An innovative role for luteolin as a natural quorum sensing inhibitor in Pseudomonas aeruginosa.

AIMS: The emergence of antibiotic tolerance was a tricky problem in the treatment of chronic Pseudomonas aeruginosa-infected cystic fibrosis and burn victims. The quorum sensing (QS) inhibitor may serve as a new tactic for the bacterial resistance by inhibiting the biofilm formation and the production of virulence factors. This study explored the potential of luteolin as a QS inhibitor against P. aeruginosa and the molecular mechanism involved. MAIN METHODS: Crystal violet staining, CLSM observation, and SEM analysis were carried out to assess the effect of luteolin on biofilm formation. The motility assays and the production of virulence factors were determined to evaluate the QS-inhibitory activity of luteolin. Acyl-homoserine lactone, RT-PCR, and molecular docking assays were conducted to explain its anti-QS mechanisms. KEY FINDINGS: The biofilm formation, the production of virulence factors, and the motility of P. aeruginosa could be efficiently inhibited by luteolin. Luteolin could also attenuate the accumulation of the QS-signaling molecules N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL) (P < 0.01) and downregulate the transcription levels of QS genes (lasR, lasI, rhlR, and rhlI) (P < 0.01). Molecular docking analysis indicated that luteolin had a greater docking affinity with LasR regulator protein compared with OdDHL. SIGNIFICANCE: This study is important as it reports the molecular mechanisms involved in the anti-biofilm formation activity of luteolin against P. aeruginosa. This study also indicated that luteolin could be helpful when used for the treatment of clinical drug-resistant infections of P. aeruginosa.

Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp.

Quorum quenching (QQ) is a promising alternative infection-control strategy to antibiotics that controls quorum-regulated virulence without killing the pathogens. Aeromonas hydrophila is an opportunistic gram-negative pathogen living in freshwater and marine environments. A. hydrophila possesses an N-acyl homoserine lactone (AHL)-based quorum-sensing (QS) system that regulates virulence, so quorum signal-inactivation (i.e., QQ) may represent a new way to combat A. hydrophila infection. In this study, an AHL lactonase gene, aiiA was cloned from Bacillus sp. strain QSI-1 and expressed in Escherichia coli strain BL21(DE3). The A. hydrophila hexanoyl homoserine lactone (C6-HSL) QS signal molecule was degraded by AiiAQSI-1, which resulted in a decrease of bacterial swimming motility, reduction of extracellular protease and hemolysin virulence factors, and inhibited the biofilm formation of A. hydrophila YJ-1 in a microtiter assay. In cell culture studies, AiiAQSI-1 decreased the ability of A. hydrophila adherence to and internalization by Epithelioma papulosum cyprini (EPC) cells. During in vivo studies, oral administration of AiiAQSI-1 via feed supplementation attenuated A. hydrophila infection in Crucian Carp. Results from this work indicate that feed supplementation with AiiAQSI-1 protein has potential to control A. hydrophila aquaculture disease via QQ.

C4-HSL aptamers for blocking qurom sensing and inhibiting biofilm formation in Pseudomonas aeruginosa and its structure prediction and analysis.

This study aimed to screen DNA aptamers against the signal molecule C4-HSL of the rhl system for the inhibition of biofilm formation of Pseudomonas aeruginosa using an improved systematic evolution of ligand by exponential enrichment (SELEX) method based on a structure-switching fluorescent activating bead. The aptamers against the C4-HSL with a high affinity and specifity were successfully obtained and evaluated in real-time by this method. Results of biofilm inhibition experiments in vitro showed that the biofilm formation of P. aeruginosa was efficiently reduced to about 1/3 by the aptamers compared with that of the groups without the aptamers. Independent secondary structure simulation and computer-aided tertiary structure prediction (3dRNA) showed that the aptamers contained a highly conserved Y-shaped structural unit. Therefore, this study benefits the search for new methods for the detection and treatment of P. aeruginosa biofilm formation.

Quorum sensing signals and related virulence inhibition of Pseudomonas aeruginosa by a potential probiotic strain's organic acid.

