scholarly journals Characterization and Complete Sequence of Lactonase Enzyme fromBacillus weihenstephanensisIsolate P65 with Potential Activity against Acyl Homoserine Lactone Signal Molecules

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Masarra Mohammed Sakr ◽  
Khaled Mohamed Anwar Aboshanab ◽  
Mohammad Mabrouk Aboulwafa ◽  
Nadia Abdel-Haleem Hassouna
Keyword(s):  
Pathogenic Bacteria ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Complete Sequence ◽  
Crude Enzyme ◽  
Signal Molecules ◽  
Acyl Homoserine Lactone ◽  
Crude Enzyme Extract ◽  
Synthetic Standard ◽  
Ph Range

Acyl homoserine lactones (AHLs) are the most common class of quorum sensing signal molecules (autoinducers) that have been reported to be essential for virulence of many relevant pathogenic bacteria such asPseudomonas aeruginosa. New approach for controlling infections of such bacteria is through quorum quenching. In this study, the acyl homoserine lactone inhibitory activity of the crude enzyme from aBacillus weihenstephanensis-isolate P65 was characterized. The crude enzyme was found to have relatively high thermal stability and was stable in pH range 6 to 9. The crude enzyme extract was found to have lactonase activity of 36.3 U/mg total protein. Maximum enzyme activity was achieved within a range of 28–50°C and pH 6–9. None of the metals used enhanced the activity neither did EDTA inhibit it. However, a concentration of 10 mM Fe+2reduced the activity to 73.8%. Catalytic activity and kinetic constants were determined using hexanoyl homoserine lactone as a substrate. Studying enzyme substrate specificity using synthetic standard signals displayed broad spectrum of activity. The enzyme was found to be constitutive. Isolation and complete nucleotide sequence of the respective lactonase gene were done and submitted to the Genbank database under accession code KC823046.

scholarly journals Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Surang Chankhamhaengdecha ◽  
Suphatra Hongvijit ◽  
Akkaraphol Srichaisupakit ◽  
Pattra Charnchai ◽  
Watanalai Panbangred
Keyword(s):  
Pathogenic Bacteria ◽  
Sequence Similarity ◽  
Soft Rot ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Amide Bond ◽  
Side Chain ◽  
Signal Molecules ◽  
Endophytic Actinomycetes

Several Gram-negative pathogenic bacteria employN-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at151.30±3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified asStreptomycesbased on16S  rRNAgene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In anin vitropathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused byPectobacterium carotovorumssp.carotovorumas demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophyticStreptomyces.

scholarly journals Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1

2006 ◽  
Vol 74 (3) ◽  
pp. 1673-1682 ◽  
Author(s):  
Charles F. Sio ◽  
Linda G. Otten ◽  
Robbert H. Cool ◽  
Stephen P. Diggle ◽  
Peter G. Braun ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Side Chain ◽  
Signal Molecules ◽  
Acyl Homoserine Lactone ◽  
Beta Lactam ◽  
Signal Molecule ◽  
Pathogenic Potential

ABSTRACT The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3′ position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.

scholarly journals Quenching of acyl-homoserine lactone-dependent quorum sensing by enzymatic disruption of signal molecules.

2009 ◽  
Vol 56 (1) ◽  
Author(s):  
Robert Czajkowski ◽  
Sylwia Jafra
Keyword(s):  
Quorum Sensing ◽  
Pathogenic Bacteria ◽  
Degradation Products ◽  
Antibiotic Production ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Animal Pathogens

Many Gram-positive and Gram-negative bacteria communicate using small diffusible signal molecules called autoinducers. This process, known as quorum sensing (QS), links cell density to the expression of genes as diverse as those associated with virulence factors production of plant and animal pathogens, bioluminescence, antibiotic production, sporulation or biofilm formation. In Gram-negative bacteria, this communication is mainly mediated by N-acyl-homoserine lactones (AHLs). It has been proven that inactivation of the signal molecules attenuates many of the processes controlled by QS. Enzymatic degradation of the signal molecules has been amply described. Two main classes of AHL-inactivating enzymes were identified: AHL lactonases which hydrolyse the lactone ring in AHLs, and AHL acylases (syn. AHL amidases) which liberate a free homoserine lactone and a fatty acid. Recently, AHL oxidoreductase, a novel type of AHL inactivating enzyme, was described. The activity of these enzymes results in silencing the QS-regulated processes, as degradation products cannot act as signal molecules. The ability to inactivate AHL (quorum quenching, QQ) might be useful in controlling virulence of many pathogenic bacteria.

scholarly journals Expanding AHL Acylases Horizon - Insights From Structural Analysis of Choloylglycine Hydrolases From Shewanella lohica PV-4

