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1.  The quorum quenching antibody RS2-1G9 protects macrophages from the cytotoxic effects of the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone 
Molecular immunology  2008;45(9):2710-2714.
The Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, uses acylhomoserine lactone-based quorum sensing systems to control its pathogenicity. One of its quorum sensing factors, N-3-oxo-dodecanoyl homoserine lactone, has been shown not only to mediate bacterial quorum sensing but also to exert cytotoxic effects on mammalian cells. The monoclonal antibody RS2-1G9 generated against a 3-oxo-dodecanoyl homoserine lactone analogue hapten was able to protect murine bone marrow-derived macrophages from the cytotoxic effects and also prevented the activation of the mitogen-activated protein kinase p38. These data demonstrate that an immunopharmacotherapeutic approach to combat P. aeruginosa infections might be a viable therapeutic option as the monoclonal antibody RS2-1G9 can readily sequester bacterial N-3-oxo-dodecanoyl homoserine lactone molecules, thus interfering with their biological effects in prokaryotic and eukaryotic systems.
doi:10.1016/j.molimm.2008.01.010
PMCID: PMC2359578  PMID: 18304641
2.  Stereochemical Insignificance Discovered in Acinetobacter baumannii Quorum Sensing 
PLoS ONE  2012;7(5):e37102.
Stereochemistry is a key aspect of molecular recognition for biological systems. As such, receptors and enzymes are often highly stereospecific, only recognizing one stereoisomer of a ligand. Recently, the quorum sensing signaling molecules used by the nosocomial opportunistic pathogen, Acinetobacter baumannii, were identified, and the primary signaling molecule isolated from this species was N-(3-hydroxydodecanoyl)-l-homoserine lactone. A plethora of bacterial species have been demonstrated to utilize 3-hydroxy-acylhomoserine lactone autoinducers, and in virtually all cases, the (R)-stereoisomer was identified as the natural ligand and exhibited greater autoinducer activity than the corresponding (S)-stereoisomer. Using chemical synthesis and biochemical assays, we have uncovered a case of stereochemical insignificance in A. baumannii and provide a unique example where stereochemistry appears nonessential for acylhomoserine lactone-mediated quorum sensing signaling. Based on previously reported phylogenetic studies, we suggest that A. baumannii has evolutionarily adopted this unique, yet promiscuous quorum sensing system to ensure its survival, particularly in the presence of other proteobacteria.
doi:10.1371/journal.pone.0037102
PMCID: PMC3358330  PMID: 22629354
3.  The Pseudomonas aeruginosa N-Acylhomoserine Lactone Quorum Sensing Molecules Target IQGAP1 and Modulate Epithelial Cell Migration 
PLoS Pathogens  2012;8(10):e1002953.
Quorum sensing (QS) signaling allows bacteria to control gene expression once a critical population density is achieved. The Gram-negative human pathogen Pseudomonas aeruginosa uses N-acylhomoserine lactones (AHL) as QS signals, which coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human cell behavior. Little is known about the mechanisms of the action of AHL on their eukaryotic targets. Here, we found that N-3-oxo-dodecanoyl-L-homoserine lactone 3O-C12-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C12-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C12-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C12-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C12-HSL with a sensor bioassay. Moreover, 3O-C12-HSL induced changes in the phosphorylation status of Rac1 and Cdc42 and the localization of IQGAP1 as evidenced by confocal and STED microscopy and Western blots. Our findings suggest that the IQGAP1 is a novel partner for P.aeruginosa 3O-C12-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C12-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial – human cell communication.
