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The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to control its virulence. One of its major signal molecules, the Pseudomonas quinolone signal PQS, has high affinity to membranes and is known to be trafficked mainly via outer membrane vesicles (OMVs). We previously reported that several 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) catalyze the cleavage of PQS and thus act as quorum quenching enzymes. Further analysis showed that, in contrast to other HQDs, the activity of HQD from Streptomyces bingchenggensis (HQDS.b.) was unexpectedly stabilized by culture supernatants of P. aeruginosa. Interestingly, the stabilizing effect was higher with supernatants from the strain PA14 than with supernatants from the strain PAO1. Heat treatment and lyophilization hardly affected the stabilizing effect; however, fractionation of the supernatant excluded small molecules as stabilizing agents. In a pull-down assay, HQDS.b. appeared to interact with several P. aeruginosa proteins previously found in the OMV proteome. This prompted us to probe the physical interaction of HQDS.b. with prepared extracellular membrane vesicles. Homo-FRET of fluorescently labeled HQDS.b. indeed indicated a spatial clustering of the protein on the vesicles. Binding of a PQS-cleaving enzyme to the OMVs of P. aeruginosa may enhance PQS degradation and is highly reconcilable with its function as a quorum quenching enzyme. [ABSTRACT FROM AUTHOR]

To obtain composite fiber materials with antibacterial properties, the samples of bacterial cellulose (BC), polylactide (PLA) and usual fibers (FM) were modified by poly-ε-caprolactone or polyhydroxybutyrate and then functionalized by the enzyme-polyelectrolyte complex of quorum-quenching enzymes, such as hexahistidine-tagged organophosphorus hydrolase with poly(glutamic acid) or by suspension of tantalum nanoparticles (Ta NPs) in ethanol. The structures of the composite fibers were analyzed using scanning electron microscopy. It was shown that the introduction of additional natural polymers into the matrix of BC, PLA and FM resulted in decreasing of the structural porosity. Comparative studies of the antibacterial activity of the composite materials were carried out using Escherichia coli and Bacillus subtilis cells. The decrease of adenosine triphosphate concentration in cell samples loaded onto fiber materials was applied as a measurable characteristic of antibacterial effect typical for the new fiber materials. The profound improvement of antibacterial activity was determined in composite materials with polyhydroxybutyrate and Ta NPs. [ABSTRACT FROM AUTHOR]

ABSTRACT: To seek viable alternatives to antibiotics, we determined the combinatorial effects of Lactobacillus and a quorum quenching enzyme (QQE) on broiler growth performance, antioxidant capacity, immune responses, and cecal microbial populations. In total, 360 one-day-old male broilers (Ross 308) were randomly allotted to 3 dietary treatments, with 12 replicate pens/treatment and 10 birds/replicate pen. Dietary treatments lasted 42 d and comprised: corn-soybean meal basal diet (control group, CON); control plus antibiotic growth promoter supplement group (AGP); and control plus Lactobacillus and QQE supplement group (LQ). Dietary LQ supplementation significantly increased final body weight (BW) and average daily gain (ADG) when compared with CON and AGP groups between 22 and 42 d and 1 to 42 d (P < 0.05). No significant differences were observed for serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels between treatments (P > 0.05). A higher concentration of total antioxidant capacity (T-AOC) was observed on d 42 in the LQ group (P = 0.06). Feeding LQ significantly increased serum immunoglobulins (IgA and IgG) levels when compared with other treatments (P < 0.05). A statistical trend was also observed for increased cecal butyrate levels (P = 0.06) in the LQ group. Bacterial α-diversity was unaffected by dietary treatments (P > 0.05). However, from principal component analysis (PCoA), the microbial community structure was different between the LQ and AGP groups. Diet supplemented with LQ significantly (P < 0.05) decreased the relative abundance of Synergistota and Proteobacteria and significantly (P < 0.05) increased the proportion of Ruminococcaceae and Faecalibacterium. Thus, supplemental LQ improved growth performance, immune status, and modulated intestinal microbial communities in broilers. We provide a new perceptive on antibiotic substitutes in the poultry industry.

