N-hexanoyl Homoserine Lactone Research Articles

Synergistic Quorum quenching activity of Cinnamaldehyde against biofilm forming Pseudomonas aeruginosa

Quorum sensing is an essential mechanism for the pathogenesis of Pseudomonas aeruginosa, which causes opportunistic infections in humans. Quorum sensing inhibition has been considered a unique strategy to treat opportunistic infections and prevent an early event of infection without killing bacterial cells. The present study has demonstrated that cinnamaldehyde is able to inhibit biofilm formation by interfering with the quorum sensing activity of P. aeruginosa and is also able to disperse biofilms if they have already formed. It was also proven that synergistic action between 2.5 μM of cinnamaldehyde and 1 μM of cephalexin was effective in controlling biofilm formation. Cephalexin combined with cinnamaldehyde repressed the virulence factors associated with biofilms in Pseudomonas aeruginosa, such as Quorum sensing mediated motility, rhamnolipid, pyocyanin, elastase and rhamnolipid activities. A combination of cinnamaldehyde and cephalexin showed better biofilm inhibition (52.80%) than individual treatments and there was a reduction in the biofilm thickness and surface coverage among P. aeruginosa strains treated with cinnamaldehyde plus cephalexin. Further, it was also reported that the N-Hexanoylhomoserine lactone concentration was lower in P. aeruginosa strains treated with cinnamaldehyde plus cephalexin, while the control set-up showed a high amount of N-Hexanoylhomoserine lactone concentration. However, cinnamaldehyde with amoxycillin, oxacillin, or streptomycin showed a comparatively weaker quorum quenching effect when tested using checkerboard assays. Combination of CAD with CEPH can be a better alternative to treat P. aeruginosa infection.

Simultaneous regulation of biocathodic γ-HCH dechlorination and CH4 production by tailoring the structure and function of biofilms based on quorum sensing

Dechlorination of chlorinated organic pollutants and methanogenesis are attractive biocathode reductions in microbial electrolysis cells (MECs). Quorum sensing (QS) is applied to regulate microbial communications. However, how acyl-homoserine lactones (AHLs)-dependent QS organize the assembly of the biocathode microbial community, and then regulate multiple biocathode reductions remains unclear. By applying N-butanoyl homoserine lactone (C4-HSL), N-hexanoyl homoserine lactone (C6-HSL) and 3-oxo-hexanoyl homoserine lactone (3OC6-HSL) in γ-hexachlorocyclohexane (γ-HCH) contaminated MECs, this study investigated the changes of biofilm microbial structure and function and the mechanisms of AHLs-QS on γ-HCH dechlorination and CH4 production. Exogenous C4-HSL and 3OC6-HSL increased cytochrome c production and enriched dechlorinators, electroactive bacteria but not methanogens to accelerate γ-HCH dechlorination and inhibit CH4 production. C6-HSL facilitated dechlorination and CH4 production by enhancing biofilm electroactivity and increasing membrane transportation. Besides, exogenous C6-HSL restored the electron transfer capacity that was damaged by the concurrent addition of acylase, an endogenous AHL quencher. From the perspective of microbial assembly, this study sheds insights into and provides an efficient strategy to selectively accelerate dechlorination and CH4 production by harnessing microbial structure based on QS systems to meet various environmental demands.

Rapid start-up of partial nitrification reactor by exogenous AHLs and Vanillin combined with intermittent aeration

