Degradation Of Acyl-homoserine Lactones Research Articles

Extracellular lactonase-mediated quorum quenching by a novel Bacillus velezensis.

Many Gram-negative bacteria coordinate their gene expression via quorum sensing (QS) mediated by small diffusible autoinducer molecules such as Acyl Homoserine Lactones (AHL). The degradation of AHLs or quorum quenching (QQ) imparts less selection pressure on the target organisms leading to a possible alternative to antimicrobial agents and traditional biofilm control methods. Here, a novel strain of Bacillus velezensis, strain PM7, exhibiting extracellular QQ activity against Gram-negative bacteria has been isolated. Analytical studies of the AHL degradation using LC-MS as well as bioreporters revealed the mechanism of QQ as homoserine lactone ring hydrolysis. Using molecular techniques, the presence of an aiiA homologues gene, specific for acyl homoserine lactonase enzyme, was confirmed in the bacterium. A biofilm inhibitory activity in the range of 36.9%-77.4% was achieved by the PM7 against three different Gram-negative bacteria. Moreover, a significant reduction (P<.05) in the QS-controlled traits such as violacein production in Chromobacterium violaceum (73%), swarming motility and prodigiosin production (67.2%) in Serratia marcescens, and exopolysaccharide production (97.9%) in Pseudomonas aeruginosa, was also observed. The extracellular and broad-spectrum lactonase of this novel strain can be beneficial for its use in QQ applications.

Leveraging quorum sensing system for automatic coordination of Escherichia coli growth and lactic acid biosynthesis

PurposeOverproduction of desired metabolites usually sacrifices cell growth. Here we report that quorum sensing (QS) can be exploited to coordinate cell growth and lactic acid production in Escherichia coli.MethodsWe engineered two QS strains: one strain overexpressing acyl-homoserine lactone (AHL) synthesis genes (“ON”), the other strain overexpressing both AHL synthesis and degradation gene (aiiA) (“ON to semi-OFF”). To clarify the impact of the QS system on lactic acid production, D-lactate dehydrogenase gene ldhA was deleted from the E. coli genome, and Enhanced Green Fluorescence Protein (eGFP) was used as the reporter.ResultsCompared to the “ON” strain, the “ON to semi-OFF” strain showed delayed log growth and decreased egfp expression at stationary phase. When egfp was replaced by ldhA for lactic acid production, compared to the wild-type strain, the “ON to semi-OFF” strain demonstrated 231.9% and 117.3% increase in D-lactic acid titer and space-time yield, respectively, while the “ON” strain demonstrated 83.6%, 31%, and 36% increase in growth rate, maximum OD600, and glucose consumption rate, respectively. Quantitative real-time PCR revealed that both ldhA and the genes for phosphotransferase system were up-regulated in ldhA-overexpressing “ON” strain compared to the strain only harboring QS system. Moreover, the “ON” strain showed considerable increase in glucose consumption after a short lag phase. Compared to the reference strain harboring only ldhA gene in vector, both the “ON” and “ON to semi-OFF” strains demonstrated synchronization between cell growth and D-lactic acid production.ConclusionsCollectively, QS can be leveraged to coordinate microbial growth and product formation.

Antiviral, Antibacterial and Anti-Quorum Sensing Activities of a Lyophilized Aqueous Pine Resin Extract

This work aimed to evaluate antiviral, antibacterial and anti-quorum sensing effects of a lyophilized aqueous pine resin extract. The pine resin toxicity was evaluated by a crystal violet assay at 492 nm for Hep-2 cells and the trypan blue method for P3HR1. The cytotoxicity expressed in CC50 was 12.5 mg/ml and 0.23 mg/ml on Hep-2 and P3HR1 cells, respectively. The antiviral effect was obtained at 4 mg/ml against coxsackievirus B4 (CVB4). Using the diffusion method discs for the antibacterial activity, the extract was more active against Gram+ than Gram−. Inhibition zones of 22 mm were obtained at a 275 mg/ml concentration. Inhibition of violacein production by pine resin extract was tested usingChomobacterium violaceum026 (CV026) in a medium supplemented with acyl-homoserine lactone (AHL); cytotoxicity was evaluated toward Artemia and its protecting effect againstVibrio harveyiBB120 and then AHL degradation was studied. Results showed that pine resin inhibited violacein production by CV026, was not toxic for Artemia and protected it againstVibrio harveyiBB120; AHL was degraded with inhibition areas from 9.6, 11.03 and 10.26 to 12.34 mm at the contact times of the mixtures “resin-AHL” respectively at 0 h, 4 h 24 h and 72 h. This study constitutes a confirmation of aqueous extract pine resin as antibacterial, by anti-quorum sensing pathway and antiviral efficacy.