Author Summary
The human pathogen Pseudomonas aeruginosa and other bacteria communicate with each other using quorum sensing (QS). This is important for their growth, virulence, motility and the formation of biofilms. Furthermore, eukaryotic cells “listen and respond” to QS signaling, but the exact mechanisms and receptors on mammalian cells have not been identified. We have previously shown that N-acylhomoserine lactones (AHL) alter epithelial barrier functions and increase chemotaxis in human neutrophils. We show here that 3O-C12-HSL modulates the migration of epithelial cells in a dose- and time-dependent manner. Using newly designed and validated biotin- and fluorescein-based 3O-C12-HSL probes we identified the IQ-motif-containing GTPase-activating protein IQGAP1 as a human target of 3O-C12-HSL. We propose that the interaction between IQGAP1 and 3O-C12-HSL provides a novel mechanism for its mode of action on eukaryotic cells, and by affecting the distribution of IQGAP1 and phosphorylation of Rac1 and Cdc42, upstream effectors of filamentous actin remodeling, also cell migration. We suggest that recognition of IQGAP1 by 3O-C12-HSL is a very early event in the communication between bacteria and human epithelial cells.
doi:10.1371/journal.ppat.1002953
PMCID: PMC3469656  PMID: 23071436
4.  The Interaction of N-Acylhomoserine Lactone Quorum Sensing Signaling Molecules with Biological Membranes: Implications for Inter-Kingdom Signaling 
PLoS ONE  2010;5(10):e13522.
Background
The long chain N-acylhomoserine lactone (AHL) quorum sensing signal molecules released by Pseudomonas aeruginosa have long been known to elicit immunomodulatory effects through a process termed inter-kingdom signaling. However, to date very little is known regarding the exact mechanism of action of these compounds on their eukaryotic targets.
Methodology/Principal Findings
The use of the membrane dipole fluorescent sensor di-8-ANEPPS to characterise the interactions of AHL quorum sensing signal molecules, N-(3-oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL), N-(3-oxododecanoyl)homoserine-L-lactone (3-oxo-C12-HSL) and N-(3-oxodecanoyl) homoserine-L-lactone (3-oxo-C10 HSL) produced by Pseudomonas aeruginosa with model and cellular membranes is reported. The interactions of these AHLs with artificial membranes reveal that each of the compounds is capable of membrane interaction in the micromolar concentration range causing significant modulation of the membrane dipole potential. These interactions fit simple hyperbolic binding models with membrane affinity increasing with acyl chain length. Similar results were obtained with T-lymphocytes providing the evidence that AHLs are capable of direct interaction with the plasma membrane. 3-oxo-C12-HSL interacts with lymphocytes via a cooperative binding model therefore implying the existence of an AHL membrane receptor. The role of cholesterol in the interactions of AHLs with membranes, the significance of modulating cellular dipole potential for receptor conformation and the implications for immune modulation are discussed.
Conclusions/ Significance
Our observations support previous findings that increasing AHL lipophilicity increases the immunomodulatory activity of these quorum compounds, while providing evidence to suggest membrane interaction plays an important role in quorum sensing and implies a role for membrane microdomains in this process. Finally, our results suggest the existence of a eukaryotic membrane-located system that acts as an AHL receptor.
doi:10.1371/journal.pone.0013522
PMCID: PMC2958149  PMID: 20975958
5.  N-Acylhomoserine Lactones Undergo Lactonolysis in a pH-, Temperature-, and Acyl Chain Length-Dependent Manner during Growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa  
Infection and Immunity  2002;70(10):5635-5646.
In gram-negative bacterial pathogens, such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis, cell-to-cell communication via the N-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures of Y. pseudotuberculosis and P. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the major P. aeruginosa [N-butanoylhomoserine lactone (C4-HSL) and N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] and Y. pseudotuberculosis [N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL), N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as the N-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, and N-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37°C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require an N-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.
doi:10.1128/IAI.70.10.5635-5646.2002
PMCID: PMC128322  PMID: 12228292
6.  Degradation of Bacterial Quorum Sensing Signaling Molecules by the Microscopic Yeast Trichosporon loubieri Isolated from Tropical Wetland Waters 
Sensors (Basel, Switzerland)  2013;13(10):12943-12957.
