LasR Protein Research Articles

Quorum quenching by 2-Hydroxyanisole extracted from Solanum torvum on Pseudomonas aeruginosa and its inhibitory action upon LasR protein

Among the list of the most dangerous superbugs, Pseudomonas aeruginosa ranks top of the table with its deadly characteristics of multi-drug resistance and ability to form biofilm during opportunistic infections to patients those are immune-compromised. MDX and XDR of this strain urged researchers to reveal better antibacterial compounds. Solanum torvum (commonly called as pea eggplant) is well known for its medicinal values however, the roots of this plant had less attention in pharmacological studies. The objective of this study is twofold. Firstly, to compare the interactions exhibited between2-Hydroxyanisole and odDHL with LasRprotein. Secondly, to explore the impact of molecule of interest on genes related to vital pathways. Three-dimensional docking studies for the two ligands were performed using surflex-dock package - Sybyl-x 2.0 whereas differentially regulated genes were identified by volcano plot, heat map prediction and KEGG pathway. It was predicted that 2-Hydroxyanisole may form three hydrogen bonds with LasR protein and revealed a decreased level of gene expression (%).After treatment with Solanum torvum Root Extract (STRE),a total of 26,418,456 reads of P. aeuroginosa were mapped to various pathways, of which, QS-regulated genes such as LasR, coIII (coxB), fliD genes along with 104 genes were down regulated. The high C Score calculated for 2-Hydroxyanisole and altered gene expression upon STRE treatment pave the pathway for designing the future in vitro experiments to validate these inhibitory effects thereby exploring the underlying mechanisms of action of 2-Hydroxyanisole to restrict the virulence of P. aeruginosa.

The outlier Pseudomonas aeruginosa strain ATCC 9027 harbors a defective LasR quorum-sensing transcriptional regulator.

Pseudomonas aeruginosa infections represent an important health problem that has been recognized by the World Health Organization as a research priority. A complex regulatory network called the quorum sensing (QS) regulates several P. aeruginosa virulence-related traits, including production of elastase, rhamnolipids and pyocyanin. The avirulent P. aeruginosa strain ATCC 9027 belongs to clade 3, which is the more distant phylogroup in relationship to the other four clades of this species. This strain does not produce QS-regulated virulence factors such as elastase and rhamnolipids when cultured in rich LB medium. We report here that ATCC 9027 harbors a defective LasR protein, presumably due to the presence of an aspartic acid in position 196 instead of a glutamic acid which is the amino acid present in this position in functional LasR proteins of the type strains PAO1 (clade 1) and PA7 (also belonging to clade 3), among others. In addition, we report that ATCC 9027 and PA7 strains present differences compared to the PAO1 strain in lasB which encodes elastase, and in the rhlR regulatory sequences that modify las-boxes, and that these mutations have a little effect in the expression of these genes by a functional LasR transcriptional regulator.

Tarragon essential oil as a source of bioactive compounds with anti-quorum sensing and anti-proteolytic activity against Pseudomonas spp. isolated from fish – in vitro, in silico and in situ approaches

The present study aimed to evaluate the anti-quorum sensing (anti-QS) and anti-proteolytic potentials of tarragon essential oil (TEO) and its major compounds against food-associated Pseudomonas spp. The activities were verified by in vitro, in silico and in situ approaches. In this work, methyl eugenol (ME)- and β-phellandrene (β-PH)-rich TEO was investigated. TEO at subMIC increased the percentage of saturated fatty acids in the bacterial membranes (from 7 to 22%) and exhibited anti-quorum sensing via decreasing the efficiency of QS autoinducer synthesis [3-oxo-C12-HSL (from 2.028 μg/mL to <LOD), C4-HSL (from 1.312 μg/mL to <LOD) and PQS (from 0.007625 μg/mL to <LOD)]. ME and β-PH were docked into LasR, RhlR and PqsR proteins, with docking scores comparable to native autoinductors. The subMICs of TEO, ME and β-PH decreased the proteolysis in the examined bacteria by 33, 29, and 21% (in TSB medium) and by 29, 26, and 19% (in fish juice medium), respectively. Almost all genes encoding proteases were downregulated by the applied agents. The ME- and β-PH-rich TEO acts as an anti-QS agent and significantly suppresses the proteolytic activity of food-associated pseudomonads. It might therefore increase the quality of fish-based products, where Pseudomonas spp. predominate.

