Substrate Report for: 3-oxo-C12-HSL

OBJECTIVE: Bacterial colonization relies on communication between bacteria via so-called "quorum-sensing molecules", which include the acyl-homoserine lactone group. Certain acyl-homoserine lactones can modulate mammalian cell function and are thought to contribute to bacterial pathogenicity. Given the role of mast cells in host defense, we investigated the ability of acyl-homoserine lactones to modulate mast cell function.
METHODS: We utilized murine primary mast cell cultures to assess the effect of acyl-homoserine lactones on degranulation and cytokine release in response to different stimuli. We also assessed cell migration in response to chemoattractants. The effect of acyl-homoserine lactones in vivo was tested using a passive cutaneous anaphylaxis model.
RESULTS: Two of the tested quorum-sensing molecules, N-3-oxo-dodecanoyl-L-homoserine lactone and N-Dodecanoyl-L-homoserine lactone, inhibited IgE dependent and independent degranulation and mediator release from primary mast cells. Further testing of N-3-oxo-dodecanoyl-L-homoserine lactone, the most potent inhibitor and a product of Pseudomonas aeruginosa, revealed that it also attenuated chemotaxis and LPS induced cytokine production. In vivo, N-3-oxo-dodecanoyl-L-homoserine lactone inhibited the passive cutaneous anaphylaxis response in mice. CONCLUSION: The ability of N-3-oxo-dodecanoyl-L-homoserine lactone to stabilize mast cells may contribute to the pathogenicity of P. aeruginosa but could potentially be exploited therapeutically in allergic disease.

The soil isolate Ochrobactrum sp. A44 inactivates N-acyl homoserine lactone (AHL) quorum sensing signal molecules and is capable of quenching the AHL-dependent virulence of Pectobacterium carotovorum in planta. To characterize this AHL inactivating activity, Ochrobactrum cell extracts were prepared and their capacity to degrade a broad range of AHLs was determined. AHLs with acyl chains ranging from C4 to C14 with or without 3-oxo or 3-hydroxy substituents were all inactivated to varying extents; long chain AHLs were generally more susceptible than short chain compounds irrespective of the three position substituent. HPLC and LC-tandem mass spectrometry of the AHL degradation products revealed that the AHL inactivating activity present in the Ochrobactrum cell extract cleaved the AHL amide bond. To identify the gene(s) responsible for AHL degradation, Ochrobactrum sp. A44 was subjected to random transposon (Tn) mutagenesis and the resulting mutants screened for the loss of AHL acylase activity. The Tn insertion in mutant A6731 was mapped to a gene termed aiiO, the translated product of which belongs to the alpha/beta hydrolase superfamily which constitutes a novel type of AHL acylase.

Many Gram-positive and Gram-negative bacteria communicate using small diffusible signal molecules called autoinducers. This process, known as quorum sensing (QS), links cell density to the expression of genes as diverse as those associated with virulence factors production of plant and animal pathogens, bioluminescence, antibiotic production, sporulation or biofilm formation. In Gram-negative bacteria, this communication is mainly mediated by N-acyl-homoserine lactones (AHLs). It has been proven that inactivation of the signal molecules attenuates many of the processes controlled by QS. Enzymatic degradation of the signal molecules has been amply described. Two main classes of AHL-inactivating enzymes were identified: AHL lactonases which hydrolyse the lactone ring in AHLs, and AHL acylases (syn. AHL amidases) which liberate a free homoserine lactone and a fatty acid. Recently, AHL oxidoreductase, a novel type of AHL inactivating enzyme, was described. The activity of these enzymes results in silencing the QS-regulated processes, as degradation products cannot act as signal molecules. The ability to inactivate AHL (quorum quenching, QQ) might be useful in controlling virulence of many pathogenic bacteria.

OBJECTIVE: Bacterial colonization relies on communication between bacteria via so-called "quorum-sensing molecules", which include the acyl-homoserine lactone group. Certain acyl-homoserine lactones can modulate mammalian cell function and are thought to contribute to bacterial pathogenicity. Given the role of mast cells in host defense, we investigated the ability of acyl-homoserine lactones to modulate mast cell function.
METHODS: We utilized murine primary mast cell cultures to assess the effect of acyl-homoserine lactones on degranulation and cytokine release in response to different stimuli. We also assessed cell migration in response to chemoattractants. The effect of acyl-homoserine lactones in vivo was tested using a passive cutaneous anaphylaxis model.
RESULTS: Two of the tested quorum-sensing molecules, N-3-oxo-dodecanoyl-L-homoserine lactone and N-Dodecanoyl-L-homoserine lactone, inhibited IgE dependent and independent degranulation and mediator release from primary mast cells. Further testing of N-3-oxo-dodecanoyl-L-homoserine lactone, the most potent inhibitor and a product of Pseudomonas aeruginosa, revealed that it also attenuated chemotaxis and LPS induced cytokine production. In vivo, N-3-oxo-dodecanoyl-L-homoserine lactone inhibited the passive cutaneous anaphylaxis response in mice. CONCLUSION: The ability of N-3-oxo-dodecanoyl-L-homoserine lactone to stabilize mast cells may contribute to the pathogenicity of P. aeruginosa but could potentially be exploited therapeutically in allergic disease.