Proteobacteria produce N-acylhomoserine lactones as signaling molecules, which will bind to their cognate receptor and activate quorum sensing-mediated phenotypes in a population-dependent manner. Although quorum sensing signaling molecules can be degraded by bacteria or fungi, there is no reported work on the degradation of such molecules by basidiomycetous yeast. By using a minimal growth medium containing N-3-oxohexanoylhomoserine lactone as the sole source of carbon, a wetland water sample from Malaysia was enriched for microbial strains that can degrade N-acylhomoserine lactones, and consequently, a basidiomycetous yeast strain WW1C was isolated. Morphological phenotype and molecular analyses confirmed that WW1C was a strain of Trichosporon loubieri. We showed that WW1C degraded AHLs with N-acyl side chains ranging from 4 to 10 carbons in length, with or without oxo group substitutions at the C3 position. Re-lactonisation bioassays revealed that WW1C degraded AHLs via a lactonase activity. To the best of our knowledge, this is the first report of degradation of N-acyl-homoserine lactones and utilization of N-3-oxohexanoylhomoserine as carbon and nitrogen source for growth by basidiomycetous yeast from tropical wetland water; and the degradation of bacterial quorum sensing molecules by an eukaryotic yeast.
doi:10.3390/s131012943
PMCID: PMC3859043  PMID: 24072030
basidiomycetous; biosensor; lactonase; N-acylhomoserine lactone; quorum sensing; quorum quenching; Rapid Resolution Liquid Chromatography; Trichosporon loubieri; yeast
7.  A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans 
PLoS ONE  2011;6(10):e26278.
In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies.
doi:10.1371/journal.pone.0026278
PMCID: PMC3202535  PMID: 22046268
8.  Substituted Lactam and Cyclic Azahemiacetals Modulate Pseudomonas aeruginosa Quorum Sensing 
Bioorganic & medicinal chemistry  2011;19(18):5500-5506.
Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence that is critical for establishing infection. The most common QS signaling molecule used by Gram-negative bacteria are acylhomoserine lactones. The development of non-native acylhomoserine lactone (AHL) ligands has emerged as a promising new strategy to inhibit QS in Gram-negative bacteria. In this work, we have synthesized a set of optically pure γ-lactams and their reduced cyclic azahemiacetal analogues, bearing the additional alkylthiomethyl substituent, and evaluated their effect on the AHL-dependent Pseudomonas aeruginosa las and rhl QS pathways. The concentration of these ligands and the simple structural modification such as the length of the alkylthio substituent has notable effect on activity. The γ-lactam derivatives with nonylthio or dodecylthio chains acted as inhibitors of las signaling with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent was found to strongly inhibit both las and rhl signaling at higher concentrations while the propylthio analogue strongly stimulated the las QS system at lower concentrations.
doi:10.1016/j.bmc.2011.07.044
PMCID: PMC3171587  PMID: 21855349
Azahemiacetals; Azasugars; Pseudomonas aeruginosa; Quorum sensing
9.  Differential Immune Modulatory Activity of Pseudomonas aeruginosa Quorum-Sensing Signal Molecules  
Infection and Immunity  2004;72(11):6463-6470.
Pseudomonas aeruginosa releases a spectrum of well-regulated virulence factors, controlled by intercellular communication (quorum sensing) and mediated through the production of small diffusible quorum-sensing signal molecules (QSSM). We hypothesize that QSSM may in fact serve a dual purpose, also allowing bacterial colonization via their intrinsic immune-modulatory capacity. One class of signal molecule, the N-acylhomoserine lactones, has pleiotropic effects on eukaryotic cells, particularly those involved in host immunity. In the present study, we have determined the comparative effects of two chemically distinct and endobronchially detectable QSSM, N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and 2-heptyl-3-hydroxy-4 (1H)-quinolone or the Pseudomonas quinolone signal (PQS), on human leukocytes exposed to a series of stimuli designed to detect differential immunological activity in vitro. 3-Oxo-C12-HSL and PQS displayed differential effects on the release of interleukin-2 (IL-2) when human T cells were activated via the T-cell receptor and CD28 (a costimulatory molecule). 3-Oxo-C12-HSL inhibited cell proliferation and IL-2 release; PQS inhibited cell proliferation without affecting IL-2 release. Both molecules inhibited cell proliferation and the release of IL-2 following mitogen stimulation. Furthermore, in the presence of Escherichia coli lipopolysaccharide, 3-oxo-C12-HSL inhibited tumor necrosis factor alpha release from human monocytes, as reported previously (K. Tateda et al., Infect. Immun. 64:37-43, 1996), whereas PQS did not inhibit in this assay. These data highlight the presence of two differentially active immune modulatory QSSM from P. aeruginosa, which are detectable endobronchially and may be active at the host/pathogen interface during infection with P. aeruginosa, should the bronchial airway lymphoid tissues prove to be accessible to QSSM.
doi:10.1128/IAI.72.11.6463-6470.2004
PMCID: PMC522992  PMID: 15501777
10.  Molecular Basis for the Recognition of Structurally Distinct Autoinducer Mimics by the Pseudomonas aeruginosa LasR Quorum Sensing Signaling Receptor 
Chemistry & biology  2009;16(9):961-970.
