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SdiA is a LuxR-type receptor that controls the virulence of Klebsiella pneumoniae, a Gram-negative bacterium that causes various infections in humans. SdiA senses exogenous acyl-homoserine lactones (AHLs) and autoinducer-2 (AI-2), two types of quorum sensing signals produced by other bacterial species. However, the molecular details of how SdiA recognizes and binds to different ligands and how this affects its function and regulation in K. pneumoniae still need to be better understood. This study uses computational methods to explore the protein-ligand binding dynamics of SdiA with 11 AHLs and 2 AI-2 ligands. The 3D structure of SdiA was predicted through homology modeling, followed by molecular docking with AHLs and AI-2 ligands. Binding affinities were quantified using MM-GBSA, and complex stability was assessed via Molecular Dynamics (MD) simulations. Results demonstrated that SdiA in Klebsiella pneumoniae exhibits a degenerate binding nature, capable of interacting with multiple AHLs and AI-2. Specific ligands, namely C10-HSL, C8-HSL, 3-oxo-C8-HSL, and 3-oxo-C10-HSL, were found to have high binding affinities and formed critical hydrogen bonds with key amino acid residues of SdiA. This finding aligns with the observed preference of SdiA for AHLs having 8 to 10 carbon-length acyl chains and lacking hydroxyl groups. In contrast, THMF and HMF demonstrated poor binding properties. Furthermore, AI-2 exhibited a low affinity, corroborating the inference that SdiA is not the primary receptor for AI-2 in K. pneumoniae. These findings provide insights into the protein-ligand binding dynamics of SdiA and its role in quorum sensing and virulence of K. pneumoniae., Competing Interests: Declarations. Conflict of interest: The authors have no competing interests to declare that are relevant to the content of this article. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Bacterial quorum sensing is a chemical language allowing bacteria to interact through the excretion of molecules called autoinducers, like N-acyl-homoserine lactones (AHLs) produced by Gram-negative Burkholderia and Paraburkholderia bacteria known as opportunistic pathogens. The AHLs differ in their acyl-chain length and may be modified by a 3-oxo or 3-hydroxy substituent, or C = C double bonds at different positions. As the bacterial signal specificity depends on all of these chemical features, their structural characterization is essential to have a better understanding of the population regulation and virulence phenomenon. This study aimed at enabling the localization of the C = C double bond on such specialized metabolites while using significantly lower amounts of biological material. The approach is based on LC-MS/MS analyses of bacterial extracts after in-solution derivatization by a photochemical Paternò-Büchi reaction, leading to the formation of an oxetane ring and subsequently to specific fragmentations when performing MS/MS experiments. The in-solution derivatization of AHLs was optimized on several standards, and then the matrix effect of bacterial extracts on the derivatization was assessed. As a proof of concept, the optimized conditions were applied to a bacterial extract enabling the localization of C = C bonds on unsaturated AHLs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

The prevention or control of bacterial infections requires continuous search for novel approaches among which bacterial quorum sensing inhibition is considered as a complementary antibacterial strategy. Quorum sensing, used by many different bacteria, functions through a cell-to-cell communication mechanism relying on chemical signals, referred to as autoinducers, such as N-acyl homoserine lactones (AHLs) which are the most common chemical signals in this system. Designing analogs of these autoinducers is one of the possible ways to interfere with quorum sensing. Since bioisosteres are powerful tools in medicinal chemistry, targeting analogs of AHLs or other signal molecules and mimics of known QS modulators built on amide bond bioisosteres is a relevant strategy in molecular design and synthetic routes. This review highlights the application of amide bond bioisosteric replacement in the design and synthesis of novel quorum sensing inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

In this study, the cadmium (Cd)-tolerant Ensifer adhaerens strain NER9 with quorum sensing (QS) systems (responsible for N-acyl homoserine lactone (AHL) production) was characterized for QS system-mediated Cd immobilization and the underlying mechanisms involved. Whole-genome sequence analysis revealed that strain NER9 contains the QS SinI/R and TraI/R systems. Strains NER9 and the NER9∆sinI/R, NER9∆traI/R, and NER9∆sinI/R-traI/R mutants were constructed and compared for QS SinI/R and TraI/R system-mediated Cd immobilization in the solution and the mechanisms involved. After 24 h of incubation, strain NER9 significantly decreased the Cd concentration in the Cd-contaminated solution compared with the NER9∆sinI/R, NER9∆traI/R, and NER9∆sinI/R-traI/R mutants. The NER9∆sinI/R mutant had a greater impact on Cd immobilization and a lower impact on the activities of AHLs than did the NER9∆traI/R mutant. The NER9∆sinI/R mutant had significantly greater Cd concentrations and lower cell wall- and exopolysaccharide (EPS)-adsorbed Cd contents than did strain NER9. Furthermore, the NER9∆sinI/R mutant presented a decrease in the number of functional groups interacting with Cd, compared with strain NER9. These results suggested that the SinI/R system in strain NER9 contributed to Cd immobilization by mediating cell wall- and EPS-adsorption in Cd-containing solution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The opportunistic pathogen Pseudomonas aeruginosa controls almost 10% of its genome, including myriad virulence genes, via a cell-to-cell chemical communication system called quorum sensing (QS). Small molecules that either inhibit or activate QS in P. aeruginosa represent useful research tools to study the role of this signaling pathway in infection and interrogate its viability as an antivirulence target. However, despite active research in this area over the past 20+ years, there are relatively few synthetic compounds known to strongly inhibit or activate QS in P. aeruginosa . Most reported QS modulators in this pathogen are of low potency or have structural liabilities that limit their application in biologically relevant environments such as mimics of the native N -acyl l-homoserine lactone (AHL) signals. Here, we report the results of a high-throughput screen for abiotic small molecules that target LasR, a key QS regulator in P. aeruginosa . We screened a 25,000-compound library and discovered four new structural classes of abiotic LasR modulators. These compounds include antagonists that surpass the potency of all known AHL-type compounds and mimetics thereof, along with an agonist with potency approaching that of LasR's native ligand. The novel structures of this compound set, along with their anticipated robust physicochemical profiles, underscore their potential value as probe molecules to interrogate the roles of QS in this formidable pathogen.

Many Gram-negative bacteria use N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm-related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to generate these improved properties using the Protein One-Stop Shop Server. The engineering of acylases is complicated by low-throughput enzymatic assays. Alleviating this challenge, we report a time-course kinetic assay for AHL acylases that monitors the real-time production of homoserine lactone. Using the assay, we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2°C, which translated into high resistance against organic solvents and increased compatibility with material coatings. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl-enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

To investigate the mechanism of aquatic pathogens in quorum sensing (QS) and decode the signal transmission of aquatic Gram-negative pathogens, this paper proposes a novel method for the intelligent matching identification of eight quorum signaling molecules (N-acyl-homoserine lactones, AHLs) with similar molecular structures, using terahertz (THz) spectroscopy combined with molecular dynamics simulation and spectral similarity calculation. The THz fingerprint absorption spectral peaks of the eight AHLs were identified, attributed, and resolved using the density functional theory (DFT) for molecular dynamics simulation. To reduce the computational complexity of matching recognition, spectra with high peak matching values with the target were preliminarily selected, based on the peak position features of AHL samples. A comprehensive similarity calculation (CSC) method using a weighted improved Jaccard similarity algorithm (IJS) and discrete Fréchet distance algorithm (DFD) is proposed to calculate the similarity between the selected spectra and the targets, as well as to return the matching result with the highest accuracy. The results show that all AHL molecular types can be correctly identified, and the average quantization accuracy of CSC is 98.48%. This study provides a theoretical and data-supported foundation for the identification of AHLs, based on THz spectroscopy, and offers a new method for the high-throughput and automatic identification of AHLs.

