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

Raphaël Billot, Laure Plener, Damien Grizard, Mikael H. Elias, Éric Chabrière, David Daudé

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Article number110092
JournalEnzyme and Microbial Technology
Volume160
DOIs
StatePublished - Oct 2022

Bibliographical note

Funding Information:
R. B. is a Ph.D. student supported by the French Agence Nationale de la Recherche et de la Technologie ( ANRT ) CIFRE Grant 2018/1543 . This work was supported by the “Investissements d’avenir” program ( Méditerranée Infection Grant 10-IAHU-03 ) of the French Agence Nationale de la Recherche (ANR). M.H.E. was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM133487 . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding Information:
R. B. is a Ph.D. student supported by the French Agence Nationale de la Recherche et de la Technologie (ANRT) CIFRE Grant 2018/1543. This work was supported by the “Investissements d'avenir” program (Méditerranée Infection Grant 10-IAHU-03) of the French Agence Nationale de la Recherche (ANR). M.H.E. was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM133487. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors would like to thank Dr. Raphael Lami for providing the P. putida KS35 reporter strain and MAPIEM lab for providing the CV026 strain. EC has patent WO2014167140 A1 licensed to Gene&GreenTK. L.P. E.C. and D.D. have filed the patent EP3941206. R.B. L.P. D.D. and E.C. report receiving personal fees from Gene&GreenTK during the study. E.C. and D.D. are shareholders in Gene&GreenTK. D.D. is CEO of Gene&GreenTK. D.G. is Director of Gene&GreenTK. M.H.E. is the co-founder, former Scientific Advisory Board member, and equity holder of Gene&GreenTK, a company that holds the license to WO2014167140 A1, FR 3068989 A1, FR19/02834. M.H.E. is an inventor of patents No. 62/816,403, WO2014167140 A1, WO2008145865A2, WO2015014971A1, FR3068989 A1, FR19/02834 and EP3941206. These interests have been reviewed and managed by the University of Minnesota in accordance with its Conflict of Interest policies.

Publisher Copyright:
© 2022 Elsevier Inc.

Keywords

  • Acyl homoserine lactones
  • Enzyme screening
  • Enzyme, engineering
  • Lactonase
  • Quorum quenching
  • Quorum sensing

PubMed: MeSH publication types

  • Journal Article

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