(473) Acetate and propionate metabolism by Pseudomonas aeruginosa contributes to significant sinus inflammation in a rabbit model of sinusitis

Background: Understanding how P. aeruginosa adapts to the in vivo nutritional environment has important implications for the treatment of recalcitrant CF airway disease. Studies have demonstrated that anaerobe-derived nutrient (e.g., acetate, propionate) acquisition by P. aeruginosa may contribute to its virulence in the CF airway epithelium. Based on our rabbit model of sinusitis, mucin-fermenting anaerobes (e.g., Bacteroidales) dominated during the acute phase, producing increased concentrations of nutrients, and pathogenic bacteria (e.g., Pseudomonas) dominated at the chronic stage. The purpose of this study is to understand the impact of P. aeruginosa acetate and propionate metabolism in the pathogenesis of sinusitis.

Methods: Rabbits’ unilateral sinuses were infected with wild type (WT) B. fragilis+WT P. aeruginosa PA14 (n=6) after blocking the sinus opening with a sterile sponge. WT B. fragilis and P. aeruginosa double mutants (ΔprpBΔacsA), lacking the genes encoding both AcsA (acetyl-coA synthetase) and PrpB (methyl isocitrate lyase) required for the metabolism of acetate and propionate, respectively, were also used (n=4). At week 1, sterile sponge was removed to restore mucociliary clearance (MCC)/oxygenation. Then rabbits were monitored another 5 more weeks (total 6 weeks) to follow sinusitis advancement. End points included CFU of P. aeruginosa and CT scores (Kerschner’s rabbit sinus CT grading (scoring each imaging based on opacification of sinus, 0 (no opacification) -10 (full opacification)), histology (epithelial (E) and submucosal (SM) thickness), and chloride (Cl^-) secretion (sinus potential difference (SPD) measurement).

Results: At week 1, the CFUs from rabbits infected with WT PA14 were significantly higher than those with double mutants (ΔprpBΔacsA) [WT PA-14=5.4x10⁵+/-3.6x10⁵, ΔprpBΔacsA=3.2x10⁴+/-1.4x10⁴, p=0.025]. There was no difference in the CT scores between the two groups at Week 1. Both groups developed fulminant acute sinusitis through blocking the sinus opening (CT scores, WT PA-14=9.74+/-0.26, ΔprpBΔacsA=8.42+/-1.57, p=0.33). At week 6, SM thickness was significantly higher in those rabbits infected with WT PA14 compared to those with double mutants (ΔprpBΔacsA). E thickness was also higher in those rabbits infected with WT PA14, but did not reach statistical significance [µm, E thickness: WT PA-14=59.18+/-5.3 vs. ΔprpBΔacsA=52.48+/-3.09, p >0.05, SM thickness: WT PA-14=265.4+/-17.75 vs. ΔprpBΔacsA=173.6+/-20.21, p< 0.01]. SPD revealed increased Cl^- transport in the double mutants (ΔprpBΔacsA) compared to those infected with WT (Cl^-free+forskolin ∆PD (mV), WT PA-14=-2.15+/-0.5, ΔprpBΔacsA=-5.42+/-173, p=0.06) at week 6.

Conclusions: After a week of hypoxia with inoculation of two bacteria strains, P. aeruginosa growth was significantly increased with acetate and propionate metabolism during the acute/hypoxic status. Even with restoration of sinus oxygenation after blocking for one week, anaerobically grown P. aeruginosa that metabolizes acetate and propionate develops significant tissue inflammation and decreases Cl^- secretion at a chronic stage.

Acknowledgements: National Institute of Diabetes and Digestive and Kidney Diseases (5P30DK072482); National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (1 R01 HL133006-05) to BW; and NIH/National Institutes of Allergy and Infectious disease (K08AI146220), Triological Society Career Development Award, and Cystic Fibrosis Foundation K08 Boost Award (CHO20A0-KB) to DYC.

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