Characterization of a luxI/luxR-type quorum sensing system and N-acyl-homoserine lactone-dependent regulation of exo-enzyme and antibacterial component production in Serratia plymuthica RVH1.

Quorum sensing by means of N-acyl-l-homoserine lactones (AHLs) is widespread in Gram-negative bacteria, where diverse AHLs influence a wide variety of functions, even in a single genus such as Serratia. Here we report the identification and characterization of the quorum sensing system of Serratia plymuthica strain RVH1. This strain isolated from a raw vegetable processing line produces at least three AHLs which were identified as N-butanoyl- (C4-HSL), N-hexanoyl- (C6-HSL) and N-(3-oxo-hexanoyl)-homoserine lactone (3-oxo-C6-HSL). The identified LuxI homolog SplI synthesizes 3-oxo-C6-HSL, and influences the production of C4-HSL and C6-HSL, as splI gene inactivation resulted in loss of 3-oxo-C6-HSL production and smaller amounts of C4-HSL and C6-HSL produced. SplI-dependent quorum sensing controls 2,3-butanediol fermentation (previously reported) and the production of an extracellular chitinase, nuclease, protease and antibacterial compound. The identity of the latter is not yet elucidated, but appears to be different from the known antibacterial compounds produced by Serratia strains. SplR, the homolog of the LuxR regulator, appears to act as a repressor of synthesis of extracellular enzymes and antibacterial compound and to autorepress its own expression, probably by binding to a 21bp lux box sequence.

Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators.

Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone (3O, C(12)-HSL), is described. Twenty-four single or combination systematic alterations of the structural components of 3O, C(12)-HSL were introduced as described. Given the already defined immunological profile of the parent compound, 3O, C(12)-HSL, these compounds were assayed for their ability to inhibit murine and human leucocyte proliferation and TNF-alpha secretion by lipopolysaccharide (LPS) stimulated human leucocytes in order to provide an initial structure-activity profile. From IC(50) values obtained with a murine splenocyte proliferation assay, it is apparent that acylated l-homoserine lactones with an 11-13 C side chain containing either a 3-oxo or a 3-hydroxy group are optimal structures for immune suppressive activity. These derivatives of 3O, C(12)-HSL with monounsaturation and/or a terminal nonpolar substituent on the side chain were also potent immune suppressive agents. However, structures lacking the homoserine lactone ring, structures lacking the l-configuration at the chiral center, and those with polar substituents were essentially devoid of activity. The ability of compounds selected from the optimal activity range to modulate mitogen-driven human peripheral blood mononuclear cell proliferation and LPS-induced TNF-alpha secretion indicates the suitability of these compounds for further investigation in relation to their molecular mechanisms of action in TNF-alpha driven immunological diseases, particularly autoimmune diseases such as psoriasis, rheumatoid arthritis, and type 1 (autoimmune) diabetes.

Quorum sensing regulates dpsA and the oxidative stress response in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal human tropical disease. The non-specific DNA-binding protein DpsA plays a key role in protecting B. pseudomallei from oxidative stress mediated, for example, by organic hydroperoxides. The regulation of dpsA expression is poorly understood but one possibility is that it is regulated in a cell population density-dependent manner via N-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) since a lux-box motif has been located within the dpsA promoter region. Using liquid chromatography and tandem mass spectrometry, it was first established that B. pseudomallei strain PP844 synthesizes AHLs. These were identified as N-octanoylhomoserine lactone (C8-HSL), N-(3-oxooctanoyl)homoserine lactone (3-oxo-C8-HSL), N-(3-hydroxyoctanoyl)-homoserine lactone (3-hydroxy-C8-HSL), N-decanoylhomoserine lactone (C10-HSL), N-(3-hydroxydecanoyl) homoserine lactone (3-hydroxy-C10-HSL) and N-(3-hydroxydodecanoyl)homoserine lactone (3-hydroxy-C12-HSL). Mutation of the genes encoding the LuxI homologue BpsI or the LuxR homologue BpsR resulted in the loss of C8-HSL and 3-oxo-C8-HSL synthesis, demonstrating that BpsI was responsible for directing the synthesis of these AHLs only and that bpsI expression and hence C8-HSL and 3-oxo-C8-HSL production depends on BpsR. In bpsI, bpsR and bpsIR mutants, dpsA expression was substantially down-regulated. Furthermore, dpsA expression in Escherichia coli required both BpsR and C8-HSL. bpsIR-deficient mutants exhibited hypersensitivity to the organic hydroperoxide tert-butyl hydroperoxide by displaying a reduction in cell viability which was restored by provision of exogenous C8-HSL (bpsI mutant only), by complementation with the bpsIR genes or by overexpression of dpsA. These data indicate that in B. pseudomallei, QS regulates the response to oxidative stress at least in part via the BpsR/C8-HSL-dependent regulation of DpsA.

Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1.

The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3' position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.

Disruption of quorum sensing in seawater abolishes attraction of zoospores of the green alga Ulva to bacterial biofilms.