Inhibition of biofilm and virulence properties of Pseudomonas aeruginosa by sub-inhibitory concentrations of aminoglycosides

Aminoglycosides are a commonly used class of antibiotics; however, their application has been discontinued due to the emergence of multi-drug resistance bacterial strains. In the present study, the subinhibitory concentrations (sub-MIC) of several aminoglycosides were determined and tested as an antibiofilm and for their anti-virulence properties against Pseudomonas aeruginosa PAO1, which is an opportunistic foodborne pathogen. P. aeruginosa PAO1 exhibits multiple mechanisms of resistance, including the formation of biofilm and production of several virulence factors, against aminoglycoside antibiotics. The sub-MIC of these antibiotics exhibited biofilm inhibition of P. aeruginosa in alkaline TSB (pH 7.9). Moreover, various concentrations of these aminoglycosides also eradicate the mature biofilm of P. aeruginosa. In the presence of sub-MIC of aminoglycosides, the morphological changes of P. aeruginosa were found to change from rod-shaped to the filamentous, elongated, and streptococcal forms. Similar growth conditions and sub-MIC of aminoglycosides were also found to attenuate several virulence properties of P. aeruginosa PAO1. Molecular docking studies demonstrate that these aminoglycosides possess strong binding properties with the LasR protein, which is a well-characterized quorum-sensing receptor of P. aeruginosa. The present study suggests a new approach to revitalize aminoglycosides as antibiofilm and antivirulence drugs to treat infections caused by pathogenic bacteria.

Inhibition of quorum sensing-associated virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1 by Mycoleptodiscus indicus PUTY1.

Pseudomonas aeruginosa is the second most emerging multidrug-resistant, opportunistic pathogen after Acinetobacter baumannii that poses a threat in nursing homes, hospitals, and patients who need devices such as ventilators and blood catheters. Its ability to form quorum sensing-regulated virulence factors and biofilm makes it more resistant to top most therapeutic agents such as carbapenems and next-generation antibiotics. In the current study, we studied the quorum quenching potential of secondary metabolites of Mycoleptodiscus indicus PUTY1 strain. In vitro observation showed a mitigation in virulence factors such as rhamnolipids, protease, elastase pyocyanin, exopolysaccharides, and hydrogen cyanide gas. Furthermore, a significant reduction in the motility such as swimming, swarming, twitching, and inhibition in biofilm formation by Pseudomonas aeruginosa PAO1 was observed. Results of in vitro studies were further confirmed by in silico studies through docking and molecular dynamic simulation of GC-MS-detected compounds of Mycoleptodiscus indicus employing LasR and RhlR proteins. Both in vitro and in silico observations indicate a new alternative approach for combating virulence of Pseudomonas aeruginosa by targeting its protein receptors LasR and RhlR. Graphical abstract.

Energy-optimized pharmacophore coupled virtual screening in the discovery of quorum sensing inhibitors of LasR protein of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an emerging opportunistic pathogen responsible for cystic fibrosis and nosocomial infections. In addition, empirical treatments are become inefficient due to their multiple-antibiotic resistance and extensive colonizing ability. Quorum sensing (QS) plays a vital role in the regulation of virulence factors in P. aeruginosa. Therefore, attenuation of virulence by QS inhibition could be an alternative and effective approach to control the infections. In this study, we sought to discover new QS inhibitors (QSIs) against LasR receptor in P. aeruginosa using chemoinformatics. Initially, a structure-based high-throughput virtual screening was performed using the LasR active site that identified 61404 relevant molecules. The e-pharmacophore (ADAHH) screening of these molecules rendered 72 QSI candidates. In standard-precision docking, only 7 compounds were found as potential QSIs based on their higher binding affinity to LasR receptor (−7.53 to −10.32 kcal/mol compared to −7.43 kcal/mol of native ligand). The ADMET properties of these compounds were suitable to be QSIs. Later, extra-precision docking and binding energy calculation suggested ZINC19765885 and ZINC72387263 as the most promising QSIs. The dynamic simulation of the docked complexes showed stable binding affinity and molecular interactions. The current study suggested that these two compounds could be used in P. aeruginosa QS inhibition to combat bacterial infections.Communicated by Ramaswamy H. Sarma

