AHL-lactonase Research Articles

N-acylhomoserine lactonase-based hybrid nanoflowers: a novel and practical strategy to control plant bacterial diseases

BackgroundThe disease caused by plant pathogenic bacteria in the production, transportation, and storage of many crops has brought huge losses to agricultural production. N-acylhomoserine lactonases (AHLases) can quench quorum-sensing (QS) by hydrolyzing acylhomoserine lactones (AHLs), which makes them the promising candidates for controlling infections of QS-dependent pathogenic bacteria. Although many AHLases have been isolated and considered as a potentially effective preventive and therapeutic agents for bacterial diseases, the intrinsically poor ambient stability has seriously restricted its application.ResultsHerein, we showed that a spheroid enzyme-based hybrid nanoflower (EHNF), AhlX@Ni3(PO4)2, can be easily synthesized, and it exhibited 10 times AHL (3OC8-HSL) degradation activity than that with free AhlX (a thermostable AHL lactonase). In addition, it showed intriguing stability even at the working concentration, and retained ~ 100% activity after incubation at room temperature (25 °C) for 40 days and approximately 80% activity after incubation at 60 °C for 48 h. Furthermore, it exhibited better organic solvent tolerance and long-term stability in a complicated ecological environment than that of AhlX. To reduce the cost and streamline production processes, CSA@Ni3(PO4)2, which was assembled from the crude supernatants of AhlX and Ni3(PO4)2, was synthesized. Both AhlX@Ni3(PO4)2 and CSA@Ni3(PO4)2 efficiently attenuated pathogenic bacterial infection.ConclusionsIn this study, we have developed N-acylhomoserine lactonase-based hybrid nanoflowers as a novel and efficient biocontrol reagent with significant control effect, outstanding environmental adaptability and tolerance. It was expected to overcome the bottlenecks of poor stability and limited environmental tolerance that have existed for over two decades and pioneered the practical application of EHNFs in the field of biological control.Graphical

Applying molecular and phenotypic screening assays to identify efficient quorum quenching lactonases

Quorum sensing (QS) is a molecular communication system used by microorganisms to adopt behaviors in a cell density-dependent manner. Lactonase enzymes, able to hydrolyze the signal molecules acyl-homoserine lactones (AHL) can counteract QS-mediated virulence in Gram-negative bacteria. Optimizing lactonases activity or specificity for AHL through enzyme engineering approaches is thus highly attractive to increase protective effect. However, only a limited number of screening methods have been developed to handle and evaluate AHL-degrading enzyme libraries. Here, a series of screening procedures were developed to identify improved lactonases using two previously reported enzymes as benchmarks, namely SsoPox and GcL. Specifically, molecular screenings using six different AHL and based on two reporter strains; i.e., Chromobacterium violaceum CV026 and Pseudomonas putida KS35, are reported. In addition, three phenotype-based screenings aiming to evaluate the ability of enzymes to quench a particular QS-related behavior are reported, using C. violaceum, Pseudomonas aeruginosa and Vibrio harveyi as pathogenic type strains. These assays were used to screen a small-sized library and allowed for the identification of various improved variants. To confirm that these variants were real “hits”, four of them were produced and purified. Their kinetic parameters against AHL substrates were found to be increased by 2–44.5 -fold as compared to the starting enzyme. Moreover, their increased activity was confirmed by measuring their ability to quench QS in different bacterial systems. These new assays will facilitate the screening of enzyme libraries and will pave the way for the development of proficient engineered QS-disrupting enzymes.

Quorum Quenching Strains Isolated from the Microbiota of Sea Anemones and Holothurians Attenuate Vibriocorallilyticus Virulence Factors and Reduce Mortality in Artemiasalina.

Interference with quorum-sensing (QS) intercellular communication systems by the enzymatic disruption of N-acylhomoserine lactones (AHLs) in Gram-negative bacteria has become a promising strategy to fight bacterial infections. In this study, seven strains previously isolated from marine invertebrates and selected for their ability to degrade C6 and C10-HSL, were identified as Acinetobacter junii, Ruegeria atlantica, Microbulbifer echini, Reinheimera aquimaris, and Pseudomonas sihuiensis. AHL-degrading activity against a wide range of synthetic AHLs were identified by using an agar well diffusion assay and Agrobacterium tumefaciens NTL4 and Chromobacterium violaceum CV026 and VIR07 as biosensors. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis indicated that this activity was not due to an AHL lactonase. All the strains degraded Vibrio coralliilyticus AHLs in coculture experiments, while some strains reduced or abolished the production of virulence factors. In vivo assays showed that strains M3-111 and M3-127 reduced this pathogen’s virulence and increased the survival rate of Artemia salina up to 3-fold, indicating its potential use for biotechnological purposes. To our knowledge, this is the first study to describe AHL-degrading activities in some of these marine species. These findings highlight that the microbiota associated with marine invertebrates constitute an important underexplored source of biological valuable compounds.