Quorum sensing (QS) and quorum quenching (QQ) are common phenomena in microbial systems and play an important role in the nitrification process. However, rapidly start up partial nitrification regulated by N-acyl-homoserine lactones (AHLs)-mediated QS or QQ has not been reported. Hence, we chose N-butyryl homoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL) as the representative AHLs, and Vanillin as the representative quorum sensing inhibitor (QSI) combined intermittent aeration to investigate their effects on the start-up process of partial nitrification. The start-up speed in the group with C4-HSL or C6-HSL addition was 1.42 or 1.26 times faster than that without addition, respectively. Meanwhile, the ammonium removal efficiency with C4-HSL or C6-HSL addition was increased by 13.87 % and 17.30 % than that of the control group, respectively. And, partial nitrification could maintain for a certain period without AHLs further addition. The increase of Nitrosomonas abundance and ammonia monooxygenase (AMO) activity, and the decrease of Nitrobacter abundance and nitrite oxidoreductase (NXR) activity were the reasons for the rapid start-up of partial nitrification in the AHLs groups. Vanillin addition reduced AMO and hydroxylamine oxidoreductase (HAO) activity, and increased Nitrobacter abundance and NXR activity, thus these were not conducive to achieving partial nitrification. Denitrifying bacteria (Hydrogenophaga, Thauera and Aquimonas) abundance increased in the Vanillin group. QS-related bacteria and gene abundance were elevated in the AHLs group, and reduced in the Vanillin group. Function prediction demonstrated that AHLs promoted the nitrogen cycle while Vanillin enhanced the carbon cycle. This exploration might provide a new technical insight into the rapid start-up of partial nitrification based on QS control.

Detection of Quorum Sensing Signal Molecules, Particularly N-Acyl Homoserine Lactones, 2-Alky-4-Quinolones, and Diketopiperazines, in Gram-Negative Bacteria Isolated From Insect Vector of Leishmaniasis

Gram-negative bacteria are known to use a quorum sensing system to facilitate and stimulate cell to cell communication, mediatedviaregulation of specific genes. This system is further involved in the modulation of cell density and metabolic and physiological processes that putatively either affect the survival of insect vectors or the establishment of pathogens transmitted by them. The process of quorum sensing generally involves N-acyl homoserine lactones and 2-alkyl-4-quinolones signaling molecules. The present study aimed to detect and identify quorum sensing signaling molecules of AHLs and AHQs type that are secreted by intestinal bacteria, and link their production to their extracellular milieu and intracellular content. Isolates for assessment were obtained from the intestinal tract ofPintomyia evansi(Leishmaniainsect vector). AHLs and AHQs molecules were detected using chromatography (TLC) assays, with the aid of specific and sensitive biosensors. For identity confirmation, ultra-high-performance liquid chromatography coupled with mass spectrometry was used. TLC assays detected quorum sensing molecules (QSM) in the supernatant of the bacterial isolates and intracellular content. Interestingly,Pseudomonas otitidis,Enterobacter aerogenes,Enterobacter cloacae, andPantoea ananatisisolates showed a migration pattern similar to the synthetic molecule 3-oxo-C6-HSL (OHHL), which was used as a control.Enterobacter cancerogenussecreted C6-HSL, a related molecules to N-hexanoyl homoserine lactone (HHL), whileAcinetobacter gyllenbergiiexhibited a migration pattern similar to 2-heptyl-4-quinolone (HHQ) molecules. In comparison to this, 3-oxo-C12-HSL (OdDHL) type molecules were produced byLysobacter soli,Pseudomonas putida,A. gyllenbergii,Acinetobacter calcoaceticus, andPseudomonas aeruginosa, whileEnterobacter cloacaeproduced molecules similar to 2-heptyl-3-hydroxy-4-quinolone (PQS). ForPseudomonas putida,Enterobacter aerogenes,P. ananatis, andPseudomonas otitidisextracts, peak chromatograms with distinct retention times and areas, consistent with the molecules described in case of TLC, were obtained using HPLC. Importantly,P. ananatisproduced a greater variety of high QSM concentration, and thus served as a reference for confirmation and identification by UHPLC-MRM-MS/MS. The molecules that were identified included N-hexanoyl-L-homoserine lactone [HHL, C10H18NO3, (M + H)], N-(3-oxohexanoyl)-L-homoserine lactone [OHHL, C10H16NO4, (M + H)], N-(3-oxododecanoyl)-L-homoserine lactone [OdDHL, C16H28NO4, (M + H)], and 2-heptyl-3-hydroxy-4(1H)-quinolone [PQS, C16H22NO2, (M + H)]. Besides this, the detection of diketopiperazines, namely L-Pro-L-Tyr and ΔAla-L-Val cyclopeptides was reported forP. ananatis.These molecules might be potentially associated with the regulation of QSM system, and might represent another small molecule-mediated bacterial sensing system. This study presents the first report regarding the detection and identification of QSM and diketopiperazines in the gut sand fly bacteria. The possible effect of QSM on the establishment ofLeishmaniamust be explored to determine its role in the modulation of intestinal microbiome and the life cycle ofPi. evansi.

