Abstract
Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, that engages the mucin 1 (MUC1) ectodomain (ED) expressed on airway epithelia, increasing association of MUC1-ED with neuraminidase 1 (NEU1) and MUC1-ED desialylation. The MUC1-ED desialylation unmasks both cryptic binding sites for Pa and a protease recognition site, permitting its proteolytic release as a hyperadhesive decoy receptor for Pa. We found here that intranasal administration of Pa strain K (PAK) to BALB/c mice increases MUC1-ED shedding into the bronchoalveolar compartment. MUC1-ED levels increased as early as 12 h, peaked at 24-48 h with a 7.8-fold increase, and decreased by 72 h. The a-type flagellin-expressing PAK strain and the b-type flagellin-expressing PAO1 strain stimulated comparable levels of MUC1-ED shedding. A flagellin-deficient PAK mutant provoked dramatically reduced MUC1-ED shedding compared with the WT strain, and purified flagellin recapitulated the WT effect. In lung tissues, Pa increased association of NEU1 and protective protein/cathepsin A with MUC1-ED in reciprocal co-immunoprecipitation assays and stimulated MUC1-ED desialylation. NEU1-selective sialidase inhibition protected against Pa-induced MUC1-ED desialylation and shedding. In Pa-challenged mice, MUC1-ED-enriched bronchoalveolar lavage fluid (BALF) inhibited flagellin binding and Pa adhesion to human airway epithelia by up to 44% and flagellin-driven motility by >30%. Finally, Pa co-administration with recombinant human MUC1-ED dramatically diminished lung and BALF bacterial burden, proinflammatory cytokine levels, and pulmonary leukostasis and increased 5-day survival from 0% to 75%. We conclude that Pa flagellin provokes NEU1-mediated airway shedding of MUC1-ED, which functions as a decoy receptor protecting against lethal Pa lung infection.
Highlights
Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, that engages the mucin 1 (MUC1) ectodomain (ED) expressed on airway epithelia, increasing association of MUC1-ED with neuraminidase 1 (NEU1) and MUC1-ED desialylation
BALB/c mice were challenged intranasally (i.n.) with Pa strain K (PAK), its flagellin-deficient PAK/fliCϪ isogenic mutant, Pa strain PAO1, its NEU-deficient PAO1/NanPsϪ isogenic mutant, or clinical Pa isolates obtained from patients diagnosed with pneumonia, at either sublethal (1.0 ϫ 105 colony-forming units (CFUs)/mouse) or lethal (2.0 ϫ 107 CFUs/ mouse) inocula
Pa CFUs in lung tissues and bronchoalveolar lavage fluid (BALF) increased at 6 h following challenge with PAK at 1.0 ϫ 105 CFUs/mouse, after which Pa lung burden peaked at 12 h, whereas BALF CFUs peaked at 24 h (Fig. S1A)
Summary
BALB/c mice were challenged intranasally (i.n.) with Pa strain K (PAK), its flagellin-deficient PAK/fliCϪ isogenic mutant, Pa strain PAO1, its NEU-deficient PAO1/NanPsϪ isogenic mutant, or clinical Pa isolates obtained from patients diagnosed with pneumonia, at either sublethal (1.0 ϫ 105 colony-forming units (CFUs)/mouse) or lethal (2.0 ϫ 107 CFUs/ mouse) inocula. Incubation of PAK and PAO1 with decreasing dilutions of anti-FlaA and anti-FlaB antisera, respectively, dose-dependently blocked Pa adhesion to human airway ECs infected with adenovirus-encoding NEU1 (Ad-NEU1) (Fig. 2, D and E) These same antisera dramatically reduced Pa-stimulated MUC1-ED shedding in mouse lungs compared with bacteria incubated with a nonimmune mouse serum (Fig. 2F). These same MUC1-ED– containing BALFs displayed no growth inhibitory activity for PAK (Fig. S3A) These findings indicate that the shed MUC1-ED endogenously generated within the murine bronchoalveolar compartment acts as decoy receptor to diminish flagellin binding and Pa adhesion to airway epithelia and disrupt Pa motility, i.e. flagellin-driven processes that contribute to Pa pathogenesis. The MUC1-ED– containing murine BALFs dose-dependently inhibited flagellin binding and Pa adhesion to NEU1-overexpressing human airway ECs, the presence of increasing concentrations of lactose did not alter BALF decoy receptor function (Fig. S4, C and D). Human rMUC1-ED had no effect on lung (Fig. 8A) or BALF (Fig. 8B) CFUs, TNF␣ (Fig. 8C) or KC (Fig. 8D) levels in the BALF, or lung MPO activity (Fig. 8E) when co-administered with 1.0 ϫ 105 CFUs/mouse of the PAK/
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