Mining Marine Metagenomes Revealed a Quorum-Quenching Lactonase with Improved Biochemical Properties That Inhibits the Food Spoilage Bacterium Pseudomonas fluorescens.

The marine environment presents great potential as a source of microorganisms that possess novel enzymes with unique activities and biochemical properties. Examples of such are the quorum-quenching (QQ) enzymes that hydrolyze bacterial quorum-sensing (QS) signaling molecules, such as N-acyl-homoserine lactones (AHLs). QS is a form of cell-to-cell communication that enables bacteria to synchronize gene expression in correlation with population density. Searching marine metagenomes for sequences homologous to an AHL lactonase from the phosphotriesterase-like lactonase (PLL) family, we identified new putative AHL lactonases (sharing 30 to 40% amino acid identity to a thermostable PLL member). Phylogenetic analysis indicated that these putative AHL lactonases comprise a new clade of marine enzymes in the PLL family. Following recombinant expression and purification, we verified the AHL lactonase activity for one of these proteins, named moLRP (marine-originated lactonase-related protein). This enzyme presented greater activity and stability at a broad range of temperatures and pH, tolerance to high salinity levels (up to 5 M NaCl), and higher durability in bacterial culture, compared to another PLL member, parathion hydrolase (PPH). The addition of purified moLRP to cultures of Pseudomonas fluorescens inhibited its extracellular protease activity, expression of the protease encoding gene, biofilm formation, and the sedimentation process in milk-based medium. These findings suggest that moLRP is adapted to the marine environment and can potentially serve as an effective QQ enzyme, inhibiting the QS process in Gram-negative bacteria involved in food spoilage. IMPORTANCE Our results emphasize the potential of sequence and structure-based identification of new QQ enzymes from environmental metagenomes, such as from the ocean, with improved stability or activity. The findings also suggest that purified QQ enzymes can present new strategies against food spoilage, in addition to their recognized involvement in inhibiting bacterial pathogen virulence factors. Future studies on the delivery and safety of enzymatic QQ strategy against bacterial food spoilage should be performed.

Bacterial Quorum-Quenching Lactonase Hydrolyzes Fungal Mycotoxin and Reduces Pathogenicity of Penicillium expansum-Suggesting a Mechanism of Bacterial Antagonism.

Penicillium expansum is a necrotrophic wound fungal pathogen that secrets virulence factors to kill host cells including cell wall degrading enzymes (CWDEs), proteases, and mycotoxins such as patulin. During the interaction between P. expansum and its fruit host, these virulence factors are strictly modulated by intrinsic regulators and extrinsic environmental factors. In recent years, there has been a rapid increase in research on the molecular mechanisms of pathogenicity in P. expansum; however, less is known regarding the bacteria–fungal communication in the fruit environment that may affect pathogenicity. Many bacterial species use quorum-sensing (QS), a population density-dependent regulatory mechanism, to modulate the secretion of quorum-sensing signaling molecules (QSMs) as a method to control pathogenicity. N-acyl homoserine lactones (AHLs) are Gram-negative QSMs. Therefore, QS is considered an antivirulence target, and enzymes degrading these QSMs, named quorum-quenching enzymes, have potential antimicrobial properties. Here, we demonstrate that a bacterial AHL lactonase can also efficiently degrade a fungal mycotoxin. The mycotoxin is a lactone, patulin secreted by fungi such as P. expansum. The bacterial lactonase hydrolyzed patulin at high catalytic efficiency, with a kcat value of 0.724 ± 0.077 s−1 and KM value of 116 ± 33.98 μM. The calculated specific activity (kcat/KM) showed a value of 6.21 × 103 s−1M−1. While the incubation of P. expansum spores with the purified lactonase did not inhibit spore germination, it inhibited colonization by the pathogen in apples. Furthermore, adding the purified enzyme to P. expansum culture before infecting apples resulted in reduced expression of genes involved in patulin biosynthesis and fungal cell wall biosynthesis. Some AHL-secreting bacteria also express AHL lactonase. Here, phylogenetic and structural analysis was used to identify putative lactonase in P. expansum. Furthermore, following recombinant expression and purification of the newly identified fungal enzyme, its activity with patulin was verified. These results indicate a possible role for patulin and lactonases in inter-kingdom communication between fungi and bacteria involved in fungal colonization and antagonism and suggest that QQ lactonases can be used as potential antifungal post-harvest treatment.