Screening of N-Hexanamide and 2H-1,3-Benzodioxol Derivatives for Quorum Sensing Modulation in Chromobacterium violaceum

The aim of the study was screening of chemical compounds library to find “quorum sensing” modulators in proteobacteria. The library included 19 molecules that contained the N-hexanamide non-polar radical or the 2H-1,3-benzodioxole polar heterocyclic group, which were partially identical to the natural autoinductor N-hexanoyl homoserine lactone (C6-AHL) chemical structure. The Chromobacterium violaceum CV026 (NCTC 13278) was used in “quorum sensing” modulation biotests. This biosensor strain is defective in the natural autoinducer C6-AHL biosynthesis, but contains the functional homodimeric protein CviR, which, after two C6-AHL molecules reception, activates the previously silent genes transcription, including the vioABCDE operon, which leads to the production of the blue-violet pigment violacein. The direct screening showed agonistic (violacein-inducing) activity in 4 compounds and the screening variant in presence of exogenously introduced C6-AHL indicated the antagonistic (violacein-inhibiting) activity in 9 tested compounds. Interestingly, the most pronounced antagonistic activity was found in molecules that proved to be C6-AHL agonists. Significantly, the effective concentrations of these compounds required for 50% violacein inhibition were 3.2–20.4-fold lower than ones causing 50% violacein induction. In addition, the absolute concentrations of the active compounds necessary for the induction of violacein were much higher than that of a natural autoinducer, and the degree of their inhibitory effect was inversely related to the concentration of C6-AHL exogenously introduced into the biotest. According to the screening results, N-1,3-benzodioxol-5-yl-oxohexanamide was identified as the most effective “quorum sensing” modulator, submitted for further in silico, in vitro and in vivo studies.

Degradation of n-hexanoyl homoserine lactone with quorum quenching bacteria immobilised magnetic nanocomposite beads

ABSTRACT N-acyl homoserine lactones (AHLs) based quorum sensing controls various phenotype expressions, including biofilm formation, hence its interruption is considered to be an ideal option for membrane biofouling control. Bead entrapped quorum quenching bacteria was reported to be an efficient approach for degradation of signal molecules in recent years. In the present study, we investigated the potential of quorum quenching (QQ) bacteria immobilised magnetic nanocomposite beads (IMN) in degradation of signalling molecule, n-hexanoyl homoserine lactone (C6-HSL). Three QQ bacteria, named Acinetobacter baumannii JYQ2, Pseudomonas nitroreducens JYQ3 and Pseudomonas JYQ4 isolated from dairy industry waste activated sludge (WAS) were immobilised in the magnetic nanocomposite (IMN) beads. The scanning electron microscopy (SEM) of the IMN beads has indicated the successful entrapment of QQ bacteria within the alginate matrix. The GC-MS analysis showed that all the QQ bacteria immobilised magnetic nanocomposite (IMN) beads degraded the signalling molecule, n-hexanoyl homoserine lactone (C6-HSL) within 72 h of incubation. The nanocomposite beads containing the QQ bacteria Pseudomonas JYQ4 showed the maximum degradation percentage of 97 ± 0.13% leaving a residual HSL of 0.7 mg/L. All the other isolates showed C6-HSL degradation percentage in the range of 87% to 95%. The data suggest the potential of C6-HSL degradation by QQ bacteria IMN beads. Hence, the study offers possibilities of controlling biofilm developed on the membrane surface during wastewater treatment processes.

Distinctive Growth and Transcriptional Changes of the Diatom Seminavis robusta in Response to Quorum Sensing Related Compounds.