Studies conducted in recent years show that pathogen bacteria are not asocial assets and they use the cell to cell communication mechanism called quorum sensing that depends on population density to adapt changing environmental conditions. This mechanism is coordinate gene expression of various bacterial factors like bioluminescence, antibiotic biosynthesis, plasmid conjugation and virulence. Bacteria communicate with each other by producing signal molecules and regulate the production of virulence factors that have importance in the pathogenity formation. Virulence mechanisms of Pseudomonas aeruginosa, which causes various types of infections in humans, are also regulated by quorum sensing. Nowadays, biotechnological researches are focused on the development of homoserine lactone antagonists. The use of these type of molecules are considered to be a new treatment approach for blocking communication between bacteria and reducing virulence, therefore improving infection control. In this study, lactic acid of a potential probiotic Pediococcus acidilactici M7 strain isolated from newborn faeces was used to evaluate the inhibitory effect on quorum sensing signal molecules and some virulence factors of clinical Pseudomonas aeruginosa isolates. Results showed that lactic acid has an inhibitory effect on short-chain HSL production and swarming-swimming-twitching motility, elastase, protease, pyocyanin, and biofilm production of Pseudomonas aeruginosa isolates in certain quantities that are regulated by the quorum sensing system.

Inhibiting bacterial quorum sensing arrests coral disease development and disease-associated microbes.

Among the greatest threats to coral reefs are coral epizootics, which are increasing in frequency and severity across many reef ecosystems. In particular, white band disease (WBD) has devastated Caribbean acroporid populations since its initial outbreak in 1979. However, despite its widespread and damaging effects, the aetiology of WBD remains largely unresolved. Here, we examine the role of quorum sensing within bacterial communities associated with WBD-infected Acropora cervicornis. Microbial communities isolated from WBD-infected corals were exposed to quorum sensing inhibitor (QSI) - a N-acyl homoserine lactone autoinducer antagonist - and then dosed onto healthy test corals. WBD-associated bacteria supplemented with QSI lost the ability to establish disease, while healthy corals exposed to uninhibited WBD bacterial communities became infected within two days. Microbial 16S rRNA metagenomic sequencing analyses were then used to identify shifts in bacterial communities due to QSI exposure on WBD-associated bacterial communities. Our results demonstrated that Vibrionaceae and Flavobacteriaceae abundances were strongly inhibited by the addition of QSI to WBD-infected corals, whereas putative coral symbiont Endozoicomonas and Halomonadaceae abundances decrease dramatically in diseased corals.

Structure based virtual screening for identification of potential quorum sensing inhibitors against LasR master regulator in Pseudomonas aeruginosa.

Inter and intracellular communication in bacteria, which is known as quorum sensing (QS), is mediated by small diffusible signaling molecules known as autoinducers. QS regulates various virulence factors responsible for pathogenesis. Increasing resistance of microorganisms against traditional antibiotics has turned the focus towards the QS as it exerts less selective pressure preventing development of resistance among microorganisms. LasR, a transcription factor that controls QS in Pseudomonas aeruginosa, is an attractive therapeutic target for inhibitors. This study aimed to screen natural compounds as potential inhibitors of LasR. About 2603 compounds from ZINC database were virtually screened against the structure of LasR. Then after qualifying compounds were filtered on the parameters of Lipinski's rule and ADME. Six novel potential QS inhibiting compounds were selected on the basis of binding energy. Structures of LasR-ligand complexes were analysed to have insight of binding between inhibitors and target. It is pertinent to mention here that all the molecules are structurally different from 3-oxo-C12HSL,a native autoinducer of LasR, that play key role in formation of LasR dimer which is an active form of the protein to facilitate QS.

The effect of burdock leaf fraction on adhesion, biofilm formation, quorum sensing and virulence factors of Pseudomonas aeruginosa.