Comparative genome analysis of Bacillus thuringiensis strain HD521 and HS18-1

Bacillus thuringiensis (Bt) is an important biological insecticide used to management of different agricultural pests by producing toxic parasporal crystals proteins. Strain HD521 has an antagonistic effect against Rhizoctonia solani AG1IA, the causal agent of rice sheath blight. This strain with three cry7 genes can the formation of bipyramidal parasporal crystals (BPCs). BPCs are used for insecticidal activities against Henosepilachna vigintioctomaculata larva (Coleoptera). Strain HS18-1 contains different types of BPCs encoding genes and has effective toxicity for Lepidoptera and Diptera insects. Here we report the whole genome sequencing and assembly of HD521 and HS18-1 strains and analyzed the genome constitution covering virulence factors, types of plasmid, insertion sequences, and prophage sequences. The results showed that the genome of strain HD521 contains a circular chromosome and six circular plasmids, encoding eight types of virulence protein factors [Immune Inhibitor A, Hemolytic Enterotoxin, S-layer protein, Phospholipase C, Zwittermicin A-resistance protein, Metalloprotease, Chitinase, and N-acyl homoserine lactonase (AiiA)], four families of insertion sequence, and comprises six pro-phage sequences. The genome of strain HS18-1 contains one circular chromosome and nine circular plasmids, encoding five types of virulence protein factors [Hemolytic Enterotoxin, S-layer protein, Phospholipase C, Chitinase, and N-acyl homoserine lactonase (AiiA)] and four families of insertion sequence, and comprises of three pro-phage sequences. The obtained results will contribute to deeply understand the B. thuringiensis strain HD521 and HS18-1 at the genomic level.

In vivo evaluation of a recombinant N-acylhomoserine lactonase formulated in a hydrogel using a murine model infected with MDR Pseudomonas aeruginosa clinical isolate, CCASUP2

Failure in the treatment of P. aeruginosa, due to its broad spectrum of resistance, has been associated with increased patient mortality. One alternative approach for infection control is quorum quenching which was found to decrease virulence of such pathogen. In this study, the efficiency of a recombinant Ahl-1 lactonase formulated as a hydrogel was investigated to control the infection of multidrug resistant (MDR) P. aeruginosa infected burn using a murine model. The recombinant N-acylhomoserine lactonase (Ahl-1) was formulated as a hydrogel. To test its ability to control the infection of MDR P. aeruginosa, a thermal injury model was used. Survival rate, and systemic spread of the infection were evaluated. Histopathological examination of the animal dorsal skin was also done for monitoring the healing and cellular changes at the site of infection. Survival rate in the treated group was 100% relative to 40% in the control group. A decrease of up to 3 logs of bacterial count in the blood samples of the treated animals relative to the control group and a decrease of up to 4 logs and 2.3 logs of bacteria in lung and liver samples, respectively were observed. Histopathological examination revealed more enhanced healing process in the treated group. Accordingly, by promoting healing of infected MDR P. aeruginosa burn and by reducing systemic spread of the infection as well as decreasing mortality rate, Ahl-1 hydrogel application is a promising strategy that can be used to combat and control P. aeruginosa burn infections.

KaAhl, a Novel N-Acylhomoserine Lactonase from Kushneria avicenniae and Attenuated Effect on the Virulence of Erwinia carotovora

KaAhl, a Novel N-Acylhomoserine Lactonase from Kushneria avicenniae and Attenuated Effect on the Virulence of Erwinia carotovora

Attenuation of Aeromonas hydrophila Infection in Carassius auratus by YtnP, a N-acyl Homoserine Lactonase from Bacillus licheniformis T-1.

