Alleviation of insulitis and moderation of diabetes in NOD mice following treatment with a synthetic Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone.

Quorum sensing signal molecules (QSSMs) from the bacterium Pseudomonas aeruginosa control bacterial population density and the expression of virulence determinants. Coincidentally, and possibly to allow this pathogen to gain a foothold in the human body, certain signal molecules also downregulate immunological responses in an apparently T-helper 1-selective manner, which would suggest their application as therapeutics to some autoimmune diseases. In the present paper, experiments are described that indicate that one particular signal molecule, a synthetic N-(3-oxododecanoyl)-L-homoserine lactone, can be used to alleviate insulitis and diabetes in non-obese diabetic (NOD) mice, suggesting that bacterial signal molecules may represent a novel source of immune modulatory compounds for the treatment of type 1 diabetes, which afflicts more than 2 million individuals in Europe and North America.

Structure, activity and evolution of the group I thiolactone peptide quorum-sensing system of Staphylococcus aureus.

In Staphylococcus aureus, the agr locus is responsible for controlling virulence gene expression via quorum sensing. As the blockade of quorum sensing offers a novel strategy for attenuating infection, we sought to gain novel insights into the structure, activity and turnover of the secreted staphylococcal autoinducing peptide (AIP) signal molecules. A series of analogues (including the L-alanine and D-amino acid scanned peptides) was synthesized to determine the functionally critical residues within the S. aureus group I AIP. As a consequence, we established that (i) the group I AIP is inactivated in culture supernatants by the formation of the corresponding methionyl sulphoxide; and (ii) the group I AIP lactam analogue retains the capacity to activate agr, suggesting that covalent modification of the AgrC receptor is not a necessary prerequisite for agr activation. Although each of the D-amino acid scanned AIP analogues retained activity, replacement of the endocyclic amino acid residue (aspartate) located C-terminally to the central cysteine with alanine converted the group I AIP from an activator to a potent inhibitor. The screening of clinical S. aureus isolates for novel AIP groups revealed a variant that differed from the group I AIP by a single amino acid residue (aspartate to tyrosine) in the same position defined as critical by alanine scanning. Although this AIP inhibits group I S. aureus strains, the producer strains possess a functional agr locus dependent on the endogenous peptide and, as such, constitute a fourth S. aureus AIP pheromone group (group IV). The addition of exogenous synthetic AIPs to S. aureus inhibited the production of toxic shock syndrome toxin (TSST-1) and enterotoxin C3, confirming the potential of quorum-sensing blockade as a therapeutic strategy.

Haemodynamic effects of the bacterial quorum sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, in conscious, normal and endotoxaemic rats.

N-acylhomoserine lactones (AHLs) are small, diffusible signalling molecules, employed by Gram-negative bacteria to coordinate gene expression with cell population density. Recent in vitro findings indicate that AHLs may function as virulence determinants per se, through modification of cytokine production by eukaryotic cells, and by stimulating the relaxation of blood vessels. In the present study, we assessed the influence of AHLs on cardiovascular function in conscious rats, and draw attention to the ability of the N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), a signal molecule produced by P. aeruginosa, to cause marked bradycardia. This bradycardic effect was blocked by atropine and atenolol, and did not occur in vitro. Furthermore, modification of the acyl side chain length resulted in the loss of activity, whereas removal of the homoserine lactone ring, did not. The bradycardic effect of 3-oxo-C12-HSL was also observed in endotoxaemic animals, albeit attenuated. In normal rats, 3-oxo-C12-HSL caused initial mesenteric and hindquarters vasoconstriction, but only slight, and delayed signs of vasodilatation in the renal and mesenteric vascular beds. Furthermore, administration of 3-oxo-C12-HSL (pre-treatment or 2 h post-treatment) together with LPS, did not modify the established regional haemodynamic effects of the LPS, 6 h after the onset of its infusion. Our observations do not provide any clear evidence for an ability of 3-oxo-C12-HSL to modify the haemodynamic responses to LPS infusion. However, they are not inconsistent with the hypothesis that some of the cardiovascular sequelae of bacterial infection may be modulated by an influence of bacterial quorum sensing signalling molecules on the host.