In aquatic habitats, diatoms are frequently found in association with Proteobacteria, many members of which employ cell-to-cell communication via N-acyl homoserine lactones (AHLs). It has been suggested that diatoms could distinguish between beneficial and algicidal bacteria in their surroundings by sensing AHLs. Although some microalgae can interfere with AHL signaling, e.g., by releasing AHL mimics or degrading them, molecular responses to AHLs in microalgae are still unclear. Therefore, we tested the effects of short-chained AHLs, i.e., N-hexanoyl homoserine lactone (C6-HSL), N-3-hydroxyhexanoyl homoserine lactone (OH-C6-HSL), and N-3-oxohexanoyl homoserine lactone (oxo-C6-HSL) and long-chained AHLs, i.e., N-tetradecanoyl homoserine lactone (C14-HSL), N-3-hydroxytetradecanoyl homoserine lactone (OH-C14-HSL), and N-3-oxotetradecanoyl homoserine lactone (oxo-C14-HSL), on growth of the benthic diatom Seminavis robusta. All tested short-chained AHLs did not affect diatom growth, while long-chained AHLs promoted (C14-HSL) or inhibited (OH-C14-HSL and oxo-C14-HSL) growth. To investigate the physiological effects of these long-chained AHLs in more detail, an RNA-seq experiment was performed during which S. robusta was treated with the growth-promoting C14-HSL and the growth-inhibiting oxo-C14-HSL. One tetramic acid was also tested (TA14), a structural rearrangement product of oxo-C14-HSL, which also induced growth inhibition in S. robusta. After 3 days of treatment, analysis revealed that 3,410 genes were differentially expressed in response to at least one of the compounds. In the treatment with the growth-promoting C14-HSL many genes involved in intracellular signaling were upregulated. On the other hand, exposure to growth-inhibiting oxo-C14-HSL and TA14 triggered a switch in lipid metabolism towards increased fatty acid degradation. In addition, oxo-C14-HSL led to downregulation of cell cycle genes, which is in agreement with the stagnation of cell growth in this treatment. Combined, our results indicate that bacterial signaling molecules with high structural similarity induce contrasting physiological responses in S. robusta.

Effect of New Analogs of Hexyloxy Phenyl Imidazoline on Quorum Sensing in Chromobacterium violaceum and In Silico Analysis of Ligand-Receptor Interactions

The increasing common occurrence of antibiotic-resistant bacteria has become an urgent public health issue. There are currently some infections without any effective treatment, which require new therapeutic strategies. An attractive alternative is the design of compounds capable of disrupting bacterial communication known as quorum sensing (QS). In Gram-negative bacteria, such communication is regulated by acyl-homoserine lactones (AHLs). Triggering of QS after bacteria have reached a high cell density allows them to proliferate before expressing virulence factors. Our group previously reported that hexyloxy phenylimidazoline (9) demonstrated 71% inhibitory activity of QS at 100 μM (IC50 = 90.9 μM) in Chromobacterium violaceum, a Gram-negative bacterium. The aim of the present study was to take 9 as a lead compound to design and synthesize three 2-imidazolines (13–15) and three 2-oxazolines (16–18), to be evaluated as quorum-sensing inhibitors on C. violaceum CV026. We were looking for compounds with a higher affinity towards the Cvi receptor of this bacterium and the ability to inhibit QS. The binding mode of the test compounds on the Cvi receptor was explored with docking studies and molecular dynamics. It was found that 8-pentyloxyphenyl-2-imidazoline (13) reduced the production of violacein (IC50 = 56.38 μM) without affecting bacterial growth, suggesting inhibition of quorum sensing. Indeed, compound 13 is apparently one of the best QS inhibitors known to date. Molecular docking revealed the affinity of compound 13 for the orthosteric site of N-hexanoyl homoserine lactone (C6-AHL) on the CviR protein. Ten amino acid residues in the active binding site of C6-AHL in the Cvi receptor interacted with 13, and 7 of these are the same as those interacting with AHL. Contrarily, 8-octyloxyphenyl-2-imidazoline (14), 8-decyloxyphenyl-2-imidazoline (15), and 9-decyloxyphenyl-2-oxazoline (18) bound only to an allosteric site and thus did not compete with C6-AHL for the orthosteric site.