The human pathogen Pseudomonas aeruginosa coordinates the expression of virulence factors using quorum sensing, a signaling cascade triggered by the activation of signal receptors by small molecule autoinducers. These homoserine lactone autoinducers stabilize their cognate receptors and activate their functions as transcription factors. As quorum sensing regulates the progression of infection and host immune resistance, significant efforts have been devoted towards the identification of small molecules that disrupt this process. Screening efforts have identified a class of triphenyl compounds that are structurally distinct from the homoserine lactone autoinducer, yet interact specifically and potently with LasR receptor to modulate quorum sensing (Muh et al., 2006a). Here we present the high-resolution crystal structures of the ligand-binding domain of LasR in complex with the autoinducer N-3-oxo-dodecanoyl homoserine lactone (1.4 Å resolution), and with the triphenyl mimics TP-1, TP-3, and TP-4 (to between 1.8-2.3 Å resolution). These crystal structures provide a molecular rationale for understanding how chemically distinct compounds can be accommodated by a highly selective receptor and provides the framework for the development of novel quorum sensing regulators, utilizing the triphenyl scaffold.
doi:10.1016/j.chembiol.2009.09.001
PMCID: PMC2778763  PMID: 19778724
11.  The Fe(III) and Ga(III) coordination chemistry of 3-(1-hydroxymethylidene) and 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione: Novel tetramic acid degradation products of homoserine lactone bacterial quorum sensing molecules 
Journal of Inorganic Biochemistry  2011;107(1):96-103.
Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C12-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding.
doi:10.1016/j.jinorgbio.2011.10.009
PMCID: PMC3258344  PMID: 22178671
Quorum sensing; tetramic acid; iron binding; Mössbauer spectroscopy
12.  N-acyl Homoserine Lactone-Producing Pseudomonas putida Strain T2-2 from Human Tongue Surface 
Sensors (Basel, Switzerland)  2013;13(10):13192-13203.
Bacterial cell-to-cell communication (quorum sensing) refers to the regulation of bacterial gene expression in response to changes in microbial population density. Quorum sensing bacteria produce, release and respond to chemical signal molecules called autoinducers. Bacteria use two types of autoinducers, namely autoinducer-1 (AI-1) and autoinducer-2 (AI-2) where the former are N-acylhomoserine lactones and the latter is a product of the luxS gene. Most of the reported literatures show that the majority of oral bacteria use AI-2 for quorum sensing but rarely the AI-1 system. Here we report the isolation of Pseudomonas putida strain T2-2 from the oral cavity. Using high resolution mass spectrometry, it is shown that this isolate produced N-octanoylhomoserine lactone (C8-HSL) and N-dodecanoylhomoserine lactone (C12-HSL) molecules. This is the first report of the finding of quorum sensing of P. putida strain T2-2 isolated from the human tongue surface and their quorum sensing molecules were identified.
doi:10.3390/s131013192
PMCID: PMC3859058  PMID: 24084113
autoinducer; bioreporter; luxS; N-acylhomoserine lactone; oral buccal cavity; quorum quenching; quorum sensing
13.  Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: Co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia 
BMC Microbiology  2011;11:51.
Background
Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novel N-acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest.