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study.

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.

Quorum sensing and the population-dependent control of virulence.

One crucial feature of almost all bacterial infections is the need for the invading pathogen to reach a critical cell population density sufficient to overcome host defences and establish the infection. Controlling the expression of virulence determinants in concert with cell population density may therefore confer a significant survival advantage on the pathogen such that the host is overwhelmed before a defence response can be fully initiated. Many different bacterial pathogens are now known to regulate diverse physiological processes including virulence in a cell-density-dependent manner through cell-cell communication. This phenomenon, which relies on the interaction of a diffusible signal molecule (e.g. an N-acylhomoserine lactone) with a sensor or transcriptional activator to couple gene expression with cell population density, has become known as 'quorum sensing'. Although the size of the 'quorum' is likely to be highly variable and influenced by the diffusibility of the signal molecule within infected tissues, nevertheless quorum-sensing signal molecules can be detected in vivo in both experimental animal model and human infections. Furthermore, certain quorum-sensing molecules have been shown to possess pharmacological and immunomodulatory activity such that they may function as virulence determinants per se. As a consequence, quorum sensing constitutes a novel therapeutic target for the design of small molecular antagonists capable of attenuating virulence through the blockade of bacterial cell-cell communication.

N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia.

Quorum sensing via an N-acyl homoserine lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, N-(3-oxohexanoyl)-L-homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 microM, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross-linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.

The Pseudomonas aeruginosa quorum-sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, inhibits porcine arterial smooth muscle contraction.

The Pseudomonas aeruginosa quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to suppress cytokine production in macrophages. We have examined the effect of OdDHL and related compounds on constrictor tone of porcine blood vessels. OdDHL (1-30 microM) caused a concentration-dependent inhibition of U46619-induced contractions of the coronary artery through a largely endothelium-independent mechanism, but was markedly less effective in the pulmonary artery. Quantitively similar effects to those produced by OdDHL were observed with N-(3-oxododecanoyl)-L-homocysteine thiolactone, a thiolactone derivative, while N-3-oxododecanamide, a lactone-free acyl analogue, possessed 1/3rd the potency as a vasorelaxant. Neither N-butanoyl-L-homoserine lactone nor L-homoserine lactone (up to 30 microM) were active. Our findings indicate that OdDHL inhibits vasoconstrictor tone of both pulmonary and coronary blood vessels from the pig. The vasorelaxant action of OdDHL appears to be primarily determined by the N-acyl chain length, with a minor contribution by the homoserine lactone moiety.

Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other gram-negative bacteria.

In cell-free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N-acylhomoserine lactone (AHL) biosensor. Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(DeltaAla-L-Val) and cyclo(L-Pro-L-Tyr) respectively. These compounds were also found in cell-free supernatants from Proteus mirabilis, Citrobacter freundii and Enterobacter agglomerans [cyclo(DeltaAla-L-Val) only]. Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes, we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo(L-Phe-L-Pro). Dose-response curves using a LuxR-based AHL biosensor indicated that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) activate the biosensor in a concentration-dependent manner, albeit at much higher concentrations than the natural activator N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL). Competition studies showed that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) antagonize the 3-oxo-C6-HSL-mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR-binding site. Similarly, DKPs were found to be capable of activating or antagonizing other LuxR-based quorum-sensing systems, such as the N-butanoylhomoserine lactone-dependent swarming motility of Serratia liquefaciens. Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.

Molecular genetics of carbapenem antibiotic biosynthesis.

Carbapenems are potent beta-lactam antibiotics with a broad spectrum of activity against both Gram positive and Gram negative bacteria. As naturally produced metabolites, they have been isolated from species of Streptomyces, Erwinia and Serratia. The latter two members of the Enterobacteriaceae have proved to be genetically amenable and a growing body of research on these organisms now exists concerning the genes responsible for carbapenem biosynthesis and the regulatory mechanisms controlling their expression. A cluster of nine carbapenem (car) genes has been identified on the chromosome of Erwinia carotovora. These genes encode the enzymes required for construction of carbapenem and the proteins responsible for a novel beta-lactam resistance mechanism, conferring carbapenem immunity in the producing host. Although sharing no homology with the well known enzymes of penicillin biosynthesis, two of the encoded proteins are apparently similar to enzymes of the clavulanic acid biosynthetic pathway implying a common mechanism for construction of the beta-lactam ring. In addition, a transcriptional activator is encoded as the first gene of the carbapenem cluster and this allows positive expression of the remaining downstream genes in response to a quorum sensing, N-acyl homoserine lactone, signalling molecule.

