Expanding dialogues: from natural autoinducers to non-natural analogues that modulate quorum sensing in Gram-negative bacteria

Bacteria are capable of "communicating" their local population densities via a process termed quorum sensing (QS). Gram-negative bacteria use N-acylated l-homoserine lactones (AHLs), in conjunction with their cognate LuxR-type receptors, as their primary signalling circuit for QS. In this critical review, we examine AHL signalling in Gram-negative bacteria with a primary focus on the design of non-natural AHLs, their structure-activity relationships, and their application in chemical biological approaches to study QS (72 references).     

Small molecule inhibitors of bacterial quorum sensing and biofilm formation

Bacteria monitor their local population densities using small molecules (or autoinducers) in a process known as quorum sensing. Here, we report a new and efficient synthetic route to naturally occurring bacterial autoinducers [N-acyl l-homoserine lactones (AHLs)] that is readily amenable to the synthesis of analogues. This route has been applied in the first synthesis of a library of non-native AHLs. Evaluation of these compounds in bacterial reporter gene and biofilm assays has revealed a potent set of quorum sensing antagonists. These ligands will serve as valuable new tools to explore the role of quorum sensing in bacterial pathogenesis.     

Antibody interference with N-acyl homoserine lactone-mediated bacterial quorum sensing

Many bacterial pathogens coordinate their virulence factor expression in a cell density-dependent manner. This population-dependent coordination of gene expression in bacteria has been termed "quorum sensing" (QS). N-Acyl homoserine lactones (AHLs) are used by over 70 Gram-negative bacterial species as autoinducers. Inhibition of QS signaling might represent a new target for antimicrobial therapy. Here we report the hapten design, synthesis, generation of monoclonal antibodies (mAbs) against AHLs, and the evaluation of these mAbs for their ability to blunt QS signaling and inhibit virulence factor expression in P. aeruginosa. The mAbs can be envisioned as a tool for future investigations into AHL-based QS, which may aid in gaining new insights into the pathogenesis of P. aeruginosa and may ultimately lead to the development of new strategies to combat bacterial diseases.     

C4-Alkoxy-HPD: A Potent Class of Synthetic Modulators Surpassing Nature in AI-2 Quorum Sensing

Bacteria have developed cell-to-cell communication mechanisms, termed quorum sensing (QS), that regulate bacterial gene expression in a cell population-dependent manner. Autoinducer-2 (AI-2), a class of QS signaling molecules derived from (4S)-4,5-dihydroxy-2,3-pentanedione (DPD), has been identified in both Gram-negative and Gram-positive bacteria. Despite considerable interest in the AI-2 QS system, the biomolecular communication used by distinct bacterial species still remains shrouded. Herein, we report the synthesis and evaluation of a new class of DPD analogues, C4-alkoxy-5-hydroxy-2,3-pentanediones, termed C4-alkoxy-HPDs. Remarkably, two of the analogues were more potent QS agonists than the natural ligand, DPD, in Vibrio harveyi. The findings presented extend insights into ligand-receptor recognition/signaling in the AI-2 mediated QS system.     

Single drop microextraction of homoserine lactones based quorum sensing signal molecules, and the separation of their enantiomers using gas chromatography mass spectrometry in the presence of biological matrices

N-acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the “quorum sensing” response of bacterial communities. AHLs are optically active components and the L-form is known to have activity in the quorum sensing. However, the knowledge regarding the stereospecific production of the AHLs in bacterial cultures is limited; therefore, there is a need for a fast and easy method for their chiral analysis. A method was developed for the preconcentration of the AHLs using single drop microextraction or liquid-liquid microphase extraction in toluene for their analysis using GC-MS. The performance of the method were determined and discussed for the chiral separation of these autoinducers using a capillary column coated with heptakis-(2,3-di-O-acetyl-6-O-t-butyldimethyl-silyl)-β-cyclodextrin. The salient feature of this study is the demonstration, that Burkholderia cepacia LA3 produced D-decanoyl-homoserine lactone beside L-decanoyl- and L-octaonyl- enantiomers.     