Results
By using a basal growth medium containing N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) as the sole source of carbon and nitrogen, the ginger rhizosphere associated bacteria were enriched for strains with AHL-degrading capabilities. Three isolates belonging to the genera Acinetobacter (GG2), Burkholderia (GG4) and Klebsiella (Se14) were identified and selected for further study. Strains GG2 and Se14 exhibited the broadest spectrum of AHL-degrading activities via lactonolysis while GG4 reduced 3-oxo-AHLs to the corresponding 3-hydroxy compounds. In GG2 and GG4, QQ was found to co-exist with AHL-dependent QS and GG2 was shown to inactivate both self-generated and exogenously supplied AHLs. GG2, GG4 and Se14 were each able to attenuate virulence factor production in both human and plant pathogens.
Conclusions
Collectively our data show that ginger rhizosphere bacteria which make and degrade a wide range of AHLs are likely to play a collective role in determining the QS-dependent phenotype of a polymicrobial community.
doi:10.1186/1471-2180-11-51
PMCID: PMC3062576  PMID: 21385437
14.  The LuxM Homologue VanM from Vibrio anguillarum Directs the Synthesis of N-(3-Hydroxyhexanoyl)homoserine Lactone and N-Hexanoylhomoserine Lactone 
Journal of Bacteriology  2001;183(12):3537-3547.
Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).
doi:10.1128/JB.183.12.3537-3547.2001
PMCID: PMC95229  PMID: 11371516
15.  N-Acylhomoserine Lactones Antagonize Virulence Gene Expression and Quorum Sensing in Staphylococcus aureus 
Infection and Immunity  2006;74(2):910-919.
Many gram-negative bacteria employ N-acylhomoserine lactone (AHL)-mediated quorum sensing to control virulence. To determine whether gram-positive bacteria such as Staphylococcus aureus respond to AHLs, we used a growth-dependent lux reporter fusion. Exposure of S. aureus to different AHLs revealed that 3-oxo-substituted AHLs with C10 to C14 acyl chains inhibited light output and growth in a concentration-dependent manner, while short-chain AHLs had no effect. N-(3-Oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) inhibited the production of exotoxins and cell wall fibronectin-binding proteins but enhanced protein A expression. Since these processes are reciprocally regulated via the S. aureus agr quorum-sensing system, which in turn, is regulated via sar, we examined the effect of AHLs on sarA and agr. At sub-growth-inhibitory concentrations of 3-oxo-C12-HSL, both sarA expression and agr expression were inhibited, indicating that the action of 3-oxo-C12-HSL is mediated at least in part through antagonism of quorum sensing in S. aureus. Spent culture supernatants from Pseudomonas aeruginosa, which produces both 3-oxo-C12-HSL and N-butanoyl-homoserine lactone (C4-HSL), also inhibited agr expression, although C4-HSL itself was inactive in this assay. Since quorum sensing in S. aureus depends on the activities of membrane-associated proteins, such as AgrB, AgrC, and AgrD, we investigated whether AHLs perturbed S. aureus membrane functionality by determining their influence on the membrane dipole potential. From the binding curves obtained, a dissociation constant of 7 μM was obtained for 3-oxo-C12-HSL, indicating the presence of a specific saturable receptor, whereas no binding was observed for C4-HSL. These data demonstrate that long-chain 3-oxo-substituted AHLs, such as 3-oxo-C12-HSL, are capable of interacting with the S. aureus cytoplasmic membrane in a saturable, specific manner and at sub-growth-inhibitory concentrations, down-regulating exotoxin production and both sarA and agr expression.
doi:10.1128/IAI.74.2.910-919.2006
PMCID: PMC1360299  PMID: 16428734
16.  The Pseudomonas aeruginosa Autoinducer N-3-Oxododecanoyl Homoserine Lactone Accelerates Apoptosis in Macrophages and Neutrophils  
Infection and Immunity  2003;71(10):5785-5793.
