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International audience; Many bacteria use quorum sensing (QS), a bacterial communication system based on the diffusion and perception of small signaling molecules, to synchronize their behavior in a cell-density dependent manner. QS regulates the expression of many genes associated with virulence factor production and biofilm formation. This latter is known to be involved in antibiotic and phage resistance mechanisms. Therefore, disrupting QS, a strategy known as quorum quenching (QQ), appears to be an interesting way to reduce bacterial virulence and increase antibiotic and phage treatment efficiency. In this study, the ability of the QQ enzyme SsoPox-W263I, a lactonase able to degrade acyl-homoserine lactones, was investigated for quenching both virulence and biofilm formation in clinical isolates of Pseudomonas aeruginosa from diabetic foot ulcers, as well as in the PA14 model strain. These strains were further evolved to resist to bacteriophage cocktails. Overall, 10 antibiotics or bacteriophage resistant strains were evaluated and SsoPox-W263I was shown to decrease pyocyanin, protease and elastase production in all strains. Furthermore, a reduction of more than 70% of biofilm formation was achieved in six out of ten strains. This anti-virulence potential was confirmed in vivo using an amoeba infection model, showing enhanced susceptibility toward amoeba of nine out of ten P. aeruginosa isolates upon QQ. This amoeba model was further used to demonstrate the ability of SsoPox-W263I to enhance the susceptibility of sensitive and phage resistant bacteria to bacteriophage and antibiotic.

International audience; Depuis quelques dizaines d’années, un nouveau modèle animal invertébré a fait son apparition dans les études toxicologiques : la planaire. Ce ver plat non parasitaire, du phylum des Plathelminthes, possède une incroyable capacité de régénération : il a été décrit comme « immortel sous la lame du couteau » en 1814 par Dalyell. Cette impressionnante capacité est due à l’abondance de cellules souches, appelées néoblastes, qui permettent à un fragment équivalent à 1/279 de la taille de ce ver de générer un animal entier. La planaire dispose d’un système nerveux semblable à celui de l’homme. Elle a ainsi été utilisée pour évaluer les perturbations de développement et la neurotoxicité engendrées par différent composés. Cette revue rassemble les principales études de toxicité réalisées chez la planaire qui illustrent le fort potentiel de ce ver en recherche.

International audience; Depuis quelques dizaines d’années, un nouveau modèle animal invertébré a fait son apparition dans les études toxicologiques : la planaire. Ce ver plat non parasitaire, du phylum des Plathelminthes, possède une incroyable capacité de régénération : il a été décrit comme « immortel sous la lame du couteau » en 1814 par Dalyell. Cette impressionnante capacité est due à l’abondance de cellules souches, appelées néoblastes, qui permettent à un fragment équivalent à 1/279 de la taille de ce ver de générer un animal entier. La planaire dispose d’un système nerveux semblable à celui de l’homme. Elle a ainsi été utilisée pour évaluer les perturbations de développement et la neurotoxicité engendrées par différent composés. Cette revue rassemble les principales études de toxicité réalisées chez la planaire qui illustrent le fort potentiel de ce ver en recherche.

International audience; Pseudomonas aeruginosa is a Gram negative pathogenic bacterium involved in many human infections including otitis, keratitis, pneumonia, and diabetic foot ulcers. P. aeruginosa uses a communication system, referred to as quorum sensing (QS), to adopt a group behavior by synchronizing the expression of certain genes. Among the regulated traits, secretion of proteases or siderophores, motility and biofilm formation are mainly involved in the pathogenicity. Many efforts have been dedicated to the development of quorum sensing inhibitors (QSI) and quorum quenching (QQ) agents to disrupt QS. QQ enzymes have been particularly considered as they may act in a catalytic way without entering the cell. Here we focus on the lactonase SsoPox which was previously investigated for its ability to degrade the signaling molecules, acyl-homoserine lactones, in particular on the engineered variant SsoPox-W263I. We highlight the potential of SsoPox-W263I to inhibit the virulence of 51 clinical P. aeruginosa isolates from diabetic foot ulcers by decreasing the secretion of two virulence factors, proteases and pyocyanin, as well as biofilm formation. We further compared the effect of SsoPox-W263I to the comprehensively described QSI, 5-fluorouracil and C-30. We found the lactonase SsoPox-W263I to be significantly more effective than the tested QSI at their respective concentration optimum and to retain its activity after immobilization steps, paving the way for future therapeutic applications.

Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver for the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of the lactonase Sso Pox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being > 1,000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, ~1,000,000-fold and beyond, since some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops, as revealed by a suite of crystal structures. This suggests that specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining an enzyme activity profile., Competing Interests: CONFLICT OF INTEREST STATEMENT DD is a shareholder and CEO of Gene&GreenTK. EC is a shareholder and a co-founder of Gene&GreenTK. PJ, RB and EC report receiving personal fees from Gene&GreenTK during the study. MHE, DD and EC have filed the patent EP3941206. MHE and CB have a patent WO2020185861A1. MHE is a co-founder, a former CEO and equity holder of Gene&GreenTK, a company that holds the license to WO2014167140 A1, FR 3068989 A1, FR 19/02834. MHE received fees from Gene&GreenTK. MHE interests with Gene&GreenTK have been reviewed and managed by the University of Minnesota in accordance with its Conflict-of-Interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.