Chitinolytic activity in Chromobacterium violaceum: substrate analysis and regulation by quorum sensing.

Quorum sensing control mediated by N-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In Chromobacterium violaceum ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL N-hexanoyl-L-homoserine lactone (HHL). In this study we show that C. violaceum produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using p-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of N-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of N-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two N-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn5 mutant of C. violaceum (CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the C. violaceum wild-type strain or 10 microM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.

Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing.

Plasmid reporter vectors have been constructed which respond to activation of LuxR and its homologues LasR and RhlR (VsmR) by N-acyl homoserine lactones (AHLs). The expression of luxCDABE from transcriptional fusions to PluxI, PlasI and PrhlI respectively, occurs in the presence of activating AHLs. A profile of structure/activity relationships is seen where the natural ligand is most potent. The characterisation of individual LuxR homologue/AHL combinations allows a comprehensive evaluation of quorum sensing signals from a test organism.

Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs.

The luxCDABE operon of Photorhabdus luminescens has been cloned and engineered as an easily mobilisable cassette flanked by sites for commonly used restriction enzymes. Constitutive and promoter probe plasmids utilising the P. luminescens luxCDABE have been constructed using a number of compatible replicons and antibiotic markers. Complementary to these plasmids, a range of promoterless and constitutive luxCDABE mini-Tn5 derivatives has been constructed. The potential of coupling mini-Tn5 luxCDABE promoter probe transposons with automated luminometry and photometry to screen for mutants that exhibit growth phase variation in gene expression is demonstrated.

Bacterial production of carbapenems and clavams: evolution of beta-lactam antibiotic pathways.

Research into two of the four classes of naturally produced beta-lactams--the clavams and carbapenems--has started to throw light upon their biochemical pathways and underlying genetics. Interesting similarities between these two classes, from their joint discovery to an apparently common beta-lactam ring-forming enzyme, are now being revealed.

In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone.

In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylhomoserine lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.

A pheromone-independent CarR protein controls carbapenem antibiotic synthesis in the opportunistic human pathogen Serratia marcescens.

Strain ATCC 39006 of Serratia marcescens makes the same carbapenem, (5R)-carbapen-2-em-3-carboxylic acid (Car), as the Erwinia carotovora strain GS101. Unlike E. carotovora, where the onset of production occurs in the late-exponential phase of growth in response to the accumulation of the small diffusible pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), in S. marcescens carbapenem is produced throughout the growth phase and does not appear to involve any diffusible pheromone molecule. Two cosmids capable of restoring antibiotic production in E. carotovora group I carbapenem mutants were isolated from an S. marcescens gene library. These cosmids were shown to contain a homologue of the E. carotovora carR gene, encoding a CarR protein with homology to the LuxR family of transcriptional regulators. The S. marcescens carR was subcloned and shown to be capable of complementing in trans, in the absence of OHHL, an E. carotovora carR carI double mutant, releasing the heterologous E. carotovora host from pheromone dependence for carbapenem production. The apparent OHHL-independence of the S. marcescens CarR explains the constitutive nature of carbapenem production in this strain of S. marcescens.

The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone has immunomodulatory activity.