In this experiment, the quorum quenching gene ytnP of Bacillus licheniformis T-1 was cloned and expressed, and the effect against infection of Aeromonas hydrophila ATCC 7966 was evaluated in vitro and vivo. The BLAST results revealed a 99% sequence identity between the ytnP gene of T-1 and its homolog in B. subtilis sub sp. BSP1, and the dendroGram showed that the similarity in the YtnP protein in T-1 was 100% in comparison with B. subtilis 3610, which was categorized as the Aidc cluster of the MBL family. The AHL lactonase activity of the purified YtnP was detected as 1.097 ± 0.7 U/mL with C6-HSL as the substrate. Otherwise, purified YtnP protein could significantly inhibit the biofilm formation of A. hydrophila ATCC 7966 with an inhibition rate of 68%. The MIC of thiamphenicol and doxycycline hydrochloride against A. hydrophila reduced from 4 μg/mL and 0.5 μg/mL to 1 μg/mL and 0.125 μg/mL, respectively, in the presence of YtnP. In addition, YtnP significantly inhibited the expression of five virulence factors hem, ahyB, ast, ep, aerA of A. hydrophila ATCC 7966 as well (p < 0.05). The results of inhibition on virulence showed a time-dependence tendency, while the strongest anti-virulence effects were within 4–24 h. In vivo, when the YtnP protein was co-injected intraperitoneally with A. hydrophila ATCC 7966, it attenuated the pathogenicity of A. hydrophila and the accumulated mortality was 27 ± 4.14% at 96 h, which was significantly lower than the average mortality of 78 ± 2.57% of the Carassius auratus injected with 108 CFU/mL of A. hydrophila ATCC 7966 only (p < 0.001). In conclusion, the AHL lactonase in B. licheniformis T-1 was proven to be YtnP protein and could be developed into an agent against infection of A. hydrophila in aquaculture.

Identification and Characterization of a New Quorum-Quenching N-acyl Homoserine Lactonase in the Plant Pathogen Erwinia amylovora.

Quorum quenching (QQ) is the ability to interfere with bacterial cell to cell communication, known as quorum sensing (QS). QQ enzymes that degrade or modify acyl homoserine lactones (AHLs) have been attracting increasing interest as promising agents for inhibiting QS-mediated bacterial pathogenicity. Plant pathogens from the genus Erwinia cause diseases in several economically important crops. Fire blight is a devastating plant disease caused by Erwinia amylovora, affecting a wide range of host species within the Rosaceae and posing a major global threat for commercial apple and pear production. While QS has been described in Erwinia species, no AHL-degrading enzymes were identified and characterized. Here, phylogenetic analysis and structural modeling were applied to identify an AHL lactonase in E. amylovora (dubbed EaAiiA). Following recombinant expression and purification, the enzyme was biochemically characterized. EaAiiA lactonase activity was dependent on metal ions and effectively degraded AHLs with high catalytic efficiency. Its highest specific activity (kcat/KM value) was observed against one of the AHLs (3-oxo-C6-homoserine lactone) secreted from E. amylovora. Exogenous addition of the purified enzyme to cultures of E. amylovora reduced the formation of levan, a QS-regulated virulence factor, by 40% and the transcription level of the levansucrase-encoding gene by 55%. Furthermore, preincubation of E. amylovora cultures with EaAiiA inhibited the progress of fire blight symptoms in immature Pyrus communis fruits. These results demonstrate the ability of the identified enzyme from E. amylovora to act as a quorum-quenching lactonase.

Directed Enzyme Evolution and Encapsulation in Peptide Nanospheres of Quorum Quenching Lactonase as an Antibacterial Treatment against Plant Pathogen.

The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N-acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta. Using directed enzyme evolution, we were able to increase the enzyme's temperature resistance (T50, the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom's infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.

An Acylase from Shewanella Putrefaciens Presents a Vibrio Parahaemolyticus Acylhomoserine Lactone-Degrading Activity and Exhibits Temperature-, Ph- and Metal-Dependences

Shewanella putrefaciens supernatant was found to increase the virulence factors of Vibrio parahaemolyticus by efficiently degrading its acylhomoserine lactone (AHL). To further reveal the regulation mechanism and its key degrading enzyme, a potential AHL-degrading enzyme acylase (Aac) from S. putrefaciens was cloned, and the influences of temperature, pH, protein modifiers, and metals on Aac were tested. Aac was significantly influenced by temperature and pH, and exhibited the highest AHL-degrading activity at temperatures of 37 °C and pH of 8. Mg2+ and Fe2+ can further increase the AHL-degrading activity. 10 mM EDTA inhibited its activity possibly by chelating the co-factors (metals) required for Aac activity. Tryptophan and arginine were identified as key components for Aac activity that are critical to its AHL-degrading activity. This study provides useful information on Aac and for V. parahaemolyticus control.