The interference of nonylphenol with bacterial cell-to-cell communication

The interference of nonylphenol (NP) with humans and animals, especially in hormone systems, has been well-studied. There is rarely any record of its effect on bacteria, which dominate in various environments. In our study, we employed Pseudomonas aeruginosa PAO1 as a model microorganism and took its common lifestyle biofilm, mainly regulated by quorum sensing (QS), as a cut-in point to investigate the effect of NP (1, 5, 10 mg L−1) on bacteria. The results showed that more than 5 mg L−1 of NP did interfere with biofilm formation and affected bacterial QS. In detail, the LasI/R circuit, but not the RhlI/R circuit, was considerably obstructed. The decrease in lasI and lasR expression resulted in a significant reduction in N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL) signals and the downstream production of elastases. Docking results indicated the binding of NP with LasR protein, simulating the binding of 3OC12-HSL with LasR protein, which explained the obstruction of the LasIR circuit. We concluded that NP competed with 3OC12-HSL and blocked 3OC12-HSL binding with the LasR protein, resulting in a direct interference in bacterial biofilm formation. This is the first report of NP interference with bacterial signaling, which is not only helpful to understand the effect of NP on various ecosystems, but is also beneficial to enrich our knowledge of inter-kingdom communication.

Effect of Quercetin Rich Onion Extracts on Bacterial Quorum Sensing.

Quorum sensing (QS) regulates bacterial gene expression and studies suggest quercetin, a flavonol found in onion, as a QS inhibitor. There are no studies showing the anti-QS activity of plants containing quercetin in its native glycosylated forms. This study aimed to evaluate the antimicrobial and anti-QS potential of organic extracts of onion varieties and its representative phenolic compounds quercetin aglycone and quercetin 3-β-D-glucoside in the QS model bacteria Chromobacterium violaceum ATCC 12472, Pseudomonas aeruginosa PAO1, and Serratia marcescens MG1. Three phenolic extracts were obtained: red onion extract in methanol acidified with 2.5% acetic acid (RO-1), white onion extract in methanol (WO-1) and white onion extract in methanol ammonium (WO-2). Quercetin 4-O-glucoside and quercetin 3,4-O-diglucoside were identified as the predominant compounds in both onion varieties using HPLC-DAD and LC-ESI-MS/MS. However, quercetin aglycone, cyanidin 3-O-glucoside and quercetin glycoside were identified only in RO-1. The three extracts showed minimum inhibitory concentration (MIC) values equal to or above 125 μg/ml of dried extract. Violacein production was significantly reduced by RO-1 and quercetin aglycone, but not by quercetin 3-β-D-glucoside. Motility in P. aeruginosa PAO1 was inhibited by RO-1, while WO-2 inhibited S. marcescens MG1 motility only in high concentration. Quercetin aglycone and quercetin 3-β-D-glucoside were effective at inhibiting motility in P. aeruginosa PAO1 and S. marcescens MG1. Surprisingly, biofilm formation was not affected by any extracts or the quercetins tested at sub-MIC concentrations. In silico studies suggested a better interaction and placement of quercetin aglycone in the structures of the CviR protein of C. violaceum ATCC 12472 than the glycosylated compound which corroborates the better inhibitory effect of the former over violacein production. On the other hand, the two quercetins were well placed in the AHLs binding pockets of the LasR protein of P. aeruginosa PAO1. Overall onion extracts and quercetin presented antimicrobial activity, and interference on QS regulated production of violacein and swarming motility.

Interaction of N-3-oxododecanoyl homoserine lactone with transcriptional regulator LasR of Pseudomonas aeruginosa: Insights from molecular docking and dynamics simulations

