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.

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.

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.

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.

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.

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.

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.

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.

Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections.

Traditional treatment of infectious diseases is based on compounds that aim to kill or inhibit bacterial growth. A major concern with this approach is the frequently observed development of resistance to antimicrobial compounds. The discovery of bacterial-communication systems (quorum-sensing systems), which orchestrate important temporal events during the infection process, has afforded a novel opportunity to ameliorate bacterial infection by means other than growth inhibition. Compounds able to override bacterial signaling are present in nature. Herein we discuss the known signaling mechanisms and potential antipathogenic drugs that specifically target quorum-sensing systems in a manner unlikely to pose a selective pressure for the development of resistant mutants.

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.

Modeling the effect of acylated homoserine lactone antagonists in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram-negative bacterium that causes serious illnesses, particularly in immunocompromised individuals, often with a fatal outcome. The finding that the acylated homoserine lactone quorum sensing (QS) system controls the production of virulence factors in P. aeruginosa makes this system a possible target for antimicrobial therapy. It has been suggested that an N-(3-oxododecanoyl)-homoserine lactone (3O-C12-HSL) antagonist, a QS blocker (QSB), would interfere efficiently with the quorum sensing system in P. aeruginosa and thus reduce the virulence of this pathogen. In this work, a mathematical model of the QS system in P. aeruginosa has been developed. The model was used to virtually add 3O-C12-HSL antagonists that differed in their affinity for the receptor protein and for their ability to mediate degradation of the receptor. The model suggests that very small differences in these parameters for different 3O-C12-HSL antagonists can greatly affect the success of QSB based inhibition of the QS system in P. aeruginosa. Most importantly, it is proposed that the ability of the 3O-C12-HSL antagonist to mediate degradation of LasR is the core parameter for successful QSB based inhibition of the QS system in P. aeruginosa. Finally, this study demonstrates that QSBs can shift the system to a low steady state, corresponding to an uninduced state and thus, suggests that the use of 3O-C12-HSL antagonists may constitute a promising therapeutic approach against P. aeruginosa involved infections.

Sexually differentiated actions of 3-PPP enantiomers on prolactin secretion.

The ability of the enantiomers of the atypical dopamine receptor agonist 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP) to counteract gamma-butyrolactone-induced hyperprolactinemia was compared in male and female rats. Following gamma-butyrolactone (GBL) pretreatment serum prolactin concentrations were higher in female than in male rats. In males (-)-3-PPP tended to be somewhat less effective than (+)-3-PPP in decreasing serum prolactin concentrations (levels after (+)-3-PPP and (-)-3-PPP: 21% and 33%, respectively, of levels in GBL-pretreated control(s). In females the (-)-form induced a much weaker response than did the (+)-form (levels after (+)-3-PPP and (-)-3-PPP: 8% and 74%, respectively, of levels in GBL pretreated controls). Parallel experiments replacing GBL by reserpine yielded similar results. Data are discussed in terms of sex differences in responsiveness of pituitary dopamine receptors.

Enterolactone and estradiol inhibit each other's proliferative effect on MCF-7 breast cancer cells in culture.

In earlier studies it has been shown that women with breast cancer and at risk for breast cancer have low excretion of urinary mammalian lignans (enterolactone and enterodiol) mainly due to low intake of whole-grain products and other fiber-rich foods. It is well known that estradiol (E2) has proliferative effects on estrogen dependent cancer cells and that antiestrogens inhibit this effect. To elucidate whether enterolactone (Enl) has antiestrogenic properties we studied, using MCF-7 breast cancer cells in culture, the in vitro effect of relatively low concentrations of Enl added both alone and in combination with E2. E2 (1 nmol/l) and Enl (0.5-2 mumol/l) separately stimulated the proliferation of MCF-7 cells, but their combination always resulted in lower stimulation than any of them alone, or the combined compounds had no stimulatory effect at all compared to the control. Higher concentrations above 10 mumol/l of Enl inhibited significantly the growth of the cells suggesting a toxic effect. The lignan was very rapidly conjugated to its monosulfate. It is suggested that one possible mechanism by which Enl may affect the growth of these estrogen sensitive cells is by competition of Enl and its sulfate with the estrogens for sulfokinases and sulfatases involved in estrogen metabolism in the cells. It is concluded that Enl inhibits E2-stimulated MCF-7 breast cancer cell growth in vitro, and vice versa. The concentrations of Enl needed for the elimination of the proliferative effect of E2 are physiologic and similar to those used in corresponding experiments utilizing tamoxifen.

Halogenated furanones from the red alga, Delisea pulchra, inhibit carbapenem antibiotic synthesis and exoenzyme virulence factor production in the phytopathogen Erwinia carotovora.

The plant pathogen Erwinia carotovora regulates expression of virulence factors and antibiotic production via an N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HSL) dependent quorum sensing mechanism. The marine alga Delisea pulchra produces halogenated furanones known to antagonise 3-oxo-C6-HSL activity. We have tested the effects of a halogenated furanone on the production of carbapenem, cellulase and protease in E. carotovora. Despite differences in the regulatory mechanisms controlling carbapenem and exoenzyme production each was inhibited by the algal metabolite. We present evidence to suggest that the furanone dependent inhibition of carbapenem production is a result of the disruption of the 3-oxo-C6-HSL dependent expression of the carABCDEFGH operon.

