Pseudomonas aeruginosa is a Gram negative pathogenic bacteria 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 behaviour 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 signalling 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 immobilisation steps, paving the way for future therapeutic applications.

The effectiveness of antibiotic molecules in treating Pseudomonas aeruginosa pneumonia is reduced as a result of the dissemination of bacterial resistance. The existence of bacterial communication systems, such as quorum sensing, has provided new opportunities of treatment. Lactonases efficiently quench acyl-homoserine lactone-based bacterial quorum sensing, implicating these enzymes as potential new anti-Pseudomonas drugs that might be evaluated in pneumonia. The aim of the present study was to evaluate the ability of a lactonase called SsoPox-I to reduce the mortality of a rat P. aeruginosa pneumonia. To assess SsoPox-I-mediated quorum quenching, we first measured the activity of the virulence gene lasB, the synthesis of pyocianin, the proteolytic activity of a bacterial suspension and the formation of biofilm of a PAO1 strain grown in the presence of lactonase. In an acute lethal model of P. aeruginosa pneumonia in rats, we evaluated the effects of an early or deferred intra-tracheal treatment with SsoPox-I on the mortality, lung bacterial count and lung damage. SsoPox-I decreased PAO1 lasB virulence gene activity, pyocianin synthesis, proteolytic activity and biofilm formation. The early use of SsoPox-I reduced the mortality of rats with acute pneumonia from 75% to 20%. Histological lung damage was significantly reduced but the lung bacterial count was not modified by the treatment. A delayed treatment was associated with a non-significant reduction of mortality. These results demonstrate the protective effects of lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use.

Enzymes able to degrade or modify acyl-homoserine lactones (AHLs) have drawn considerable interest for their ability to interfere with the bacterial communication process referred to as quorum sensing. Many proteobacteria use AHL to coordinate virulence and biofilm formation in a cell density–dependent manner; thus, AHL-interfering enzymes constitute new promising antimicrobial candidates. Among these, lactonases and acylases have been particularly studied. These enzymes have been isolated from various bacterial, archaeal, or eukaryotic organisms and have been evaluated for their ability to control several pathogens. Engineering studies on these enzymes were carried out and successfully modulated their capacity to interact with specific AHL, increase their catalytic activity and stability, or enhance their biotechnological potential. In this review, special attention is paid to the screening, engineering, and applications of AHL-modifying enzymes. Prospects and future opportunities are also discussed with a view to developing potent candidates for bacterial control.

The human opportunistic pathogen Pseudomonas aeruginosa orchestrates the expression of many genes in a cell density-dependent manner by using quorum sensing (QS). Two acyl-homoserine lactones (AHLs) are involved in QS circuits and contribute to the regulation of virulence factors production, biofilm formation, and antimicrobial sensitivity. Disrupting QS, a strategy referred to as quorum quenching (QQ) can be achieved using exogenous AHL-degrading lactonases. However, the importance of enzyme specificity on quenching efficacy has been poorly investigated. Here, we used two lactonases both targeting the signal molecules N-(3-oxododecanoyl)-L-Homoserine lactone (3-oxo-C12 HSL) and butyryl-homoserine lactone (C4 HSL) albeit with different efficacies on C4 HSL. Interestingly, both lactonases similarly decreased AHL concentrations and comparably impacted the expression of AHL-based QS genes. However, strong variations were observed in Pseudomonas Quinolone Signal (PQS) regulation depending on the lactonase used. Both lactonases were also found to decrease virulence factors production and biofilm formation in vitro, albeit with different efficiencies. Unexpectedly, only the lactonase with lower efficacy on C4 HSL was able to inhibit P. aeruginosa pathogenicity in vivo in an amoeba infection model. Similarly, proteomic analysis revealed large variations in protein levels involved in antibiotic resistance, biofilm formation, virulence and diverse cellular mechanisms depending on the chosen lactonase. This global analysis provides evidences that QQ enzyme specificity has a significant impact on the modulation of QS-associated behavior in P. aeruginosa PA14.