Background: In 2017, the World Health Organization announced a list of the most dangerous superbugs. Among them is Pseudomonas aeruginosa, an opportunistic human pathogen with high levels of resistance to antibiotics that is listed as one of the ‘ESKAPE’ pathogens, which are the leading cause of nosocomial infections. A major issue is that it mostly affects vulnerable patients such as those suffering from AIDS, cystic fibrosis, cancer and severe burns. P. aeruginosa creates and inhabits surface-associated biofilms which increase resistance to antibiotics and host immune responses and contribute to the ineffectiveness of current antibacterial treatments. It is therefore imperative to find new antibacterial treatment strategies against P. aeruginosa. The LasR protein is a major transcriptional activator of P. aeruginosa and plays a pivotal role in biofilm formation and the activation of many virulence genes, although detailed characteristics of the LasR protein are not currently known. In the present study, we aimed to analyse the molecular properties of the LasR protein as well as its interactions with the signalling molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL). Methods: We used a combination of molecular docking, molecular dynamics (MD) simulations and machine learning techniques to study the interaction of the LasR protein with the 3OC12-HSL ligand. We assessed conformational changes occurring upon their interaction and analysed the molecular details of their binding. Results: A new possible interaction site for 3OC12-HSL and LasR was found, involving conserved residues from the ligand binding domain (LBD), beta turns in the short linker region (SLR) and the DNA-binding domain (DBD). This interaction is referred to as the LBD-SLR-DBD bridge or ‘the bridge’ interaction. Conclusions: This study may enable future experimental studies to detect the interaction of signalling molecules with “the bridge” of the LasR protein and suggests a potential new interaction site to assist antibacterial drug design.

Virulence of Pseudomonas aeruginosa exposed to carvacrol: alterations of the Quorum sensing at enzymatic and gene levels.

The main goal of this study was to evaluate the inhibition of Pseudomonas aeruginosa virulence factors and Quorum Sensing during exposure to carvacrol. P. aeruginosa (ATCC 10154) was exposed to carvacrol determining changes in biofilm development, motility, acyl-homoserine lactones (AHL) synthesis and relative expression of lasI/lasR. Docking analysis was used to determinate interactions between carvacrol with LasI and LasR proteins. P. aeruginosa produced 60% lower AHLs when exposed to carvacrol (1.9mM) compared to control, without affecting cellular viability, indicating a reduction on the LasI synthase activity. AHL-C12, C6, and C4 were detected and related to biofilm development, motility, and pyocyanin production, respectively. The presence of carvacrol reduced the expression of lasR, without affecting lasI gen. Moreover, computational docking showed interactions of carvacrol with amino acids in the active site pocket of LasI (-5.6kcalmol-1) and within the binding pocket of LasR (-6.7kcalmol-1) of P. aeruginosa. These results demonstrated that virulence of P. aeruginosa was reduced by carvacrol, by inhibiting LasI activity with the concomitant reduction on the expression of lasR, biofilm and swarming motility. This study provides relevant information about the effect of carvacrol against quorum sensing to inhibit virulence factors of P. aeruginosa at enzymatic and gene levels. These findings can contribute to the development of natural anti-QS products, which can affect pathogenesis.

An Autoinducer Analogue Reveals an Alternative Mode of Ligand Binding for the LasR Quorum-Sensing Receptor

Bacteria use a cell-cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.

Antioxidant, anti-quorum sensing and anti-biofilm potential of ethanolic leaf extract of Phrynium capitatum and Dryptes indica

Objective: To investigate the antioxidant and anti-infective potential of Phrynium capitatum and Dryptes indica extract. Methods: The antioxidant potentials were determined by DPPH radical scavenging, reducing power, hydroxyl radical scavenging and total antioxidant assays. We further examined anti-quorum sensing activity and inhibition of synthesis of pathogenic factor of Chromobacterium violaceum and Pseudomonas aeruginosa PAO1. Bioactive compounds were determined using gas chromatography–mass spectrometry analysis. In silico analysis was conducted to determine the binding affinity of bioactive compounds of plant extracts for the quorum sensing regulatory receptor LasR. Results: DPPH assay showed that the ethanolic extract of Phrynium capitatum and Dryptes indica at 500 μg/mL showed (86.96 ± 4.07)% and (74.83 ± 3.47)% inhibition, respectively. Hydroxyl radical scavenging assay showed (73.17 ± 3.03)% and (62.63 ± 4.59)% activity, respectively. The ethanolic extract of Phrynium capitatum and Dryptes indica showed high level of attenuation of quorum sensing regulated pyocyanin production. Confocal laser scanning microscopic analysis revealed that the extracts had the potential to effectively inhibit biofilm formation of Pseudomonas aeruginosa. Molecular docking analysis showed a better binding affinity of bioactive compounds from the extracts for the structure of LasR protein of Pseudomonas aeruginosa. Conclusions: The ethanolic extracts of Phrynium capitatum and Dryptes indica possess antioxidant activity and the potential to inhibit the quorum sensing system and its regulatory irulence traits in Pseudomonas aeruginosa PAO1.