Design, synthesis and biological evaluation of non-natural modulators of quorum sensing in Pseudomonas aeruginosa

Many species of bacteria employ a mechanism of intercellular communication known as quorum sensing which is mediated by small diffusible signalling molecules termed autoinducers. The most common class of autoinducer used by Gram-negative bacteria are N-acylated-L-homoserine lactones (AHLs). Pseudomonas aeruginosa is a clinically important bacterium which is known to use AHL-mediated quorum sensing systems to regulate a variety of processes associated with virulence. Thus the selective disruption of AHL-based quorum sensing represents a strategy to attenuate the pathogenicity of this bacterium. Herein we describe the design, synthesis and biological evaluation of a collection of structurally novel AHL mimics. A number of new compounds capable of modulating the LasR-dependent quorum sensing system of P. aeruginosa were identified, which could have value as molecular tools to study and manipulate this signalling pathway. Worthy of particular note, this research has delivered novel potent quorum sensing antagonists, which strongly inhibit the production of virulence factors in a wild type strain of this pathogenic bacterium.     

超高效液相色谱-串联质谱法检测微氧生物脱氮菌群酰基高丝氨酸内酯信号分子

为揭示处理低碳氮比废水的微氧活性污泥系统的生物脱氮机制,了解脱氮功能菌群的群体生长和代谢规律,建立了超高效液相色谱-串联质谱同时定量检测介导革兰氏阴性(G-)细菌群体感应信号分子酰基高丝氨酸内酯( AHLs)的方法。取自升流式微氧活性污泥反应器的泥水混合物,使用乙酸乙酯液液萃取,旋转蒸干后以甲醇定容,经C18色谱柱分离。以5 mmol/L乙酸铵(含0.1%甲酸)和甲醇为流动相进行梯度洗脱,采用多反应离子监测模式,使用配有电喷雾离子源的三重四极杆质谱进行检测。对9种AHLs的检测结果表明,在0.5~100μg/L范围内呈现良好的线性关系,检出限为0.01~0.5μg/L,回收率为62.5%~118.1%,相对标准偏差为2.9%~12.1%,分析时间为6.5 min。本方法具有快速、准确和精密等特点,可及时反映活性污泥功能菌群的生长状态和代谢活性,对了解生物脱氮系统的生物学机制和废水生物处理系统的运行调控具有重要意义。     

Robust routes for the synthesis of N-acylated-l-homoserine lactone (AHL) quorum sensing molecules with high levels of enantiomeric purity

The ready availability of native quorum sensing molecules and related structural analogues is of significant biological interest in the development of methods to manipulate bacterial quorum sensing systems in a useful fashion. In this Letter we report robust routes for the synthesis of a range of N-acylated-l-homoserine lactone (AHL) quorum sensing molecules. Crucially, we have analysed the enantiopurity of the final AHLs and in all cases, excellent levels were observed.     

Uptake, degradation and chiral discrimination of N-acyl-D/L-homoserine lactones by barley (Hordeum vulgare) and yam bean (Pachyrhizus erosus) plants

Bacterial intraspecies and interspecies communication in the rhizosphere is mediated by diffusible signal molecules. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as autoinducers in the quorum sensing response. While bacterial signalling is well described, the fate of AHLs in contact with plants is much less known. Thus, adsorption, uptake and translocation of N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL) and N-decanoyl-homoserine lactone (C10-HSL) were studied in axenic systems with barley (Hordeum vulgare L.) and the legume yam bean (Pachyrhizus erosus (L.) Urban) as model plants using ultra-performance liquid chromatography (UPLC), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tritium-labelled AHLs. Decreases in AHL concentration due to abiotic adsorption or degradation were tolerable under the experimental conditions. The presence of plants enhanced AHL decline in media depending on the compounds' lipophilicity, whereby the legume caused stronger AHL decrease than barley. All tested AHLs were traceable in root extracts of both plants. While all AHLs except C10-HSL were detectable in barley shoots, only C6-HSL was found in shoots of yam bean. Furthermore, tritium-labelled AHLs were used to determine short-term uptake kinetics. Chiral separation by GC-MS revealed that both plants discriminated D-AHL stereoisomers to different extents. These results indicate substantial differences in uptake and degradation of different AHLs in the plants tested.     

HPLC-MS法检测N-酰基-高丝氨酸内酯类信号分子

建立了HPLC-MS检测群体感应信号分子N-酰基-高丝氨酸内酯类(N-acyl-homoserine lactones,AHLs)化合物的方法。利用该方法对细菌发酵液中的AHLs进行分析,结果表明,样品只需简单的处理,在没有标样的条件下,可以对样品中任意的AHLs分子进行定性分析。该方法简单、高效,检测的灵敏度明显大于经典的TLC-B iosensor检测AHLs的方法,为从复杂混合物中鉴定AHLs提供了有效手段。     

Perceiving the chemical language of Gram-negative bacteria: listening by high-resolution mass spectrometry