Quorum-sensing systems are critical regulators of the expression of virulence factors of various organisms, including Pseudomonas aeruginosa. Las and Rhl are two major quorum-sensing components, and they are regulated by their corresponding autoinducers, N-3-oxododecanoyl homoserine lactone (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL). Recent progress has demonstrated the potential of quorum-sensing molecules, especially 3-oxo-C12-HSL, for modulation of the host immune system. Here we show the specific ability of 3-oxo-C12-HSL to induce apoptosis in certain types of cells. When bone marrow-derived macrophages were incubated with synthetic 3-oxo-C12-HSL, but when they were incubated not C4-HSL, significant loss of viability was observed in a concentration (12 to 50 μM)- and incubation time (1 to 24 h)-dependent manner. The cytotoxic activity of 3-oxo-C12-HSL was also observed in neutrophils and monocytic cell lines U-937 and P388D1 but not in epithelial cell lines CCL-185 and HEp-2. Cells treated with 3-oxo-C12-HSL revealed morphological alterations indicative of apoptosis. Acceleration of apoptosis in 3-oxo-C12-HSL-treated cells was confirmed by multiple criteria (caspases 3 and 8, histone-associated DNA fragments, phosphatidylserine expression). Structure-activity correlation experiments demonstrated that the fine structure of 3-oxo-C12-HSL, the HSL backbone, and side chain length are required for maximal activity. These data suggest that Pseudomonas 3-oxo-C12-HSL specifically promotes induction of apoptosis, which may be associated with 3-oxo-C12-HSL-induced cytotoxicity in macrophages and neutrophils. Our data suggest that the quorum-sensing molecule 3-oxo-C12-HSL has critical roles in the pathogenesis of P. aeruginosa infection, not only in the induction of bacterial virulence factors but also in the modulation of host responses.
doi:10.1128/IAI.71.10.5785-5793.2003
PMCID: PMC201082  PMID: 14500500
17.  Analysis of the Pseudomonas aeruginosa elastase (lasB) regulatory region. 
Journal of Bacteriology  1996;178(4):1134-1140.
The enzyme elastase is an important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa. Previous studies have shown that expression of the P. aeruginosa elastase gene (lasB) requires both an activator protein, LasR, and an N-acylhomoserine lactone compound termed Pseudomonas autoinducer (PAI). In this study, we analyzed the lasB promoter region to learn more about lasB activation by LasR and PAI. We report that the lasB transcriptional start is located 141 nucleotides upstream from the lasB translational start. It was also discovered that the lasB promoter region contains two putative operator sequences (OP1 and OP2) that are similar to each other and the Vibrio fischeri lux operator. OP1 is located directly upstream from, and may overlap with, the lasB promoter region, and OP2 is centered 102 nucleotides upstream from the lasB transcriptional start site. To study the effects of these putative operators and other sequences upstream from the lasB transcriptional start site on lasB activation, a series of transcriptional lasBp-lacZ gene fusions was constructed. Data from these fusions indicate that both putative operators are involved in LasR- and PAI-mediated lasB activation, with OP1 being more important than OP2.
PMCID: PMC177776  PMID: 8576049
18.  Mechanism of Pseudomonas aeruginosa RhlR Transcriptional Regulation of the rhlAB Promoter 
Journal of Bacteriology  2003;185(20):5976-5983.
Pseudomonas aeruginosa contains two transcription regulators (LasR and RhlR) that, when complexed with their specific autoinducers (3-oxo-dodecanoyl-homoserine lactone and butanoyl-homoserine lactone, respectively) activate transcription of different virulence-associated traits. We studied the RhlR-dependent transcriptional regulation of the rhlAB operon encoding rhamnosyltransferase 1, an enzyme involved in the synthesis of the surfactant monorhamnolipid, and showed that RhlR binds to a specific sequence in the rhlAB regulatory region, both in the presence and in the absence of its autoinducer. Our data suggest that in the former case it activates transcription, whereas in the latter it acts as a transcriptional repressor of this promoter. RhlR seems to repress the transcription of other quorum-sensing-regulated genes; thus, RhlR repressor activity might be of importance in the finely regulated expression of P. aeruginosa virulence-associated traits.
doi:10.1128/JB.185.20.5976-5983.2003
PMCID: PMC225020  PMID: 14526008
19.  Ellagic Acid Derivatives from Terminalia chebula Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate Pseudomonas aeruginosa PAO1 Virulence 
PLoS ONE  2013;8(1):e53441.
Background
Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors.
Methods and Results
Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C12HSL and C4HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C4HSL. F7 also showed antagonistic activity against 3-oxo-C12HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract.