The halogenation of quorum sensing molecules (QSMs) is known to be catalyzed by enzymes such as haloperoxidase (HPO) as well as cerium dioxide nanocrystals (NC), which mimic enzymes. Those enzymes and mimics can influence biological processes such as biofilm formation, where bacteria use QSMs for the "chemical" communication between each other and the coordination of surface colonization. However, not much is known about the degradation behavior of a broad spectrum of QSMs, especially for HPO and its mimics. Therefore, in this study, the degradation of three QSMs with different molecule moieties was elucidated. For this purpose, different batch experiments were carried out with HPOs, NCs and free active bromine (FAB). For N -β-ketocaproyl-homoserine lactone (3-Oxo-C 6 -AHL), N - cis -tetradec-9 Z -enoyl-homoserine lactone (C 14:1 -AHL) and 2-heptyl-4-quinolone (HHQ) a fast degradation and moiety-specific transformations were observed. The HPO vanadium bromoperoxidase as well as cerium dioxide NCs catalyzed the formation of the same brominated transformation products (TPs). Since the same TPs are formed in batch experiments with FAB it is very likely that FAB is playing a major role in the catalytical reaction mechanism leading to the transformation of QSMs. In this study in total 17 TPs could be identified in different levels of confidence and the catalytic degradation processes for two QS groups (unsaturated AHLs and alkyl quinolones) with cerium dioxide NCs and vanadium bromoperoxidase were expanded.

The effective prevention and control of non-filamentous bulking is a significant challenge. In this study, the underlying effect of quorum sensing (QS) on inducing non-filamentous bulking and the maintenance effect of silver nanoparticles (AgNPs) on sludge floc stability, aggregation and settleability based on the quorum quenching (QQ) activity during non-filamentous bulking were investigated. The results showed that the concentration of N-acyl homoserine lactone (AHL) increased significantly in the activated sludge system at a high organic load rate (OLR), triggering the AHL-mediated QS. Additionally, the triggered QS promoted exopolysaccharide secretion, reducing the surface charge and hydrophobicity of the sludge aggregates, and further deteriorating the settleability of the sludge aggregates. AgNPs, a quorum sensing inhibitor (QSI), inhibited the AHL-QS based on QQ activity under high OLR, which maintained the physicochemical properties of extracellular polymeric substances (EPS). AgNPs-QQ maintained the surface energy barrier and electrostatic barrier of sludge aggregates and the gel properties of exopolysaccharides, which is favorable for microbial aggregation. The appropriate concentrations of AgNPs (≤10 mg/L) had no negative effect on biological nutrient removal in the sequencing batch reactors (SBRs) at the high organic loading. Therefore, AgNPs effectively prevent and control non-filamentous bulking by their QQ activity in the activated sludge process. Thus, the present study provided new insights into controlling non-filamentous bulking during the activated sludge process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Acyl-homoserine lactone synthases make specific AHL quorum sensing signals to aid virulence in Gram-negative bacteria. Here, we use solution NMR spectroscopy to demonstrate that the carrier protein-enzyme interface accurately reveals substrate recognition mechanisms in two quorum signal synthases.

Salmonella is an important foodborne pathogen, and it is unable to produce the quorum sensing signaling molecules called acyl-homoserine lactones (AHLs). However, it synthesizes the SdiA protein, detecting AHL molecules, also known as autoinducer-1 (AI-1), in the external environment. Exogenous AHLs can regulate specific genes related to virulence and stress response in Salmonella. Thus, interfering with quorum sensing can be a strategy to reduce virulence and help elucidate the cell-to-cell communication role in the pathogens' response to extracellular signals. This study aimed to evaluate the influence of the quorum sensing inhibitors furanone and phytol on phenotypes regulated by N-dodecanoyl homoserine lactone (C12-HSL) in Salmonella enterica serovar Enteritidis. The furanone C30 at 50 nM and phytol at 2 mM canceled the alterations promoted by C12-HSL on glucose consumption and the levels of free cellular thiol in Salmonella Enteritidis PT4 578 under anaerobic conditions. In silico analysis suggests that these compounds can bind to the SdiA protein of Salmonella Enteritidis and accommodate in the AHL binding pocket. Thus, furanone C30 and phytol act as antagonists of AI-1 and are likely inhibitors of the quorum sensing mechanism mediated by AHL in Salmonella., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Bacterial biofilm formation is a huge problem in industry and medicine. Therefore, the discovery of anti-biofilm agents may hold great promise. Biofilm formation is usually a consequence of bacterial cell-cell communication, a process called quorum sensing (QS). CeO 2 nanocrystals (NCs) have been established as haloperoxidase (HPO) mimics and ecologically beneficial biofilm inhibitors. They were suggested to interfere with QS, a mechanism termed quorum quenching (QQ), but their molecular mechanism remained elusive. We show that CeO 2 NCs are effective QQ agents, inactivating QS signals by bromination. Catalytic bromination of 3-oxo-C 12 -AHL a QS signaling compound used by Pseudomonas aeruginosa , was detected in the presence of CeO 2 NCs, bromide ions, and hydrogen peroxide. Brominated acyl-homoserine lactones (AHLs) no longer act as QS signals but were not detected in the bacterial cultures. Externally added brominated AHLs also disappeared in P. aeruginosa cultures within minutes of their addition, indicating that they are rapidly degraded by the bacteria. Moreover, we detected the catalytic bromination of 2-heptyl-1-hydroxyquinolin-4(1 H )-one (HQNO), a multifunctional non-AHL QS signal from P. aeruginosa with antibacterial and algicidal properties controlling the expression of many virulence genes. Brominated HQNO was not degraded by the bacteria in vivo. The repression of the Pseudomonas quinolone signal (PQS) production and biofilm formation in P. aeruginosa through the catalytic formation of Br-HQNO on surfaces with coatings containing CeO 2 enzyme mimics validates the non-toxic strategy for the development of anti-infectives.

