket of LsrK, a key enzyme in AI-2 processing, were tested in an LsrK inhibition assay, providing 36 hits. The same set of compounds was tested by the AI-2-mediated QS interference assay, resulting in 24 active compounds. Among those, six were also found to be active against LsrK, whereas 18 might target other components of the pathway. Thus, this AI-2-mediated QS interference cell-based assay is an effective tool for complementing target-based assays, yet also stands as an independent assay for primary screening.
Highlights
Bacterial infections are one the biggest threats to human health. [1] spontaneous mutations, combined with the general misuse of antibiotics, have compromised the efficacy many antibiotics, exposing humans to multidrug-resistant bacteria. [2,3,4] Traditional antibiotics are bactericidal or bacteriostatic, targeting essential cellular functions which are related to bacterial growth and survival
The plasmid was constructed by replacing the PesaR, a Quorum sensing (QS) responsive promoter induced by 3-oxo-hexanoyl-homoserine lactone, with the lsr promoter in the pET-PesaRlux plasmid and the new construct was transformed into single-gene knock-out E. coli JW5503 which, due to efflux pump defect, is unable to expel xenobiotics from the cell. [28, 29] Since extrusion by the efflux pump system is one of the main obstacles for the discovery of new antibacterial agents and even limits the identification of target-active compounds to be further optimized, we considered the reduced efflux activity of the strain as an advantage to identify candidate molecules which can be further optimized
To support the discovery of new QS inhibitors, we developed a new AI-2-mediated QS interference assay based on the detection of luciferase produced in E. coli JW5503 pET-Plsrlux
Summary
A new cell-based AI-2-mediated quorum sensing interference assay in screening of LsrK-targeted inhibitors. Viviana Gatta[a], Tihomir Tomašič[b], Janez Ilaš[b], Nace Zidar[b], Lucija Peterlin Mašič[b], Michaela Barančoková[b], Rok Frlan[b], Marko Anderluh[b], Danijel Kikelj[b], Päivi Tammela*[a]
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