Conclusions
This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.
doi:10.1371/journal.pone.0053441
PMCID: PMC3539995  PMID: 23320085
20.  Mouse and Human Cell Activation by N-Dodecanoyl-dl-Homoserine Lactone, a Chromobacterium violaceum Autoinducer▿  
Infection and Immunity  2006;74(12):7029-7031.
Chromobacterium violaceum produces autoinducers, including homoserine lactones (HSLs), for genetic regulation. Among the seven HSLs derived from C. violaceum we evaluated, only C12-HSL stimulated the production of inflammatory cytokines in mammalian monocytic cell lines through the activation of the NF-κB signaling pathway besides their quorum-sensing role, like 3-oxo-C12-HSL from Pseudomonas aeruginosa.
doi:10.1128/IAI.00038-06
PMCID: PMC1698062  PMID: 16982829
21.  At a Supra-Physiological Concentration, Human Sexual Hormones Act as Quorum-Sensing Inhibitors 
PLoS ONE  2013;8(12):e83564.
N-Acylhomoserine lactone (AHL)-mediated quorum-sensing (QS) regulates virulence functions in plant and animal pathogens such as Agrobacterium tumefaciens and Pseudomonas aeruginosa. A chemolibrary of more than 3500 compounds was screened using two bacterial AHL-biosensors to identify QS-inhibitors (QSIs). The purity and structure of 15 QSIs selected through this screening were verified using HPLC MS/MS tools and their activity tested on the A. tumefaciens and P. aeruginosa bacterial models. The IC50 value of the identified QSIs ranged from 2.5 to 90 µg/ml, values that are in the same range as those reported for the previously identified QSI 4-nitropyridine-N-oxide (IC50 24 µg/ml). Under the tested culture conditions, most of the identified QSIs did not exhibit bacteriostatic or bactericidal activities. One third of the tested QSIs, including the plant compound hordenine and the human sexual hormone estrone, decreased the frequency of the QS-regulated horizontal transfer of the tumor-inducing (Ti) plasmid in A. tumefaciens. Hordenine, estrone as well as its structural relatives estriol and estradiol, also decreased AHL accumulation and the expression of six QS-regulated genes (lasI, lasR, lasB, rhlI, rhlR, and rhlA) in cultures of the opportunist pathogen P. aeruginosa. Moreover, the ectopic expression of the AHL-receptors RhlR and LasR of P. aeruginosa in E. coli showed that their gene-regulatory activity was affected by the QSIs. Finally, modeling of the structural interactions between the human hormones and AHL-receptors LasR of P. aeruginosa and TraR of A. tumefaciens confirmed the competitive binding capability of the human sexual hormones. This work indicates potential interferences between bacterial and eukaryotic hormonal communications.
doi:10.1371/journal.pone.0083564
PMCID: PMC3871529  PMID: 24376718
22.  The Global Posttranscriptional Regulator RsmA Modulates Production of Virulence Determinants and N-Acylhomoserine Lactones in Pseudomonas aeruginosa 
Journal of Bacteriology  2001;183(22):6676-6683.
Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria. Here we report the isolation of a Pseudomonas aeruginosa gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites). Overexpression of rsmA resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin. While inactivation of rsmA in P. aeruginosa had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase. The influence of RsmA on N-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and N-butanoylhomoserine lactone (C4-HSL) produced by the rsmA mutant and the rsmA-overexpressing strain. RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using lasI and rhlI translational fusions. These data also highlighted the temporal expression control of the lasI gene, which was induced much earlier and to a higher level during the exponential growth phase in an rsmA mutant. To investigate whether RsmA modulates HCN production solely via quorum-sensing control, hcn translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (hcnABC). RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the hcnA ribosome-binding site. This suggests that, in P. aeruginosa, RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry.
doi:10.1128/JB.183.22.6676-6683.2001
PMCID: PMC95500  PMID: 11673439
23.  Lyngbyoic acid, a “tagged” fatty acid from a marine cyanobacterium, disrupts quorum sensing in Pseudomonas aeruginosa† 
Molecular bioSystems  2011;7(4):1205-1216.