Quorum sensing (QS) is a molecular communication system used by microorganisms to adopt behaviors in a cell density-dependent manner. Lactonase enzymes, able to hydrolyze the signal molecules acyl-homoserine lactones (AHL) can counteract QS-mediated virulence in Gram-negative bacteria. Optimizing lactonases activity or specificity for AHL through enzyme engineering approaches is thus highly attractive to increase protective effect. However, only a limited number of screening methods have been developed to handle and evaluate AHL-degrading enzyme libraries. Here, a series of screening procedures were developed to identify improved lactonases using two previously reported enzymes as benchmarks, namely SsoPox and GcL. Specifically, molecular screenings using six different AHL and based on two reporter strains; i.e., Chromobacterium violaceum CV026 and Pseudomonas putida KS35, are reported. In addition, three phenotype-based screenings aiming to evaluate the ability of enzymes to quench a particular QS-related behavior are reported, using C. violaceum, Pseudomonas aeruginosa and Vibrio harveyi as pathogenic type strains. These assays were used to screen a small-sized library and allowed for the identification of various improved variants. To confirm that these variants were real "hits", four of them were produced and purified. Their kinetic parameters against AHL substrates were found to be increased by 2-44.5 -fold as compared to the starting enzyme. Moreover, their increased activity was confirmed by measuring their ability to quench QS in different bacterial systems. These new assays will facilitate the screening of enzyme libraries and will pave the way for the development of proficient engineered QS-disrupting enzymes., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cell-to-cell signaling, or quorum sensing (QS), in many Gram-negative bacteria is governed by small molecule signals (N-acyl-L-homoserine lactones, AHLs) and their cognate receptors (LuxR-type proteins). The mechanistic underpinnings of QS in these bacteria are severely limited due to the challenges of isolating and manipulating most LuxR-type proteins. Reports of quantitative direct-binding experiments on LuxR-type proteins are scarce, and robust and generalizable methods that provide such data are largely nonexistent. We report herein a Förster resonance energy transfer (FRET) assay that leverages (1) conserved tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore-AHL conjugates, and (2) isolation of the proteins bound to weak agonists. The FRET assay permits straightforward measurement of ligand-binding affinities with receptor-either in vitro or in cells-and was shown to be compatible with six LuxR-type proteins. These methods will advance fundamental investigations of LuxR-type protein mechanism and the development of small molecule QS modulators., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Quorum sensing (QS), a type of bacterial cell-cell communication, produces autoinducers which help in biofilm formation in response to cell population density. In this review, biofilm formation, the role of QS in biofilm formation and development with reference to biological wastewater treatment are discussed. Autoinducers, for example, acyl-homoserine lactones (AHLs), auto-inducing oligo-peptides (AIPs) and autoinducer 2, present in both Gram-negative and Gram-positive bacteria, with their mechanism, are also explained. Over the years, wastewater treatment (WWT) by QS-regulated biofilms and their optimization for WWT have gained much attention. This article gives a comprehensive review of QS regulation methods, QS enrichment methods and QS inhibition methods in biological waste treatment systems. Typical QS enrichment methods comprise adding QS molecules, adding QS accelerants and cultivating QS bacteria, while typical QS inhibition methods consist of additions of quorum quenching (QQ) bacteria, QS-degrading enzymes, QS-degrading oxidants, and QS inhibitors. Potential applications of QS regulated biofilms for WWT have also been summarized. At last, the knowledge gaps present in current researches are analyzed, and future study requirements are proposed.

The most studied mechanism of quorum sensing in Gram-negative bacteria is mediated by autoinducer 1 (AI-1), namely, acyl-homoserine lactone (AHL). This system allows communication among different bacterial species and regulates the expression of virulence genes in many pathogens. Although AHL-producing bacteria have been detected in the intestines of humans and other animals, no report was found about AHL-producing bacteria in the insect gut and the possible effects of these autoinducers on enteropathogenic bacteria. Therefore, this study aimed to identify AHL-producing bacteria in the gut of larvae of Galleria mellonella and to evaluate the influence of this quorum sensing signal on the regulation of adhesion and motility phenotypes in the intestinal pathogen Salmonella. Sequencing of the 16S rRNA gene, 16S rRNA gene-based phylogenetic analyses, and phenotypic characterization of gut isolates was performed. The profile of AHLs produced by the isolates was determined using thin-layer chromatography (TLC) and revealed with the biosensor strain Chromobacterium violaceum CV026. Sequencing, phylogenetic analyses and phenotypic characterization of gut isolates showed that the three AHL-producing strains belong to the species Rahnella inusitata, named GM34, GM56, and GM60. The TLC showed that R. inusitata produces a six-carbon AHL. In the presence of cell-free extract of R. inusitata containing AHL and under anaerobic conditions, Salmonella enterica increased the adhesion to stainless steel coupons and presented swarming motility. Extracts from the culture medium of R. inusitata isolates containing AHL increased the adhesion on stainless steel coupons and swarming motility of Salmonella enterica serovar Enteritidis PT4 under anaerobic conditions. The results suggest the possibility of communication between members of the G. mellonella intestinal microbiota with pathogens such as Salmonella., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The gut microbiome can produce metabolic products that exert diverse activities, including effects on the host. Short chain fatty acids and amino acid derivatives have been the focus of many studies, but given the high microbial density in the gastrointestinal tract, other bacterial products such as those released as part of quorum sensing are likely to play an important role for health and disease. In this review, we provide of an overview on quorum sensing (QS) in the gastrointestinal tract and summarise what is known regarding the role of QS molecules such as auto-inducing peptides (AIP) and acyl-homoserine lactones (AHL) from commensal, probiotic, and pathogenic bacteria in intestinal health and disease. QS regulates the expression of numerous genes including biofilm formation, bacteriocin and toxin secretion, and metabolism. QS has also been shown to play an important role in the bacteria-host interaction. We conclude that the mechanisms of action of QS at the intestinal neuro-immune interface need to be further investigated.

Quorum sensing (QS) is a unique mechanism for microorganisms to coordinate their activities through intercellular communication, including four main types of autoinducer-1 (AI-1, namely, N -acyl homoserine lactone [AHL]), AI-2, AI-3, and diffusible signaling factor [DSF]) based on signaling molecules. Quorum quenching (QQ) enzymes can disrupt the QS phenomenon by inactivating signaling molecules. QS is proposed to regulate biofilm formation in extremely acidic environments, but the QS/QQ-related genomic features in most acidophilic bacteria are still largely unknown. Here, genome annotation of 83 acidophiles from the genera Acidithiobacillus , Leptospirillum , Sulfobacillus , and Acidiphilium altogether revealed the existence of AI-1, AI-3, DSF, and AhlD (AHL degradation enzyme). The conservative investigation indicated that some QS/QQ-related proteins harbored key residues or motifs, which were necessary for their activities. Phylogenetic analysis showed that LuxI/R (AI-1 synthase/receptor), QseE/F (two-component system of AI-3), and RpfC/G (two-component system of DSF) exhibited similar evolutionary patterns within each pair. Meanwhile, proteins clustered approximately according to the species taxonomy. The widespread Acidithiobacillus strains, especially A. ferrooxidans, processed AI-1, AI-3, and DSF systems as well as the AhlD enzyme, which were favorable for their mutual information exchange and collective regulation of gene expression. Some members of the Sulfobacillus and Acidiphilium without AHL production capacity contained the AhlD enzyme, which may evolve for niche competition, while DSF in Leptospirillum and Acidithiobacillus could potentially combine with the cyclic diguanylate (c-di-GMP) pathway for self-defense and niche protection. This work will shed light on our understanding of the extent of communication networks and adaptive evolution among acidophiles via QS/QQ coping with environmental changes. IMPORTANCE Understanding cell-cell communication QS is highly relevant for comprehending the regulatory and adaptive mechanisms among acidophiles in extremely acidic ecosystems. Previous studies focused on the existence and functionality of a single QS system in several acidophilic strains. Four representative genera were selected to decipher the distribution and role of QS and QQ integrated with the conservative and evolutionary analysis of related proteins. It was implicated that intra- or intersignaling circuits may work effectively based on different QS types to modulate biofilm formation and energy metabolism among acidophilic microbes. Some individuals could synthesize QQ enzymes for specific QS molecular inactivation to inhibit undesirable acidophile species. This study expanded our knowledge of the fundamental cognition and biological roles underlying the dynamical communication interactions among the coevolving acidophiles and provided a novel perspective for revealing their environmental adaptability.

