Novel Paraoxonase 2-Dependent Mechanism Mediating the Biological Effects of the Pseudomonas aeruginosa Quorum-Sensing Molecule N -(3-Oxo-Dodecanoyl)- l -Homoserine Lactone

Abstract

cPseudomonas aeruginosa produces N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12), a crucial signaling molecule that elicits diverse biological responses in host cells thought to subvert immune defenses. The mechanism mediating many of these responses remains unknown. The intracellular lactonase paraoxonase 2 (PON2) hydrolyzes and inactivates 3OC12 and is therefore considered a component of host cells that attenuates 3OC12-mediated responses. Here, we demonstrate in cell lines and in primary human bronchial epithelial cells that 3OC12 is rapidly hydrolyzed intracellularly by PON2 to 3OC12 acid, which becomes trapped and accumulates within the cells. Subcellularly, 3OC12 acid accumulated within the mitochondria, a compartment where PON2 is localized. Treatment with 3OC12 caused a rapid PON2-dependent cytosolic and mitochondrial pH decrease, calcium release, and phosphorylation of stress signaling kinases. The results indicate a novel, PON2-dependent intracellular acidification mechanism by which 3OC12 can mediate its biological effects. Thus, PON2 is a central regulator of host cell responses to 3OC12, acting to decrease the availability of 3OC12 for receptor-mediated effects and acting to promote effects, such as calcium release and stress signaling, via intracellular acidification. P seudomonas aeruginosa is a common pathogen causing serious infections in immunocompromised and ill individuals due to the bacteria's ability to evade host immune responses and acquire antibiotic resistance (1). Many Gram-negative bacteria, including P. aeruginosa, produce acyl-homoserine lactone (AHL) signaling molecules which regulate the cell density-dependent expression of virulence factors in a process termed quorum sensing (QS) (1). The AHL N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) is a key P. aeruginosa QS signal that has been shown to be necessary for biofilm maturation and full expression of virulence in P. aeruginosa animal infection models (2-5). Concentrations up to 600 M 3OC12 have been measured in P. aeruginosa biofilms in vitro (6). Concentrations of over 6 M 3OC12 have been detected in the sputum of individuals with pulmonary P. aeruginosa infections (7), suggesting active 3OC12 signaling in the human disease as well.In addition to modulating bacterial gene expression, 3OC12 elicits a multitude of biological responses in diverse mammalian cell types (8). Depending upon the cell type and dose, 3OC12 (10 to 100 M) can induce apoptosis, endoplasmic reticulum (ER) stress, chemotaxis, and proinflammatory gene expression (8-10). Conversely, 3OC12 inhibited lipopolysaccharide (LPS) induction of proinflammatory mediators in macrophages, fibroblasts, and epithelial cells and in vivo by repressing nuclear factor -light chain enhancer of activated B-cell (NF-B) signaling (11). In antigen-stimulated T-lymphocytes, 3OC12 inhibits cell proliferation and production of gamma interferon and interleukin-4 (IL-4), critical regulators of immunity (8,12). These diverse responses suggest that 3OC12 acts through multiple, and cell-...

Paraoxonase 2 Facilitates Pancreatic Cancer Growth and Metastasis by Stimulating GLUT1-Mediated Glucose Transport

“…Neither the PON2-interaction with GLUT1 nor the PON2-mediated increase in GLUT1 activity was affected by the H114Q or H133Q mutation, which abrogates PON2 lactonase activity (Altenhofer et al, 2010; Horke et al, 2015), nor was there an effect of the commonly occurring polymorphism, S311C (Altenhofer et al, 2010) (Figure S6D and S6E). Furthermore, ectopic expression of GLUT1 restored the ability of PANC1 cells expressing PON2 shRNAs to form colonies in soft agar (Figure S6F).…”

Peroxisome proliferator‐activated receptor‐γ agonists attenuate biofilm formation by Pseudomonas aeruginosa

“…PON‐2 is a mitochondrial protein that plays an important role in modulating bacterial QS and host cell responses to bacterial homoserine lactone signaling molecules (48). We recently published that PPARγ modulates expression of PON‐2 (20); therefore, we wanted to determine the effects of PAO1 and 3O‐C12‐HSL on the expression of PON‐2 in epithelial cells.…”

Enhanced Clearance of Pseudomonas aeruginosa by Peroxisome Proliferator-Activated Receptor Gamma

“…Recent studies have demonstrated that increased cellular calcium mediates degradation of PON-2 protein and mRNA. Since 3-oxo-C 12 -HSL can increase cytosolic calcium, it can be hypothesized that 3-oxo-C 12 -HSL may mediate changes in PPAR␥ and PON-2 levels by modulating the activity of key transcriptional factors (16,17,72,73). The induction of NF-B and interleukin-10 may also play a role in the inhibition of PPAR␥, as these are upregulated by 3-oxo-C 12 -HSL (74,75).…”

“…All three PON enzymes degrade oxidized lipids, protect against oxidative stress, and act to suppress inflammation. Paraoxonases have been shown to hydrolyze and thereby inactivate bacterial QS molecules, or N-acyl-homoserine lactones, such as 3-oxo-C 12 -HSL (13)(14)(15)(16)(17). In particular, PON-2 has the highest catalytic activity among the three PON proteins and is expressed in almost all human tissues (16).…”