Quorum sensing (QS) is a mechanism of bacterial gene regulation in response to increases in population density. Perhaps most studied are QS pathways mediated by acylhomoserine lactones (AHLs) in Gram-negative bacteria. Production of small molecule QS signals, their accumulation within a diffusion-limited environment and their binding to a LuxR-type receptor trigger QS-controlled gene regulatory cascades. In Pseudomonas aeruginosa, for example, binding of AHLs to their cognate receptors (LasR, RhlR) controls production of virulence factors, pigments, antibiotics and other behaviors important for its interactions with eukaryotic hosts and other bacteria. We have previously shown that marine cyanobacteria produce QS-inhibitory molecules, including 8-epi-malyngamide C (1), malyngamide C (2) and malyngolide (3). Here we isolated a new small cyclopropane-containing fatty acid, lyngbyoic acid (4), as a major metabolite of the marine cyanobacterium, Lyngbya cf. majuscula, collected at various sites in Florida. We screened 4 against four reporters based on different AHL receptors (LuxR, AhyR, TraR and LasR) and found that 4 most strongly affected LasR. We also show that 4 reduces pyocyanin and elastase (LasB) both on the protein and transcript level in wild-type P. aeruginosa, and that 4 directly inhibits LasB enzymatic activity. Conversely, dodecanoic acid (9) increased pyocyanin and LasB, demonstrating that the fused cyclopropane “tag” is functionally relevant and potentially confers resistance to β-oxidation. Global transcriptional effects of 4 in some ways replicate the gene expression changes of P. aeruginosa during chronic lung infections of cystic fibrosis patients, with reduced lasR signaling, increased biofilm and expression of the virulence locus HSI-I. Compound 4 may therefore prove to be a useful tool in the study of P. aeruginosa adaption during such chronic infections.
doi:10.1039/c0mb00180e
PMCID: PMC3802547  PMID: 21258753
24.  Microbial Regulation in Gorgonian Corals  
Marine Drugs  2012;10(6):1225-1243.
Gorgonian corals possess many novel natural products that could potentially mediate coral-bacterial interactions. Since many bacteria use quorum sensing (QS) signals to facilitate colonization of host organisms, regulation of prokaryotic cell-to-cell communication may represent an important bacterial control mechanism. In the present study, we examined extracts of twelve species of Caribbean gorgonian corals, for mechanisms that regulate microbial colonization, such as antibacterial activity and QS regulatory activity. Ethanol extracts of gorgonians collected from Puerto Rico and the Florida Keys showed a range of both antibacterial and QS activities using a specific Pseudomonas aeruginosa QS reporter, sensitive to long chain AHLs and a short chain N-acylhomoserine lactones (AHL) biosensor, Chromobacterium violaceium. Overall, the gorgonian corals had higher antimicrobial activity against non-marine strains when compared to marine strains. Pseudopterogorgia americana, Pseusopterogorgia acerosa, and Pseudoplexuara flexuosa had the highest QS inhibitory effect. Interestingly, Pseudoplexuara porosa extracts stimulated QS activity with a striking 17-fold increase in signal. The stimulation of QS by P. porosa or other elements of the holobiont may encourage colonization or recruitment of specific microbial species. Overall, these results suggest the presence of novel stimulatory QS, inhibitory QS and bactericidal compounds in gorgonian corals. A better understanding of these compounds may reveal insight into coral-microbial ecology and whether a therapeutic potential exists.
doi:10.3390/md10061225
PMCID: PMC3397436  PMID: 22822369
gorgonian corals; quorum sensing (QS); antimicrobial activity; microbial regulation
25.  Pseudomonas aeruginosa Quorum-Sensing Signal Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone Inhibits Expression of P2Y Receptors in Cystic Fibrosis Tracheal Gland Cells 
Infection and Immunity  1999;67(10):5076-5082.
ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by Pseudomonas aeruginosa. As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible N-acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated. Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF. Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs N-butanoyl-l-homoserine lactone (BHL), N-hexanoyl-l-homoserine lactone (HHL), N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL), and N-(3-oxohexanoyl)-l-homoserine lactone (OHHL), with special reference to P2Y receptors. All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells. The 50% inhibitory concentrations were as high as 10 and 5 μM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively. Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors. Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this. These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.
PMCID: PMC96855  PMID: 10496880

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