AIMS: This study aimed to evaluate the effect of a fraction of burdock (Arctium lappa L.) leaf on the initial adhesion, biofilm formation, quorum sensing and virulence factors of Pseudomonas aeruginosa. METHODS AND RESULTS: Antibiofilm activity of the burdock leaf fraction was studied by the method of crystal violet staining. When the concentration of the burdock leaf fraction was 2·0 mg ml-1 , the inhibition rates on biofilm formation of P. aeruginosa were 100%. The burdock leaf fraction was found to inhibit the formation of biofilm by reducing bacterial surface hydrophobicity, decreasing bacterial aggregation ability and inhibiting swarming motility. Interestingly, the burdock leaf fraction inhibited the secretion of quorum-sensing (QS) signalling molecule 3-oxo-C12-HSL and interfered quorum sensing. Moreover, the QS-regulated pyocyanin and elastase were also inhibited. Chemical composition analysis by UPLC-MS showed 11 active compounds in the burdock leaf fraction. CONCLUSIONS: The burdock leaf fraction significantly inhibited the formation of biofilm and quorum sensing, as well as significantly decreased the content of virulence factors. SIGNIFICANCE AND IMPACT OF THE STUDY: This study introduces a natural and effective bacterial biofilm inhibitor, which could also significantly decrease the content of virulence factors and the drug resistance of P. aeruginosa.

Discovery of new diketopiperazines inhibiting Burkholderia cenocepacia quorum sensing in vitro and in vivo.

Burkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia.

Small molecule screen yields inhibitors of Pseudomonas homoserine lactone-induced host responses.

Pseudomonas aeruginosa infections are commonly associated with cystic fibrosis, pneumonias, neutropenia and burns. The P. aeruginosa quorum sensing molecule N-(3-oxo-dodecanoyl) homoserine lactone (C12) cause multiple deleterious host responses, including repression of NF-κB transcriptional activity and apoptosis. Inhibition of C12-mediated host responses is predicted to reduce P. aeruginosa virulence. We report here a novel, host-targeted approach for potential adjunctive anti-Pseudomonal therapy based on inhibition of C12-mediated host responses. A high-throughput screen was developed to identify C12 inhibitors that restore NF-κB activity in C12-treated, lipopolysaccharide (LPS)-stimulated cells. Triazolo[4,3-a]quinolines with nanomolar potency were identified as C12-inhibitors that restore NF-κB-dependent luciferase expression in LPS- and TNF-stimulated cell lines. In primary macrophages and fibroblasts, triazolo[4,3-a]quinolines inhibited C12 action to restore cytokine secretion in LPS-stimulated cells. Serendipitously, in the absence of an inflammatory stimulus, triazolo[4,3-a]quinolines prevented C12-mediated responses, including cytotoxicity, elevation of cytoplasmic calcium, and p38 MAPK phosphorylation. In vivo efficacy was demonstrated in a murine model of dermal inflammation involving intradermalC12 administration. The discovery of triazolo[4,3-a]quinolines provides a pharmacological tool to investigate C12-mediated host responses, and a potential host-targeted anti-Pseudomonal therapy.

Identification of catechin as one of the flavonoids from Combretum albiflorum bark extract that reduces the production of quorum-sensing-controlled virulence factors in Pseudomonas aeruginosa PAO1.

Quorum-sensing (QS) regulates the production of key virulence factors in Pseudomonas aeruginosa and other important pathogenic bacteria. In this report, extracts of leaves and bark of Combretum albiflorum (Tul.) Jongkind (Combretaceae) were found to quench the production of QS-dependent factors in P. aeruginosa PAO1. Chromatographic fractionation of the crude active extract generated several active fractions containing flavonoids, as shown by their typical spectral features. Purification and structural characterization of one of the active compounds led to the identification of the flavan-3-ol catechin [(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol]. The identity of catechin as one of the active molecules was confirmed by comparing the high-pressure liquid chromatography profiles and the mass spectrometry spectra obtained for a catechin standard and for the active C. albiflorum fraction. Moreover, standard catechin had a significant negative effect on pyocyanin and elastase productions and biofilm formation, as well as on the expression of the QS-regulated genes lasB and rhlA and of the key QS regulatory genes lasI, lasR, rhlI, and rhlR. The use of RhlR- and LasR-based biosensors indicated that catechin might interfere with the perception of the QS signal N-butanoyl-l-homoserine lactone by RhlR, thereby leading to a reduction of the production of QS factors. Hence, catechin, along with other flavonoids produced by higher plants, might constitute a first line of defense against pathogenic attacks by affecting QS mechanisms and thereby virulence factor production.

Structural understanding of quorum-sensing inhibitors by molecular modeling study in Pseudomonas aeruginosa.