N -Acyl-l-homoserine lactones (AHLs) are a large family of signaling molecules in "quorum sensing" communication. This mechanism is present in a number of bacterial physiological phenomena, including pathogenic phenomena. In this study, we described a simple and accessible way to detect, annotate, and quantify these compounds from bacterial culture media. Analytical standards and ethyl acetate bacterial extracts containing AHLs were analyzed by an ultra-high-performance liquid chromatography system coupled to a mass spectrometer using a nontargeted FullMS data-dependent MS 2 method. The results were processed in MZmine2 and then analyzed by a Feature-Based Molecular Networking (FBMN) workflow in the Global Natural Products Social Networking (GNPS) platform for the discovery and annotation of known and unknown AHLs. Our group analyzed 31 AHL standards and included the MS 2 spectra in the spectral library of the GNPS platform. We also provide the 31 standard AHL spectrum list for inclusion in molecular networking analyses. FBMN analysis annotated 30 out of 31 standards correctly. Then, as an example, a set of five bacterial extracts was prepared for AHL annotation. Following the method described in this Article, 5 known and 11 unknown AHLs were properly annotated using the FBMN-based molecular network approach. This study offers the possibility for the automatic annotation of known AHLs and the search for nonreferenced AHLs in bacterial extracts in a somewhat straightforward approach even without acquiring analytical standards. The method also provides relative quantification information.

To obtain fiber materials with pronounced chemical-biological protection, metal (Zn or Ta) nanoparticles were jointly applied with polyelectrolyte complexes of enzymes and polypeptides being their stabilizers. Computer modeling revealed the preferences between certain polyelectrolyte partners for N -acyl-homoserine lactone acylase and hexahistidine-tagged organophosphorus hydrolase (His 6 -OPH) possessing the quorum quenching (QQ) behavior with bacterial cells. The combinations of metal nanoparticles and enzymes appeared to function better as compared to the combinations of the same QQ-enzymes with antibiotics (polymyxins), making it possible to decrease the applied quantities by orders of magnitude while giving the same effect. The elimination of Gram-positive and Gram-negative bacterial cells from doubly modified fiber materials notably increased (up to 2.9-fold), whereas His 6 -OPH retained its hydrolytic activity in reaction with organophosphorus compounds (up to 74% of initially applied activity). Materials with the certain enzyme and Zn nanoparticles were more efficient against Bacillus subtilis cells (up to 2.1-fold), and Ta nanoparticles acted preferentially against E scherichia coli (up to 1.5-fold). Some materials were proved to be more suitable for combined modification by metal nanoparticles and His 6 -OPH complexes as antimicrobial protectants.

In this study, we investigated the activation of TRPV1 and TRPA1 by N-acyl homoserine lactones, quorum sensing molecules produced by Gram-negative bacteria, and the inhibitory effect of TRPV1 and TRPA1 by autoinducing peptides (AIPs), quorum sensing molecules produced by Gram-positive bacteria, using human embryonic kidney 293T cell lines stably expressing human TRPV1 and TRPA1, respectively. As a result, we found that some N-acyl homoserine lactones, such as N-octanoyl-L-homoserine lactone (C8-HSL), N-nonanoyl-L-homoserine lactone (C9-HSL) and N-decanoyl-L-homoserine lactone (C10-HSL), activated both TRPV1 and TRPA1. In addition, we clarified that some N-acyl homoserine lactones, such as N-3-oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL), only activated TRPV1 and N-acyl homoserine lactones having saturated short acyl chain, such as N-acetyl-L-homoserine lactone (C2-HSL) and N-butyryl-L-homoserine lactone (C4-HSL), only activated TRPA1. Furthermore, we found that an AIP, simple linear peptide CHWPR, inhibited both TRPV1 and TRPA1 and peptide having thiolactone ring DICNAYF, the thiolactone ring were formed between C3 to F7, strongly inhibited only the TRPV1. Although the specificity of TRPV1 and TRPA1 for quorum sensing molecules was different, these data suggest that both TRPV1 and TRPA1 would function as receptors for quorum sensing molecule produced by bacteria. Graphical Abstract., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Phyllanthus emblica is a traditional medicinal plant that is endowed with curative properties including anti-bacterial, anti-fungal, anti-viral, and analgesic properties. Bacteria make use of cell-cell signaling system known as quorum sensing (QS) and respond to their own population. In most gram-negative bacteria, the transcriptional regulators belonging to the Lux R protein play a crucial role in the QS mechanism by detecting the presence of signaling molecules known as N-acyl homoserine lactones (AHLs). In this present work, the anti-quorum sensing activity of Phyllanthus emblica was evaluated against Pseudomonas aeruginosa. Anti-quorum sensing efficacy of Phyllanthus emblica was estimated with reference to QS bio-monitoring strain Chromobacterium violaceum. The binding efficacy of the phytochemicals of Phyllanthus emblica against CviR protein from Chromobacterium violaceum and LasR protein from Phyllanthus emblica were studied., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Pathogenic bacteria use an intercellular chemical communication system called quorum sensing (QS) to control the expression of cellular functions such as virulence factors, biofilm formation, toxin production, and antibiotic resistance in a manner that is highly dependent on population density. Hence, since the emergence of QS, there has been a great interest in exploiting the QS mechanism as a new drug target. Therefore, blocking the QS mechanism can be an effective strategy to control infection and solve the problem of drug resistance. So far, there is no clinically approved anti-QS drug that can disable the circuits of QS systems. This review discusses the quorum-sensing network systems and novel anti-QS inhibitors in some Gram-negative bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Natural products are an essential source of bioactive compounds. Isotopic labeling is an effective way to identify natural products that incorporate a specific precursor; however, this approach is limited by the availability of isotopically enriched precursors. We used an inverse stable isotopic labeling approach to identify natural products by growing bacteria on a 13 C-carbon source and then identifying 12 C-precursor incorporation by mass spectrometry. We applied this approach to methylotrophs, ecologically important bacteria predicted to have significant yet underexplored biosynthetic potential. We demonstrate that this method identifies N- acyl homoserine lactone quorum sensing signals produced by diverse methylotrophs grown on three different one-carbon compounds. We then apply this approach to simultaneously detect five previously unidentified signals produced by a methylotroph and link these compounds to their synthases. We envision that this method can be used to identify other natural product classes synthesized by methylotrophs and other organisms that grow on relatively inexpensive 13 C-carbon sources.

Background: Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C 4 /C 6 -HSL synthase., Results: In this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C 4 /C 6 -ACP substrates. These predictions were further supported by a computational Ala mutation assay., Conclusions: In this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C 4 /C 6 -ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.

Many bacteria, such as Pseudomonas aeruginosa, regulate phenotypic switching in a population density-dependent manner through a phenomenon known as quorum sensing (QS). For Gram-negative bacteria, QS relies on the synthesis, transmission, and perception of low-molecular-weight signal molecules that are predominantly N-acyl-l-homoserine lactones (AHLs). Efforts to disrupt AHL-mediated QS have largely focused on the development of synthetic AHL analogues (SAHLAs) that are structurally similar to native AHLs. However, like AHLs, these molecules tend to be hydrophobic and are poorly soluble under aqueous conditions. Water-soluble macrocycles, such as cyclodextrins (CDs), that encapsulate hydrophobic guests have long been used by both the agricultural and pharmaceutical industries to overcome the solubility issues associated with hydrophobic compounds of interest. Conveniently, CDs have also demonstrated anti-AHL-mediated QS effects. Here, using fluorescence spectroscopy, NMR spectrometry, and mass spectrometry, we evaluate the affinity of SAHLAs, as well as their hydrolysis products, for β-CD inclusion. We also evaluated the ability of these complexes to inhibit wild-type P. aeruginosa virulence in a Caenorhabditis elegans host infection study, for the first time. Our efforts confirm the potential of β-CDs for the improved delivery of SAHLAs at the host/microbial interface, expanding the utility of this approach as a strategy for probing and controlling QS., (© 2020 Wiley-VCH GmbH.)