The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to control its virulence. One of its major signal molecules, the Pseudomonas quinolone signal PQS, has high affinity to membranes and is known to be trafficked mainly via outer membrane vesicles (OMVs). We previously reported that several 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) catalyze the cleavage of PQS and thus act as quorum quenching enzymes. Further analysis showed that, in contrast to other HQDs, the activity of HQD from Streptomyces bingchenggensis (HQDS.b.) was unexpectedly stabilized by culture supernatants of P. aeruginosa. Interestingly, the stabilizing effect was higher with supernatants from the strain PA14 than with supernatants from the strain PAO1. Heat treatment and lyophilization hardly affected the stabilizing effect; however, fractionation of the supernatant excluded small molecules as stabilizing agents. In a pull-down assay, HQDS.b. appeared to interact with several P. aeruginosa proteins previously found in the OMV proteome. This prompted us to probe the physical interaction of HQDS.b. with prepared extracellular membrane vesicles. Homo-FRET of fluorescently labeled HQDS.b. indeed indicated a spatial clustering of the protein on the vesicles. Binding of a PQS-cleaving enzyme to the OMVs of P. aeruginosa may enhance PQS degradation and is highly reconcilable with its function as a quorum quenching enzyme.

Quorum quenching (QQ) is proposed as a new strategy for mitigating microbe-associated problems (e.g., fouling, biocorrosion). However, most QQ agents reported to date have not been evaluated for their quenching efficacies under conditions representative of seawater desalination plants, cooling towers or marine aquaculture. In this study, bacterial strains were isolated from Saudi Arabian coastal environments and screened for acyl homoserine lactone (AHL)-quenching activities. Five AHL quenching bacterial isolates from the genera Pseudoalteromonas, Pontibacillus, and Altererythrobacter exhibited high AHL-quenching activity at a salinity level of 58 g/L and a pH of 7.8 at 50°C. This result demonstrates the potential use of these QQ bacteria in mitigating microbe-associated problems under saline and alkaline conditions at high (>37°C) temperatures. Further characterizations of the QQ efficacies revealed two bacterial isolates, namely, Pseudoalteromonas sp. L11 and Altererythrobacter sp. S1-5, which could possess enzymatic QQ activity. The genome sequences of L11 and S1-5 with a homologous screening against reported AHL quenching genes suggest the existence of four possible QQ coding genes in each strain. Specifically, two novel AHL enzymes, AiiAS1-5 and EstS1-5 from Altererythrobacter sp. S1-5, both contain signal peptides and exhibit QQ activity over a broad range of pH, salinity, and temperature values. In particular, AiiAS1-5 demonstrated activity against a wide spectrum of AHL molecules. When tested against three bacterial species, namely, Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio alginolyticus, AiiAS1-5 was able to inhibit the motility of all three species under saline conditions. The biofilm formation associated with P. aeruginosa was also significantly inhibited by AiiAS1-5. AiiAS1-5 also reduced the quorum sensing-mediated virulence traits of A. hydrophila, P. aeruginosa, and V. alginolyticus during the mid and late exponential phases of cell growth. The enzyme did not impose any detrimental effects on cell growth, suggesting a lower potential for the target bacterium to develop resistance over long-term exposure. Overall, this study suggested that some QQ enzymes obtained from the bacteria that inhabit saline environments under high temperatures have potential applications in the mitigation of microbe-associated problems.

Quorum quenching (QQ) is proposed as a new strategy for mitigating microbe-associated problems (e.g., fouling, biocorrosion). However, most QQ agents reported to date have not been evaluated for their quenching efficacies under conditions representative of seawater desalination plants, cooling towers or marine aquaculture. In this study, bacterial strains were isolated from Saudi Arabian coastal environments and screened for acyl homoserine lactone (AHL)-quenching activities. Five AHL quenching bacterial isolates from the genera Pseudoalteromonas, Pontibacillus , and Altererythrobacter exhibited high AHL-quenching activity at a salinity level of 58 g/L and a pH of 7.8 at 50°C. This result demonstrates the potential use of these QQ bacteria in mitigating microbe-associated problems under saline and alkaline conditions at high (>37°C) temperatures. Further characterizations of the QQ efficacies revealed two bacterial isolates, namely, Pseudoalteromonas sp. L11 and Altererythrobacter sp. S1-5, which could possess enzymatic QQ activity. The genome sequences of L11 and S1-5 with a homologous screening against reported AHL quenching genes suggest the existence of four possible QQ coding genes in each strain. Specifically, two novel AHL enzymes, AiiAS1-5 and EstS1-5 from Altererythrobacter sp. S1-5, both contain signal peptides and exhibit QQ activity over a broad range of pH, salinity, and temperature values. In particular, AiiAS1-5 demonstrated activity against a wide spectrum of AHL molecules. When tested against three bacterial species, namely, Aeromonas hydrophila, Pseudomonas aeruginosa , and Vibrio alginolyticus , AiiAS1-5 was able to inhibit the motility of all three species under saline conditions. The biofilm formation associated with P. aeruginosa was also significantly inhibited by AiiAS1-5. AiiAS1-5 also reduced the quorum sensing-mediated virulence traits of A. hydrophila, P. aeruginosa , and V. alginolyticus during the mid and late exponential phases of cell growth. The enzyme did not impose any detrimental effects on cell growth, suggesting a lower potential for the target bacterium to develop resistance over long-term exposure. Overall, this study suggested that some QQ enzymes obtained from the bacteria that inhabit saline environments under high temperatures have potential applications in the mitigation of microbe-associated problems. [ABSTRACT FROM AUTHOR]