Inhibitors of 3OC12, an initial signal molecule of the quorum sensing (QS) signaling cascade in Pseudomonas aeruginosa have been developed. Eight inhibitor candidates were synthesized by substituting the head part of 3-oxododecanoyl-homoserine lactone (3OC12) with different aromatic rings, and their docking poses and scores (binding energies) were predicted by in silico modeling study. All compounds gave better docking scores than 3OC12 and good inhibition effects on LasR activity in the in vivo bioassay. Like the modifications in the tail part of 3OC12 in our previous study Kim et al. (2008), the head-part modifications also showed inhibition activity in a fairly good proportion to the docking scores from the modeling analysis. This implies that the head part of 3OC12 also contributes significantly to forming the active conformation of the LasR-3OC12 complex, and its modification could effectively induce the inactive conformation of the complex. We suggest that the head part of 3OC12 is also a good target moiety to develop the structure-based Pseudomonas QS inhibitors.

Bacterial quorum sensing: a new target for anti-infective immunotherapy.

BACKGROUND: Cell-to-cell communication via exchange of small molecules, 'autoinducers', is a widespread phenomenon among Gram-negative and -positive bacteria. This intercellular signaling that synchronizes population-wide gene expression in a cell-density-dependent manner is termed 'quorum sensing' (QS). The discovery that Gram-negative bacteria employ non-peptide structures, N-acyl homoserine lactones, to globally regulate production of secondary metabolites and proteins, initiated a new area of research. Subsequently, other quorum-sensing systems and small signaling molecules were identified. With the emergence of antibiotic-resistant bacteria, most prominently methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, new approaches for combating infections are needed. Inhibition of QS results in attenuation of virulence rather than direct killing of microbes. OBJECTIVE: We highlight current trends in preventing bacterial infections using quorum-quenching strategies. METHODS: We mainly focus on P. aeruginosa and S. aureus and their QS systems as targets for intervention. RESULTS/CONCLUSION: New research strongly suggests that QS systems represent attractive targets for discovery of novel anti-infective agents, including immunotherapeutic strategies.

Making bacteria behave: new agonists and antagonists of quorum sensing.

Small-molecule agonists and antagonists of bacterial quorum sensing can enhance our understanding of this form of cell-cell communication. A recent effort has discovered effective modulators of the autoinducer-1 circuit for bacterial quorum sensing by the synthesis and evaluation of a small library of aryl-substituted acyl-homoserine lactone analogues. This series highlights the sensitivity to structure of the contrasting responses of agonism and antagonism of the natural signal and identifies an analogue that provokes the same response as the natural signal but at 10-fold lower concentration, a "superagonist".

N-phenylacetanoyl-L-homoserine lactones can strongly antagonize or superagonize quorum sensing in Vibrio fischeri.

Bacteria monitor their population densities using low-molecular-weight ligands in a process known as quorum sensing. At sufficient cell densities, bacteria can change their mode of growth and behave as multicellular communities that play critical roles in both beneficial symbioses and the pathogenesis of infectious disease. The development of non-native ligands that can block quorum-sensing signals has emerged as a promising new strategy to attenuate these divergent outcomes. Here, we report that N-phenylacetanoyl-L-homoserine lactones are capable of either inhibiting or, in some cases, strongly inducing quorum sensing in the bacterial symbiont Vibrio fischeri. Moreover, simple structural modifications to these ligands have remarkable effects on activity. These studies have revealed one of the first synthetic superagonists of quorum sensing, N-(3-nitro-phenylacetanoyl)-L-homoserine lactone. Together, these ligands represent a powerful new class of chemical probes with the potential to significantly expand the current understanding of quorum sensing and its role in host/bacteria interactions.

Inhibition of quorum sensing in Pseudomonas aeruginosa by N-acyl cyclopentylamides.