Directed evolution of RhlI to generate new and increased quorum sensing signal molecule catalytic activities

Quorum sensing is a population density-dependent gene expression regulation mechanism in bacteria. The substrate specificity of RhlI, an enzyme in the RhlI–RhlR quorum sensing system of Pseudomonas aeruginosa, was explored by directed evolution to gain insight into the molecular mechanisms of quorum sensing. RhlI catalyzes S-adenosyl methionine and butanoyl or hexanoyl acyl carrier protein to form N-butanoyl homoserine lactone (BHL) and or N-hexanoyl homoserine lactone (HHL), respectively, none of which contain 3-oxo groups. We developed high-throughput genetic screening and selection methods to identify RhlI mutants via four rounds of directed evolution and identified RhlI-4M1 as the mutant that generated new catalytic activity and synthesized 3-oxo-hexanoyl homoserine lactone (OHHL) containing the 3-oxo group in Escherichia coli. Additionally, the synthesizing activities of BHL and HHL were improved by 3.98- and 3.01-fold, respectively. RhlI-4M1 contains five amino acid substitutions (A15D, K31R, T92S, Y129N, and L184Q) and one stop codon (Q193*) mutations. The deletion of nine amino acids in the C-terminus was crucial for OHHL production by RhlI mutants. This work demonstrates that the genetic screen/selection should be useful in the development of applications involving the manipulation of bacterial quorum sensing. The new catalytic activity of these RhlI mutants will prove beneficial in elucidating the mechanistic understanding of bacterial quorum sensing and similarly, may prove beneficial in the development of new drugs including antimicrobial compounds.

Inhibition of quorum sensing in opportunistic pathogen, Serratia marcescens, using cyclodextrin-immobilized, multiple parallel gel filaments fabricated with dynamic flow of polymer blend solution.

Prodigiosin production in the model opportunistic human pathogen, Serratia marcescens AS-1, depends on quorum sensing (QS), the activation of which is induced by an increase in N-hexanoyl homoserine lactone (C6HSL) concentration. When mixed with C6HSL in the broth culture of S. marcescens immobilized cyclodextrin (CD) effectively formed an inclusion complex resulting in suppression of the QS-mediated prodigiosin production. Thousands of parallel gel filament bundles could be fabricated by the dynamic flow of aqueous sodium alginate blended with α-CD or hydroxypropyl-β-CD (HP-β-CD) immobilized hydroxypropyl cellulose (HPC) inside a co-axial microfluidic device. Shear-induced fibrous domains of HPC-alginate complex assembly were immediately jacketed by calcium alginate gel formed by a sheath flow containing Ca2+ ions. Immobilized α-CD and HP-β-CD on the HPC/alginate gel fibers could inhibit the C6HSL-mediated prodigiosin production to approximately 10%. Furthermore, the swarming motility of S. marcescens AS-1 was effectively suppressed in the presence of free or immobilized α-CD or HP-β-CD. The CD-immobilized gel filament bundle is a novel type of preventive material against bacterial infections.

Strengthening of aerobic sludge granulation by the endogenous acylated homoserine lactones-secreting strain Aeromonas sp. A-L3

Increasing the quorum sensing effect has become an effective means of strengthening the sludge granulation process in recent years, but most of the current methods for achieving this effect involve the direct addition of signal molecules. To identify a more environmentally friendly biological method for accelerating the formation of aerobic granular sludge (AGS) and further reducing the operating cost, an Aeromonas sp. strain (A-L3) screened from AGS seed sludge was added to strengthen the granulation of aerobic sludge in a sequencing batch reactor (SBR). The results showed that strain A-L3 could secrete N-butyryl homoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL) and that these secretions were related to the OD600. The formation time of AGS could be shortened, and the aerobic granules could develop a larger size and denser structure. Furthermore, the addition of the endogenous strain A-L3 promoted the secretion of extracellular polymeric substances (EPS), and the efficiencies of chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) removal were improved. In addition, the contents of C4-HSL and C6-HSL were 27.77% and 20.46% higher, respectively, than those in the control on day 42. In summary, the addition of the AHLs-secreting strain A-L3 may be a novel economically and environmentally favorable strategy for strengthening the formation of AGS.