Quorum-sensing inhibitor potential of trans-anethole aganist Pseudomonas aeruginosa.

Quorum sensing (QS) is a cell-cell communication system used by a broad spectrum of pathogenic bacteria to control the expression of their virulence genes. The interruption of QS systems of pathogenic bacteria has been considered as a novel way to fight bacterial diseases. In this study, trans-anethole, the main component of anise (Pimpinella anisum) oil was examined for its QS inhibitor (QSI) potential in an attempt to identify novel QSI compound effective against opportunistic pathogen Pseudomonas aeruginosa. The preliminary screening of QSI capacity of trans-anethole was determined using a quorum-sensing inhibitor screen (QSIS) assay. The QSIS assay indicated that trans-anethole has QSI properties. QSI capacity of trans-anethole was further confirmed by lasB-gfp fussion assay and virulence factor assays. A sub-MIC of trans-anethole reduced the expression of lasB by 57%, elastase production by 59%, protease production by 56%, pyocyanin production by 95% and swarming motility by 68% without inhibiting growth of Pseudomonas aeruginosa PA01. Molecular docking and protein-ligand interaction studies were performed to understand the molecular mechanism underlying inhibitory activity of trans-anethole. The results of these analysis suggested that trans-anethole fits within the binding site of the LasR protein of P. aeruginosa. Trans-anethole has the potential to inhibit QS-regulated virulence factors in P. aeruginosa by binding to LasR protein, similar to its natural ligand N-(3-oxododecanoyl)-l-homoserine lactone. In this study, for the first time, it was demonstrated that trans-anethole has the potential to disrupt bacterial communication and can be developed as a novel QSI to combat with P. aeruginosa and other clinically significant pathogens.

Biofilm production by clinical isolates of Pseudomonas aeruginosa and structural changes in LasR protein of isolates non biofilm-producing

IntroductionBiofilm production is an important mechanism for the survival of Pseudomonas aeruginosa and its relationship with antimicrobial resistance represents a challenge for patient therapeutics. P. aeruginosa is an opportunistic pathogen frequently associated to nosocomial infections, especially in imunocompromised hosts. ObjectivesAnalyze the phenotypic biofilm production in P. aeruginosa isolates, describe clonal profiles, and analyze quorum sensing (QS) genes and the occurrence of mutations in the LasR protein of non-biofilm producing isolates. MethodsIsolates were tested for biofilm production by measuring cells adherence to the microtiter plates. Clonal profile analysis was carried out through ERIC-PCR, QS genes were by specific PCR. ResultsThe results showed that 77.5% of the isolates were considered biofilm producers. The results of genotyping showed 38 distinct genetic profiles. As for the occurrence of the genes, 100% of the isolates presented the lasR, rhlI and rhlR genes, and 97.5%, presented the lasI gene. In this study nine isolates were not biofilm producers. However, all presented the QS genes. Amplicons related to genes were sequenced in three of the nine non-biofilm-producing isolates (all presenting different genetic similarity profile) and aligned to the sequences of those genes in P. aeruginosa strain PAO1 (standard biofilm-producing strain). Alignment analysis showed an insertion of three nucleotides (T, C and G) causing the addition of an amino acid valine in the sequence of the LasR protein, in position 53. ConclusionThe modeling of the resulting LasR protein showed a conformational change in its structure, suggesting that this might be the reason why these isolates are unable to produce biofilm.

Inhibition of quorum sensing related virulence factors of Pseudomonas aeruginosa by pyridoxal lactohydrazone