Quorum sensing (QS) is a bacterial communication process mediated by both native and non-native small-molecule quorum sensing modulators (QSMs), many of which have been synthesized to disrupt QS pathways. While structure-activity relationships have been developed to relate QSM structure to the activation or inhibition of QS receptors, less is known about the transport mechanisms that enable QSMs to cross the lipid membrane and access intracellular receptors. In this study, we used atomistic MD simulations and an implicit solvent model, called COSMOmic, to analyze the partitioning and translocation of QSMs across lipid bilayers. We performed umbrella sampling at atomistic resolution to calculate partitioning and translocation free energies for a set of naturally occurring QSMs, then used COSMOmic to screen the water-membrane partition and translocation free energies for 50 native and non-native QSMs that target LasR, one of the LuxR family of quorum-sensing receptors. This screening procedure revealed the influence of systematic changes to head and tail group structures on membrane partitioning and translocation free energies at a significantly reduced computational cost compared to atomistic MD simulations. Comparisons with previously determined QSM activities suggest that QSMs that are least likely to partition into the bilayer are also less active. This work thus demonstrates the ability of the computational protocol to interrogate QSM-bilayer interactions which may help guide the design of new QSMs with engineered membrane interactions., Competing Interests: The authors have declared that no competing interests exist.

Considered a key taxon in soil and marine microbial communities, myxobacteria exist as coordinated swarms that utilize a combination of lytic enzymes and specialized metabolites to facilitate predation of microbes. This capacity to produce specialized metabolites and the associated abundance of biosynthetic pathways contained within their genomes have motivated continued drug discovery efforts from myxobacteria. Of all myxobacterial biosynthetic gene clusters deposited in the antiSMASH database, only one putative acylhomoserine lactone (AHL) synthase, agpI, was observed, in genome data from Archangium gephyra. Without an AHL receptor also apparent in the genome of A. gephyra, we sought to determine if AgpI was an uncommon example of an orphaned AHL synthase. Herein we report the bioinformatic assessment of AgpI and discovery of a second AHL synthase from Vitiosangium sp. During axenic cultivation conditions, no detectible AHL metabolites were observed in A. gephyra extracts. However, heterologous expression of each synthase in Escherichia coli provided detectible quantities of 3 AHL signals including 2 known AHLs, C8-AHL and C9-AHL. These results suggest that A. gephyra AHL production is dormant during axenic cultivation. The functional, orphaned AHL synthase, AgpI, is unique to A. gephyra, and its utility to the predatory myxobacterium remains unknown.

Quorum sensing (QS) is the ability of some bacteria to detect and to respond to population density through signalling molecules. QS molecules are involved in motility and cell aggregation mechanisms in diseases such as sepsis. Few biomarkers are currently available to diagnose sepsis, especially in high-risk conditions. The aim of this study was the development of new analytical methods based on liquid chromatography-mass spectrometry for the detection and quantification of QS signalling molecules, including N-acyl homoserine lactones (AHL) and hydroxyquinolones (HQ), in biofluids. Biological samples used in the study were Pseudomonas aeruginosa bacterial cultures and plasma from patients with sepsis. We developed two MS analytical methods, based on neutral loss (NL) and product ion (PI) experiments, to identify and characterize unknown AHL and HQ molecules. We then established a multiple-reaction-monitoring (MRM) method to quantify specific QS compounds. We validated the HPLC-MS-based approaches (MRM-NL-PI), and data were in accord with the validation guidelines. With the NL and PI MS-based methods, we identified and characterized 3 and 13 unknown AHL and HQ compounds, respectively, in biological samples. One of the newly found AHL molecules was C12-AHL, first quantified in Pseudomonas aeruginosa bacterial cultures. The MRM quantitation of analytes in plasma from patients with sepsis confirmed the analytical ability of MRM for the quantification of virulence factors during sepsis. Graphical abstract.

Background: Since acyl-homoserine lactone (AHL) profiling has been described in the gut of healthy subjects and patients with inflammatory bowel disease (IBD), the potential effects of these molecules on host cells have raised interest in the medical community. In particular, natural AHLs such as the 3-oxo-C12-HSL exhibit anti-inflammatory properties. Our study aimed at finding stable 3-oxo-C12-HSL-derived analogues with improved anti-inflammatory effects on epithelial and immune cells., Methods: We first studied the stability and biological properties of the natural 3-oxo-C12-HSL on eukaryotic cells and a bacterial reporter strain. We then constructed and screened a library of 22 AHL-derived molecules. Anti-inflammatory effects were assessed by cytokine release in an epithelial cell model, Caco-2, and a murine macrophage cell line, RAW264.7, (respectively, IL-8 and IL-6) upon exposure to the molecule and after appropriate stimulation (respectively, TNF-α 50 ng/mL and IFN-γ 50 ng/mL, and LPS 10 ng/mL and IFN-γ 20 U/mL)., Results: We found two molecules of interest with amplified anti-inflammatory effects on mammalian cells without bacterial-activating properties in the reporter strain. The molecules furthermore showed improved stability in biological medium compared to the native 3-oxo-C12-HSL., Conclusions: We provide new bio-inspired AHL analogues with strong anti-inflammatory properties that will need further study from a therapeutic perspective.

Background: Schistosomiasis is a prevalent neglected tropical disease that affects approximately 300 million people worldwide. Its treatment is through a single class chemotherapy, praziquantel. Concerns surrounding the emergence of praziquantel insensitivity have led to a need for developing novel anthelmintics., Methodology/principle Findings: Through evaluating and screening fourteen compounds (initially developed for anti-cancer and anti-viral projects) against Schistosoma mansoni, one of three species responsible for most cases of human schistosomiasis, a racemic N-acyl homoserine (1) demonstrated good efficacy against all intra mammalian lifecycle stages including schistosomula (EC50 = 4.7 μM), juvenile worms (EC50 = 4.3 μM) and adult worms (EC50 = 8.3 μM). To begin exploring structural activity relationships, a further 8 analogues of this compound were generated, including individual (R)- and (S)- enantiomers. Upon anti-schistosomal screening of these analogues, the (R)- enantiomer retained activity, whereas the (S)- lost activity. Furthermore, modification of the lactone ring to a thiolactone ring (3) improved potency against schistosomula (EC50 = 2.1 μM), juvenile worms (EC50 = 0.5 μM) and adult worms (EC50 = 4.8 μM). As the effective racemic parent compound is structurally similar to quorum sensing signaling peptides used by bacteria, further evaluation of its effect (along with its stereoisomers and the thiolactone analogues) against Gram+ (Staphylococcus aureus) and Gram- (Escherichia coli) species was conducted. While some activity was observed against both Gram+ and Gram- bacteria species for the racemic compound 1 (MIC 125 mg/L), the (R) stereoisomer had better activity (125 mg/L) than the (S) (>125mg/L). However, the greatest antimicrobial activity (MIC 31.25 mg/L against S. aureus) was observed for the thiolactone containing analogue (3)., Conclusion/significance: To the best of our knowledge, this is the first demonstration that N-Acyl homoserines exhibit anthelmintic activities. Furthermore, their additional action on Gram+ bacteria opens a new avenue for exploring these molecules more broadly as part of future anti-infective initiatives., Competing Interests: The authors have declared that no competing interests exist.