Interference in quorum sensing and virulence of the phytopathogen Pseudomonas syringae pv. passiflorae by Bacillus and Variovorax species

Pathogenic bacteria often engage in a form of cell-to-cell communication termed quorum sensing (QS) to coordinate the expression of multiple virulence factors. Therefore, interference of QS has potential as a means of controlling bacterial-mediated plant diseases. Accordingly, this study was aimed at: (1) identifying QS signals produced by the phytopathogen, Pseudomonas syringae pv. passiflorae, and (2) evaluating interference in QS and virulence of the pathogen by putative N-acyl homoserine lactone (AHL)-degrading Bacillus and Variovorax species as a biocontrol strategy. Detection of AHLs using the biosensor strain and high-resolution mass spectroscopy suggested that P. syringae pv. passiflorae produced N-tetradecanoyl homoserine lactone and N-hexanoyl homoserine lactone as quorum-sensing signal molecules. Evaluation of putative AHL-degrading bacteria as biocontrol agents, through a series of inhibition assays (inhibition of motility, biofilm and virulence factors), suggested that bacteria which targets AHLs could be used to control P. syringae pv. passiflorae. We further demonstrated that putative AHL-degrading Bacillus and Variovorax species prevented hypersensitivity in tomato plants by P. syringae pv. passiflorae. Taken together, these results indicated that putative AHL-degrading bacteria were potential biocontrol agents against P. syringae pv. passiflorae and reinforced the idea that disrupting QS and associated virulence factors could be an effective method in controlling plant pathogens.

Application of molecularly imprinted polymer nanoparticles for degradation of the bacterial autoinducer N-hexanoyl homoserine lactone.

A novel bacterial quorum quenching system is presented. For the first time the degradation of N-l-hexanoyl homoserine lactone (C6-AHL), a Gram-negative quorum sensing autoinducer, has been enhanced using molecularly imprinted nanoparticles (MIP NPs) which were prepared using transition state analogue of the γ-lactone ring hydrolysis as template.

Screening of quorum quenching activity of the bacteria isolated from dairy industry waste activated sludge

Intercellular bacterial communication process via exchange of signalling molecules acyl homoserine lactone results in various group activities like bioluminescence, antibiotic production, biofilm formation, sporulation, and virulence. The signalling molecules are targeted, and the communication is interrupted by a group of bacteria termed quorum quenching bacteria. The present study aims to isolate the quorum quenching bacteria from the waste activated sludge collected from the dairy industry effluent treatment plant and explore for its quorum quenching potential. The bacteria were cultured in the KG medium containing n-hexanoyl homoserine lactone as a sole source of carbon and nitrogen. The isolates were identified by the 16S ribosomal deoxyribonucleic acid analysis and subsequently were evaluated for its quorum quenching activity through Chromobacterium violaceum CV026 biosensor assay. The n-hexanoyl homoserine lactone degradation was quantified by GC–MS analysis. The 16S ribosomal deoxyribonucleic acid analysis revealed the isolated bacteria as Klebsiella pneumoniae (JYQ1 and JYQ5), Acinetobacter baumannii JYQ2, Pseudomonas nitroreducens JYQ3, and Pseudomonas JYQ4. The biosensor strain assay and GC–MS analysis indicated that all the isolates possessed an inherent ability to degrade N-hexanoyl homoserine lactone. The strain Pseudomonas JYQ4 exhibited the highest quorum quenching activity of 84 ± 3.3% within 6 h of incubation. The strain A. baumannii JYQ2 acted both as quorum sensing and as quorum quenching bacteria as evidenced by the decrease in quorum quenching from 79 ± 3.1 to 76.8 ± 2.5%.

The Quorum Sensing System of Yersinia enterocolitica 8081 Regulates Swimming Motility, Host Cell Attachment, and Virulence Plasmid Maintenance.

Although Yersinia enterocolitica genomes are highly heterogeneous, they contain a conserved N-acylhomoserine lactone-dependent (AHL) quorum sensing (QS) system consisting of the luxR and luxI orthologs yenR and yenI respectively. Certain hypervirulent strains also contain a putative orphan luxR gene, ycoR, that is not linked to an AHL synthase. To explore the contribution of yenR/yenI/ycoR to QS-dependent phenotypes in Yersinia enterocolitica strain 8081, single and multiple mutants were constructed. AHL profiling identified N-(3-oxohexanoyl) homoserine lactone, N-hexanoylhomoserine lactone, and N-(3-oxoseptanoyl) homoserine lactone as the most abundant. The AHL profiles of the yenR, ycoR and yenR/ycoR mutants were similar to the parent suggesting that the two LuxR homologues do not regulate AHL production while the yenI mutants were AHL-negative. A role for QS in swimming motility and cell attachment was demonstrated. Down-regulation of the virulence plasmid partition gene, spyA, in yenI and yenI/yenR/ycoR mutants is consistent with the greater loss of the Y. enterocolitica pYVe virulence plasmid in the yenI mutant during serial passage at 37 °C but not at 22 °C. A role for QS-regulated spyA in virulence plasmid maintenance is suggested.