Many pathogenic bacteria that infect humans, animals and plants rely on a quorum-sensing (QS) system to produce virulence factors. N-Acyl homoserine lactones (AHLs) are the best-characterized cell-cell communication signals in QS. The concentration of AHL plays a key role in regulating the virulence-gene expression and essential biological functions of pathogenic bacteria. N-Acyl homoserine lactonases (AHL-lactonases) have important functions in decreasing pathogenicity by degrading AHLs. Here, structures of the AHL-lactonase from Ochrobactrum sp. (AidH) in complex with N-hexanoyl homoserine lactone, N-hexanoyl homoserine and N-butanoyl homoserine are reported. The high-resolution structures together with biochemical analyses reveal convincing details of AHL degradation. No metal ion is bound in the active site, which is different from other AHL-lactonases, which have a dual Lewis acid catalysis mechanism. AidH contains a substrate-binding tunnel between the core domain and the cap domain. The conformation of the tunnel entrance varies with the AHL acyl-chain length, which contributes to the binding promiscuity of AHL molecules in the active site. It also supports the biochemical result that AidH is a broad catalytic spectrum AHL-lactonase. Taken together, the present results reveal the catalytic mechanism of the metal-independent AHL-lactonase, which is a typical acid-base covalent catalysis.

The pathogen Pseudomonas aeruginosa causes serious damage in immunocompromised patients by secretion of various virulence factors, among them the quorum sensing N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) and the redox-active pyocyanin (PCN). Paraoxonase-2 (PON2) may protect against P. aeruginosa infections, as it efficiently inactivates 3OC12 and diminishes PCN-induced oxidative stress. This defense could be circumvented because 3OC12 mediates intracellular Ca(2+)-rise in host cells, which causes rapid inactivation and degradation of PON2. Importantly, we recently found that the PON2 paralogue PON3 prevents mitochondrial radical formation. Here we investigated its role as additional potential defense mechanism against P. aeruginosa infections. Our studies demonstrate that PON3 diminished PCN-induced oxidative stress. Moreover, it showed clear anti-inflammatory potential by protecting against NF-kappaB activation and IL-8 release. The latter similarly applied to PON2. Furthermore, we observed a Ca(2+)-mediated inactivation and degradation of PON3, again in accordance with previous findings for PON2. Our results suggest that the anti-oxidative and anti-inflammatory functions of PON2 and PON3 are an important part of our innate defense system against P. aeruginosa infections. Furthermore, we conclude that P. aeruginosa circumvents PON3 protection by the same pathway as for PON2. This may help identifying underlying mechanisms in order to sustain the protection afforded by these enzymes.

The soil isolate Ochrobactrum sp. A44 inactivates N-acyl homoserine lactone (AHL) quorum sensing signal molecules and is capable of quenching the AHL-dependent virulence of Pectobacterium carotovorum in planta. To characterize this AHL inactivating activity, Ochrobactrum cell extracts were prepared and their capacity to degrade a broad range of AHLs was determined. AHLs with acyl chains ranging from C4 to C14 with or without 3-oxo or 3-hydroxy substituents were all inactivated to varying extents; long chain AHLs were generally more susceptible than short chain compounds irrespective of the three position substituent. HPLC and LC-tandem mass spectrometry of the AHL degradation products revealed that the AHL inactivating activity present in the Ochrobactrum cell extract cleaved the AHL amide bond. To identify the gene(s) responsible for AHL degradation, Ochrobactrum sp. A44 was subjected to random transposon (Tn) mutagenesis and the resulting mutants screened for the loss of AHL acylase activity. The Tn insertion in mutant A6731 was mapped to a gene termed aiiO, the translated product of which belongs to the alpha/beta hydrolase superfamily which constitutes a novel type of AHL acylase.

Many Gram-positive and Gram-negative bacteria communicate using small diffusible signal molecules called autoinducers. This process, known as quorum sensing (QS), links cell density to the expression of genes as diverse as those associated with virulence factors production of plant and animal pathogens, bioluminescence, antibiotic production, sporulation or biofilm formation. In Gram-negative bacteria, this communication is mainly mediated by N-acyl-homoserine lactones (AHLs). It has been proven that inactivation of the signal molecules attenuates many of the processes controlled by QS. Enzymatic degradation of the signal molecules has been amply described. Two main classes of AHL-inactivating enzymes were identified: AHL lactonases which hydrolyse the lactone ring in AHLs, and AHL acylases (syn. AHL amidases) which liberate a free homoserine lactone and a fatty acid. Recently, AHL oxidoreductase, a novel type of AHL inactivating enzyme, was described. The activity of these enzymes results in silencing the QS-regulated processes, as degradation products cannot act as signal molecules. The ability to inactivate AHL (quorum quenching, QQ) might be useful in controlling virulence of many pathogenic bacteria.

Quorum sensing system is a cell-to-cell communication system that plays a pivotal role in virulence expression in bacteria. Recent advances have demonstrated that the Pseudomonas aeruginosa quorum sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC(12)-HSL), exerts effects on mammalian cells and modulates host immune response. Mast cells (MCs) are strategically located in the tissues that are constantly exposed to external stimulus. Therefore, it is very much possible that 3OC(12)-HSL may interact with MCs. Little is known, however, about specific effects of 3OC(12)-HSL on MCs. To address this, we investigated the influence of 3OC(12)-HSL on cell viability, apoptosis, intracellular calcium and cytokine release in MCs. We found that at high concentrations (100 microM), 3OC(12)-HSL inhibited proliferation and induced apoptosis in P815. The 3OC(12)-HSL treatment significantly increased intracellular calcium release in both P815 and HMC-1. We also observed that 3OC(12)-HSL-induced histamine release and degranulation in HMC-1 cells. Furthermore, 3OC(12)-HSL-induced IL-6 production at lower concentrations (6.25-12.5 microM) but steadily reduced IL-6 production at high concentration (50-100 muM). These data demonstrate that P. aeruginosa 3OC(12)-HSL affects MCs function.