Diverse gram-negative bacterial cells communicate with each other by using diffusible N-acyl homoserine lactone (AHL) signal molecules to coordinate gene expression with cell population density. Accumulation of AHLs above a threshold concentration renders the population "quorate," and the appropriate target gene is activated. In pathogenic bacteria, such as Pseudomonas aeruginosa, AHL-mediated quorum sensing is involved in the regulation of multiple virulence determinants. We therefore sought to determine whether the immune system is capable of responding to these bacterial signal molecules. Consequently the immunomodulatory properties of the AHLs N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) were evaluated in murine and human leukocyte immunoassays in vitro. OdDHL, but not OHHL, inhibited lymphocyte proliferation and tumor necrosis factor alpha production by lipopolysaccharide-stimulated macrophages. Furthermore, OdDHL simultaneously and potently down-regulated the production of IL-12, a Th-1-supportive cytokine. At high concentrations (>7 x 10(-5) M) OdDHL inhibited antibody production by keyhole limpet hemocyanin-stimulated spleen cells, but at lower concentrations (<7 x 10(-5) M), antibody production was stimulated, apparently by increasing the proportion of the immunoglobulin G1 (IgG1) isotype. OdDHL also promoted IgE production by interleukin-4-stimulated human peripheral blood mononuclear cells. These data indicate that OdDHL may influence the Th-1-Th-2 balance in the infected host and suggest that, in addition to regulating the expression of virulence determinants, OdDHL may contribute to the pathogenesis of P. aeruginosa infections by functioning as a virulence determinant per se.

The rap and hor proteins of Erwinia, Serratia and Yersinia: a novel subgroup in a growing superfamily of proteins regulating diverse physiological processes in bacterial pathogens.

The enteric bacterium Serratia marcescens is an opportunistic human pathogen. The strain ATCC39006 makes the red pigment, prodigiosin (Pig), and the beta-lactam antibiotic carbapenem (Car). Mutants were isolated that were concomitantly defective for Pig and Car production. These mutants were found to have a mutation in the rap gene (Regulation of Antibiotic and Pigment). Sequence analysis of the rap gene revealed a predicted protein product showing strong homology to SlyA, originally thought to be a haemolytic virulence determinant in Salmonella typhimurium. Homologues of rap were detected in several bacterial genera, including Salmonella, Yersinia, Enterobacter, and species of the plant pathogen, Erwinia. The Erwinia hoeEr (homologue of rap) and the Yersinia horYe genes were also found to be very similar to rap and slyA. Marker exchange mutagenesis of horEr revealed that it encoded a regulatory protein controlling the production of antibiotic and exoenzyme virulence determinants in the phytopathogen, Erwinia carotovora subspecies carotovora. We have shown that these new homologues of SlyA form a highly conserved subgroup of a growing superfamily of bacterial regulatory proteins controlling diverse physiological processes in human, animal and plant pathogens.

Analysis of the carbapenem gene cluster of Erwinia carotovora: definition of the antibiotic biosynthetic genes and evidence for a novel beta-lactam resistance mechanism.

Members of two genera of Gram-negative bacteria, Serratia and Erwinia, produce a beta-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have reported previously the cloning and sequencing of the genes responsible for production of this carbapenem in Erwinia carotovora. These genes are organized as an operon, carA--H, and are controlled by a LuxR-type transcriptional activator, encoded by the linked carR gene. We report in this paper the genetic dissection of this putative operon to determine the function of each of the genes. We demonstrate by mutational analysis that the products of the first five genes of the operon are involved in the synthesis of the carbapenem molecule. Three of these, carABC, are absolutely required. In addition, we provide evidence for the existence of a novel carbapenem resistance mechanism, encoded by the CarF and carG genes. Both products of these overlapping and potentially translationally coupled genes have functional, N-terminal signal peptides. Removal of these genes from the Erwinia chromosome results in a carbapenem-sensitive phenotype. We assume that these novel beta-lactam resistance genes have evolved in concert with the biosynthetic genes to ensure 'self-resistance' in the Erwinia carbapenem producer.

Bacterial N-acyl-homoserine-lactone-dependent signalling and its potential biotechnological applications.

N-acyl homoserine lactones are bacterial signalling molecules involved in regulating diverse metabolic functions, particularly those relating to virulence, in concert with cell density. Each aspect of the signalling pathway, from production and recognition of the signal to expression of the target genes, offers a potential opportunity for exploitation. Attention is now focusing on the development of novel methods for bacterial enumeration, modulation of bacterial virulence and flexible, coordinated expression of heterologous genes through the use of N-acyl-homoserine-lactone-based systems.