Bacterial biofilms are responsible for persistent infections and / or antibiotics resistance. Biofilm formation and expression of virulence factors by the pathogenic bacteria is 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 as therapeutics due to their immunogenicity derived from their prokaryotic origin. Here we applied 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 its extruded hydrophobic helixes with small short linker peptide libraries, huPON2 clones able to be solubly expressed were successfully isolated. In addition to lactone hydrolysis activities toward 3-oxo-C12-HSL, these isolated huPON2 variants also clearly demonstrated their quorum quenching function in cell based assays. To further improve huPON2’s potency, a high-throughput screening method based on a bioluminescent reporter was successfully established. And directed evolution has been applied for enhancing their enzymatic activities toward specific QS lactones found in Pseudomonas aeruginosa, Enterobacter cloacae, etc clinically significant pathogens. Keywords: quorum quenching, homoserine lactones, human paraoxase

Highly soluble and active human paraoxase variants for quorum quenching

249th National Meeting (2015)

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