Gram-negative bacteria use N-acylhomoserine lactones (AHLs) as their command language to coordinate population behavior during invasion and colonization of higher organisms. Although many different bacterial bioreporters are available for AHLs monitoring, in which a phenotypic response, e.g. bioluminescence, violacin production, and β-galactosidase activity, is exploited, mass spectrometry (MS) is the most versatile detector for rapid analysis of AHLs in complex microbial samples, with or without prior separation steps. In this paper we critically review recent advances in the application of high-resolution MS to analysis of the quorum sensing (QS) signaling molecules used by Gram-negative bacteria, with much emphasis on AHLs. A critical review of the use of bioreporters in the study of AHLs is followed by a short methodological survey of the capabilities of high-resolution mass spectrometry (HRMS), including Fourier-transform ion cyclotron resonance (FTICR) MS and quadrupole time-of-flight (qTOF) MS. Use of infusion electrospray ultrahigh-resolution FTICR MS (12 Tesla) enables accurate mass measurements for determination of the elemental formulas of AHLs in Acidovorax sp. N35 and Burkholderia ubonensis AB030584. Results obtained by coupling liquid chromatography with a hybrid quadrupole linear ion trap-FTICR mass spectrometer (LC–LTQ-FTICRMS, 7-T) for characterization of acylated homoserine lactones in the human pathogen Pseudomonas aeruginosa are presented. UPLC–ESI-qTOF MS has also proved to be suitable for identification of 3O-C₁₀HSL in Pseudomonas putida IsoF cell culture supernatant. Aspects of sample preparation and the avoidance of analytical pitfalls are also emphasized.

Modulation of bacterial quorum sensing with synthetic ligands: systematic evaluation of N-acylated homoserine lactones in multiple species and new insights into their mechanisms of action

Bacteria use a language of low molecular weight ligands to assess their population densities in a process called quorum sensing. This chemical signaling process plays a pivotal role both in the pathogenesis of infectious disease and in beneficial symbioses. There is intense interest in the development of synthetic ligands that can intercept quorum-sensing signals and attenuate these divergent outcomes. Both broad-spectrum and species-selective modulators of quorum sensing hold significant value as small-molecule tools for fundamental studies of this complex cell-cell signaling process and for future biomedical and environmental applications. Here, we report the design and synthesis of focused collections of non-native N-acylated homoserine lactones and the systematic evaluation of these approximately 90 ligands across three Gram-negative bacterial species: the pathogens Agrobacterium tumefaciens and Pseudomonas aeruginosa; the model symbiont Vibrio fischeri. This study is the first to report and compare the activities of a set of ligands across multiple species and has revealed some of the most potent synthetic modulators of quorum sensing to date. Moreover, several of these ligands exhibit agonistic or antagonistic activity in all three species, while other ligands are only active in one or two species. Analysis of the screening data revealed that at least a subset of these ligands modulate quorum sensing via a partial agonism mechanism. We also demonstrate that selected ligands can either inhibit or promote the production of elastase B, a key virulence factor in wild-type P. aeruginosa, depending on their concentrations. Overall, this work provides broad insights into the molecular features required for small-molecule inhibition or activation of quorum sensing in Gram-negative bacteria. In addition, this study has supplied an expansive set of chemical tools for the further investigation of quorum-sensing pathways and responses.     

Identification of bacterial <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones (AHLs) with a combination of ultra-performance liquid chromatography (UPLC), ultra-high-resolution mass spectrometry,and in-situ biosensors

N-Acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the “quorum sensing” response of bacterial communities. Several reports have recently been published on the identification of AHLs from different species and attempts have been made to study their role in natural habitats, for example the surface of plant roots in the rhizosphere. In this article, different analytical methods, including bacterial biosensors and chromatographic techniques, are reviewed. A concept for assignment of the structures of AHLs is also presented. The retention behaviour of derivatives of AHLs containing β-keto or hydroxyl groups and/or double bonds has been evaluated in relation to the separation behaviour of AHLs with saturated and unsubstituted alkanoyl chains. Samples have also been analysed by high resolution mass spectrometry (Fourier-transform ion-cyclotron-resonance mass spectrometry, FTICR-MS), nano liquid chromatography–electrospray ionization ion trap mass spectrometry (nano-LC–MS) and by the aid of a biosensor. The results obtained from ultra performance liquid chromatography (UPLC), FTICR-MS, nano-LC–MS, and bioassays have been compared to attempt structural characterisation of AHL without chemical synthesis of analytical standards. The method was used to identify the major AHL compound produced by the rhizosphere bacterium Acidovorax sp. N35 as N-(3-hydroxydecanoyl)homoserine lactone.     