Pseudomonas aeruginosa quorum sensing (QS) system is a cell to cell signaling mechanism that regulates virulence factors and pathogenicity. Therefore, the QS system in P. aeruginosa may be an important target for pharmacological intervention. The present study aimed to investigate the effects of sub-MIC concentrations of (S,E)-2-hydroxy-N-(3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)propane hydrazide (pyridoxal lactohydrazone) against P. aeruginosa QS related virulence factors. We investigated the effect of sub-MIC concentrations of chiral pyridoxal lactohydrazone, which formed by the reaction of chiral lactic acid hydrazide and pyridoxal (one form of Vitamin B6) as bioactive reagents, on virulence factors. Treated PAO1 cultures in the presence of tested compound at 1/4 and 1/16 MIC (32 and 8 μg/mL respectively) showed significant inhibition of virulence factors including motility, alginate and pyocyanin production and susceptibility to H2O2 (P < 0.001). Also, the pyridoxal lactohydrazone showed anti-QS activity in Chromobacterium violaceum CV026 biosensor bioassay. Because of quorum sensing is a promising target for anti-virulence therapy and also important role of LasR regulatory protein in the initiation of P. aeruginosa QS system, we carried out molecular docking for understanding the interactions of pyridoxal lactohydrazone with the LasR receptor. The results of docking study suggested that the pyridoxal lactohydrazone has potential to inhibit the LasR protein. The results indicated that sub-MIC concentrations of this compound exhibited inhibitory effect on P. aeruginosa QS related virulence factors.

Inhibition of quorum sensing-controlled biofilm formation in Pseudomonas aeruginosa by quorum-sensing inhibitors

Antimicrobial therapy against extensively drug-resistant (XDR) P. aeruginosa biofilms is less efficient compared to the treatment of equal bacterial counts of free-floating planktonic cells, which has become a serious threat in hospital environment. P. aeruginosa regulate their cooperative activities and physiological processes through a cell to cell chemical communication process called Quorum sensing (QS). This attracted our interest to synthesize, and to chemically characterize two anti-QS compounds, N-(4-{4-fluoroanilno} butanoyl) -l-homoserine lactone (FABHL) and N-(4-{4-chlororoanilno} butanoyl) -l-homoserine lactone (CABHL) to inhibit biofilm formation via disabling the QS circuits. Structural and morphological properties of these compounds were characterized by 1H Nuclear Magnetic Resonance (NMR), 13C NMR and High-resolution mass spectrometry (HRMS). Two biofilm forming XDR P. aeruginosa isolates were included in this study. Anti-biofilm property of FABHL or CABHL was confirmed by biofilm formation assay and it was shown to occur without affecting the bacterial growth. Anti-QS property of FABHL or CABHL was determined by evaluating the expression levels of QS genes (lasR and rhlR) by quantitative real time PCR (qRT-PCR). Although, FABHL and CABHL downregulates the expression levels of QS genes, lasR expression was significantly reduced. Molecular modeling studies revealed that the binding energy of FABHL and CABHL with LasR protein was −4.27 and −4.51, respectively. Hence, the synthesized compounds have the potential to serve as a potent anti-biofilm agent via disabling the QS systems. Lethality of FABHL and CABHL against PBMCs was assessed by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphynyl tetrazolium bromide (MTT) assay. Cell viability was observed for both the compounds.

Anti-quorum sensing activity of Pistacia atlantica against Pseudomonas aeruginosa PAO1 and identification of its bioactive compounds

Pseudomonas aeruginosa is a multidrug resistant opportunistic pathogen and an important cause of nosocomial infections. Quorum-sensing (QS) is a process in which bacterial cell-cell communication can regulates production of many virulence factors including pigment formation and the ability to form biofilm which is essential for establishment of chronic infections. We examined the inhibitory effect of Pistacia atlantica (Anacardiaceae) methanolic leaf extract and its bioactive components on biofilm formation and pigment production by P. aeruginosa PAO1. Fractionation of the methanolic leaf extract was carried out using HPLC based activity profiling. Identification of the active compounds was carried out by the integrated approach of HPLC-DAD and LC-MS followed by molecular docking analysis.Pistacia atlantica crude extract at 2 and 1 mg/mL, inhibited 92% and 79% biofilm formation, respectively. Minimum biofilm inhibitory concentration (MBIC) determined by microbroth dilution was 0.25 mg/mL with 39% inhibition. Pyocyanin production measured by spectrophotometry showed 100% and 83% inhibition at 2 and 1 mg/mL and minimum inhibitory concentration (MIC) was 0.5 mg/mL with 40% inhibition. Four active HPLC fractions (11, 15, 16 and 19) showed MBIC values of 0.06, 0.16, 0.10, 0.15 mg/mL, and MICs for pyocyanin production of 0.49, 0.31, 0.76, >0.30 mg/mL, respectively. The active compounds were identified as rutin (1), myricetin, 3-O-rutinoside (2) and kaempferol-3-O-rutinoside (4), all belonging to the flavonoid family. Molecular docking simulation of the active compounds showed that all had high affinity for LasR protein which is an important quorum-sensing signal receptor. The results of this study suggest that the active components of P. atlantica have high anti-QS activities and may have the potential for treatment of chronic infections caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa quorum-sensing response in the absence of functional LasR and LasI proteins: the case of strain 148, a virulent dolphin isolate.

