MomL, a Novel Marine-Derived N -Acyl Homoserine Lactonase from Muricauda olearia

Abstract

bGram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short-and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-␤-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C 6 -HSL is ϳ2.9 ؋ 10 5 s ؊1 M ؊1 . Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the "HXHXDH" motif with other AHL lactonases belonging to the metallo-␤-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent. N-Acyl homoserine lactones (AHLs) are quorum-sensing (QS) signaling molecules that are used by many Gram-negative bacteria to communicate within species, to regulate gene expression and to synchronize social behaviors, such as biofilm formation, bioluminescence, and secretion of virulence factors (1, 2). An AHL molecule typically consists of a homoserine lactone and an acyl chain with an even number of carbons, with an occasional modification (hydroxy or olefinic double bond) at the C-3 position (1). It has been well established that AHL-dependent QS regulates virulence factor production in many bacterial pathogens, such as Pseudomonas aeruginosa, Erwinia carotovora, Vibrio spp., and Burkholderia spp. (1). Interference with QS has been recognized as a promising antivirulence therapy. Disturbing the QS systems in these pathogens would attenuate virulence rather than kill the bacteria and thereby weaken the selective pressure imposed on the pathogens and reduce the potential for evolution of drug resistance (3). QS inhibitors (QSIs; small molecules) and quorum-quenching (QQ) enzymes can both be used to interfere with QS. QSIs generally act to inactivate autoinducer synthases or receptors through competitive binding, whereas QQ enzymes switch off signal transmission through degradation of the signaling molecules. It has been demonstrated th...

Aii20J, a wide-spectrum thermostable N-acylhomoserine lactonase from the marine bacterium Tenacibaculum sp. 20J, can quench AHL-mediated acid resistance in Escherichia coli

“…The denaturation and refolding process strongly affected the thermo resistance of the enzyme. Residual enzymatic activity was found in the purified, full sequence Aii20J after a treatment of 80°C for 10 min, but only when reconstituted in the presence of metallic ions, which indicates that the presence of metal ions is beneficial for the correct folding and catalytic activity of the protein, as described previously for other metallohydrolases (Cao et al 2012;Chen et al 2010;Momb et al 2008;Tang et al 2015;Wang et al 2012) and confirms that correct folding is a key factor in the thermostability of the denaturized enzyme. Activity was still present in the purified enzyme after a treatment at 60°C during 10 min but was significantly reduced in comparison with the untreated enzyme (Fig.…”

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

“…The resuspended cells were lysed using Sonics Vibracell TM sonicator (30% amplitude for 3 minutes at alternating intervals of 9 seconds) and supernatant and cell content were collected after centrifugation at 10,000 rpm for 10 min at 4°C. 20μl of culture supernatant, supernatant after sonication or cell pellet after sonication were assayed individually for AHL degradation by mixing with 80μl of 25μM C6HSL and incubating at 30°C for 2 hours under static conditions followed by the biosensor assay described above [25]. Reaction mixture containing whole cell of Lysinibacillus sp.…”

“…amyloliquefaciens , Geobacillus sp.) [13–18]; and also in the Actinobacteria ( Arthrobacter sp ., Microbacterium testaceum , Rhodococcus erythropolis ) [19–21]; Proteobacteria ( Agrobacterium , Ochrobactrum sp., Klebsiella pneumoniae ) [22, 23, 19] and Bacteroidetes) ( Chryseobacterium sp., Muricauda olearia ) [24, 25]. The AHL lactonases produced by all these bacteria belong to the metallo- β-lactamase, phosphotriesterase (PTE) and α/β hydrolase-fold families of proteins.…”

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

“…Sequencing and bioinformatic results demonstrate that the genome contains a gene encoding AHL lactonase named MomL. MomL consists of 294 amino acids with a molecular weight of approximately 38.4 KDa [29]. MomL belongs to the metallo-β-lactamase family, and the homolog exhibits a 24.5% similarity with AiiA.…”

“…In vitro enzyme assays indicated that MomL possesses high activity and broad substrate selectivity. Kinetic results indicated MomL had 10-fold increased C6-HSL (C6-homoserine lactones) degrading activity compared with AiiA protein [29]. …”