Production and properties of an inhibitor of the Pseudomonas autoinducer by Pseudomonas aeruginosa

An inhibitor was found in the culture fluid of Pseudomonas aeruginosa PAO1, which could inhibit the activity of the Pseudomonas autoinducer (PAI). The maximal inhibitory activity occurred in stationary phase culture sup ernatant. The PAI inhibitor did not influence the cell growth and the PAI production by P. aeruginosa PAO1 when the PAI inhibitor was added into culture medium. The induced expression of lacZ in the reporter strain Agrobacterium tumefaciens NT1 was suppressed by this PAI inhibitor, whereas inhibition could be relieved by increasing the auto inducer concentration. The quorum sensing of P. aeruginosa was inhibited presumably by inhibiting the inducing activity of Pseudomonas autoinducer but not by inhibiting the production of Pseudomonas autoinducer. It was demonstrated that the structure of the PAI inhibitor was different from that of acyl-homoserine lactones.     

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.     

Metagenome-derived clones encoding two novel lactonase family proteins involved in biofilm inhibition in Pseudomonas aeruginosa

Here we report the isolation and characterization of three metagenome-derived clones that interfere with bacterial quorum sensing and degrade N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C(8)-HSL). By using a traI-lacZ gene fusion, the metagenome-derived clones were identified from a soil DNA library and analyzed. The open reading frames linked to the 3-oxo-C(8)-HSL-degrading activities were designated bpiB01, bpiB04, and bpiB07. While the BpiB07 protein was similar to a known lactonase, no significant similarities were observed for the BpiB01 and BpiB04 proteins or the deduced amino acid sequences. High-performance liquid chromatography-mass spectrometry analyses confirmed that the identified genes encode novel lactone-hydrolyzing enzymes. The original metagenome-derived clones were expressed in Pseudomonas aeruginosa and employed in motility and biofilm assays. All clones were able to reproducibly inhibit motility in P. aeruginosa. Furthermore, these genes clearly inhibited biofilm formation in P. aeruginosa when expressed in P. aeruginosa PAO1. Thus, this is the first study in which metagenome-derived proteins have been expressed in P. aeruginosa to successfully inhibit biofilm formation.     

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.     

Quorum Sensing Inhibition in Pseudomonas aeruginosa PAO1 by Antagonistic Compound Phenylacetic Acid

In Pseudomonas aeruginosa, quorum sensing (QS) autoinducer known as acyl homoserine lactone (AHL) acts as a key regulator in the expression of pathogenic characters. In this work, the efficiency of phenylacetic acid (PAA) in reducing the production of AHL-dependent factors in P. aeruginosa PAO1 was studied. PAA at a concentration of 200?μg?ml(-1) displayed significant reduction in QS-dependent pyocyanin, exopolysaccharide, and protease and elastase production in PAO1. In swimming inhibition assay, PAA-treated PAO1 cells exhibited poor motility in swimming agar plate. In in vivo analysis, PAO1-preinfected Caenorhabditis elegans showed enhanced survival when treated with PAA. PAA at the QS inhibitory concentration showed no growth inhibitory activity on PAO1. Results of the present study revealed the potential of PAA as antipathogenic compound to prevent QS-dependent pathogenicity of P. aeruginosa.     

Characterization of wetland quorum quenching Pseudomonas aeruginosa strain 2SW8 and its 2-heptyl-3-hydroxy-4-quinolone production

Most Proteobacteria produce N-acylhomoserine lactones for bacterial cell-to-cell communication, a process called quorum sensing. Interference of quorum sensing, commonly known as quorum quenching, represents an important way to control quorum sensing. This work reports the isolation of quorum quenching bacterium strain 2WS8 from Malaysia tropical wetland water (2°11'8"N, 102°15'2"E, in 2007) by using a modified version of a previously reported KG medium. Strain 2WS8 was isolated based on its ability to utilize N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) as the sole source of energy. This bacterium clustered closely to Pseudomonas aeruginosa PAO1. Strain 2SW8 possesses both quiP and pvdQ homologue acylase genes. Rapid Resolution Liquid Chromatography analysis confirmed that strain 2SW8 preferentially degraded N-acylhomoserine lactones with 3-oxo group substitution but not those with unsubstituted groups at C3 position in the acyl side chain. Strain 2SW8 also showed 2-heptyl-3-hydroxy-4-quinolone production.     