The prevalence of substrate cross-reactivity between AHL acylases and β-lactam acylases provides a glimpse of probable links between quorum sensing and antibiotic resistance in bacteria. Both these enzyme classes belong to the N-terminal nucleophile (Ntn)-hydrolase superfamily. Penicillin V acylases alongside bile salt hydrolases constitute the cholylglycine hydrolase (CGH) group of the Ntn-hydrolase superfamily. Here we report the ability of two acylases, Slac1 and Slac2, from the marine bacterium Shewanella loihica-PV4 to hydrolyze AHLs. Three-dimensional structure of Slac1reveals the conservation of the Ntn hydrolase fold and CGH active site, making it a unique CGH exclusively active on AHLs. Slac1homologs phylogenetically cluster separate from reported CGHs and AHL acylases, thereby representing a functionally distinct sub-class of CGH that might have evolved as an adaptation to the marine environment. We hypothesize that Slac1 could provide the structural framework for understanding this subclass, and further our understanding of the evolutionary link between AHL acylases and β-lactam acylases., Competing Interests: Declaration of conflict of interests The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

This study investigated the effects of exogenous N-acyl-homoserine lactone (AHL) signal molecules, N-hexanoyl- l -homoserine lactone (C6-HSL) and N-octanoyl- l -homoserine lactone (C8-HSL), on treatment performance, sludge properties and microbial community structures in activated sludge systems. Results showed that the nitrification and denitrification efficiencies were enhanced with the addition of signal molecules. The particle size, irregularity, and internal mass transfer resistance of activated sludge flocs (ASFs) increased, primarily because dosing AHLs led to a content increase and chemical composition variation of extracellular polymeric substances (EPS) in sludge. Microbial analysis indicated an increase in both the bacterial richness and diversity of the systems. The relative abundances of the key functional groups, including bacteria related to C and N removal and EPS production, varied correspondingly. This study presents an insight into the comprehensive understanding of the effects of AHL-based quorum sensing on activated sludge treatment process., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Quorum sensing in bacteria is a cell density-dependent phenomenon in which, a community of cells communicate with each other using signalling molecules belonging to various families of which N-acyl homoserine lactone (AHL) is one. AHL acts via ligand-receptor interaction where receptors of AHL differ from species to species, and possess great degree of similarity in conformation at the active site. A macromolecule, LasR, is a receptor protein that binds to N-(3-oxododecanoyl)-L-homoserinelactone (OdDHL), a type of AHL, viz. responsible for biofilm formation in Pseudomonas aeruginosa. Similar macromolecule LuxR, like LasR, found in Vibrio sp. identifies a different AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OhHSL), responsible for the phenomenon of bioluminescence. In silico study depicted that curcumin could bind to both LasR and LuxR by unique sets of hydrogen bonding and hydrophobic interactions that can lead to the inactivation of these proteins, enabling this plant-derived organic AHL antagonist to be categorized as a quorum sensing inhibitor (QSI). To prove this hypothesis, curcumin was treated on P. aeruginosa to access the reduction in biofilm formation and on V. alginolyticus to check its efficacy to reduction in bioluminescence by inhibition of QS. The results of these studies proved curcumin to be an efficient QSI.

We report aqueous emulsions of thermotropic liquid crystals (LCs) that can intercept and report on the presence of N -acyl-l-homoserine lactones (AHLs), a class of amphiphiles used by pathogenic bacteria to regulate quorum sensing (QS), monitor population densities, and initiate group activities, including biofilm formation and virulence factor production. The concentration of AHL required to promote "bipolar" to "radial" transitions in micrometer-scale droplets of the nematic LC 4'-pentyl-cyanobiphenyl (5CB) decreases with increasing carbon number in the acyl tail, reaching a threshold concentration of 7.1 μM for 3-oxo-C12-AHL, a native QS signal in the pathogen Pseudomonas aeruginosa . The LC droplets in these emulsions also respond to biologically relevant concentrations of the biosurfactant rhamnolipid, a virulence factor produced by communities of P. aeruginosa under the control of QS. Systematic studies using bacterial mutants support the conclusion that these emulsions respond selectively to the production of rhamnolipid and AHLs and not to other products produced by bacteria at lower (subquorate) population densities. Finally, these emulsions remain configurationally stable in growth media, enabling them to be deployed either in bacterial supernatants or in situ in bacterial cultures to eavesdrop on QS and report on changes in bacterial group behavior that can be detected in real time using polarized light. Our results provide new tools to detect and report on bacterial QS and virulence and a materials platform for the rapid and in situ monitoring of bacterial communication and resulting group behaviors in bacterial communities.

Quorum sensing (QS), a bacterial cell-to-cell communication system mediated by small molecules and peptides, has received significant interest as a potential target to block infection. The common pathogen Pseudomonas aeruginosa uses QS to regulate many of its virulence phenotypes at high cell densities, and the LasR QS receptor plays a critical role in this process. Small molecule tools that inhibit LasR activity would serve to illuminate its role in P. aeruginosa virulence, but we currently lack highly potent and selective LasR antagonists, despite considerable research in this area. V-06-018, an abiotic small molecule discovered in a high-throughput screen, represents one of the most potent known LasR antagonists but has seen little study since its initial report. Herein, we report a systematic study of the structure-activity relationships (SARs) that govern LasR antagonism by V-06-018. We synthesized a focused library of V-06-018 derivatives and evaluated the library for bioactivity using a variety of cell-based LasR reporter systems. The SAR trends revealed by these experiments allowed us to design probes with 10-fold greater potency than that of V-06-018 and 100-fold greater potency than other commonly used N -acyl-l-homoserine lactone (AHL)-based LasR antagonists, along with high selectivities for LasR. Biochemical experiments to probe the mechanism of antagonism by V-06-018 and its analogues support these compounds interacting with the native ligand-binding site in LasR and, at least in part, stabilizing an inactive form of the protein. The compounds described herein are the most potent and efficacious antagonists of LasR known and represent robust probes both for characterizing the mechanisms of LuxR-type QS and for chemical biology research in general in the growing QS field.

In recent years, use of supercritical-fluid chromatography (SFC) with CO 2 as the mobile phase has been expanding in the research laboratory and industry since it is considered to be a green analytical method. This technique offers numerous advantages, such as good separation and sensitive detection, short analysis times, and stability of analytes. In this study, a method for quantification of N-acyl homoserine lactones (AHLs), signaling molecules responsible for cell-to-cell communication initially discovered in bacteria, by SFC coupled with high-resolution mass spectrometry (HRMS) was developed. The SFC conditions and MS ionization settings were optimized to obtain the best separation and greatest sensitivity. The optimal analysis conditions allowed quantification of up to 30 AHLs in a single run within 16 min with excellent linearity (R 2 > 0.998) and sensitivity (picogram level). This method was then applied to study AHL production by one Gram-negative endophytic bacterium, Paraburkholderia sp. BSNB-0670. Nineteen known AHLs were detected, and nine abundant HSLs were quantified. To further investigate the production of uncommon AHLs, a molecular networking approach was applied on the basis of the SFC-HRMS/MS data. This led to additional identification of four unknown AHLs annotated as N-3-hydroxydodecanoylol homoserine lactone, N-3-hydroxydodecadienoyl homoserine lactone, and N-3-oxododecenoyl homoserine lactones (two isomers).

A series of carbamate, thiocarbamate, and hydrazide analogues of acylhomoserine lactones (AHLs) were synthesized and their ability to modulate Vibrio fischeri -quorum sensing was evaluated. The compounds in the series exhibit variable side chain length and the possible presence of a diversely substituted phenyl substituent. Biological evaluation on the Vibrio fischeri quorum sensing system revealed that the ethyl substituted carbamate ( 1 ) display a weak agonistic activity whereas compounds with longer chain length or benzyl substituents display significant antagonistic activity. The most active compounds in the series were the 4-nitrobenzyl carbamate and thiocarbamate 7 and 11 which exhibited an IC 50 value of about 20 µM. These activities are in the range of other reported of AHL-structurally related quorum sensing (QS) inhibitors. Docking experiments conducted on the LuxR model showed that, compared to the natural ligand OHHL, the additional heteroatom of the carbamate group induces a new hydrogen bond with Tyr70 leading to a different global hydrogen-bond network. Tyr70 is an important residue in the binding site and is strictly conserved in the LuxR family. For the 4-nitrobenzyl carbamate and thiocarbamate analogues, the docking results highlight an additional hydrogen bond between the nitro group and Lys178. For hydrazide analogues, which are deprived of any activity, docking shows that the orientation of the carbonyl group is opposite as compared with the natural ligand, leading to the absence of a H-bond between the C=O with Tyr62. This suggests that, either this later interaction, or the influence of the C=O orientation on the overall ligand conformation, are essential for the biological activity.

