Towards the harnessing of quorum quenching lactonases to fight plant bacterial pathogens

Many gram-negative bacterial pathogens can synchronize their behavior and control the expression of their virulence factor secretion and biofilm formation via quorum-sensing (QS) signaling, a population density-dependent regulatory mechanism. The link between cell density and gene regulation is mediated by the secretion and sensing of auto-inducer molecules, such as N-acyl-homoserine lactones (AHLs). Different bacterial species produce different AHLs detected by AHL receptors, this lead to genes expression at certain AHLs concentration threshold. Strategies to attenuate QS, dubbed quorum quenching (QQ). AHL lactonases, named quorum quenching (QQ) lactonases, catalyze the opening of the AHL ring and hydrolyze the lactones, therefore disrupt their sensing, and their potential anti-bacterial properties are of great interest in agriculture, food safety and medicine. However, such enzymes with high substrate specificity as well as stability are in demand. Erwinia Amylovora is the cause of "Fire blight", a devastating plant disease with significant economic losses in agriculture. This plant pathogen uses QS to regulate its virulence factors expression. We have determined the specific activity of PTE-Like–Lactonase, PPH from M. tuberculosis with the AHL secreted by Erwinia Amylovora, as well as tested its ability to decrease virulence factors in culture and in planta. Using directed enzyme evolution we have increased PPH`s thermal stability, with 50% residual activity at 65ºC, and increased it`s shelf life, as 50% of its residual activity was maintained for more than two weeks since purification. Moreover, these variants maintained high activity with k_cat/K_M values up to 3.07*10⁵ M^-1s^-1. These evolved stable and active variants can serve as a better starting point for the development of biological sustainable anti-bacterial treatment based on quorum quenching lactonases.