Design, synthesis, and biological evaluation of abiotic, non-lactone modulators of LuxR-type quorum sensing

Quorum sensing (QS) is a cell-cell signaling mechanism that allows bacteria to monitor their population size and alter their behavior at high cell densities. Gram-negative bacteria use N-acylated L-homoserine lactones (AHLs) as their primary signals for QS. These signals are susceptible to lactone hydrolysis in biologically relevant media, and the ring-opened products are inactive QS signals. We have previously identified a range of non-native AHLs capable of strongly agonizing and antagonizing QS in Gram-negative bacteria. However, these abiotic AHLs are also prone to hydrolysis and inactivation and thereby have a relatively short time window for use (～12-48 h). Non-native QS modulators with reduced or no hydrolytic instability could have enhanced potencies and would be valuable as tools to study the mechanisms of QS in a range of environments (for example, on eukaryotic hosts). This study reports the design and synthesis of two libraries of new, non-hydrolyzable AHL mimics. The libraries were screened for QS modulatory activity using LasR, LuxR, and TraR bacterial reporter strains, and several new, abiotic agonists and antagonists of these receptors were identified.     

Bacterial quorum sensing in symbiotic and pathogenic relationships with hosts*

Gram-negative bacteria communicate with each other by producing and sensing diffusible signaling molecules. This mechanism is called quorum sensing (QS) and regulates many bacterial activities from gene expression to symbiotic/pathogenic interactions with hosts. Therefore, the elucidation and control of bacterial QS systems have been attracted increasing attention over the past two decades. The most common QS signals in Gram-negative bacteria are N-acyl homoserine lactones (AHLs). There are also bacteria that employ different QS systems, for example, the plant pathogen Ralstonia solanacearum utilizes 3-hydroxy fatty acid methyl esters as its QS signals. The QS system found in the endosymbiotic bacterium associated with the fungus Mortierella alpina, the development of an affinity pull-down method for AHL synthases, and the elucidation of a unique QS circuit in R. solanacearum are discussed herein.     

Methods for detecting acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics

In the process of evaluating the role of acylated homoserine lactones (AHLs) in food-spoiling Gram-negative bacteria, we have combined a range of bacterial AHL monitor systems to determine the AHL-profile and the kinetics of AHL-production. AHL production from 148 strains of Enterobacteriaceae isolated from foods was tested using Escherichia coli pSB403 (LuxR), Agrobacterium tumefaciens A136 (TraR) and both induction and inhibition of Chromobacterium violaceum CV026 (CviR). All strains except one was found to produce AHL(s). In no case could a single monitor system identify more than 64% of the Enterobacteriaceae as AHL-producers, showing that the simultaneous use of monitor strains is required in the process of screening bacterial populations for AHL-production. AHLs from 20 selected strains were profiled by thin layer chromatography. Most strains produced more than one AHL with 3-N-oxo-hexanoyl homoserine lactone being the most prominent. It was found that the simultaneous use of monitor strains in the top-layer was necessary for the detection of (presumably) all the AHLs. An agar well-diffusion assay based on A. tumefaciens pDZLR4 was used for quantifying AHLs from bacterial supernatants and enabled an assessment of the kinetics of AHL-production of 3 strains (Serratia proteamaculans strain B5a, Erwinia carotovora ATCC 39048 and V. fischeri strain MJ-1). As expected, the production of AHL (OHHL) and luminescence in Vibrio fischeri strain MJ-1 increased faster than growth indicating up-regulation of the AHL regulated phenotype and auto-induction of AHL production. In contrast, production kinetics of AHL (OHHL) in the two Enterobacteriaceae indicated lack of auto-induction.     

A LuxR homologue of Xanthomonas oryzae pv. oryzae is required for optimal rice virulence

In Gram-negative bacteria a typical quorum sensing (QS) system usually involves the production and response to acylated homoserine lactones (AHLs). An AHL QS system is most commonly mediated by a LuxI family AHL synthase and a LuxR family AHL response regulator. This study reports for the first time the presence of a LuxR family-type regulator in Xanthomonas oryzae pv. oryzae ( Xoo ), which has been designated as OryR. The primary structure of OryR contains the typical signature domains of AHL QS LuxR family response regulators: an AHL-binding and a HTH DNA binding motif. The oryR gene is conserved among 26 Xoo strains and is also present in the genomes of close relatives X. campestris pv. campestris and X. axonopodis pv. citri . Disrupting oryR in three Xoo strains resulted in a significant reduction of rice virulence. The wild-type Xoo strains do not seem to produce AHLs and analysis of the Xoo sequenced genomes did not reveal the presence of a LuxI-family AHL synthase. The OryR protein was shown to be induced by macerated rice and affected the production of two secreted proteins: a cell-wall-degrading cellobiosidase and a 20-kDa protein of unknown function. By expressing and purifying OryR it was then observed that it was solubilized when grown in the presence of rice extract indicating that there could be a molecule(s) in rice which binds OryR. The role of OryR as a possible in planta induced LuxR family regulator is discussed.     

Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors

Many Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum-sensing (QS) signal molecules. AHL QS has been the subject of extensive investigation in the last decade and has become a paradigm for bacterial intercellular signaling. Research in AHL QS has been considerably aided by simple methods devised to detect AHLs using bacterial biosensors that phenotypically respond when exposed to exogenous AHLs. This article reviews and discusses the currently available bacterial biosensors which can be used in detecting and studying the different AHLs.     

AHL-dependent quorum sensing inhibition: synthesis and biological evaluation of α-(N-alkyl-carboxamide)-γ-butyrolactones and α-(N-alkyl-sulfonamide)-γ-butyrolactones

New N-acylhomoserine lactone (AHL) analogues in which the amide function is replaced by a reverse-amide one have been studied as AHL QS modulators. The series of compounds consists of α-(N-alkyl-carboxamide)-γ-butyrolactones, α-(N-alkyl-sulfonamide)-γ-butyrolactones, and 2-(N-alkyl-carboxamide)-cyclopentanones and cyclopentanols. Most active compounds exhibited antagonist activities against LuxR reaching the 30 μM range.     

Evaluation of a focused library of N-aryl L-homoserine lactones reveals a new set of potent quorum sensing modulators

A focused library of N-aryl l-homoserine lactones was designed around known lactone leads and evaluated for antagonistic and agonistic activity against quorum-sensing receptors in Agrobacterium tumefaciens, Pseudomonas aeruginosa, and Vibrio fischeri. Several compounds were identified with significantly heightened activities relative to the lead compounds, and new structure-activity relationships (SARs) were delineated. Notably, 4-substituted N-phenoxyacetyl and 3-substituted N-phenylpropionyl l-homoserine lactones were identified as potent antagonists of TraR and LuxR, respectively.     

Presence of quorum-sensing inhibitor-like compounds from bacteria isolated from the brown alga Colpomenia sinuosa

Aims:  Several Gram-negative bacterial species use N -acyl homoserine lactone (AHL) molecules as quorum-sensing (QS) signals to regulate various biological functions. Similarly, various bacteria can stimulate, inhibit or inactivate QS signals in other bacteria by producing molecules called as quorum-sensing inhibitors (QSI). Our aim was to screen and identify the epibiotic bacteria associated with brown algae for their ability of producing QS-inhibiting activity.
Methods and Results:  QSI screenings were conducted on several epibiotic bacteria isolated from a marine brown alga Colpomenia sinuosa , using Serratia rubidaea JCM 14263 as an indicator organism. Strain JCM 14263 controls the production of red pigment, prodigiosin by AHL QS. Out of 96 bacteria, which were isolated from the surface of the brown alga, 12% of strains showed the ability to produce QSI, which was observed from the pigmentation inhibition on Ser. rubidaea JCM 14263 without affecting its growth. Phylogenetic analysis using 16S rRNA gene sequencing method demonstrated bacterial isolates showing QS inhibition-producing bacteria belonging to the Bacillaceae (Firmicutes), Pseudomonadaceae (Proteobacteria), Pseudoalteromonadaceae (Proteobacteria) and Vibrionaceae (Proteobacteria).
Conclusion:  An appreciable percentage of bacteria isolated from the brown alga produced QSI-like compounds.
Significance and Impact of the Study:  The screening method using Ser. rubidaea described in this report will facilitate the rapid identification of QSI-producing bacteria from marine environment. This study reveals new avenue for future environmental applications. This study also suggests that these algal epibiotic bacteria may play a role in the defensive mechanism for their host by producing QSI or QSI-like compounds to suppress the settlement of other competitive bacteria.     

Substituted lactam and cyclic azahemiacetals modulate Pseudomonas aeruginosa quorum sensing

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence that is critical for establishing infection. The most common QS signaling molecule used by Gram-negative bacteria are acylhomoserine lactones. The development of non-native acylhomoserine lactone (AHL) ligands has emerged as a promising new strategy to inhibit QS in Gram-negative bacteria. In this work, we have synthesized a set of optically pure γ-lactams and their reduced cyclic azahemiacetal analogues, bearing the additional alkylthiomethyl substituent, and evaluated their effect on the AHL-dependent Pseudomonas aeruginosa las and rhl QS pathways. The concentration of these ligands and the simple structural modification such as the length of the alkylthio substituent has notable effect on activity. The γ-lactam derivatives with nonylthio or dodecylthio chains acted as inhibitors of las signaling with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent was found to strongly inhibit both las and rhl signaling at higher concentrations while the propylthio analogue strongly stimulated the las QS system at lower concentrations.     

