Dominant Role of Paraoxonases in Inactivation of the Pseudomonas aeruginosa Quorum-Sensing Signal N -(3-Oxododecanoyl)- l -Homoserine Lactone

Abstract

The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-Oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosa's quorum-sensing system and regulates the expression of many virulence factors. 3OC12-HSL was previously shown to be hydrolytically inactivated by the paraoxonase (PON) family of calcium-dependent esterases, consisting of PON1, PON2, and PON3. Here we determined the specific activities of purified human PONs for 3OC12-HSL hydrolysis, including the common PON1 polymorphic forms, and found they were in the following order: PON2 Ͼ Ͼ PON1 192R > PON1 192Q > PON3. PON2 exhibited a high specific activity of 7.6 ؎ 0.4 mols/min/mg at 10 M 3OC12-HSL, making it the best PON2 substrate identified to date. By use of class-specific inhibitors, approximately 85 and 95% of the 3OC12-HSL lactonase activity were attributable to PON1 in mouse and human sera, respectively. In mouse liver homogenates, the activity was metal dependent, with magnesium-and manganese-dependent lactonase activities comprising 10 to 15% of the calcium-dependent activity. In mouse lung homogenates, all of the activity was calcium dependent. The calcium-dependent activities were irreversibly inhibited by extended EDTA treatment, implicating PONs as the major enzymes inactivating 3OC12-HSL. In human HepG2 and EA.hy 926 cell lysates, the 3OC12-HSL lactonase activity closely paralleled the PON2 protein levels after PON2 knockdown by small interfering RNA treatment of the cells. These findings suggest that PONs, particularly PON2, could be an important mechanism by which 3OC12-HSL is inactivated in mammals.Pseudomonas aeruginosa is an opportunistic bacterium which causes serious infections in immunocompromised and cystic fibrosis patients (10). As with many gram-negative bacteria, P. aeruginosa produces acyl-homoserine lactone (AHL) quorumsensing (QS) signaling molecules termed autoinducers which allow the single-celled organisms to coordinate their actions (36). N-(3-Oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a key autoinducer synthesized by P. aeruginosa which regulates the expression of extracellular virulence factors and biofilm formation (5, 36). Rats and mice experimentally infected with P. aeruginosa mutants deficient in the ability to produce or respond to 3OC12-HSL exhibited significantly diminished lung pathology, bacterial dissemination, and morbidity and accelerated bacterial clearance compared to animals infected with wild-type bacteria, demonstrating the importance of 3OC12-HSL for P. aeruginosa pathogenicity (14,21,27,31,40). 3OC12-HSL also has an array of immunomodulatory effects on eukaryotic cells, including the induction of apoptosis, inhibition of leukocyte proliferation, activation of neutrophils and macrophages, and induction of proinflammatory mediators (7,15,34,37,39,43). Recently, it was shown that a number of mammalian cell lines were able to inactive 3OC12-HSL (5), providing a possible mechanism for reduction of bacterial virulence.Mam...

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Interestingly, in addition to differential expression at various sites within the host (PON1 and PON3 are expressed in the liver and sera, whereas PON2 is a cell-associated enzyme expressed in various tissues [103]), the different PON enzymes appear to have unique enzymatic activities. All three purified PONs had AHL lactonase activity in vitro, but each enzyme was determined to have a distinct specificity against a range of substrates, with PON1 and PON3 acting on the widest range of substrates but PON2 being the most active against AHLs (76,(103)(104)(105). In vitro results have supported a role for PON2 in AHL degradation by murine tracheal epithelial cells (104), and other experiments have demonstrated that both serum PON1 and purified PON1 reduce P. aeruiginosa biofilm formation in vitro (103).…”

HDL-associated paraoxonase-1 can redistribute to cell membranes and influence sensitivity to oxidative stress

“…This is an important function that may link paraoxonases to innate immunity, as lactonases can act as quorum quenching agents to limit bacterial virulence. It has been clearly demonstrated that the paraoxonases, by hydrolysis of HSL, limit infection and biofilm formation by Pseudomonas aeruginosa [34]. All three members of the paraoxonase gene family can neutralize HSL, with paraoxonase-2 being the most active [34].…”

“…It has been clearly demonstrated that the paraoxonases, by hydrolysis of HSL, limit infection and biofilm formation by Pseudomonas aeruginosa [34]. All three members of the paraoxonase gene family can neutralize HSL, with paraoxonase-2 being the most active [34]. However, it is an intracellular enzyme, and under conditions where diffusion of HSLs into cells may be rate limiting for their inactivation activity, PON1 assumes greater importance [35].…”

“…However, it is an intracellular enzyme, and under conditions where diffusion of HSLs into cells may be rate limiting for their inactivation activity, PON1 assumes greater importance [35]. Thus, the quorum quenching activity of serum is essentially due to PON1 [34]. In this context, extracellularly located PON1 could serve as an initial line of defense against P. aeruginosa infection.…”

Functionality of HDL: Antioxidation and Detoxifying Effects

“…Whilst the intracellular PON2 variant shows the strongest lactonase activity, PON1is also active towards HSL (Ozer et al 2005) and is able to protect against Pseudomonas infection (Stoltz et al 2008). Indeed, PON1 is the major source of HSL neutralising activity in serum (Teiber et al 2008). …”