Pseudomonas aeruginosa is a significant contributor to recalcitrant multi-drug resistant infections especially in immunocompromised and hospitalized patients. Multi-drug and totally drug resistant strains of P. aeruginosa are increasing threats that contribute to high mortality in these patients (1, 2). Hence there is an urgent need to develop new strategies to combat P. aeruginosa and other resistant pathogens. The pathogenic profile of P. aeruginosa is related to its ability to secrete a variety of virulence factors and promote biofilm formation. Quorum sensing (QS) is a mechanism wherein P. aeruginosa secretes small diffusible molecules, specifically acyl homo serine lactones (AHL) such as 3O-C12-HSL that promote inter-bacterial communication, virulence, and biofilm formation (3, 4). Strategies that strengthen the ability of the host to inhibit these virulence factors would enhance host defenses and improve treatment. Over the previous funding cycle, we examined how lipid mediators regulate the lung host innate immune response to P. aeruginosa virulence factors (5-8). Using biochemical and genetic approaches, we discovered that prostaglandin D2 (PGD2), a product of L-PGD synthase, and its downstream metabolite, 15d-PGJ2, stimulate host response to P. aeruginosa (9-11). Further, we found that these immune-stimulatory effects are dependent on the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPAR?), a ligand-activated transcription factor with a wide spectrum of biological functions that includes metabolism, inflammation and redox balance (12). Our preliminary data provide novel evidence that P. aeruginosa (strain PAO1) QS genes and molecules induce select miRNAs (including miR-27a and miR-130a) which reduce PPAR? levels in host cells? Furthermore treatment with PPAR? agonists induces host expression of paraoxonase 2 (PON-2), a mitochondrial enzyme with lactonase activity that hydrolyzes QS molecules. Induction of PON-2 inhibits biofilm formation by PAO1 on epithelial cells, improves epithelial integrity and enhances clearance of PAO1 in mouse lungs through inhibition of QS effects on cells. These findings support the novel hypothesis that P. aeruginosa evades host defenses by inhibiting PPAR? and downstream immunomodulatory effectors in host cells and that pharmacological PPAR? activation provides a complementary therapeutic approach to the treatment of P.aeruginosa infection. This hypothesis will be examined in three interrelated specific aims: 1) Investigate the molecular mechanisms by which P. aeruginosa attenuates expression of PPAR?. miRs-27a and 130a, as post- transcriptional mechanisms will be interrogated to define the molecular underpinnings as to how QS systems attenuate PPAR? in host cells. 2) Determine the mechanisms by which PPAR? enhances immune defenses. PON-2 knockout or overexpressing lung epithelial cells infected with PAO1 and treated with PPAR? agonists or antagonists will further define the link between PON-2 induction and PPAR? functional effects. Analysis of tight junction proteins and biofilm formation will determine the functional impact of PPAR? on P. aeruginosa infected cells. 3) Define the impact of PPAR? modulation in P. aeruginosa lung infection in vivo. PPAR? activation will be accomplished using a currently available PPAR? agonist (pioglitazone), in mice infected intra-nasally with PAO1 and its mutants lacking QS systems. The goal of this proposal is to define novel molecular approaches to stimulate immune response to virulent pathogens e.g. P. aeruginosa. Since pioglitazone is an FDA approved PPAR? agonist used in management of patients with type II diabetes, the results from these studies can be readily translated to human clinical trials. Completion of the proposed work will have fundamentally important implications and a major impact on the management of patients with resistant P. aeruginosa infections and will lay the ground work for testing therapies to treat difficult infections in human trials.
Pseudomonas aeruginosa is a significant contributor to recalcitrant multi-drug resistant infections especially in immunocompromised and hospitalized patients. Multi-drug and totally drug resistant strains of P. aeruginosa are increasing threats that contribute to high mortality in these patients. Drug resistant infections are a threat to our Veteran population. Hence there is an urgent need to develop new strategies to combat P. aeruginosa and other resistant pathogens. The pathogenic profile of P. aeruginosa is related to its ability to secrete a variety of virulence factors and promote biofilm formation. Strategies that strengthen the ability of the host to inhibit these virulence factors would enhance host defenses and improve treatment. The goal of this proposal is to define novel molecular approaches to stimulate immune response through pioglitazone (PPAR? agonist) to inhibit bacterial virulence and enhance host defenses. The results from these studies can be readily translated to human clinical trials since pioglitazone is already FDA approved for treatment of diabetes.
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