Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

Abstract

N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography-mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach.

Figure 1

AHL production of 1% (w/v) L-arabinose induced E. coli MG1655 [pBAD-rhlI] treated with 10 pure compounds with the concentration 3 mg/ml detected by C. violaceum CV026 (A).Salicylic acid, tannic acid and trans-cinnamaldehyde are potential AHL synthase inhibitors (B).

Figure 2. LCMS analysis of C4-HSL production treated by salicylic acid, tannic acid and trans-cinnamaldehyde.

Grey arrows indicate the parental ion of C4-HSL (m/z: 172).

Figure 3. LCMS analysis of 3-oxo-C12-HSL production treated by salicylic acid, tannic acid and trans-cinnamaldehyde.

Grey arrows indicate the parental ion of 3-oxo-C12-HSL (m/z: 298).

Figure 4

Swarming assay of P. aeruginosa PAO1on semi-solid agar without inhibitors as non-treated control, agar containing with DMSO and Trans-cinnamaldehyde (A). Pyocyanin assay of P. aeruginosa PAO1without inhibitor as non-treated control, with DMSO as solvent control and treated with 0.1 mg/ml, 0.2 mg/ml and 0.3 mg/ml of trans-cinnamaldehyde (B).

Figure 5. The binding of trans-cinnamaldehyde with LasI (A) and EsaI (B).

The protein was represented by ribbon, the carbon atoms of trans-cinnamaldehyde were coloured in orange, the carbon atoms on amino acids were coloured in green, the oxygen atoms were coloured in red and the nitrogen atoms were coloured in blue.