Heterologous expression of AHL lactonase AiiK by Lactobacillus casei MCJ\u03941 with great quorum quenching ability against Aeromonas hydrophila AH-1 and AH-4

BackgroundNowadays, microbial infections have caused increasing economic losses in aquaculture industry and deteriorated worldwide environments. Many of these infections are caused by opportunistic pathogens through cell-density mediated quorum sensing (QS). The disruption of QS, known as quorum quenching (QQ), is an effective and promising way to prevent and control pathogens, driving it be the potential bio-control agents. In our previous studies, AHL lactonase AiiK was identified with many characteristics, and constitutive expression vector pELX1 was constructed to express heterologous proteins in Lactobacillus casei MCJΔ1 (L. casei MCJΔ1). In this study, recombinant strain pELCW-aiiK/L. casei MCJΔ1 (LcAiiK) and wild-type Aeromonas hydrophila (A. hydrophila) were co-cultured to test the QQ ability of LcAiiK against A. hydrophila.ResultsA cell wall-associated expression vector pELCW for L. casei MCJΔ1 was constructed. Localization assays revealed that the expressed AiiK was anchored at the surface layer of LcAiiK via vector pELCW-aiiK. LcAiiK (OD600 = 0.5) degraded 24.13 μM of C6-HSL at 2 h, 40.99 μM of C6-HSL at 12 h, and 46.63 μM of C6-HSL at 24 h. Over 50% LcAiiK cells maintained the pELCW-aiiK plasmid after 15 generations of cultivation without erythromycin. Furthermore, LcAiiK inhibited the swimming motility, extracellular proteolytic activity, haemolytic activity and biofilm formation of A. hydrophila AH-1 and AH-4.ConclusionThe AHL lactonase AiiK is firstly and constitutively expressed at the surface layer of L. casei MCJΔ1. LcAiiK displayed considerable AHL lactonase activity and great QQ abilities against A. hydrophila AH-1 and AH-4 by attenuating their QS processes instead of killing them. Therefore, the LcAiiK can be exploited as an anti-pathogenic drug or a bio-control agent to control the AHL-mediated QS of pathogenic bacteria.

Identification and Characterization of Quorum-Quenching Activity of N-Acylhomoserine Lactonase from Coagulase-Negative Staphylococci.

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signals in Gram-negative bacteria. Many genes encoding AHL-degrading enzymes have been cloned and characterized in various microorganisms. Coagulase-negative staphylococci (CNS) are present on the skin of animals and are considered low-virulent species. The AHL-lactonase gene homologue, ahlS, was present in the genomes of the CNS strains Staphylococcus carnosus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, and Staphylococcus sciuri. We cloned the candidate ahlS homologue from six CNS strains into the pBBR1MCS5 vector. AhlS from the CNS strains showed a higher degrading activity against AHLs with short acyl chains compared to those with long acyl chains. AhlS from S. sciuri was expressed and purified as a maltose-binding protein (MBP) fusion. Pseudomonas aeruginosa is an opportunistic pathogen that regulates several virulence factors such as elastase and pyocyanin by quorum-sensing systems. When MBP-AhlS was added to the culture of P. aeruginosa PAO1, pyocyanin production and elastase activity were substantially reduced compared to those in untreated PAO1. These results demonstrate that the AHL-degrading activity of AhlS from the CNS strains can inhibit quorum sensing in P. aeruginosa PAO1.

Improving the production of AHL lactonase AiiO-AIO6 from Ochrobactrum sp. M231 in intracellular protease-deficient Bacillus subtilis

Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway. To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis 1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrN and ywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrN and BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 1416.47 U/mL/OD600, which was about 121% higher than that of the wild-type strain. Furthermore, LC–MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6 was classified as a member of the α/β hydrolase family with a conserved “nucleophile-acid-histidine” catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

Spectroscopic, Structural, DFT and Molecular Docking Studies on Novel Cocrystal Salt Hydrate of Chromotropic Acid and Its Antibiofilm Activity

A novel organic cocrystal salt hydrate (CTPTH) of Chromotropic acid with 1,10-phenanthroline was successfully obtained using both traditional solution method and liquid-assisted grinding method, a green approach. The formation and characterization were done by Powder x-ray diffraction, Fourier-transform infrared spectroscopy, thermogravimetric analysis, and the structural confirmation by single crystal x-ray diffraction. The molecular properties were studied using density functional theory, B3LYP/def2-SVP including dispersion correction (D3) to calculate the HOMO and LUMO energies using Orca and Gaussian packages. The calculated geometric parameters were in good agreement with those of experimentally observed. The non-bonding interaction between the CTPTH and active site of the crystal structure of quorum-quenching N-Acyl homoserine lactone lactonase (PDB: 2BR6) in bacterium was studied by molecular docking. The total energy of ligand interaction of CTPTH with the receptor is found to be − 42.78 kcal mol−1. The Hirshfeld surfaces were mapped over dnorm for the identification and quantification of robust synthons for a better understanding of the overall packing pattern of CTPTH. In addition, the antibiofilm efficacy of CTPTH against the pathogenic bacterium Pseudomonas aeruginosa was also determined.

