Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

Abstract

Many pathogenic bacteria utilize quorum sensing (QS) systems to regulate the expression of their virulence genes and promote the formation of biofilm, which renders pathogens with extreme resistance to conventional antibiotic treatments. As a novel approach for attenuating antibiotic resistance and in turn fighting chronic infections, enzymatic inactivation of QS signaling molecules, such as N-acyl homoserine lactones (AHLs), holds great promises. Instead of using bacterial lactonases that can evoke immune response when administered, we focus on the human paraoxonase 2 (huPON2). However, insolubility when heterologously overexpressed hinders its application as anti-infection therapeutics. In this study, huPON2 was engineered for soluble expression with minimal introduction of foreign sequences. On the basis of structure modeling, degenerate linkers were exploited for the removal of hydrophobic helices of huPON2 without disrupting its folding structure and thus retaining its enzymatic function. High soluble expression levels were achieved with a yield of 76 mg of fully human PON2 variants per liter of culture media. Particularly, two clones, D2 and E3, showed significant quorum quenching (QQ) bioactivities and effectively impeded Pseudomonas aeruginosa swimming and swarming motilities, signs of an early stage of biofilm formation. In addition, by correlating QQ with luminescence signal readouts, quantitative analysis of QQ toward natural or non-natural AHL-regulator combinations suggested that D2 and E3 exhibited strong lactone hydrolysis activities toward five AHLs of different side chain lengths and modifications widely utilized by a variety of biomedically important pathogens.

Catalytic Characteristics of New Antibacterials Based on Hexahistidine-Containing Organophosphorus Hydrolase

“…That is, having a similar activity pattern as His 6 -OPH and being suggested as another prospective quorum quenching enzyme [7], PON1 seems to be much more sensitive to inhibition by such common antibiotics. Thus, His 6 -OPH has additional catalytic advantages as compared to PON1.…”

Engineering acyl-homoserine lactone-interfering enzymes toward bacterial control

“…huPON2 D2 and huPON2 E3 variants have been obtained and tested on swimming and swarming motilities in P. aeruginosa PAO1. Inhibition of P. aeruginosa motilities has been observed; however, huPON2 D2 and huPON2 E3 variants were not better than AiiA lactonase (80).…”

“…Three highly hydrophobic helixes, unlinked to lactone hydrolysis, have been replaced by hydrophilic polypeptide linkers, leading to two mutants (D2 and E3) with higher soluble expression (6.2 and 3.2 mg/liter of culture). The latter have then been fused to maltose-binding protein (MBP) to lead to a final protein yield of 320 and 200 mg/liter of culture for MBP-D2 and MBP-E3, respectively, whereas MBP-huPON2 has only been expressed in its insoluble form (80).…”

“…By preincubating the bacteria and the variant before injection in Galleria mellonella larvae, drastic enhancement in survival was obtained as compared with larvae treated with WT enzyme; nearly 100% of larvae survived with PvdQ F24Y/L146W and around 20% of larvae survived with PvdQ WT (38). One of the limitations of the thera-peutic use of QQ enzymes is their potential to trigger an immune response (80). To that end, the huPON2, as described previously, has been engineered for QQ purposes to benefit from minimal immunogenicity and a soluble expression.…”

Engineering quorum quenching enzymes: progress and perspectives

“…Of the characterised human PONs, PON2 has the highest activity towards AHLs [40]. However, it is largely insoluble when heterologously overexpressed in Escherichia coli [41]. This has limited its potential as a therapeutic, as protein yields can be an important factor for industrial-scale production [23,24].…”