In the Gram-negative bacterium Aeromonas hydrophila , N -acyl homoserine lactone (AHL)-mediated quorum sensing (QS) influences pathogenicity, protein secretion, and motility. However, the catalytic mechanism of AHL biosynthesis and the structural basis and substrate specificity for AhyI members remain unclear. In this study, we cloned the ahyI gene from the isolate A. hydrophila HX-3, and the overexpressed AhyI protein was confirmed to produce six types of AHLs by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, contrasting with previous reports that AhyI only produces N -butanoyl-l-homoserine lactone (C 4 -HSL) and N -hexanoyl-l-homoserine lactone (C 6 -HSL). The results of an in vitro biosynthetic assay showed that purified AhyI can catalyze the formation of C 4 -HSL using S -adenosyl-l-methionine (SAM) and butyryl-acyl carrier protein (ACP) as substrates and indicated that the fatty acyl substrate used in AhyI-mediated AHL synthesis is derived from acyl-ACP rather than acyl-CoA. The kinetic data of AhyI using butyryl-ACP as an acyl substrate indicated that the catalytic efficiency of the A. hydrophila HX-3 AhyI enzyme is within an order of magnitude compared to other LuxI homologues. In this study, for the first time, the tertiary structural modeling results of AhyI and those of molecular docking and structural and functional analyses showed the importance of several crucial residues, as well as the secondary structure with respect to acylation. A Phe125-Phe152 clamp grasps the terminal methyl group to assist in stabilizing the long acyl chains in a putative binding pocket. The stacking interactions within a strong hydrophobic environment, a hydrogen-bonding network, and a β bulge presumably stabilize the ACP acyl chain for the attack of the SAM α-amine toward the thioester carbon, offering a relatively reasonable explanation for how AhyI can synthesize AHLs with diverse acyl-chain lengths. Moreover, Trp34 participates in forming the binding pocket for C 4 -ACP and becomes ordered upon SAM binding, providing a good basis for catalysis. The novel finding that AhyI can produce both short- and long-chain AHLs enhances current knowledge regarding the variety of AHLs produced by this enzyme. These structural data are expected to serve as a molecular rationale for AHL synthesis by AhyI.

With the emergence of microbial resistance pathogens, recent research aims at studying new mechanisms of action of antibiotics. This review discusses the mechanisms and types of quorum sensing (QS) inhibitors in Gram negative bacteria. It illustrates all published data available in literature pertaining to novel compounds that showed activity against different targets in the quorum sensing pathways in Gram negative bacteria. A systemic overview has been conducted by searching PubMed, Medline, and the Cochrane Library and data extraction of all quorum sensing inhibitors with their mechanisms of action have been collected. This review will focus on signaling autoinducer AI-1 in Gram negative bacteria. The biological activity of the antagonists is mainly reported as IC 50 (the concentration of an inhibitor where the response is reduced by half)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The plant pathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) regulates the expression of virulence factors by N-acylhomoserine lactone (AHL)-mediated quorum sensing. The LuxI family protein, ExpI, catalyzes AHL biosynthesis in Pcc. The structure of the predominant AHL produced by ExpI differs among Pcc strains, which may be divided into two quorum-sensing classes (QS classes) based on the AHL produced. In the present study, AHL produced by 282 Pcc strains were extracted and identified by LC-MS/MS. Seventy Pcc strains produced N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL) as the predominant AHL and were categorized into QS class I. Two hundred Pcc strains produced N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL) as the predominant AHL, and were categorized into QS class II-1. Twelve Pcc strains produced only small amounts of 3-oxo-C6-HSL, and were categorized into QS class II-2. The phylogenetic analysis revealed that the amino acid sequences of ExpI may be divided into two major clades (I and II). The Pcc strains categorized into ExpI clades I and II entirely matched QS classes I and II, respectively. A multiple alignment analysis demonstrated that only 6 amino acid substitutions were observed among ExpI from QS classes II-1 and II-2. Furthermore, many amino acid substitutions between QS classes I and II were concentrated at the C-terminal region. These amino acid substitutions are assumed to cause significant reductions in 3-oxo-C6-HSL in QS class II-2 or affect the substrate specificity of ExpI between QS classes I and II.

Quorum sensing (QS) refers to chemical signalling between micro-organisms and defines a social concord among them. Once a threshold of signal is accumulated, certain virulent traits are regulated within bacteria in response to the surrounding environment. These virulence traits are known to contribute in the pathogenicity of bacterial diseases. To prevent the activation of virulence factors, QS is inhibited in different ways through a strategy known as quorum quenching. Various types of quorum-quenching strategies have already been used and characterized, as discussed in this review. The phenomenon of quorum quenching has long been considered as an alternative therapy to circumvent the ill-effects of the overuse of antibiotics. Considering the need to compare and evaluate various strategies, selected quorum-quenching paradigms are detailed along with their pros and cons in this review. A rationale has been drawn between naturally evolved quorum-quenching strategies and synthetically modified approaches adopted to abrogate QS.

Virulence in the Gram-negative pathogen Pseudomonas aeruginosa relies in part on the efficient functioning of two LuxI/R dependent quorum sensing (QS) cascades, namely, the LasI/R and RhlI/R systems that generate and respond to N- (3-oxo)-dodecanoyl-l-homoserine lactone and N- butyryl-l-homoserine lactone, respectively. The two acyl homoserine lactone (AHL) synthases, LasI and RhlI, use 3-oxododecanoyl-ACP and butyryl-ACP, respectively, as the acyl-substrates to generate the corresponding autoinducer signals for the bacterium. Although AHL synthases represent excellent targets for developing QS modulators in P. aeruginosa , and in other related bacteria, the identification of potent and signal synthase specific inhibitors has represented a significant technical challenge. In the current study, we sought to test the utility of AHL analogs as potential modulators of an AHL synthase and selected RhlI in P. aeruginosa as an initial target. We systematically varied the chemical functionalities of the AHL headgroup, acyl chain tail, and head-to-tail linkage to construct a small library of signal analogs and evaluated them for RhlI modulatory activity. Although the native N- butyryl-l-homoserine lactone did not inhibit RhlI, we discovered that several of our long-chain, unsubstituted acyl-d-homoserine lactones and acyl-d-homocysteine thiolactones inhibited while a few of the 3-oxoacyl-chain counterparts activated the enzyme. Additional mechanistic investigations with acyl-substrate analogs and docking experiments with AHL analogs revealed two distinct inhibitor and activator binding pockets in the enzyme. This study provides the first evidence of the yet untapped potential of AHL analogs as signal synthase modulators of QS pathways.