Engineered cells as biosensing systems in biomedical analysis

Over the past two decades there have been great advances in biotechnology, including use of nucleic acids, proteins, and whole cells to develop a variety of molecular analytical tools for diagnostic, screening, and pharmaceutical applications. Through manipulation of bacterial plasmids and genomes, bacterial whole-cell sensing systems have been engineered that can serve as novel methods for analyte detection and characterization, and as more efficient and cost-effective alternatives to traditional analytical techniques. Bacterial cell-based sensing systems are typically sensitive, specific and selective, rapid, easy to use, low-cost, and amenable to multiplexing, high-throughput, and miniaturization for incorporation into portable devices. This critical review is intended to provide an overview of available bacterial whole-cell sensing systems for assessment of a variety of clinically relevant analytes. Specifically, we examine whole-cell sensing systems for detection of bacterial quorum sensing molecules, organic and inorganic toxic compounds, and drugs, and for screening of antibacterial compounds for identification of their mechanisms of action. Methods used in the design and development of whole-cell sensing systems are also reviewed.     

Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays

The Burkholderia cepacia complex (Bcc) employs a quorum sensing (QS) mechanism which is a cell density-dependent bacterial communication system to regulate certain gene expressions. As with many other Gram-negative bacteria, Burkholderia cepacia species use (N-acyl-)homoserine lactones (AHLs or HSLs) as signalling molecules. Because of the essential role of QS in bacterial behavior, the aim of this study was to demonstrate the applicability of our in-house-developed enzyme-linked immunosorbent assays (ELISAs) for the detection of bacterial activities via HSLs in B. cepacia strain LA3 culture supernatants. For this purpose the previously developed monoclonal antibodies (mAbs) HSL1/2-2C10 and HSL1/2-4H5 were exploited. N-3-Oxo-decanoyl-L-homoserine lactone (3-oxo-C10-HSL) was used as main analyte throughout all experiments. With the bacterial culture medium (named ABC medium) a matrix effect in both ELISAs was visible (slight increase in optical density, shift in test midpoints (IC₅₀) and working ranges). For example, ELISA with mAb HSL1/2-2C10 and enzyme tracer HSL3-HRP (HSL derivative conjugated to horseradish peroxidase) had an IC₅₀ of 120 μg L⁻¹ for 3-oxo-C10-HSL in phosphate-buffered saline versus 372 μg L⁻¹ in ABC medium. A significant increase of HSLs in B. cepacia strain LA3 culture supernatants after 12 h to 48 h of growth was observed. Although the analytical result of these immunoassays cannot distinguish HSLs from homoserines (HSs), the appearance of these compounds can be easily followed. Hydrolysis and spiking experiments were carried out with these biological samples. According to our knowledge, these are the first immunoassays for the detection of quorum sensing molecules in biological culture supernatants. This study provides a cost-effective, fast, and sensitive analytical method for detection of HSLs/HSs in biological samples without complex sample preparation and will offer a quick idea about B. cepacia activities. The low sample amount requirement (less than 1 mL) constitutes a tremendous advantage for many analytical questions with biological samples.     

Convergence of hormones and autoinducers at the host/pathogen interface

Most living organisms possess sophisticated cell-signaling networks in which lipid-based signals modulate biological effects such as cell differentiation, reproduction and immune responses. Acyl homoserine lactone (AHL) autoinducers are fatty acid-based signaling molecules synthesized by several Gram-negative bacteria that are used to coordinate gene expression in a process termed “quorum sensing” (QS). Recent evidence shows that autoinducers not only control gene expression in bacterial cells, but also alter gene expression in mammalian cells. These alterations include modulation of proinflammatory cytokines and induction of apoptosis. Some of these responses may have deleterious effects on the host’s immune response, thereby leading to increased bacterial pathogenesis. Prokaryotes and eukaryotes have cohabited for approximately two billion years, during which time they have been exposed to each others’ soluble signaling molecules. We postulate that organisms from the different kingdoms of nature have acquired mechanisms to sense and respond to each others signaling molecules, and we have named this process interkingdom signaling. We further propose that autoinducers, which exhibit structural and functional similarities to mammalian lipid-based hormones, are excellent candidates for mediating this interkingdom communication. Here we will compare and contrast bacterial QS systems with eukaryotic endocrine systems, and discuss the mechanisms by which autoinducers may exploit mammalian signal transduction pathways.     