Quorum quenching activity in Anabaena sp. PCC 7120: identification of AiiC, a novel AHL-acylase

Many bacteria use quorum sensing (QS) to coordinate responses to environmental changes. In Gram-negative bacteria, the most extensively studied QS systems rely on the use of N -acylhomoserine lactones (AHLs) signal molecules. Some bacteria produce enzymes that are able to inactivate AHL signals produced by other bacteria and hence interfere with QS-mediated processes via a phenomenon known as quorum quenching. Acylase-type AHL degradation activity has been found in the biomass of the filamentous nitrogen-fixing cyanobacterium Anabaena ( Nostoc ) sp. PCC 7120, being absent from the culture media. The gene all3924 has been identified and cloned whose product exhibits homology to the acylase QuiP of Pseudomonas aeruginosa PAO1, demonstrating that it is at least partially responsible for the AHL-acylase activity. The recombinant enzyme, which was named auto-inducer inhibitor from Cyanobacteria (AiiC), shows broad acyl-chain length specificity. Because the presence of AHLs in the biomass of nitrogen-fixing cultures of Anabaena sp. PCC 7120 has been described recently, AiiC could represent a self-modulatory system to control the response to its own QS signals but could also be involved in the interference of signalling within complex microbial communities in which Cyanobacteria are present.     

AHL类似物作为细菌群体感应系统促进剂的研究进展

应用N-酰基高丝氨酸内酯（N-acyl-L-homoserine lactones，AHL）介导的群体感应（quorum sensing，QS）系统调控生物膜形成和次级代谢物合成具有巨大的商业价值，但自然界中许多微生物能够产生群体淬灭（Quorum Quenching，QQ）酶，QQ酶能够降解天然AHL信号分子，使外源天然 AHL 信号分子的半衰期缩短，限制了天然AHL 信号分子的应用范围。化学合成的AHL类似物作为QS促进剂，通过与天然信号分子类似的结合方式形成转录二聚体，激活下游基因表达，但与天然AHL信号分子相比，化学合成的QS促进剂具有活性高、半衰期长等优点。本文综述了化学合成AHL类似物的设计思路、种类、作用机制及其在提高次级代谢物产量和生物浸矿方面的应用，并讨论了QS促进剂今后主要的研究方向，以期为QS促进剂的合成设计和实际应用提供参考。     

The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor

Many bacteria use quorum sensing (QS) as an intercellular signaling mechanism to regulate gene expression in local populations. Plant and algal hosts, in turn, secrete compounds that mimic bacterial QS signals, allowing these hosts to manipulate QS-regulated gene expression in bacteria. Lumichrome, a derivative of the vitamin riboflavin, was purified and chemically identified from culture filtrates of the alga Chlamydomonas as a QS signal-mimic compound capable of stimulating the Pseudomonas aeruginosa LasR QS receptor. LasR normally recognizes the N-acyl homoserine lactone (AHL) signal, N-3-oxo-dodecanoyl homoserine lactone. Authentic lumichrome and riboflavin stimulated the LasR receptor in bioassays and lumichrome activated LasR in gel shift experiments. Amino acid substitutions in LasR residues required for AHL binding altered responses to both AHLs and lumichrome or riboflavin. These results and docking studies indicate that the AHL binding pocket of LasR recognizes both AHLs and the structurally dissimilar lumichrome or riboflavin. Bacteria, plants, and algae commonly secrete riboflavin or lumichrome, raising the possibility that these compounds could serve as either QS signals or as interkingdom signal mimics capable of manipulating QS in bacteria with a LasR-like receptor.     

Development and comparison of whole-cell assay systems for quorum-sensing inhibitors based on TraR, LasR, and QscR

Quorum sensing (QS) is a cell density-dependent signaling system that is used by bacteria to coordinate gene expression within their population. In this study, the authors describe the development and characterization of various cell-based bioassay systems for detecting QS inhibitors based on three LuxR family proteins, TraR, LasR, and the recently identified QscR. Three different gram-negative bacteria, Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa, were employed as reporter strains to overproduce one of the aforementioned QS activator proteins and respond to inhibitors. The nine different whole-cell assay systems (three reporter strains × three QS proteins) were evaluated for their applicability and reliability by studying quantitative responses to various furanones, which are potent inhibitors of the LuxR family proteins. These results demonstrate that the cell-based bioassay systems are sensitive and reliable tools for screening of QS activators and inhibitors. This study also suggests that furanones are potentially important QS inhibitors for many LuxR-type activator proteins.