Despite the conservative DNA sequences among LuxI (Acyl Homoserine Lactones synthase gene) homologs, structure-product relationship of AHL synthase remains to be elucidated. In this study, through degenerate primers and in vitro expression methods, we collected the information of the gene sequences and AHL profiles from nine LuxIs among Ensifer adhaerens strains. The chromosome-encoded LuxI (C-LuxI) distinguished themselves from the plasmid-encoded ones (P-LuxI) not only in the DNA sequences, but also in AHL profiles. The C-LuxIs produced only C14-HSL, while the P-LuxIs produced predominantly C8-HSL and 3-oxo-C8-HSL. Sequence-product relationship analysis updated our recognition of the role of T140 (EsaI) in the 3-oxo-HSL production. Computational calculation based on 3D structures of these LuxIs revealed the linear relationship between the chain length and the affinity of amides to AHL synthase in C-LuxI, which was not found in the P-LuxI. We hereby proposed the linear docking affinity as a criterion for the prediction of long-chain AHL production by an AHL synthase. This study extends our understanding on the structure-product relationship of AHL synthases and revealed the distinct chromosome and plasmid origin of this enzyme among E. adhaerens., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Quorum quenching lactonases are enzymes capable of hydrolyzing lactones, including N-acyl homoserine lactones (AHLs). AHLs are molecules known as signals in bacterial communication dubbed quorum sensing. Bacterial signal disruption by lactonases was previously reported to inhibit behavior regulated by quorum sensing, such as the expression of virulence factors and the formation of biofilms. Herein, we report the enzymatic and structural characterization of a novel lactonase representative from the metallo-β-lactamase superfamily, dubbed GcL. GcL is a broad spectrum and highly proficient lactonase, with k cat /K M values in the range of 10 4 to 10 6  m -1  s -1 . Analysis of free GcL structures and in complex with AHL substrates of different acyl chain length, namely, C4-AHL and 3-oxo-C12-AHL, allowed their respective binding modes to be elucidated. Structures reveal three subsites in the binding crevice: 1) the small subsite where chemistry is performed on the lactone ring; 2) a hydrophobic ring that accommodates the amide group of AHLs and small acyl chains; and 3) the outer, hydrophilic subsite that extends to the protein surface. Unexpectedly, the absence of structural accommodation for long substrate acyl chains seems to relate to the broad substrate specificity of the enzyme., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

N -Acylhomoserine lactonase degrades the lactone ring of N -acylhomoserine lactones (AHLs) and has been widely suggested as a promising candidate for use in bacterial disease control. While a number of AHL lactonases have been characterized, none of them has been developed as a commercially available enzymatic product for in vitro AHL quenching due to their low stability. In this study, a highly stable AHL lactonase (AhlX) was identified and isolated from the marine bacterium Salinicola salaria MCCC1A01339. AhlX is encoded by a 768-bp gene and has a predicted molecular mass of 29 kDa. The enzyme retained approximately 97% activity after incubating at 25 °C for 12 days and ~100% activity after incubating at 60 °C for 2 h. Furthermore, AhlX exhibited a high salt tolerance, retaining approximately 60% of its activity observed in the presence of 25% NaCl. In addition, an AhlX powder made by an industrial spray-drying process attenuated Erwinia carotovora infection. These results suggest that AhlX has great potential for use as an in vitro preventive and therapeutic agent for bacterial diseases., Competing Interests: The authors declare no conflicts of interest.

Vibrio fischeri possesses a complex AHL-mediated Quorum-sensing (QS) system including two pathways, LuxI/R (3-oxo-C6-HSL and C6-HSL) and AinS/R (C8-HSL), which are important for the regulation of physiological traits. Diverse QS-dependent functional phenotypes have been described in V. fischeri; however, AHL diversity is still underestimated. In the present study, we investigated AHL diversity in five symbiotic V. fischeri strains with distinct phenotypic properties using UHPLC-HRMS/MS. The results obtained (1) revealed an unexpectedly high diversity of signaling molecules, (2) emphasized the complexity of QS in V. fischeri, and (3) highlight the importance of understanding the specificity of AHL-mediated QS.

Biofilm formation and maturation have been demonstrated to be regulated by distinct forms of cell-cell communication factors such as chemical and physical signals. However, whether the Geobacter sp. biofilms, which are typical electroactive biofilms, are affected by chemical signals is poorly understood. This research investigated the effects and corresponding mechanisms of endogenous and exogenous chemical signals (i.e., N-acylhomoserine lactones, AHLs) on the Geobacter soli biofilm. The results showed that Geobacter soli GSS01 secreted detectable endogenous AHLs to facilitate the formation and electrochemical activity of the biofilm, and that exogenous AHLs could further promoted these activities. Analyses of surface proteins revealed that the mechanisms promoted by endogenous and exogenous AHLs were somewhat different. Endogenous AHLs improved the relative abundance of external membrane proteins, while exogenous AHLs further facilitated the formation of amide II and a stronger H-bond between the carbonyl group and the amide. Furthermore, the proteomics analysis indicated that endogenous AHLs enhanced extracellular polymeric substance production by up-regulating the expression of key enzymes participating in EPS production, and simultaneously affected the physiological performance of individual cells. These results demonstrate, for the first time, the importance of chemical signals in Geobacter sp. and provide a comprehensive understanding of the chemical signals involved in biofilm formation and electrochemical activity of Geobacter sp.., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The present work combines molecular docking calculations, 3D-QSAR, molecular dynamics simulations and free binding energy calculations (MM/PBSA and MM/GBSA) in a set of 28 structural analogues of acyl homoserine lactones with Quorum Sensing antagonist activity. The aim of this work is to understand how ligand binds and is affected by the molecular microenvironment in the active site of the LasR receptor for pseudomonas aeruginosa. We also study the stability of the interaction to find key structural characteristics that explain the antagonist activities of this set of ligands. This information is relevant for the rational modification or design of molecules and their identification as powerful LasR modulators. The analysis of molecular docking simulations shows that the 28 analogues have a similar binding mode compared to the native ligand. The carbonyl groups belonging to the lactone ring and the amide group of the acyl chain are oriented towards the amino acids forming hydrogen bond like interactions. The difference in antagonist activity is due to location and orientation of the LasR side chains within the hydrophobic pocket in its binding site. Additionally, we carried out molecular dynamics simulations to understand the conformational changes in the ligand-receptor interaction and the stability of each complex. Results show a direct relationship among the interaction energies of the ligands and the activities as an antagonist of the LasR receptor., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Plant-associated bacteria produce quorum sensing (QS) signals for community (biofilm) formation and functioning in the rhizosphere. The QS-positive biofilm-forming rhizobacteria that excel benefits to the plants are now gaining increased importance for agricultural use due to their high competitiveness. However, there is no method available to distinguish these bacteria from the roots of a plant to ease the isolation. Currently, all the plant-associated bacteria have to be isolated, purified and subsequently screened for the QS activity using biosensor strains. This study describes a direct isolation method for N-acyl-homoserine lactone (AHL) type quorum sensing signal producing bacteria from the plant root. In this method, the root sample collected from the field was overlaid directly with the bacterial growth medium seeded with the biosensor reporter, Chromobacterium violaceum (CV026). The AHL produced by QS positive rhizobacteria residing on the surface of the root will be recognized by violacein production of CV026. The bacterial isolates recovered from rice root using this method were further confirmed for the QS activity and biofilm formation. All the QS-positive strains produced N-butyryl DL-homoserine lactone (a C4-AHL type) signal in the culture medium and had biofilm formation during in vitro culturing. The 16S rRNA gene sequences of these QS-positive biofilm-forming rhizobacteria revealed that these strains are phylogenetically close to Pseudomonas siluiensis, Aeromonas hydrophila and A. caviae. Therefore, this could be a simple, rapid and straightforward procedure for isolation and characterization of quorum-sensing rhizobacteria from plant roots., (Copyright © 2018 Elsevier B.V. All rights reserved.)