Quorum quenching activity of AiiA lactonase KMMI17 from endophytic Bacillus thuringiensis KMCLO7 on AHL-mediated pathogenic phenotype in Pseudomonas aeruginosa

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation to disrupt quorum-sensing in these bacteria could pave the way for the new development in decreasing resistance strains and are of significant interest for clinical, agricultural, and industrial applications. Isolated endophytic Bacillus thuringiensis strain KMCL07 showing quorum quenching activity on Pseudomonas aeruginosa PAO1 has been studied. AiiA lactonase KMMI17 identified belongs to metallo- beta-lactamase superfamily preserving conserved regions of (HXDH)-H-106-59 amino acids-H-169-21 amino acids-D-191 motif, significantly inhibits the biofilm formation and attenuates virulence factor pyocyanin production of PAO1. Insilico molecular docking analysis of lactonase (KMMI17) using alternative catalytic site (PDB entry: 3DHA) with the AHL-based QS system regulators of PAO-1, C4 AHL, C6 AHL and 3-oxo-C12 AHL molecules showed good binding affinity between the protein and ligands, Phe111 and Tyr198 residues plays an important role in binding them. Crude enzyme extract was found to have K-m value for C6-HSL: 134.2702 +/- 34.83 mu M-1, C4-HSL: 308.217 +/- 139.9 mu M-1 and 3-oxo-C12-HSL: 760.463 +/- 251.3 mu M-1. LCMS analysis confirms the degradation activity of lactonase (KMMI17) on AHL molecules and its hydrolytic process, which indicates the potential application of lactonase (KMMII7) as a biocontrol agent or an anti-pathogenic drug.