(582bg) Engineering Human Paraoxases for Quorum Sensing Quenching

Bacterial biofilms are responsible for persistent infections such as cystic fibrosis, chronic wounds, etc.  Mounting evidence implicated that formation of biofilms and expression of virulence factors by the pathogenic bacteria are controlled through intercellular communication mediated by quorum sensing (QS). QS signal molecules, e.g. acyl homoserine lactones (AHLs), have been therefore recognized as attractive pharmaceutical targets for fighting antibiotics resistance. Unlike antimicrobials that inhibit cell growth, enzymatic hydrolysis/degradation of QS molecules cause less selection pressure for bacterial resistance. Toward the development of QS-quenching enzymes, bacterial lactonases have been extensively studied but present a daunting challenge caused by their immunogenicity. Here we apply both rational and combinatorial approaches to engineer human paraoxase 2 (huPON2) as a novel agent to fight bacterial infections.  Particularly, wild type huPONs are extremely difficult for expression in their soluble format and exhibit low activities.  By replacing the extruded hydrophobic helixes with short linker peptide libraries, huPON2 clones able to be solubly expressed were successfully isolated. In addition, these clones clearly demonstrated their lactonase activities toward 3-oxo-C₁₂-HSL. To further engineer huPON2s as potent therapeutics, directed evolution has been applied to improve their enzymatic specific activities toward targeted QS lactones found in Pseudomonas aeruginosa, Enterobacter cloacae, etc clinically significant pathogens.