Using directed enzyme evolution towards a stable and specific Quorum Quenching lactonases to disrupt the signaling of pathogenic bacteria

Many gram-negative bacterial pathogens synchronize their population size with behavior and control the expression of their virulence and biofilm formation via quorum-sensing (QS). 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 combinations of AHLs, detected by LuxR receptors, leading to genes expression at certain threshold of AHLs concentration. Strategies to attenuate QS, dubbed quorum quenching (QQ), are of prime interest for the development of therapeutic alternatives to antibiotics. One of them is using AHL lactonases (QQ lactonases), to catalyze the opening of the AHL ring and disrupt their sensing. While the role of QQ lactonases in bacteria is still under debate, their potential anti-bacterial properties are of great interest in agriculture, food safety and medicine. However, for such applications there is a demand for enzymes that will be both stable and specific. We have identified new QQ lactonases as well as used directed enzyme evolution to increase the thermal stability up to 50% residual activity at 65ºC, while maintaining 50% of its residual activity for more than three weeks. Moreover, the evolved variants exhibit high activity with k_cat/K_M values up to 3.07*10⁵ M^-1s^-1 with AHLs secreted from Erwinia and Pseudomonas species. These evolved variants can serve as a better starting point for the development of specific biological anti-bacterial treatment by degrading the signaling molecules secreted by pathogenic bacteria, without interfering with the cell-to-cell communication signals of symbiotic bacteria.