Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.     

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

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

Influence of Pseudomonas autoinducer N-3-oxododecanoyl homoserine lactone on human corneal epithelial cells

The quorum-sensing (QS) signaling-dependent extracellular virulence factors of Pseudomonas aeruginosa can cause infections such as P. aeruginosa keratitis. P. aeruginosa communicates by secreting and sensing small chemical molecules called autoinducers in QS system. The key QS signal molecule, N-3-oxododecanoyl-homoserine lactone (3OC12HSL), can affect the behavior of host cells and initiate immune response. In this report we investigated the influence of 3OC12HSL on human corneal epithelial cells (HCECs) and the mechanisms of 3OC12HSL on activated toll-like receptor 2 (TLR2)-dependent interleukin-8 (IL-8) secretion in HCECs. Cells were cultured under different concentrations of 3OC12HSL. Cell viability was assessed using Crystal violet staining and the cell counting kit-8 assay. We demonstrated the administration of 3OC12HSL decreased HCEC viability and survival in a concentration- and time-dependent manner. At high concentrations, 3OC12HSL rapidly promoted a time-dependent increase in the expressions of TLR2 and TLR4. It was found that the nuclear translocation and expression of nuclear factor-κB (NF-κB) were also increased in response to 3OC12HSL treatment. The significantly elevated expressions of TLR2, TLR4, and NF-κB, encouraged us to further test their mechanisms that cause inflammatory response. Among the inflammatory factors examined (IL-6, IL-8, IL-10, and TNF-α), we found that IL-8 was significantly increased after treatment with 3OC12HSL and its expression was inhibited when TLR2 was specifically blocked or silenced. These results indicated that the QS signaling molecule 3OC12HSL could be recognized by the host innate immune system in HCECs. This recognition then triggered an immune inflammatory response involving the activation of TLR2 and an increase in expression of IL-8. This crosstalk between 3OC12HSL and host immunity in HCECs contributes to the development and progression of P. aeruginosa keratitis.     

The BpsIR quorum-sensing system of Burkholderia pseudomallei

BpsIR, a LuxIR quorum-sensing homolog, is required for optimal expression of virulence and secretion of exoproducts in Burkholderia pseudomallei. Cell density-dependent expression of bpsI and bpsR, the positive regulation of bpsIR expression by BpsR, and the synthesis of N-octanoyl-homoserine lactone (C8HSL) by BpsI are described in this report.     

Probing the impact of ligand binding on the acyl-homoserine lactone-hindered transcription factor EsaR of Pantoea stewartii subsp. stewartii

The quorum-sensing regulator EsaR from Pantoea stewartii subsp. stewartii is a LuxR homologue that is inactivated by acyl-homoserine lactone (AHL). In the corn pathogen P. stewartii, production of exopolysaccharide (EPS) is repressed by EsaR at low cell densities. However, at high cell densities when high concentrations of its cognate AHL signal are present, EsaR is inactivated and derepression of EPS production occurs. Thus, EsaR responds to AHL in a manner opposite to that of most LuxR family members. Depending on the position of its binding site within target promoters, EsaR serves as either a repressor or activator in the absence rather than in the presence of its AHL ligand. The effect of AHL on LuxR homologues has been difficult to study in vitro because AHL is required for purification and stability. EsaR, however, can be purified without AHL enabling an in vitro analysis of the response of the protein to ligand. Western immunoblots and pulse-chase experiments demonstrated that EsaR is stable in vivo in the absence or presence of AHL. Limited in vitro proteolytic digestions of a biologically active His-MBP tagged version of EsaR highlighted intradomain and interdomain conformational changes that occur in the protein in response to AHL. Gel filtration chromatography of the full-length fusion protein and cross-linking of the N-terminal domain both suggest that this conformational change does not impact the multimeric state of the protein. These findings provide greater insight into the diverse mechanisms for AHL responsiveness found within the LuxR family.     

Directed evolution of LuxI for enhanced OHHL production

Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.     

NMR detection of adventitious xylose binding to the quorum-sensing protein SdiA of Escherichia coli

During the solution structure determination of the Escherichia coli quorum-sensing protein SdiA in the presence of N-octanoyl-l-homoserine lactone (HSL), NMR signals were detected in (13)C-filter-(13)C-filter spectra for the bound HSL molecule. An additional set of coupled signals, independent of those of HSL, were also detected, indicating the presence of another unlabeled molecule, also bound to the labeled SdiA. Analysis of the NMR spectrum of this ligand and of the mass spectrum of the dissociated components indicates that the ligand is most likely xylose. Further analysis of xylose-bound SdiA defines a site close to the C terminus, remote from the HSL binding site. These observations provide an example of the sensitivity of high-resolution NMR experiments and their ability to detect, identify, and map the adventitious binding of a small organic molecule to a protein.     

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.     

Utilization of Acyl-Homoserine Lactone Quorum Signals for Growth by a Soil Pseudomonad and Pseudomonas aeruginosa PAO1

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42°C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other γ-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.