Non-native autoinducer analogs capable of modulating the SdiA quorum sensing receptor in Salmonella enterica serovar Typhimurium

Abstract

Quorum sensing (QS) allows many common bacterial pathogens to coordinate group behaviors such as virulence factor production, host colonization, and biofilm formation at high population densities. This cell–cell signaling process is regulated by N -acyl L-homoserine lactone (AHL) signals, or autoinducers, and LuxR-type receptors in Gram-negative bacteria. SdiA is an orphan LuxR-type receptor found in Escherichia, Salmonella, Klebsiella, and Enterobacter genera that responds to AHL signals produced by other species and regulates genes involved in several aspects of host colonization. The inhibition of QS using non-native small molecules that target LuxR-type receptors offers a non-biocidal approach for studying, and potentially controlling, virulence in these bacteria. To date, few studies have characterized the features of AHLs and other small molecules capable of SdiA agonism, and no SdiA antagonists have been reported. Herein, we report the screening of a set of AHL analogs to both uncover agonists and antagonists of SdiA and to start to delineate structure–activity relationships (SARs) for SdiA:AHL interactions. Using a cell-based reporter of SdiA in Salmonella enterica serovar Typhimurium, several non-natural SdiA agonists and the first set of SdiA antagonists were identified and characterized. These compounds represent new chemical probes for exploring the mechanisms by which SdiA functions during infection and its role in interspecies interactions. Moreover, as SdiA is highly stable when produced in vitro, these compounds could advance fundamental studies of LuxR-type receptor:ligand interactions that engender both agonism and antagonism.

Autoinducer-Fluorophore Conjugates Enable FRET in LuxR Proteins in Vitro and in Cells

“…Likewise, CviR and CepR have similarly sized native ligands (N-hexanoyl L-homoserine lactone (HHL) and N-octanoyl L-homoserine lactone (OHL), respectively) but are maximally activated by differently sized FRET-probes: long chain CU6 and DA6 probes for CviR and the short chain DA1 probe for CepR. As observed in prior work demonstrating their promiscuity with regards to AHL-type ligands, 12,30 SdiAEC and SdiASE were strongly activated by most of the FRET-probes. RhlR showed little to no activation by any of the compounds in the probe library at 10 µM; the probe DA6, with 15% activation at 100 µM, was the most efficacious agonist of RhlR found.…”

“…S6) in E. coli b-galactosidase reporter strains for the following seven LuxRtype receptors: LasR, 6 RhlR, 28 and QscR 27 from P. aeruginosa; CviR from Chromobacterium violaceum; CepR from Burkholderia cenocepacia; 29 SdiA from E. coli (referred to as SdiAEC); 12 and SdiA from Salmonella enterica (referred to as SdiASE). 30 Efficacious agonists were identified in almost every receptor (Fig. 2B), with the coumarin derivatives generally less active than their dansyl counterparts.…”

“…The FRET assay has the potential to determine whether a change in potency in these reporter assays reflects a change in affinity, allowing for more educated design and tailoring of ligands. As an example, we explored the activity of a set of thiolactone AHL analogs that have been explored as QS modulators 14 and reported to have increased potency relative to their parent lactone analogs in cell-based reporter assays of several LuxR-type receptors -i.e., in SdiASE, 30 SdiAEC, 12 RhlR, 15 and LasR. 10 One explanation, aside from their increased hydrolytic stability over the time course of the assay, would be increased ligand affinity.…”

N-Acyl l-Homocysteine Thiolactones Are Potent and Stable Synthetic Modulators of the RhlR Quorum Sensing Receptor in Pseudomonas aeruginosa

“…More recently, Bassler and co-workers reported that a meta-bromo aryl homocysteine thiolactone (i.e., mBTL; Figure 1B) was a RhlR partial agonist (11). Homocysteine thiolactones have been examined in AHL analogs previously (11,(18)(19)(20)(21)(22), but except for mBTL, have not been explored as modulators of RhlR. Together, these prior studies indicated that RhlR can accommodate non-lactone head groups (assuming these close AHL mimetics target the BHL-binding site) and that further research into such compound scaffolds could be fruitful for new ligand design.…”

Effects of Quorum Sensing AHL Signaling on the Biological Characteristics of Porcine Derived F4ac+ Enterotoxigenic Escherichia coli

“…E. coli cannot synthesize AHL molecule on its own, but it has SdiA, the molecular receptor of AHL, which can monitor the AHL signal secreted by adjacent flora in the infection pathway through quorum sensing eavesdropping, so as to further regulate its own virulence (Culler et al, 2018;Styles and Blackwell, 2018;Dyszel et al, 2010;Yakhnin et al, 2011). In previous study of our laboratory, the AHL positive strains in the pig intestine has been screened, and its regulation on the virulence of Shiga toxin-producing E. coli (STEC), Enterohemorrhagic E. coli (EHEC) and avian pathogenic E. coli (APEC) has been verified (Smith et al, 2011;Yang et al, 2013).…”