Pseudomonas aeruginosa is an opportunistic pathogen that presents a complex regulatory network called 'quorum-sensing', which is responsible for the transcription of genes coding for several traits implicated in its pathogenicity. Strain 148 is a dolphin isolate that has been shown to produce quorum-sensing-regulated virulence traits and to be virulent in a mouse model, despite the fact that it contains a 20-kbp deletion that eliminates from the chromosome the lasR gene and the lasI promoter. LasR is a key quorum-sensing transcriptional regulator that, when coupled with the autoinducer 3-oxo-dodecanoyl homoserine lactone (3O-C12-HSL) produced by LasI, activates transcription of genes coding for some virulence-associated traits such as elastase, lasI, rhlI and rhlR. RhlR is also a key quorum-sensing transcriptional regulator that, when interacting with the autoinducer butanoyl homoserine lactone (C4-HSL) that is produced by the synthase RhlI, activates the genes involved in the synthesis of some virulence-associated traits, as rhamnolipids and pyocyanin. We describe that in P. aeruginosa 148, the LasR/3O-C12-HSL-independent rhlR transcriptional activation is due to the release of the negative effect of Vfr (a CRP-ortholog) caused by the insertion of an IS element in vfr, and that rhlI transcription is driven from the rhlR promoter, forming the rhlR-I operon.

Purification and characterization of Pseudomonas aeruginosa LasR expressed in acyl-homoserine lactone free Escherichia coli cultures

Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 μM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL. The α-helical content as determined by CD spectroscopy was found to be in agreement with the corresponding value derived from the homology model. Analytical ultracentrifugation studies show that LasR is a mixture of monomers and dimers and that AHL binding does not alter its oligomeric state. Thermal unfolding studies indicate that LasR has a significant thermal stability and that AHL binding does not significantly alter the unfolding temperature. Two LasR-DNA complexes were observed in electrophoretic mobility shift assays using the hcnABC promoter that has two lux boxes. Taken together, data indicate that the presence of AHLs is not a requisite for correct LasR protein folding. The protein is able to bind AHL ligands in a reversible manner, revising initial concepts of this regulator. The availability of AHL-free protein will permit further studies to determine more precisely its mode of action.

LasR Variant Cystic Fibrosis Isolates Reveal an Adaptable Quorum-Sensing Hierarchy in Pseudomonas aeruginosa.

ABSTRACTChronic Pseudomonas aeruginosa infections cause significant morbidity in patients with cystic fibrosis (CF). Over years to decades, P. aeruginosa adapts genetically as it establishes chronic lung infections. Nonsynonymous mutations in lasR, the quorum-sensing (QS) master regulator, are common in CF. In laboratory strains of P. aeruginosa, LasR activates transcription of dozens of genes, including that for another QS regulator, RhlR. Despite the frequency with which lasR coding variants have been reported to occur in P. aeruginosa CF isolates, little is known about their consequences for QS. We sequenced lasR from 2,583 P. aeruginosa CF isolates. The lasR sequences of 580 isolates (22%) coded for polypeptides that differed from the conserved LasR polypeptides of well-studied laboratory strains. This collection included 173 unique lasR coding variants, 116 of which were either missense or nonsense mutations. We studied 31 of these variants. About one-sixth of the variant LasR proteins were functional, including 3 with nonsense mutations, and in some LasR-null isolates, genes that are LasR dependent in laboratory strains were nonetheless expressed. Furthermore, about half of the LasR-null isolates retained RhlR activity. Therefore, in some CF isolates the QS hierarchy is altered such that RhlR quorum sensing is independent of LasR regulation. Our analysis challenges the view that QS-silent P. aeruginosa is selected during the course of a chronic CF lung infection. Rather, some lasR sequence variants retain functionality, and many employ an alternate QS strategy involving RhlR.