Role of dopamine storage function in the control of rat striatal tyrosine hydroxylase activity.

Pretreatment of rats with reserpine prevents and post-treatment with RO4-1284 depletes the gamma-butyrolactone (GBL)-induced increase of striatal dopamine (DA) levels. This suggests that the accumulation of DA in striatal nerve endings that normally follows GBL-induced cessation of nigrostriatal impulse flow is in reserpine-sensitive sites. Three days after a single injection of reserpine, the ability of either haloperidol, a DA receptor blocker, or GBL to enhance DA synthesis is greatly reduced and these responses recover slowly over a two week period. Similarly, the ability of haloperidol to elevate striatal DA metabolite concentrations shows a similar pattern of inhibition. The rate of recovery after reserpine of haloperidol effects on DA metabolite concentrations and the activation of striatal tyrosine hydroxylase (measured in vivo by the 30 min L-DOPA accumulation after decarboxylase inhibition with NSD-1015) after either haloperidol or GBL parallels the rate of recovery of basal DA levels. The accumulation of DA after GBL proceeds for 60 min before beginning to plateau in normal rats, but 3 days after reserpine the DA elevation stops after 15 min and lasts for only 30 min in 10 day reserpinized animals. The initial 15 min accumulation of DA after GBL is the same in normal, 3 day and 10 day reserpinized rats, indicating that the initial enzymic rate of activity is the same, but the duration of activation is less. Thus, inhibition of DA storage function by reserpine alters the coupling of DA autoreceptor activity with tyrosine hydroxylase activity. It is suggested that DA storage function modulates tyrosine hydroxylase activity by controlling the amount of DA available for attachment to and inhibition of tyrosine hydroxylase enzyme. This hypothesis is consistent with recent immunocytochemical observations which suggest an association of tyrosine hydroxylase with synaptic vesicles in DA neuronal terminal areas.

The GABAB antagonist, CGP 35348, antagonizes the effects of baclofen, gamma-butyrolactone and HA 966 on rat striatal dopamine synthesis.

The effects of the new GABAB antagonist, CGP 35348 (3-aminopropane-diethoxymethylphosphinic acid), on rat striatal dopamine synthesis and the increases thereof, caused by (-)-baclofen, gamma-butyrolactone (GBL), and HA 966 (3-amino-1-hydroxypyrrolid-2-one), were investigated. CGP 35348 did not alter dopamine synthesis on its own up to the highest dose tested (500 mg/kg i.p.). However, it antagonized the increase elicited by 50 mg/kg s.c. (-)-baclofen at doses above 100 mg/kg i.p.; at 500 mg/kg i.p. this antagonism disappeared within about 6 h of interval between the administration of the compound and (-)-baclofen. CGP 35348 also clearly and significantly attenuated the effects of graded doses of GBL and HA 966 at 500 mg/kg i.p., but was unable to alter the responses elicited by 0.3 mg/kg i.p. haloperidol or 10 mg/kg i.p. tetrabenazine. This indicates that the compound did not generally attenuate increases of dopamine synthesis. It is likely that its GABAB antagonistic properties are responsible for the attenuation of the effect of (-)-baclofen, and our results suggest that this compound is useful for the characterization of the role of GABAB receptors in vivo, e.g. in behaviour. On the other hand, they also suggest the possibility that GBL and HA 966 elicit their effects on dopamine synthesis by means of an interaction with GABAB receptors; a weak in vitro interaction with the latter in radioligand binding experiments has been found for GBL, but not for HA 966.

A simple screening protocol for the identification of quorum signal antagonists.

Quorum sensing (QS) is a mechanism by which diverse microorganisms can control specific processes in response to population density. A relatively well-known form of QS among Proteobacteria involves production and subsequent response to acylated homoserine lactones (AHLs). Quorum sensing inhibition (QSI), targeting AHL-dependent signaling, has been reported as a strategy for the control of biofilm formation used by several marine organisms. We developed a simple soft agar overlay protocol, based on pigmentation inhibition, to rapidly screen for the presence of potential QSI by bacteria and plants. For bacterial screens, test organisms are first streaked onto their appropriate media and incubated overnight. For plant screens, the plant material (leaf, stem, flower, etc.) is placed onto LB agar. The bacterial growth or plant samples are then covered with an overlay of LB soft agar containing an inoculum of either Pseudomonas aureofaciens 30-84 or Chromobacterium violaceum ATCC 12472 (indicator cultures) and then incubated overnight. These indicator bacteria regulate pigment production by N-hexanoyl-HSL (C6-HSL) QS and are readily inhibited by AHL analogues and other antagonists. QSI is indicated by the lack of pigment production of the indicator culture in the vicinity of the test sample. Growth inhibition of the indicator culture indicates possible antibiotic production. Two different biosensor organisms based on derivatives of Agrobacterium tumefaciens and C. violaceum, capable of detecting a range of AHLs were used to determine whether QSI is due to the production of interfering AHLs competing with the C6-HSL regulation of C. violaceum and P. aureofaciens pigment production. This simple protocol will facilitate the screening of multiple organisms for the production of potential antifouling compounds.