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

Autor: Billot R; Gene&GreenTK, 19-21 Boulevard Jean Moulin, 13005 Marseille, France; Aix Marseille Univ, IRD, APHM, MEPHI, IHU-Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France., Plener L; Gene&GreenTK, 19-21 Boulevard Jean Moulin, 13005 Marseille, France., Grizard D; Gene&GreenTK, 19-21 Boulevard Jean Moulin, 13005 Marseille, France; Proxis Développement, Levallois-Perret, France., Elias MH; Department of Biochemistry, Molecular Biology and Biophysics - BioTechnology Institute, University of Minnesota, St. Paul, MN, United States., Chabrière É; Aix Marseille Univ, IRD, APHM, MEPHI, IHU-Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France., Daudé D; Gene&GreenTK, 19-21 Boulevard Jean Moulin, 13005 Marseille, France. Electronic address: david.daude@gene-greentk.com.
Jazyk: angličtina
Zdroj: Enzyme and microbial technology [Enzyme Microb Technol] 2022 Oct; Vol. 160, pp. 110092. Date of Electronic Publication: 2022 Jul 01.
DOI: 10.1016/j.enzmictec.2022.110092
Abstrakt: 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.
(Copyright © 2022 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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