2019 ◽  
Author(s):  
Pushparani D Philem ◽  
Yashpal Yadav ◽  
Avinash V Sunder ◽  
Deepanjan Ghosh ◽  
Asmita Prabhune ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Active Site ◽  
Acyl Chain ◽  
Complex Structure ◽  
Evolutionary Relationship ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Acyl Homoserine Lactone ◽  
Structural Framework

AbstractAcyl homoserine lactone acylases are quorum quenching enzymes that degrade the Gram negative bacterial autoinducer N-acyl homoserine lactone (AHL) and belong to the Ntn-hydrolases superfamily of enzymes. Recent findings reported AHL acylase activity of pencillin V acylases (PVA) which, alongside bile salt hydrolases, are members of the cholyolglycine hydrolase (CGH) family of the Ntn-hydrolases superfamily. The present study reports the unique activity profile of two CGHs from a marine bacterium Shewanella loihica-PV4, designated here as SlCGH1 and SlCGH2, including the structural analysis of SlCGH1. Both the enzymes exhibit AHL acylase activity while unexpectedly being inactive on standard CGH substrates PenV and bile salts. SlCGH1 differs from known homotetrameric CGHs in being a homodimer displaying a reduced active site volume attributed to loop orientation, which subsequently directs the substrate specificity. Moreover a ligand bound complex structure revealed an unusual bent conformation of the saturated acyl chain bound to the active site and also predicts a single oxyanion hole forming residue during catalysis instead of the usual two residues. Phylogenetic analysis reveals SlCGH1 homologs cluster separate from reported CGHs and AHL acylases. On the whole, SlCGH1 could represent a functionally distinct new sub-class of CGH as an adaptation to the marine environment and its structure could provide the structural framework for understanding such a novel subclass. We also make a modest proposal of a probable evolutionary link between AHL acylases and β lactam acylases based on the overlap in activity and structural features.SignificanceCross-reactivity between AHL acylases and b lactam acylases has been recently identified giving us a vivid glimpse of a possible evolutionary relationship between the phenomena of quorum sensing and antibiotic resistance. We report here the first AHL acylase of the CGH structural framework. SlCGH1 from Shewanella loihica PV-4 is also the first report of a marine CGH with a unique activity and a new structural subclass of CGH family with AHL acylase activity. This finding highlights the vast diversity of AHL acylases and by extension quorum quenching enzymes as adaptation to different habitats. The results from this study also bolster the link between signal molecules and antibiotics, extending our understanding of the inadequately understood physiological roles of b-lactam acylases.

Novel approach to quorum quenching: rational design of antibacterials in combination with hexahistidine-tagged organophosphorus hydrolase

2018 ◽  
Vol 399 (8) ◽  
pp. 869-879 ◽  
Author(s):  
Aysel Aslanli ◽  
Ilya Lyagin ◽  
Elena Efremenko
Keyword(s):  
Molecular Docking ◽  
Rational Design ◽  
Pathogenic Bacteria ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Catalytic Action ◽  
Organophosphorus Hydrolase ◽  
Signal Molecules ◽  
Bacterial Strains ◽  
Native Form

Abstract N-acyl homoserine lactones (AHLs) are quorum sensing (QS) signal molecules used by most Gram-negative pathogenic bacteria. In this article the lactonase activity of the preparations based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) towards AHLs was studied. Initially, three of the most interesting β-lactam antibiotics were selected from seven that were trialed during molecular docking to His6-OPH. Combinations of antibiotics (meropenem, imipenem, ceftriaxone) and His6-OPH taken in the native form or in the form of non-covalent enzyme-polyelectrolyte complexes (EPCs) with poly(glutamic acid) or poly(aspartic acid) were obtained and investigated. The lactonase activity of the preparations was investigated under different physical-chemical conditions in the hydrolysis of AHLs [N-butyryl-D,L-homoserine lactone, N-(3-oxooctanoyl)-D,L-homoserine lactone, N-(3-oxododecanoyl)-L-homoserine lactone]. An increased efficiency of catalytic action and stability of the lactonase activity of His6-OPH was shown for its complexes with antibiotics and was confirmed in trials with bacterial strains. The broadening of the catalytic action of the enzyme against AHLs was revealed in the presence of the meropenem. Results of molecular docking of AHLs to the surface of the His6-OPH dimer in the presence of antibiotics allowed proposing the mechanism of such interference based on a steric repulsion of the carbon chain of hydrolyzed AHLs by the antibiotics bounded to the enzyme surface.

scholarly journals A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

2021 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth
Keyword(s):  
Single Point ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Single Amino Acid Residue