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 (C₆-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 (C₉-CPA), had a strong inhibitory effect on prodigiosin production. C₉-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C₉-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C₆-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C₉-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Quorum sensing regulates denitrification in Pseudomonas aeruginosa PAO1

Anaerobic growth of Pseudomonas aeruginosa PAO1 was affected by quorum sensing. Deletion of genes that produce N-acyl-l-homoserine lactone signals resulted in an increase in denitrification activity, which was repressed by exogenous signal molecules. The effect of the las quorum-sensing system was dependent on the rhl quorum-sensing system in regulating denitrification.     

A new quorum-sensing inhibitor attenuates virulence and decreases antibiotic resistance in Pseudomonas aeruginosa

Quorum sensing (QS) has been a novel target for the treatment of infectious diseases. Here structural analogs of Pseudomonas aeruginosa autoinducer N-acyl homoserine lactone (AHL) were investigated for QS inhibitor (QSI) activity and a novel QSI was discovered, N-decanoyl-L-homoserine benzyl ester (C2). Virulence assays showed that C2 down-regulated total protease and elastase activities, as well as the production of rhamnolipid, that are controlled by QS in P. aeruginosa wild-type strain PAO1 without affecting growth. C2 was also shown to inhibit swarming motility of PAO1. Using a microdilution checkerboard method, we identified synergistic interactions between C2 and several antibiotics, tobramycin, gentamycin, cefepime, and meropenem. Data from real-time RT-PCR suggested that C2 inhibited the expression of lasR (29.67%), lasI (21.57%), rhlR (28.20%), and rhlI (29.03%).     

Quorum sensing system lactones do not increase invasiveness of a MexAB-OprM efflux mutant but do play a partial role in Pseudomonas aeruginosa invasion

We studied the quorum sensing (QS) system and the related homoserine lactones (HSLs) observing Pseudomonas aeruginosa invasion using the epithelial cell monolayer penetration assay model. Compared to the PAO1 wild-type, the QS mutants, DeltalasI and DeltarhlI, were compromised in their capacity to invade. The decreased invasiveness of DeltarhlI was restored by adding 100 microM exogenous C(4)-HSL. However, the decreased invasiveness of an efflux mutant, DeltamexAB-oprM, was not restored in the presence of exogenous HSLs. The QS system partially plays a role in P. aeruginosa invasion; however, C(4)-HSL and 3-O-C(12)-HSL are not the essential determinants for invasiveness for P. aeruginosa.     

Quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone influences siderophore biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa

Siderophore synthesis of Pseudomonas putida F1 was found to be regulated by quorum sensing since normalized siderophore production (per cell) increased 4.2-fold with cell density after the cells entered middle exponential phase; similarly, normalized siderophore concentrations in Pseudomonas aeruginosa JB2 increased 28-fold, and a 5.5-fold increase was seen for P. aeruginosa PAO1. Further evidence of the link between quorum sensing and siderophore synthesis of P. putida F1 was that the quorum-sensing-disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the marine red alga Delisea pulchra was found to inhibit the formation of the siderophore produced by P. putida F1 in a concentration-dependent manner, with 57% siderophore synthesis repressed by 100 g/ml furanone. In contrast, this furanone did not affect the siderophore synthesis of Burkholderia cepacia G4 at 20–40 g/ml, and stimulated siderophore synthesis of P. aeruginosa JB2 2.5- to 3.7-fold at 20–100 g/ml. Similarly, 100 g/ml furanone stimulated siderophore synthesis in P. aeruginosa PAO1 about 3.5-fold. The furanone appears to interact with the quorum-sensing machinery of P. aeruginosa PAO1 since it stimulates less siderophore synthesis in the P. aeruginosa qscR quorum-sensing mutant (QscR is a negative regulator of LasI, an acylated homoserine lactone synthase).     

Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.