Immobilizing a visible light-responsive photocatalyst on a recyclable polymeric composite for floating and suspended applications in water treatment.

A visible light responsive TiO2/Ag3PO4 (10 : 1) nanocomposite was prepared and successfully immobilized (12 wt%) in a spherical polymeric matrix consisting of polysulfone and alginate (10 : 6). The resulted beads featured a sponge-like structure with interconnected macrovoids and micropores, and showed high adsorption and visible-light photocatalytic activity towards various wastewater pollutants, including the widely used dye – methylene blue (k = 0.0321 min−1), and two emerging pharmaceutical contaminants – diclofenac (k = 0.018 min−1) and triclosan (k = 0.052 min−1). As determined, the ˙OH radical and h+ are the primary reactive oxygen species responsible for the photodegradation. The composite photocatalytic beads are also effective in bacterial inactivation and degradation of acyl-homoserine lactones (AHLs), the bacterial quorum sensing autoinducers triggering biofilms, thus exhibiting a promising future in wastewater disinfection and biofilm retardation. Additionally, these beads could be used in inter-switchable suspended or buoyant forms, and be effectively regenerated by H2O2 treatment, and used for multiple cycles without any significant loss in photoactivity. With these unique features, the prepared visible-light photocatalytic beads could be easily applied in large-scale water and wastewater treatment systems.

Attenuation of Quorum Sensing Regulated Virulence of Pectobacterium carotovorum subsp. carotovorum through an AHL Lactonase Produced by Lysinibacillus sp. Gs50.

Quorum sensing (QS) is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs) and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ), is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp) designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- β- lactamase superfamily, has a conserved “HXHXDH” motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 μM and the specific activity was 0.8 picomol min-1μg-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc), a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent.

Engineering of a thermostable esterase Est816 to improve its quorum-quenching activity and the underlying structural basis.

N-acyl-homoserine lactones (AHLs) are small diffusible molecules called autoinducers that mediate cell-to-cell communications. Enzymatic degradation of AHLs is a promising bio-control strategy known as quorum-quenching. To improve the quorum-quenching activity of a thermostable esterase Est816, which had been previously cloned, we have engineered the enzyme by random mutagenesis. One of the mutants M2 with double amino acid substitutions (A216V/K238N) showed 3-fold improvement on catalytic efficiency. Based on the crystal structure determined at 2.64 Å, rational design of M2 was conducted, giving rise to the mutant M3 (A216V/K238N/L122A). The kcat/KM value of the mutant M3 is 21.6-fold higher than that of Est816. Furthermore, activity assays demonstrated that M3 reached 99% conversion of 10-μM N-octanoyl-DL-homoserine lactone (C8-HSL) to N-octanoyl- DL-homoserine (C8-Hse) in 20 min, in contrast to the 8 h required by wild type Est816. The dramatic activity enhancement may be attributed to the increased hydrophobic interactions with the lactone ring by the mutation A216V, and the reduced steric clashes between the long side chain of L122 and the aliphatic tail of HSL by the mutation L122A, according to the crystal structure. This study sheds lights on the activity-structure relationship of AHL-lactonases, and may provide useful information in engineering AHL-degrading enzymes.

Colostrum Hexasaccharide, a Novel Staphylococcus aureus Quorum-Sensing Inhibitor

The discovery of quorum-sensing (QS) systems regulating antibiotic resistance and virulence factors (VFs) has afforded a novel opportunity to prevent bacterial pathogenicity. Dietary molecules have been demonstrated to attenuate QS circuits of bacteria. But, to our knowledge, no study exploring the potential of colostrum hexasaccharide (CHS) in regulating QS systems has been published. In this study, we analyzed CHS for inhibiting QS signaling in Staphylococcus aureus. We isolated and characterized CHS from mare colostrum by high-performance thin-layer chromatography (HPTLC), reverse-phase high-performance liquid chromatography evaporative light-scattering detection (RP-HPLC-ELSD), (1)H and (13)C nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). Antibiofilm activity of CHS against S. aureus and its possible interference with bacterial QS systems were determined. The inhibition and eradication potentials of the biofilms were studied by microscopic analyses and quantified by 96-well-microtiter-plate assays. Also, the ability of CHS to interfere in bacterial QS by degrading acyl-homoserine lactones (AHLs), one of the most studied signal molecules for Gram-negative bacteria, was evaluated. The results revealed that CHS exhibited promising inhibitory activities against QS-regulated secretion of VFs, including spreading ability, hemolysis, protease, and lipase activities, when applied at a rate of 5 mg/ml. The results of biofilm experiments indicated that CHS is a strong inhibitor of biofilm formation and also has the ability to eradicate it. The potential of CHS to interfere with bacterial QS systems was also examined by degradation of AHLs. Furthermore, it was documented that CHS decreased antibiotic resistance in S. aureus. The results thus give a lead that mare colostrum can be a promising source for isolating a next-generation antibacterial.