Cadmium ion inhibition of quorum signalling in Chromobacterium violaceum.

Single-celled bacteria are capable of acting as a community by sensing and responding to population density via quorum signalling. Quorum signalling in Chromobacterium violaceum, mediated by the luxI/R homologue, cviI/R, regulates a variety of phenotypes including violacein pigmentation, virulence and biofilm formation. A number of biological and organic molecules have been described as quorum signalling inhibitors but, to date, metal-based inhibitors have not been widely tested. In this study, we show that quorum sensing is inhibited in C. violaceum in the presence of sub-lethal concentrations of cadmium salts. Notable Cd2+-inhibition was seen against pigmentation, motility, chitinase production and biofilm formation. Cd-inhibition of quorum-signalling genes occurred at the level of transcription. There was no direct inhibition of chitinase activity by Cd2+ at the concentrations tested. Addition of the cognate quorum signals, N-hexanoyl homoserine lactone or N-decanoyl homoserine lactone, even at concentrations in excess of physiological levels, did not reverse the inhibition, suggesting that Cd-inhibition of quorum signaling is irreversible. This study represents the first description of heavy metal-based quorum inhibition in C. violaceum.

Effect of small chain N acyl homoserine lactone quorum sensing signals on biofilms of food-borne pathogens.

Quorum sensing or cell to cell communication which includes inter- and intra-cellular communication has been implicated in the production of virulence factor and formation of biofilm in food-borne pathogens. In the present study, the effect of quorum sensing signals on the biofilms of food-borne pathogens has been elucidated. N-butryl homoserine lactone and N-hexanoyl homoserine lactone belonging to acyl homoserine lactone (AHL) family of signaling molecules were investigated for their effect on the biofilm formation (attachment and exopolymeric substance production) in the food-borne pathogens Escherichia coli, Salmonella enterica serovar Typhimurium and Vibrio parahemolyticus. The signaling molecules at a concentration of 1µM were capable of increasing biofilm formation in all the tested pathogens. There was an increase in the attachment of the bacterial cells and biomass as observed by microtiter plate assay and exopolymeric substances production in the biofilms in presence of the AHLs. Further, it needs to be elucidated if the effect of AHLS on the biofilms of E. coli and S. enterica serovar Typhimurium is SdiA dependent.

Switch of SpnR function from activating to inhibiting quorum sensing by its exogenous addition

The opportunistic human pathogen Serratia marcescens AS-1 produces the N-hexanoylhomoserine lactone (C6HSL) receptor SpnR, a homologue of LuxR from Vibrio fischeri, which activates pig clusters to produce the antibacterial prodigiosin. In this study, we attempted to artificially regulate quorum sensing (QS) by changing the role of SpnR in N-acylhomoserine lactone (AHL)-mediated QS. SpnR was obtained as a fusion protein tagged with maltose-binding protein (MBP) from overexpression in Escherichia coli, and its specific affinity to C6HSL was demonstrated by quartz crystal microbalance analysis and AHL-bioassay with Chromobacterium violaceum CV026. Prodigiosin production was effectively inhibited by externally added MBP-SpnR in both wild-type AS-1 and the AHL synthase-defective mutant AS-1(ΔspnI). For the mutant, the induced amount of prodigiosin was drastically reduced to approximately 4% with the addition of 18 μM MBP-SpnR to the liquid medium, indicating 81% trapping of C6HSL. A system for inhibiting QS can be constructed by adding exogenous AHL receptor to the culture broth to keep the concentration of free AHL low, whereas intracellular SpnR naturally functions as the activator in response to QS.

Degradation intermediates of polyhydroxy butyrate inhibits phenotypic expression of virulence factors and biofilm formation in luminescent Vibrio sp. PUGSK8.