Directed Computational Evolution of Quorum-Quenching Lactonases from the Amidohydrolase Superfamily

In this work, we present a generalizable directed computational evolution protocol to effectively reduce the sequence space to be explored in rational enzyme design. The protocol involves in silico mutation modeling and substrate docking to rapidly identify mutagenesis hotspots that may enhance an enzyme's substrate binding and overall catalysis. By applying this protocol to a quorum-quenching Geobacillus kaustophilus lactonase, GKL, we generated 1,881 single mutants and docked high-energy intermediates of nine acyl homoserine lactones onto them. We found that Phe28 and Tyr99 were two hotspots that produced most of the predicted top 20 mutants. Of the 180 enzyme-substrate combinations (top 20 mutants × 9 substrates), 51 (28%) exhibited enhanced substrate binding and 22 (12%) had better overall activity when compared with wild-type GKL. X-ray crystallographic studies of Y99C and Y99P provided rationalized explanations for the enhancement in enzyme function and corroborated the utility of the protocol.

Investigation of the quorum-sensing regulon of the biocontrol bacterium Pseudomonas chlororaphis strain PA23.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of “phz-box” sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.

Computational prediction of active sites and ligands in different AHL quorum quenching lactonases and acylases

With the emergence of multidrug-resistant 'superbug', conventional treatments become obsolete. Quorum quenching (QQ), enzyme-dependent alteration of quorum sensing (QS), is now considered as a promising antimicrobial therapy because of its potentiality to impede virulence gene expression without resulting in growth inhibition and antibiotic resistance. In our study, we intended to compare between two major QQ enzyme groups (i.e., AHL lactonases and AHL acylases) in terms of their structural and functional aspects. The amino acid composition-based principal component analysis (PCA) suggested that probably there is no structural and functional overlapping between the two groups of enzymes as well as within the lactonase enzymes but the acylases may functionally be affected by one another. In subcellular localization analysis, we also found that most lactonases are cytoplasmic while acylases are periplasmic. Investigation on the secondary structural features showed random coil dominates over alpha-helix and beta-sheet in all evaluated enzymes. For structural comparison, the tertiary structures of the selected proteins were modelled and submitted to the PMDB database (Accession ID: PM0081007 to PM0081018). Interestingly, sequence alignment revealed the presence of several conserved domains important for functions in both protein groups. In addition, three amino acid residues, namely aspartic acid, histidine, and isoleucine, were common in the active sites of all protein models while most frequent ligands were found to be 3C7, FEO, and PAC. Importantly, binding interactions of predicted ligands were similar to that of native QS signal molecules. Furthermore, hydrogen bonds analysis suggested six proteins are more stable than others. We believe that the knowledge of this comparative study could be useful for further research in the development of QSbased universal antibacterial strategies.

Influence of N-acylhomoserine lactonase silver nanoparticles on the quorum sensing system of Helicobacter pylori: A potential strategy to combat biofilm formation.

The treatment of Helicobacter pylori usually fails due to their ability to form biofilms and resistance to antibiotics. This might potentially lead to gastric carcinoma and mucosa-associated lymphoid tissue lymphoma. In the present study, we elucidate the potential role of N-acylhomoserine lactonase stabilized silver nanoparticles (AiiA-AgNPs) in treating biofilms produced by H. pylori. AiiA-AgNPs inhibited quorum sensing (QS) by degradation of QS molecules, thereby reducing biofilm formation, urease production, and altering cell surface hydrophobicity of H. pylori. AiiA-AgNPs showed no cytotoxic effects on RAW 264.7 macrophages at the effective concentration (1-5 µM) of antibiofilm activity. In addition, AiiA-AgNP in high concentration (80-100 µM) exhibited cytotoxicity against HCT-15 carcinoma cells, depicting its therapeutic role in treating cancer.