Identification and Characterization of a New Quorum-Quenching N-acyl Homoserine Lactonase in the Plant Pathogen Erwinia amylovora.

Quorum quenching (QQ) is the ability to interfere with bacterial cell to cell communication, known as quorum sensing (QS). QQ enzymes that degrade or modify acyl homoserine lactones (AHLs) have been attracting increasing interest as promising agents for inhibiting QS-mediated bacterial pathogenicity. Plant pathogens from the genus Erwinia cause diseases in several economically important crops. Fire blight is a devastating plant disease caused by Erwinia amylovora, affecting a wide range of host species within the Rosaceae and posing a major global threat for commercial apple and pear production. While QS has been described in Erwinia species, no AHL-degrading enzymes were identified and characterized. Here, phylogenetic analysis and structural modeling were applied to identify an AHL lactonase in E. amylovora (dubbed EaAiiA). Following recombinant expression and purification, the enzyme was biochemically characterized. EaAiiA lactonase activity was dependent on metal ions and effectively degraded AHLs with high catalytic efficiency. Its highest specific activity (kcat/KM value) was observed against one of the AHLs (3-oxo-C6-homoserine lactone) secreted from E. amylovora. Exogenous addition of the purified enzyme to cultures of E. amylovora reduced the formation of levan, a QS-regulated virulence factor, by 40% and the transcription level of the levansucrase-encoding gene by 55%. Furthermore, preincubation of E. amylovora cultures with EaAiiA inhibited the progress of fire blight symptoms in immature Pyrus communis fruits. These results demonstrate the ability of the identified enzyme from E. amylovora to act as a quorum-quenching lactonase.

Directed Enzyme Evolution and Encapsulation in Peptide Nanospheres of Quorum Quenching Lactonase as an Antibacterial Treatment against Plant Pathogen.

The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N-acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta. Using directed enzyme evolution, we were able to increase the enzyme's temperature resistance (T50, the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom's infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.