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

scholarly journals Signal Integration in Quorum Sensing Enables Cross-Species Induction of Virulence in Pectobacterium wasabiae

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Rita S. Valente ◽  
Pol Nadal-Jimenez ◽  
André F. P. Carvalho ◽  
Filipe J. D. Vieira ◽  
Karina B. Xavier
Keyword(s):  
Signal Transduction ◽  
Quorum Sensing ◽  
Plant Pathogen ◽  
Bacterial Species ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Acyl Homoserine Lactone ◽  
Interspecies Interactions ◽  
Major Factors

ABSTRACT Bacterial communities can sense their neighbors, regulating group behaviors in response to cell density and environmental changes. The diversity of signaling networks in a single species has been postulated to allow custom responses to different stimuli; however, little is known about how multiple signals are integrated and the implications of this integration in different ecological contexts. In the plant pathogen Pectobacterium wasabiae (formerly Erwinia carotovora), two signaling networks—the N-acyl homoserine lactone (AHL) quorum-sensing system and the Gac/Rsm signal transduction pathway—control the expression of secreted plant cell wall-degrading enzymes, its major virulence determinants. We show that the AHL system controls the Gac/Rsm system by affecting the expression of the regulatory RNA RsmB. This regulation is mediated by ExpR2, the quorum-sensing receptor that responds to the P. wasabiae cognate AHL but also to AHLs produced by other bacterial species. As a consequence, this level of regulation allows P. wasabiae to bypass the Gac-dependent regulation of RsmB in the presence of exogenous AHLs or AHL-producing bacteria. We provide in vivo evidence that this pivotal role of RsmB in signal transduction is important for the ability of P. wasabiae to induce virulence in response to other AHL-producing bacteria in multispecies plant lesions. Our results suggest that the signaling architecture in P. wasabiae was coopted to prime the bacteria to eavesdrop on other bacteria and quickly join the efforts of other species, which are already exploiting host resources. IMPORTANCE Quorum-sensing mechanisms enable bacteria to communicate through small signal molecules and coordinate group behaviors. Often, bacteria have various quorum-sensing receptors and integrate information with other signal transduction pathways, presumably allowing them to respond to different ecological contexts. The plant pathogen Pectobacterium wasabiae has two N-acyl homoserine lactone receptors with apparently the same regulatory functions. Our work revealed that the receptor with the broadest signal specificity is also responsible for establishing the link between the main signaling pathways regulating virulence in P. wasabiae. This link is essential to provide P. wasabiae with the ability to induce virulence earlier in response to higher densities of other bacterial species. We further present in vivo evidence that this novel regulatory link enables P. wasabiae to join related bacteria in the effort to degrade host tissue in multispecies plant lesions. Our work provides support for the hypothesis that interspecies interactions are among the major factors influencing the network architectures observed in bacterial quorum-sensing pathways. IMPORTANCE Quorum-sensing mechanisms enable bacteria to communicate through small signal molecules and coordinate group behaviors. Often, bacteria have various quorum-sensing receptors and integrate information with other signal transduction pathways, presumably allowing them to respond to different ecological contexts. The plant pathogen Pectobacterium wasabiae has two N-acyl homoserine lactone receptors with apparently the same regulatory functions. Our work revealed that the receptor with the broadest signal specificity is also responsible for establishing the link between the main signaling pathways regulating virulence in P. wasabiae. This link is essential to provide P. wasabiae with the ability to induce virulence earlier in response to higher densities of other bacterial species. We further present in vivo evidence that this novel regulatory link enables P. wasabiae to join related bacteria in the effort to degrade host tissue in multispecies plant lesions. Our work provides support for the hypothesis that interspecies interactions are among the major factors influencing the network architectures observed in bacterial quorum-sensing pathways.

scholarly journals Cross-Species GacA-Controlled Induction of Antibiosis in Pseudomonads

2006 ◽  
Vol 73 (2) ◽  
pp. 650-654 ◽  
Author(s):  
Christophe Dubuis ◽  
Dieter Haas
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pseudomonas Fluorescens ◽  
Small Rnas ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Acyl Homoserine Lactone ◽  
Pseudomonas Corrugata ◽  
Interspecies Communication ◽  
Antibiotic Compounds ◽  
Culture Supernatants

ABSTRACT Signal extracts prepared from culture supernatants of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa PAO stimulated GacA-dependent expression of small RNAs and hence of antibiotic compounds in both hosts. Pseudomonas corrugata LMG2172 and P. fluorescens SBW25 also produced signal molecules stimulating GacA-controlled antibiotic synthesis in strain CHA0, illustrating a novel, N-acyl-homoserine lactone-independent type of interspecies communication.

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