Zoospores of the eukaryotic green seaweed Ulva respond to bacterial N-acylhomoserine lactone (AHL) quorum sensing signal molecules for the selection of surface sites for permanent attachment. In this study we have investigated the production and destruction of AHLs in biofilms of the AHL-producing marine bacterium, Vibrio anguillarum and their stability in seawater. While wild type V. anguillarum NB10 was a strong attractor of zoospores, inactivation of AHL production in this strain by either expressing the recombinant Bacillus lactonase coding gene aiiA, or by mutating the AHL biosynthetic genes, resulted in the abolition of zoospore attraction. In seawater, with a pH of 8.2, the degradation of AHL molecules was temperature-dependent, indicating that the AHLs produced by marine bacterial biofilms have short half-lives. The Ulva zoospores sensed a range of different AHL molecules and in particular more zoospores settled on surfaces releasing AHLs with longer (>six carbons) N-linked acyl chains. However, this finding is likely to be influenced by the differential diffusion rates of AHLs from the experimental surface matrix. Molecules with longer N-acyl chains, such as N-(3-oxodecanoyl)- L-homoserine lactone, diffused more slowly than those with shorter N-acyl chains such as N-(3-hydroxy-hexanoyl)- L-homoserine lactone. Image analysis using GFP-tagged V. anguillarum biofilms revealed that spores settle directly on bacterial cells and in particular on microcolonies which we show are sites of concentrated AHL production.

Identification of a quorum-sensing signal molecule in the facultative intracellular pathogen Brucella melitensis.

Brucella melitensis is a gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades and survives within both professional and nonprofessional phagocytes. A dichloromethane extract of spent culture supernatant from B. melitensis induces bioluminescence in an Escherichia coli acyl-homoserine lactone (acyl-HSL) biosensor strain based upon the activity of the LasR protein of Pseudomonas aeruginosa. HPLC fractionation of the extract, followed by mass spectrometry, identified the major active molecule as N-dodecanoylhomoserine lactone (C12-HSL). This is the first report of the production of an acyl-HSL by an intracellular pathogen. The addition of synthetic C12-HSL to an early log phase culture of either B. melitensis or Brucella suis 1330 reduces the transcription of the virB operon, which contains virulence genes known to be required for intracellular survival. This mimics events seen during the stationary phase of growth and suggests that quorum sensing may play a role in the control of virulence in Brucella.

Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones.

Quorum sensing relies upon the interaction of a diffusible signal molecule with a transcriptional activator protein to couple gene expression with cell population density. In Gram-negative bacteria, such signal molecules are usually N-acylhomoserine lactones (AHLs) which differ in the structure of their N-acyl side chains. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein. Previously the authors described a violacein-negative, mini-Tn5 mutant of C. violaceum (CV026) in which pigment production can be restored by incubation with supernatants from the wild-type strain. To develop this mutant as a general biosensor for AHLs, the natural C. violaceum AHL molecule was first chemically characterized. By using solvent extraction, HPLC and mass spectrometry, a single AHL, N-hexanoyl-L-homoserine lactone (HHL), was identified in wild-type C. violaceum culture supernatants which was absent from CV026. Since the production of violacein constitutes a simple assay for the detection of AHLs, we explored the ability of CV026 to respond to a series of synthetic AHL and N-acylhomocysteine thiolactone (AHT) analogues. In CV026, violacein is inducible by all the AHL and AHT compounds evaluated with N-acyl side chains from C4 to C8 in length, with varying degrees of sensitivity. Although AHL compounds with N-acyl side chains from C10 to C14 are unable to induce violacein production, if an activating AHL (e.g. HHL) is incorporated into the agar, these long-chain AHLs can be detected by their ability to inhibit violacein production. The versatility of CV026 in facilitating detection of AHL mixtures extracted from culture supernatants and separated by thin-layer chromatography is also demonstrated. These simple bioassays employing CV026 thus greatly extend the ability to detect a wide spectrum of AHL signal molecules.

The LuxR family protein SpnR functions as a negative regulator of N-acylhomoserine lactone-dependent quorum sensing in Serratia marcescens.

Serratia marcescens SS-1 produces at least four N-acylhomoserine lactones (AHLs) which were identified using high-resolution mass spectrometry and chemical synthesis, as N-(3-oxohexanoyl) homo-serine lactone (3-oxo-C6-HSL), N-hexanoyl- (C6-HSL), N-heptanoyl (C7-HSL) and N-octanoyl- (C8-HSL) homoserine lactone. These AHLs are synthesized via the LuxI homologue SpnI, and regulate via the LuxR homologue SpnR, the production of the red pigment, prodigiosin, the nuclease, NucA, and a biosurfactant which facilitates surface translocation. spnR overexpression and spnR gene deletion show that SpnR, in contrast to most LuxR homologues, acts as a negative regulator. spnI overexpression, the provision of exogenous AHLs and spnI gene deletion suggest that SpnR is de-repressed by 3-oxo-C6-HSL. In addition, long chain AHLs antagonize the biosurfactant-mediated surface translocation of S. marcescens SS-1. Upstream of spnI there is a gene which we have termed spnT. spnI and spnT form an operon and although database searches failed to reveal any spnT homologues, overexpression of this novel gene negatively affected both sliding motility and prodigiosin production.