N-octanoyl cyclopentylamide (C8-CPA) was found to moderately inhibit quorum sensing in Pseudomonas aeruginosa PAO1. To obtain more powerful inhibitors, a series of structural analogs of C8-CPA were synthesized and examined for their ability to inhibit quorum sensing in P. aeruginosa PAO1. The lasB-lacZ and rhlA-lacZ reporter assays revealed that the chain length and the ring structure were critical for C8-CPA analogs to inhibit quorum sensing. N-decanoyl cyclopentylamide (C10-CPA) was found to be the strongest inhibitor, and its concentrations required for half-maximal inhibition for lasB-lacZ and rhlA-lacZ expression were 80 and 90 microM, respectively. C10-CPA also inhibited production of virulence factors, including elastase, pyocyanin, and rhamnolipid, and biofilm formation without affecting growth of P. aeruginosa PAO1. C10-CPA inhibited induction of both lasI-lacZ by N-(3-oxododecanoyl)-L-homoserine lactone (PAI1) and rhlA-lacZ by N-butanoyl-L-homoserine lactone (PAI2) in the lasI rhlI mutant of P. aeruginosa PAO1, indicating that C10-CPA interferes with the las and rhl quorum-sensing systems via inhibiting interaction between their response regulators (LasR and RhlR) and autoinducers.

Bacterial quorum sensing and interference by naturally occurring biomimics.

Bacteria are able to coordinate gene expression as a community through the secretion and detection of signalling molecules so that the members of the community can simultaneously express specific behaviours. This mechanism of regulation of behaviour appears to be a key trait for adaptation to specific environments and has been shown to regulate a variety of important phenotypes, from virulence factor production to biofilm formation to symbiosis related behaviours such as bioluminescence. The ability to communicate and communally regulate gene expression is hypothesised to have evolved as a way for organisms to delay expression of phenotypes until numerical supremacy is reached. For example, in the case of infection, if an invading microorganism were to express virulence factors too early, the host may be able to mount a successful defence and repel the invaders. There is growing evidence that bacterial quorum sensing (QS) systems are involved in cross-kingdom signalling with eukaryotic organisms and that eukaryotes are capable of actively responding to bacteria in their environment by detecting and acting upon the presence of these signalling molecules. Likewise, eukaryotes produce compounds that can interfere with QS systems in bacteria by acting as agonists or antagonists. An exciting new field of study, biomimetics, takes inspiration from nature's models and attempts to design solutions to human problems, and biomimics of QS systems may be one such solution. This article presents the acylated homoserine lactone and autoinducer 2 QS systems in bacteria, the means of intercepting or interfering with bacterial QS systems evolved by eukaryotes, and the rational design of synthetic antagonists.

Quorum-sensing inhibitors as anti-pathogenic drugs.

Quorum-sensing (QS) signalling systems of pathogens are central regulators for the expression of virulence factors and represent highly attractive targets for the development of novel therapeutics. In Pseudomonas aeruginosa, QS systems are also involved in elevated antibiotic tolerance of biofilms as well as elevated tolerance to the activity of the innate immune system. Gram-negative bacteria commonly use N-acyl homoserine lactones (AHL) as QS signal molecules. The use of signal molecule based drugs to attenuate bacterial pathogenecity rather than bacterial growth is attractive for several reasons, particularly considering the emergence of increasingly antibiotic-resistant bacteria. Compounds capable of this type of interference have been termed anti-pathogenic drugs. A large variety of synthetic AHL analogues and natural products libraries have been screened and a number of QS inhibitors (QSI) have been identified. Promising QSI compounds have been shown to make biofilms more susceptible to antimicrobial treatments, and are capable of reducing mortality and virulence as well as promoting clearance of bacteria in experimental animal models of infection.

Antibody interference with N-acyl homoserine lactone-mediated bacterial quorum sensing.

Many bacterial pathogens coordinate their virulence factor expression in a cell density-dependent manner. This population-dependent coordination of gene expression in bacteria has been termed "quorum sensing" (QS). N-Acyl homoserine lactones (AHLs) are used by over 70 Gram-negative bacterial species as autoinducers. Inhibition of QS signaling might represent a new target for antimicrobial therapy. Here we report the hapten design, synthesis, generation of monoclonal antibodies (mAbs) against AHLs, and the evaluation of these mAbs for their ability to blunt QS signaling and inhibit virulence factor expression in P. aeruginosa. The mAbs can be envisioned as a tool for future investigations into AHL-based QS, which may aid in gaining new insights into the pathogenesis of P. aeruginosa and may ultimately lead to the development of new strategies to combat bacterial diseases.