Abstract
Bacterial quorum sensing (QS) is a cell-to-cell communication system that governs the expression of a large set of genes involved in bacterial–host interactions, including the production of virulence factors. Conversely, the hosts can produce anti-QS compounds to impair virulence of bacterial pathogens. One of these inhibitors is the plant flavonoid naringenin, which impairs the production of QS-regulated Pseudomonas aeruginosa virulence factors. In the present work, we analyze the molecular basis for such inhibition. Our data indicate that naringenin produces its effect by directly binding the QS regulator LasR, hence competing with its physiological activator, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12-HSL). The in vitro analysis of LasR binding to its cognate target DNA showed that the capacity of naringenin to outcompete 3OC12-HSL, when the latter is previously bound to LasR, is low. By using an E. coli LasR-based biosensor strain, which does not produce 3OC12-HSL, we determined that the inhibition of LasR is more efficient when naringenin binds to nascent LasR than when this regulator is already activated through 3OC12-HSL binding. According to these findings, at early exponential growth phase, when the amount of 3OC12-HSL is low, naringenin should proficiently inhibit the P. aeruginosa QS response, whereas at later stages of growth, once 3OC12-HSL concentration reaches a threshold enough for binding LasR, naringenin would not efficiently inhibit the QS response. To test this hypothesis, we analyze the potential effect of naringenin over the QS response by adding naringenin to P. aeruginosa cultures at either time zero (early inhibition) or at stationary growth phase (late inhibition). In early inhibitory conditions, naringenin inhibited the expression of QS-regulated genes, as well as the production of the QS-regulated virulence factors, pyocyanin and elastase. Nevertheless, in late inhibitory conditions, the P. aeruginosa QS response was not inhibited by naringenin. Therefore, this time-dependent inhibition may compromise the efficiency of this flavonoid, which will be effective just when used against bacterial populations presenting low cellular densities, and highlight the importance of searching for QS inhibitors whose mechanism of action does not depend on the QS status of the population.
Highlights
Quorum sensing (QS) is a cell-to-cell communication system that enables to coordinate a global response of a bacterial population when a certain cell density is reached (Nealson et al, 1970; Fuqua et al, 1994)
To analyze the putative capacity of naringenin to inhibit the binding of LasR to the lasI promoter, whose activity is directly dependent of LasR binding, we started by establishing the purification conditions required to obtain an active LasR protein
Protein extracts obtained in each of the conditions were used for performing electrophoretic mobility shift assays (EMSAs) using the complete intergenic region upstream of lasI, containing the LasR-binding site (Schuster et al, 2004), as the DNA probe
Summary
Quorum sensing (QS) is a cell-to-cell communication system that enables to coordinate a global response of a bacterial population when a certain cell density is reached (Nealson et al, 1970; Fuqua et al, 1994). The lasR and rhlR genes, which encode the receptors of 3OC12-HSL and C4-HSL respectively, are located upstream the lasI and rhlI genes These two LuxRtype transcriptional regulators contain both, a DNA binding domain and a ligand binding domain (Lamb et al, 2003; Bottomley et al, 2007) and control the expression of hundreds of genes in P. aeruginosa (Wagner et al, 2003; Schuster and Greenberg, 2007). It has been described that the disruption of the QS response reduces P. aeruginosa virulence (Tan et al, 1999; Diggle et al, 2007) and impairs biofilm formation (Wagner and Iglewski, 2008) This indicates that QS is a fundamental element in the success of this opportunistic pathogen for colonizing and infecting its hosts, from plants to humans. The QS system transcriptional regulators, such as LasR, have become attractive targets to be inhibited (Williams et al, 2000; Hurley et al, 2012; Kalia et al, 2017)
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