Novel approach of using homoserine lactone-degrading and poly-β-hydroxybutyrate-accumulating bacteria to protect Artemia from the pathogenic effects of Vibrio harveyi

Homoserine lactone-degrading and poly-β-hydroxybutyrate-accumulating enrichment cultures (ECs) were obtained using glycerol as C-source and homoserine lactones as N-source, at a high C/N ratio (78). The kinetics of acyl homoserine lactone (AHL) degradation was established and the poly-β-hydroxybutyrate (PHB) content of the obtained ECs was determined. The ECs were characterised through denaturing gradient gel electrophoresis (DGGE) and the main bands were genetically identified by sequence analysis. In a Vibrio challenge test using Artemia as target organism, a distinct difference in survival of challenged Artemia between the treatments with and without the presence of ECs was observed (53% ± 6; 69% ± 5 and 58% ± 8 versus 4% ± 5; 4% ± 5 and 25% ± 21) in three independent experiments. There was a strong positive correlation between the relative percentage survival values of Artemia and the AHL degradation rate and the PHB content of the ECs. Challenge data with a quorum sensing mutant suggested that the quorum quenching ability of the ECs was less relevant in this Artemia model system, pointing to the importance of PHB. The potential of growing these beneficial ECs on glycerol released into the hatching medium of Artemia was an asset that still requires validation at hatchery level.

Central Role of Quorum Sensing in Regulating the Production of Pathogenicity Factors in PSeudomonas Aeruginosa

Pseudomonas aeruginosa is an important opportunistic human pathogen, causing various infections that are often very persistent. P. aeruginosa infections are the major cause of death in cystic fibrosis patients. Infections are difficult to treat since P. aeruginosa is resistant to most antibiotics and its antibiotic susceptibility is decreased when it is present in biofilms. P. aeruginosa produces many exoproducts (including toxins and hydrolytic enzymes) that are involved in virulence. Recent research has elucidated many mechanisms and pathways that regulate the production of these virulence factors. The regulation is extremely complex and many components are influenced by environmental conditions. Quorum sensing is a key regulatory system, which itself is affected by many other regulators. Targeting the regulation of pathogenicity factors provides a novel strategy for combating P. aeruginosa infections. Degradation of acyl homoserine lactones, the signaling molecules of the quorum-sensing system, is a promising therapeutic treatment option.

Quorum quenching enzyme activity is widely conserved in the sera of mammalian species

Acyl-homoserine lactone (AHL) quorum sensing signals play a key role in synchronizing virulence gene expression in Pseudomonas aeruginosa, which could cause fatal bloodstream infections. We showed that AHL inactivation activity, albeit with variable efficiency, was conserved in the serum samples of all the 6 tested mammalian animals. High-performance liquid chromatography and mass spectrometry analyses revealed that mammalian sera had a lactonase-like enzyme(s), which hydrolyzed the lactone ring of AHL to produce acyl homoserine, with enzyme properties reminiscent of paraoxonases (PONs). We further showed that the animal cell lines expressing three mouse PON genes, respectively, displayed strong AHL degradation activities.

Microbial diversity and prevalence of virulent pathogens in biofilms developed in a water reclamation system

Bacterial biofilm is a common phenomenon in both natural and engineered systems which often becomes a source of contamination and microbially influenced corrosion. It is thought that formation of biofilm in the monoculture of several bacterial species is regulated by acylhomoserine lactone (AHL) quorum-sensing signals. In this study, we investigated the microbial diversity and existence of AHL-producing and AHL-degrading bacterial species in the biofilm samples from a water reclamation system located in a tropical environment. 16S ribosomal DNA sequencing analysis indicated the presence of at least 11 bacterial species, including the frequently encountered bacterial pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae, and several rare pathogens. We showed that only two groups of isolates, belonging to P. aeruginosa and Enterobacter agglomerans, produced AHL signals. We also found that three bacterial isolates, i.e., Agrobacterium tumefaciens XJ01, Bacillus cereus XJ08, and Ralstonia sp. XJ12, expressed AHL degradation enzymes. Furthermore, we showed that P. aeruginosa isolate HL43 was virulent against animal model Caenorhabditis elegans and released 2–6-fold more pyocyanin cytotoxin than P. aeruginosa strains PA01 and PA14, the two commonly used laboratory strains. These data indicate the complexity and importance of biofilm research in water reclamation.