[Display omitted] • Effluent reflux enhanced AOB and suppressed NOB. • Rapid and effective nitrite accumulation was achieved via effluent reflux. • Quorum quenching enzyme limited the growth of AOB but slightly affected its activity. • Quorum quenching enzyme induced denitrification which then consumed the nitrite. • 43.2% of TN was removed via autotrophic or endogenous denitrification. Nitrite accumulation is crucial in nitrogen removal processes that treat wastewater with a low carbon-to-nitrogen (C/N) ratio. This study investigates the effectiveness of effluent reflux in accelerating nitrite accumulation, considering quorum sensing, and explores the impact of quorum quenching enzymes. The results demonstrate that effluent reflux successfully accelerates the nitrite accumulation rate to 92.2% and increases the abundance of Nitrosomonas from 0.2% to 19.5%, as well as hydroxylamine oxidoreductase (HAO) from 0.285 Eu g−1 SS to 0.475 Eu g−1 SS. The quenching enzyme suppresses the growth of aerobic ammonia-oxidizing bacteria, leading to a decrease in Nitrosomonas abundance to 4.2% and HAO to 0 Eu g−1 SS. However, it enhances denitrification, which consumes nitrite and results in the deterioration of nitrite accumulation. Consequently, the total nitrogen (TN) removal efficiency increases from 15.5% to 43.2%, and the abundance of Hyphomicrobium increases to 33.7% accordingly. Quorum sensing proves to be an effective strategy for regulating ammonia removal and nitrite accumulation, and effluent reflux has a positive effect on enhancing quorum sensing. [ABSTRACT FROM AUTHOR]

MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.

Quorum sensing (QS) is closely associated with the production of multiple virulence factors in bacterial pathogens. N-acyl homoserine lactones (AHLs) are important QS signal molecules that modulate the virulence of gram-negative pathogenic bacteria. Enzymatic degradation of AHLs to interrupt QS, termed quorum quenching (QQ), has been considered a novel strategy for reduction of pathogenicity and prevention of bacterial disease. However, the low expression levels of QQ proteins in the original host bacteria has affected the applications of these proteins. Previously, we identified a novel marine QQ enzyme, named MomL, with high activity and promising biocontrol function. In this study, we linked the target fragment momL to pNCMO2, which provided a basis for the first heterologous expression of MomL in the antifungal and anti-gram-positive-bacteria biocontrol strain Bacillus brevis, and obtaining the recombinant strain named BbMomL. The QQ activity of BbMomL was confirmed using a series of bioassays. BbMomL could not only degrade the exogenous signal molecule C6-HSL, but also the AHL signal molecules produced by the gram-negative pathogens Pectobacterium carotovorum subsp. carotovorum (Pcc) and Pseudomonas aeruginosa PAO1. In addition, BbMomL significantly reduced the secretion of pathogenic factors and the pathogenicity of Pcc and P. aeruginosa PAO1. We tested the biocontrol function of BbMomL for prevention of plant diseases in vitro. The result indicates that BbMomL has a broad antibacterial spectrum. Compared with wild-type B. brevis, BbMomL not only inhibited fungi and gram-positive bacterial pathogens but also considerably inhibited gram-negative bacterial pathogens. Moreover, the Bacillus brevis expression system has good application prospects and is an ideal host for expression and secretion of foreign proteins.

