(491) Pel Overexpression by Pseudomonas Aeruginosa Reduces Induction of Heme-Oxygenase 1 in Macrophages.

Background: Heme-oxygenase-1 (HO-1) is an enzyme induced in host lungs in response to infection and inflammation that results in production of carbon monoxide (CO), a molecule with anti-inflammatory and anti-bacterial properties. The induction of HO-1 after exposure to Pseudomonas aeruginosa (PA) is reduced in CF macrophages (MF) compared to wild type. This contributes to the hyper-inflammatory state associated with infection in the CF lung. In this study we tested whether phenotypically different clinical PA CF isolates, that are adapted to survival in the CF lung, differentially induce HO-1 in MF. To this end, we tested clinical strains with increased expression of the exopolysaccharide (EPS) Pel which is produced by PA in CF sputum. We then used the results of these assays to examine laboratory strains with mutations that alter Pel expression.

Methods: Phenotypic bacterial screening of respiratory isolates of PA from patients with CF were performed to test swimming motility in 0.3% agar LB plates and swimming and colony morphology on EPS agar (LB supplemented with Congo Red and Coomassie blue) for the selected isolates and laboratory strains. MFs were generated by culturing bone marrow cells from CF (B6-KOCftrtm1UNC) and wild-type (WT) littermate mice in media containing M-CSF. MFs were challenged with PA laboratory strains in the PA14 background that express different levels of Pel: PA14(WT), PA15 (↑Pel), PA33 (∆Pel). Clinical isolates were selected with reduced swimming or increased Pel expression based on the EPS agar phenotype. Bacteria were incubated with the MFs at a multiplicity of infection of 10:1 for 1 hour, followed by extracellular bacteria killed with gentamicin (50µg/ml). MFs were incubated for an additional 16 hours prior to protein extraction to test HO-1 induction, and RNA to measure pro-inflammatory cytokine gene expression.

Results: A clinical isolate of PA (CF80) showed increased Pel expression as its morphology on EPS agar was similar to the laboratory isolate that constitutively produces Pel, PA15 (↑Pel), with the formation of highly wrinkled and dark red colonies. HO-1 induction was reduced in CF and WT Mfs, when challenged with PA15 (↑Pel) and CF80, compared to PA14 (WT) and PA33 (∆Pel) (Figure 1). Additionally, preliminary data showed a slight decrease in the gene expression of IL-12, IL-6 and CXCL1 when WT Mfs were challenged with PAO15 (↑Pel) compared with PA14 (WT).

Conclusions: Overexpression of Pel in a laboratory strain and a clinical isolate resulted in decreased induction of HO-1 in both CF and WT Mfs. Reducing HO-1 dependent CO induction by overexpression of Pel in PA may be a mechanism by which PA avoids being killed by the host immune response and establishes persistence in the CF lung. Ongoing work will determine if this reduction is directly due to Pel overexpression or an indirect effect due to changes in other surface molecules as a result of increased Pel production.Acknowledgements:

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