Investigating the effect of antibiotics on quorum sensing with whole-cell biosensing systems

Quorum sensing (QS) allows bacteria to communicate with one another by means of QS signaling molecules and control certain behaviors in a group-based manner, including pathogenicity and biofilm formation. Bacterial gut microflora may play a role in inflammatory bowel disease pathogenesis, and antibiotics are one of the available therapeutic options for Crohn’s disease. In the present study, we employed genetically engineered bioluminescent bacterial whole-cell sensing systems as a tool to evaluate the ability of antibiotics commonly employed in the treatment of chronic inflammatory conditions to interfere with QS. We investigated the effect of ciprofloxacin, metronidazole, and tinidazole on quorum sensing. Several concentrations of individual antibiotics were allowed to interact with two different types of bacterial sensing cells, in both the presence and absence of a fixed concentration of N-acylhomoserine lactone (AHL) QS molecules. The antibiotic effect was then determined by monitoring the biosensor’s bioluminescence response. Ciprofloxacin, metronidazole, and tinidazole exhibited a dose-dependent augmentation in the response of both bacterial sensing systems, thus showing an AHL-like effect. Additionally, such an augmentation was observed, in both the presence and absence of AHL. The data obtained indicate that ciprofloxacin, metronidazole, and tinidazole may interfere with bacterial communication systems. The results suggest that these antibiotics, at the concentrations tested, may themselves act as bacterial signaling molecules. The beneficial effect of these antibiotics in the treatment of intestinal inflammation may be due, at least in part, to their effect on QS-related bacterial behavior in the gut.     

Heterocyclic Chemistry Applied to the Design of N-Acyl Homoserine Lactone Analogues as Bacterial Quorum Sensing Signals Mimics

N-acyl homoserine lactones (AHLs) are small signaling molecules used by many Gram-negative bacteria for coordinating their behavior as a function of their population density. This process, based on the biosynthesis and the sensing of such molecular signals, and referred to as Quorum Sensing (QS), regulates various gene expressions, including growth, virulence, biofilms formation, and toxin production. Considering the role of QS in bacterial pathogenicity, its modulation appears as a possible complementary approach in antibacterial strategies. Analogues and mimics of AHLs are therefore biologically relevant targets, including several families in which heterocyclic chemistry provides a strategic contribution in the molecular design and the synthetic approach. AHLs consist of three main sections, the homoserine lactone ring, the central amide group, and the side chain, which can vary in length and level of oxygenation. The purpose of this review is to summarize the contribution of heterocyclic chemistry in the design of AHLs analogues, insisting on the way heterocyclic building blocks can serve as replacements of the lactone moiety, as a bioisostere for the amide group, or as an additional pattern appended to the side chain. A few non-AHL-related heterocyclic compounds with AHL-like QS activity are also mentioned.     

Bacterial quorum sensing and interference by naturally occurring biomimics

Bacteria are able to coordinate gene expression as a community through the secretion and detection of signalling molecules so that the members of the community can simultaneously express specific behaviours. This mechanism of regulation of behaviour appears to be a key trait for adaptation to specific environments and has been shown to regulate a variety of important phenotypes, from virulence factor production to biofilm formation to symbiosis related behaviours such as bioluminescence. The ability to communicate and communally regulate gene expression is hypothesised to have evolved as a way for organisms to delay expression of phenotypes until numerical supremacy is reached. For example, in the case of infection, if an invading microorganism were to express virulence factors too early, the host may be able to mount a successful defence and repel the invaders. There is growing evidence that bacterial quorum sensing (QS) systems are involved in cross-kingdom signalling with eukaryotic organisms and that eukaryotes are capable of actively responding to bacteria in their environment by detecting and acting upon the presence of these signalling molecules. Likewise, eukaryotes produce compounds that can interfere with QS systems in bacteria by acting as agonists or antagonists. An exciting new field of study, biomimetics, takes inspiration from nature’s models and attempts to design solutions to human problems, and biomimics of QS systems may be one such solution. This article presents the acylated homoserine lactone and autoinducer 2 QS systems in bacteria, the means of intercepting or interfering with bacterial QS systems evolved by eukaryotes, and the rational design of synthetic antagonists. Figure Natural products, furanones, from the red alga Delisea pulchra inhibit the quorum sensing regulated production of violacein by Chromobacterium violaceum “The proof of evolution lies in those adaptations that arise from improbable foundations”—Stephen Jay Gould