Engineering Soluble Human Paraoxonase 2 for Quorum Quenching.

Area of Science:

• Microbiology
• Biochemistry
• Biotechnology

Background:

• Pathogenic bacteria use quorum sensing (QS) to control virulence and biofilm formation, leading to antibiotic resistance.
• Enzymatic inactivation of QS molecules (N-acyl homoserine lactones - AHLs) is a promising strategy against chronic infections.
• Human paraoxonase 2 (huPON2) shows potential for QS inhibition but suffers from poor solubility during overexpression.

Purpose of the Study:

• To engineer a soluble and functionally active form of human paraoxonase 2 (huPON2) for anti-infective applications.
• To assess the quorum quenching (QQ) activity and biofilm inhibition capabilities of engineered huPON2 variants.
• To characterize the enzymatic hydrolysis of various AHL molecules by the engineered huPON2.

Main Methods:

• Structure-based design utilizing degenerate linkers to remove hydrophobic regions of huPON2.
• Heterologous overexpression and purification of engineered huPON2 variants.
• Assay of QQ bioactivity against Pseudomonas aeruginosa motility and AHL hydrolysis.

Main Results:

• Achieved high soluble expression of huPON2 variants (76 mg/L).
• Identified two clones (D2 and E3) with significant QQ bioactivities.
• Demonstrated inhibition of bacterial motility and effective hydrolysis of five different AHLs by D2 and E3 variants.

Conclusions:

• Engineered huPON2 variants (D2, E3) exhibit enhanced solubility and retained enzymatic function for QS inhibition.
• These variants effectively reduce bacterial virulence factor expression and biofilm precursor activities.
• Soluble huPON2 represents a promising therapeutic candidate for combating antibiotic-resistant bacterial infections.