Hydrogels have become promising materials for food packaging due to their unique microstructure. However, hydrogel materials suitable for seafood preservation have rarely been reported. In this study, a tamarind polysaccharide-polyvinyl alcohol hydrogel with the ability to maintain seafood freshness was prepared and characterized. The hydrogel possesses quick self-healing, good tissue fitting, and freezing tolerance capability. Moreover, a peeling force of only 0.1 N between the hydrogel and the fillet tissue confirmed the non-stick properties. The FTIR characteristic peak at 1600 cm−1 and 1450 cm−1 proved the ester bond-based chemical cross-linking of the hydrogel. Release profiles at pH 6.0 to 8.0 verified the pH-responsive release of quorum-quenching (QQ) enzymes over 120 h, which enabled the hydrogel to achieve biofilm and protease inhibitory activities. In vivo spoilage tests showed that the shelf life of hydrogel-coated red snapper fillets was extended by >3 days. These results illustrate the potential of the prepared hydrogel as functional packaging for seafood preservation. [Display omitted] [ABSTRACT FROM AUTHOR]

Membrane biofouling is a process that can impede the development of membrane bioreactor (MBR), which constitutes an important system of the wastewater treatment process. Membrane biofouling is governed by quorum sensing (QS), a communication system heavily dependent on the activities of signal molecules. Certain bacteria, known as quorum quenching (QQ) bacteria, can quench the QS process by destroying the signal molecules. These QQ bacteria are considered a sustainable and feasible way of mitigating membrane biofouling in MBR. In this study, a QQ enzyme (designated as AisZ) from a Serratia sp. was first identified and characterized. Escherichia coli BL21 expressing AisZ was able to degrade different QS signal molecules. Furthermore, these cells could also mitigate membrane biofouling in MBR during a 29-day operation by reducing the transmembrane pressure from 31 to 21 kPa. The metal ions Co2+ and Ni2+ were relatively important to AisZ in that they could significantly enhance the activity of AisZ and restore the EDTA-inactivated AisZ. Expression of the ais A gene was not influenced by Co2+, Ni2+ and QS signal molecules. AisZ might, therefore, extend the diversity of potential candidates for the mitigation of biofouling associated with membrane filtration technologies. [Display omitted] • A quorum-quenching enzyme (AisZ) was first identified in Serratia sp. • AisZ mitigated the membrane biofouling in a membrane bioreactor. • The expression of ais Z was constitutive. • The activity of AisZ was enhanced by Co2+ and Ni2+. [ABSTRACT FROM AUTHOR]

To seek viable alternatives to antibiotics, we determined the combinatorial effects of Lactobacillus and a quorum quenching enzyme (QQE) on broiler growth performance, antioxidant capacity, immune responses, and cecal microbial populations. In total, 360 one-day-old male broilers (Ross 308) were randomly allotted to 3 dietary treatments, with 12 replicate pens/treatment and 10 birds/replicate pen. Dietary treatments lasted 42 d and comprised: corn-soybean meal basal diet (control group, CON); control plus antibiotic growth promoter supplement group (AGP); and control plus Lactobacillus and QQE supplement group (LQ). Dietary LQ supplementation significantly increased final body weight (BW) and average daily gain (ADG) when compared with CON and AGP groups between 22 and 42 d and 1 to 42 d (P < 0.05). No significant differences were observed for serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels between treatments (P > 0.05). A higher concentration of total antioxidant capacity (T-AOC) was observed on d 42 in the LQ group (P = 0.06). Feeding LQ significantly increased serum immunoglobulins (IgA and IgG) levels when compared with other treatments (P < 0.05). A statistical trend was also observed for increased cecal butyrate levels (P = 0.06) in the LQ group. Bacterial α-diversity was unaffected by dietary treatments (P > 0.05). However, from principal component analysis (PCoA), the microbial community structure was different between the LQ and AGP groups. Diet supplemented with LQ significantly (P < 0.05) decreased the relative abundance of Synergistota and Proteobacteria and significantly (P < 0.05) increased the proportion of Ruminococcaceae and Faecalibacterium. Thus, supplemental LQ improved growth performance, immune status, and modulated intestinal microbial communities in broilers. We provide a new perceptive on antibiotic substitutes in the poultry industry. [ABSTRACT FROM AUTHOR]

