Molecular insights into quorum sensing in the human pathogen Pseudomonas aeruginosa from the structure of the virulence regulator LasR bound to its autoinducer

Many Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C(12)-acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an alpha-beta-alpha fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-A resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors.     

Quorum-sensing molecules: Sampling,identification and characterization of N-acyl-homoserine lactone in Vibrio sp

Quorum sensing (QS) is a mechanism by which gram-negative bacteria regulate their gene expression by making use of cell density. QS is triggered by a small molecule known as an autoinducer. Typically, gram-negative bacteria such as Vibrio produce signaling molecules called acyl homoserine lactones (AHLs). However, their levels are very low, making them difficult to detect. We used thin layer chromatography (TLC) to examine AHLs in different Vibrio species, such as Vibrio alginolyticus, Vibrio parahemolyticus, and Vibrio cholerae, against a standard- Chromobacterium violaceum. Further, AHLs were characterised by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC–MS). C4-HSL (N- butanoyl- L- homoserine lactone), C6-HSL (N- hexanoyl- L- homoserine lactone), 3-oxo-C8-HSL (N-(3-Oxooctanoyl)-DL-homoserine lactone), C8-HSL (N- octanoyl- L- homoserine lactone), C₁10-HSL (N- decanoyl- L- homoserine lactone), C12-HSL (N- dodecanoyl- L- homoserine lactone) and C14-HSL (N- tetradecanoyl- L- homoserine lactone) were identified from Vibrio. These results may provide a basis for blocking the AHL molecules of Vibrio, thereby reducing their pathogenicity and eliminating the need for antimicrobials.     

Mutation of pfm affects the adherence of Pseudomonas aeruginosa to host cells and the quorum sensing system

The Pseudomonas aeruginosa quorum sensing (QS) system is controlled by the signal molecules acyl homoserine lactones (AHLs) that are synthesized from acyl enoyl-acyl carrier proteins (acyl-ACPs) provided by the fatty acid biosynthesis cycle. Pfm (PA2950), an enoyl-CoA reductase, has previously been shown to affect swimming mobility and fatty acid biosynthesis. In this report, we further show that pfm influences bacterial adherence to human cells. Microarray assay results suggest that pfm affects bacterial adherence through its influence on the QS system. Further experiments confirmed that the pfm mutant strain produces significantly less QS signal molecules than the corresponding wild-type strain. Using strains Escherichia coli DH5α(pECP64, lasB'-lacZ) and E.?coli DH5α(pECP61.5, rhlA'-lacZ), biosensors for N-(3-oxododecanoyl) homoserine lactone (3O-C(12) -HSL) and N-butyryl homoserine lactone (C(4) -HSL), respectively, we found that pfm mutant strain produces decreased amounts of both signal molecules. Elastase activity and pyocyanin measurements further confirmed the reduced levels of 3O-C(12) -HSL and C(4) -HSL in the pfm mutant. Finally, bacterial virulence, as assessed by the Caenorhabditis elegans worm killing assay, is decreased in the pfm mutant. Taken together, these data indicate that pfm can be an important target for the control of P.?aeruginosa infectivity.     

The Burkholderia mallei BmaR3-BmaI3 quorum-sensing system produces and responds to N-3-hydroxy-octanoyl homoserine lactone

Burkholderia mallei has two acyl-homoserine lactone (acyl-HSL) signal generator-receptor pairs and two additional signal receptors, all of which contribute to virulence. We show that B. mallei produces N-3-hydroxy-octanoyl HSL (3OHC8-HSL) but a bmaI3 mutant does not. Recombinant Escherichia coli expressing BmaI3 produces hydroxylated acyl-HSLs, with 3OHC8-HSL being the most abundant compound. In recombinant E. coli, BmaR3 responds to 3OHC8-HSL but not to other acyl-HSLs. These data indicate that the signal for BmaR3-BmaI3 quorum sensing is 3OHC8-HSL.     

Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for disruption of quorum sensing

SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower K(m) values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (k(cat)/K(m) = 5.5 × 10(3) M(-1)·s(-1)), but SsoPox can also degrade N-butyryl homoserine lactone (C(4)-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C(12)-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C(4)-HSL- and oxo-C(12)-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.     

A hierarchical quorum-sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping

In cell-free Yersinia pseudotuberculosis culture supernatants, we have chemically characterized three N-acyl homoserine lactone (AHL) molecules, N-octanoyl homoserine lactone (C8-HSL), N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoyl homoserine lactone (C6-HSL). We have identified, cloned and sequenced two pairs of LuxR/I homologues termed YpsR/I and YtbR/I. In Escherichia coli at 37 degrees C, YpsI and YtbI both synthesize C6-HSL, although YpsI is responsible for 3-oxo-C6-HSL and YtbI for C8-HSL synthesis respectively. However, in a Y. pseudotuberculosis ypsI-negative background, YtbI appears capable of adjusting the AHL profile from all three AHLs at 37 degrees C and 22 degrees C to the absence of 3-oxo-C6-HSL at 28 degrees C. Insertion deletion mutagenesis of ypsR leads to the loss of C8-HSL at 22 degrees C, which suggests that at this temperature the YpsR protein is involved in the hierarchical regulation of the ytbR/I locus. When compared with the parent strain, the ypsR and ypsI mutants exhibit a number of phenotypes, including clumping (ypsR mutant), overexpression of a major flagellin subunit (ypsR mutant) and increased motility (both ypsR and ypsI mutants). The clumping and motility phenotypes are both temperature dependent. These data are consistent with a hierarchical quorum-sensing cascade in Y. pseudotuberculosis that is involved in the regulation of clumping and motility.     

Inhibition of quorum sensing in Serratia marcescens AS-1 by synthetic analogs of N-acylhomoserine lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C(6)-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C(9)-CPA), had a strong inhibitory effect on prodigiosin production. C(9)-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C(9)-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C(6)-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C(9)-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.     

Producing mechanism of an algicidal compound against red tide phytoplankton in a marine bacterium γ-proteobacterium

Strain MS-02-063, γ-proteobacterium, isolated from a coast area of Nagasaki, Japan, produced a red pigment which belongs to prodigiosin members. This pigment, PG-L-1, showed potent algicidal activity against various red tide phytoplanktons in a concentration-dependent manner. An understanding of a mechanism of PG-L-1 production by this marine bacterium may yield important new insights and strategies for preventing blooms of harmful flagellate algae in natural marine environments. Therefore, we analyzed the mechanisms of PG-L-1 production. In our previous study, the pigment production by this marine bacterium was completely inhibited at 1.56 μg/ml of erythromycin or 3.13 μg/ml of chloramphenicol, while minimal inhibitory concentrations for cell growth of erythromycin and chloramphenicol against this bacterium were >100 and 25 μg/ml, respectively. It is interesting to note that the ability of the pigment production in erythromycin-treated bacterium recovered by an addition of homoserine lactone. In fact, the pigment production was inhibited by β-cyclodextrin that inhibits autoinducer activities by a complex with N-acyl homoserine lactones. N-acyl homoserine lactones with autoinducer activities are ubiquitous bacterial signaling molecules that regulate gene expression in a cell density dependent process known as quorum sensing. Therefore, it was suggested that PG-L-1 produced by strain MS-02-063 is controlled by the homoserine lactone quorum sensing. It is speculated that this quorum sensing is involved in the production of algicidal agents of other marine bacteria. This bacterium and other algicidal bacteria might be concerned in regulating the blooms of harmful flagellate algae through the quorum sensing system.     

The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone

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).     

Quorum Sensing in <Emphasis Type="Italic">Aeromonas</Emphasis> Species Isolated from Patients in Malaysia

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C₆. Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.