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.