Bacteria are the main cause of spoilage for fish and fishery products. Through the inactivation of the quorum sensing (QS) system, quorum quenching (QQ) enzymes can block the synthesis of bacterial virulence factors and effectively inhibit bacteria-induced food spoilage. This study analyzed the changes of microbiota in red sea bream filets during refrigerated storage. The results showed a decrease in microbial diversity with storage time, with Aeromonas veronii becoming the dominant bacteria on day 4. A novel N -acyl homoserine lactones (AHL) acylase PF2571, from the screened QQ bacterium Pseudomonas fluorescens PF08, was identified and expressed in Escherichia coli to evaluate its QQ efficiency and effects on spoilage potential. Spoilage-related QS factors of A. veronii BY-8, including biofilm formation, motility, and protease, lipase, and alginate production, were inhibited by PF2571. Its inhibitory effect on red sea bream spoilage was demonstrated by the lower freshness indicators for PF2571 treated filets. Our study demonstrates the potential of the QQ enzyme for prolonging the shelf life of fish and fishery products. • The dynamics and composition of the microbial communities in red sea bream filets during storage at 4 °C were analyzed. • The quorum quenching enzyme PF2571 was identified and heterologous expressed. • The purified PF2571 enzyme showed good QS inhibitory activity against A. veronii BY-8. • The purified PF2571 enzyme effectively improved the quality of red sea bream filets during storage. [ABSTRACT FROM AUTHOR]

MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase. [ABSTRACT FROM AUTHOR]

Quorum sensing (QS) is closely associated with the production of multiple virulence factors in bacterial pathogens. N-acyl homoserine lactones (AHLs) are important QS signal molecules that modulate the virulence of gram-negative pathogenic bacteria. Enzymatic degradation of AHLs to interrupt QS, termed quorum quenching (QQ), has been considered a novel strategy for reduction of pathogenicity and prevention of bacterial disease. However, the low expression levels of QQ proteins in the original host bacteria has affected the applications of these proteins. Previously, we identified a novel marine QQ enzyme, named MomL, with high activity and promising biocontrol function. In this study, we linked the target fragment momL to pNCMO2, which provided a basis for the first heterologous expression of MomL in the antifungal and anti-gram-positive-bacteria biocontrol strain Bacillus brevis, and obtaining the recombinant strain named BbMomL. The QQ activity of BbMomL was confirmed using a series of bioassays. BbMomL could not only degrade the exogenous signal molecule C6-HSL, but also the AHL signal molecules produced by the gram-negative pathogens Pectobacterium carotovorum subsp. carotovorum (Pcc) and Pseudomonas aeruginosa PAO1. In addition, BbMomL significantly reduced the secretion of pathogenic factors and the pathogenicity of Pcc and P. aeruginosa PAO1. We tested the biocontrol function of BbMomL for prevention of plant diseases in vitro. The result indicates that BbMomL has a broad antibacterial spectrum. Compared with wild-type B. brevis, BbMomL not only inhibited fungi and gram-positive bacterial pathogens but also considerably inhibited gram-negative bacterial pathogens. Moreover, the Bacillus brevis expression system has good application prospects and is an ideal host for expression and secretion of foreign proteins. [ABSTRACT FROM AUTHOR]

The nosocomial pathogen Pseudomonas aeruginosa regulates its virulence via a complex quorum sensing network, which, besides N -acylhomoserine lactones, includes the alkylquinolone signal molecules 2-heptyl-3-hydroxy-4(1 H )-quinolone ( Pseudomonas quinolone signal [PQS]) and 2-heptyl-4(1 H )-quinolone (HHQ). Mycobacteroides abscessus subsp. abscessus , an emerging pathogen, is capable of degrading the PQS and also HHQ. Here, we show that although M. abscessus subsp. abscessus reduced PQS levels in coculture with P. aeruginosa PAO1, this did not suffice for quenching the production of the virulence factors pyocyanin, pyoverdine, and rhamnolipids. However, the levels of these virulence factors were reduced in cocultures of P. aeruginosa PAO1 with recombinant M. abscessus subsp. massiliense overexpressing the PQS dioxygenase gene aqdC of M. abscessus subsp. abscessus , corroborating the potential of AqdC as a quorum quenching enzyme. When added extracellularly to P. aeruginosa cultures, AqdC quenched alkylquinolone and pyocyanin production but induced an increase in elastase levels. When supplementing P. aeruginosa cultures with QsdA, an enzyme from Rhodococcus erythropolis which inactivates N -acylhomoserine lactone signals, rhamnolipid and elastase levels were quenched, but HHQ and pyocyanin synthesis was promoted. Thus, single quorum quenching enzymes, targeting individual circuits within a complex quorum sensing network, may also elicit undesirable regulatory effects. Supernatants of P. aeruginosa cultures grown in the presence of AqdC, QsdA, or both enzymes were less cytotoxic to human epithelial lung cells than supernatants of untreated cultures. Furthermore, the combination of both aqdC and qsdA in P. aeruginosa resulted in a decline of Caenorhabditis elegans mortality under P. aeruginosa exposure., (Copyright © 2019 American Society for Microbiology.)