Luminescent vibrios are ubiquitous in the marine environment and are the causative agents of vibriosis and mass mortality in many aquatic animals. In aquatic environments, treatments cannot be limited to the diseased population alone, therefore treatment of the entire aquatic system is the only possible approach. Thus, the use of antibiotics to treat part of the infected animals requires a dose based on the entire biomass, which results in the treatment of uninfected animals as well as non-target normal microbial flora. A treatment method based on anti-virulence or quorum quenching has recently been proposed as an effective treatment strategy for aquatic animals. Polyhydroxy butyrates (PHB) are bacterial storage molecules, which accumulate in cells under nutritional stress. The degradation of PHB releases short-chain β-hydroxy butyric acid, which may act as anti-infective molecule. To date, there is very limited information on the potential anti-infective and anti-virulence mechanisms involving PHB. In this study, we aim to examine the effect of PHB on inhibition of the virulence cascade of Vibrio such as biofilm formation, luminescence, motility behaviour, haemolysin and quorum sensing. A luminescent Vibrio PUGSK8, tentatively identified as Vibrio campbellii PUGSK8 was tested in vitro for production of extracellular virulence factors and then established as a potential shrimp pathogen based on in vivo challenge experiments. The ability of Vibrio PUGSK8 to form biofilms and the effect of PHB on biofilm formation was tested in a 96-well microtitre-plate assay system. The motility behaviour of Vibrio PUGSK8 was evaluated using twitching, swimming and swarming plate assays. Reporter strains such as Chromobacterium violaceum CV026 and Agrobacterium tumefaciens were used to detect quorum-sensing molecules. Gas chromatography–mass spectrometry spectral analysis was performed to elucidate the fragmentation pattern and structure of N-hexanoyl homoserine lactone. PHB depolymerase activity in Vibrio PUGSK8 was quantified as the amount of the enzyme solution to hydrolyse 1 μg of PHB per min. An in vivo challenge experiment was performed using a gnotobiotic Artemia assay. Of the 27 isolates tested, the Vibrio PUGSK8 strain was selected for target-specific assays based on the high intensity of luminescence and production of virulence factors. The virulence cascade detected in Vibrio PUGSK8 include luminescence, motility behaviour, biofilm formation, quorum sensing and haemolysin production. Thus inhibition/degradation of the virulence cascade would be an effective approach to contain Vibrio infections in aquatic animals. In this report, we demonstrate that the degradation intermediate of PHB effectively inhibits biofilm formation, luminescence, motility behaviour, haemolysin production and the N-acyl-homoserine lactone (AHL)-mediated quorum-sensing pathway in PUGSK8. Interestingly, the growth of Vibrio PUGSK8 remains unaffected in the presence of PHB, with PHB degradation being detected in the media. PHB depolymerase activity in Vibrio PUGSK8 results in the release of degradation intermediates include a short-chain β-hydroxy butyric acid, which inhibits the virulence cascade in Vibrio PUGSK8. Thus, a molecule that targets quorum sensing and the virulence cascade and which is species/strain-specific could prove to be an effective alternative to antimicrobial agents to control the pathogenesis of Vibrio, and thereby help to contain Vibrio outbreaks in aquatic systems.

Targeting quorum sensing by designing azoline derivatives to inhibit the N-hexanoyl homoserine lactone-receptor CviR: Synthesis as well as biological and theoretical evaluations

To counteract bacterial resistance, we investigated the interruption of quorum sensing mediated by non-classical bioisosteres of the N-hexanoyl homoserine lactone with an azoline core. For this purpose, a set of selected 2-substituted azolines was synthesized, establishing the basis for a new protocol to synthesize 2-amino imidazolines. The synthesized compounds were evaluated as inhibitors of violacein production in Chromobacterium violaceum. Theoretical studies on bioisostere–protein interactions were performed using CviR. The results show that some azolines decreased violacein production, suggesting an antiquorum sensing profile against Gram-negative bacteria. Docking and molecular dynamic simulations together with binding free energy calculations revealed the exact binding and inhibitory profiles. These theoretical results show relationship with the in vitro activity of the azoline series.