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. 2019 Jan 11;294(2):662-678.
doi: 10.1074/jbc.RA118.006022. Epub 2018 Nov 14.

Neuraminidase 1-mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection

Erik P Lillehoj  1 , Wei Guang  2 , Sang W Hyun  3   4 , Anguo Liu  3   4 , Nicolas Hegerle  3   5 , Raphael Simon  3   5 , Alan S Cross  3   5 , Hideharu Ishida  6 , Irina G Luzina  3   4 , Sergei P Atamas  3   4 , Simeon E Goldblum  3   4   7
Affiliations

Neuraminidase 1-mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection

Erik P Lillehoj et al. J Biol Chem. .

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.

Keywords: Pseudomonas aeruginosa (P. aeruginosa); adhesin; cell surface associated (MUC1); decoy protein; flagellin; mucin 1; neuraminidase; sialic acid; sialidase; virulence.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Flagellin-expressing Pa induces MUC1-ED shedding in vivo. A, BALB/c mice were administered i.n. with 1.0 × 105 CFUs/mouse of WT PAK or the flagellin-deficient PAK/fliC− mutant, or the PBS vehicle. At increasing times postinfection, BALF was collected, and MUC1-ED levels quantified by ELISA and normalized to BALF protein. B, at 24 h postinfection with increasing inocula of WT PAK, MUC1-ED levels in BALF were quantified and normalized to BALF protein. C and D, BALB/c mice were infected i.n. with 1.0 × 105 CFUs of the indicated strains of Pa, or were administered i.n. with PBS, 10 ng of Pa or STm flagellins, 10 ng of Pa rFlaA or rFlaB expressed in E. coli, 100 ng of Pa LPS, 10 μg of Pam3Cys-Ser-(Lys)4, or 10 μg of CpG ODN 1826. At 24 h postinfection/administration, MUC1-ED levels (C) and TNFα levels (D) in BALF were quantified and normalized to BALF protein. C and D, the dashed lines indicate the levels of MUC1-ED shedding and TNFα in mice administered with PBS for comparison with the other experimental groups. Error bars represent mean ± S.E. values (n = 3–7). *, increased MUC1-ED/TNFα levels compared with uninfected or PBS controls at p < 0.05. **, decreased MUC1-ED levels following infection with the flagellin-deficient PAK/fliC− strain compared with flagellin-expressing WT PAK at p < 0.05. The results are representative of three independent experiments.
Figure 2.
Figure 2.
Purified Pa flagellin recapitulates Pa-provoked MUC1-ED shedding in vivo. A, BALB/c mice were administered i.n. with 10 ng/mouse of PAK flagellin or the PBS vehicle. At increasing times posttreatment, MUC1-ED levels in BALF were quantified and normalized to BALF protein. B, mice were administered increasing doses of PAK flagellin. At 24 h posttreatment, MUC1-ED levels in BALF were quantified and normalized to BALF protein. C, lysates of PAK and PAO1 were processed for flagellin immunoblotting using mouse anti-FlaA (lane 1) or anti-FlaB (lane 2) polyclonal antisera. Molecular masses in kDa are indicated on the left. D and E, PAK (D) or PAO1 (E) (1.0 × 107 CFUs) were incubated for 30 min at 4 °C with undiluted or decreasing dilutions of 100 μl of anti-FlaA (D) or anti-FlaB (E) antisera or the PBS control. The bacteria were washed and processed for adhesion to Ad-NEU1–infected (multiplicity of infection (m.o.i.) = 100) A549 cells. F, PAK (1.0 × 105 CFU) was preincubated with 10 μl of anti-FlaA antiserum or nonimmune mouse serum, and PAO1 (1.0 × 105 CFUs) was preincubated with 10 μl of anti-FlaB antiserum or nonimmune mouse serum for 30 min at 4 °C and washed. BALB/c mice were infected i.n. with the bacteria or administered the PBS vehicle. At 24 h postinfection, MUC1-ED levels in BALF were quantified and normalized to BALF protein. Error bars represent the mean ± S.E. values (n = 3). *, increased MUC1-ED levels compared with (A and B) PBS controls or (F) nonimmune mouse serum at p < 0.05. **, (E) decreased Pa adhesion compared with PBS controls, or (F) decreased MUC1-ED levels compared with nonimmune Ab, at p < 0.05. The results are representative of three independent experiments.
Figure 3.
Figure 3.
Pa lung infection increases NEU1-MUC1 and PPCA-MUC1 association and MUC1-ED desialylation. A–E and G–J, BALB/c mice were administered i.n. with 1.0 × 105 CFUs/mouse of PAK or the PBS vehicle. At 24 h postchallenge, BALF and lung tissues were collected, and lungs were homogenized. A, lung homogenates were immunoprecipitated with anti-MUC1-CD (lanes 1, 2, 5, 6), anti-NEU1 (lanes 3, 4), or anti-PPCA (lanes 7, 8) Abs. The MUC1-CD immunoprecipitates were processed for NEU1 (lanes 1, 2) or PPCA (lanes 5, 6) immunoblotting, and the NEU1 (lanes 3, 4) and PPCA (lanes 7, 8) immunoprecipitates were processed for MUC1-CD immunoblotting (upper panels). To control for protein loading and transfer, the immunoblots were stripped and reprobed with the immunoprecipitating Ab (lower panels). B–E, densitometric analyses of the blots in (A). Error bars represent mean ± S.E. MUC1-CD, NEU1, or PPCA signal normalized to the immunoprecipitate signal in the same lane on the same stripped and reprobed blot (n = 3). *, increased normalized lung NEU1, MUC1-CD, or PPCA signal compared with PBS controls at p < 0.05. F, to validate PNA selectivity, the negative control, fetuin (lane 1), and the positive control, asialofetuin (lane 2), 1.0 μg each, were processed for PNA lectin blotting. G, BALF (lanes 1 and 2) and lung homogenates (lanes 3 and 4) from mice administered i.n. with PAK or the PBS vehicle were processed for MUC1-ED immunoblotting. H, BALF (lanes 1 and 2) and lung homogenates (lanes 3 and 4) were incubated with PNA-agarose and the PNA-binding proteins processed for MUC1-ED immunoblotting. I and J, densitometric analyses of the blots in (H). Error bars represent mean ± S.E. BALF/lung desialylated MUC1-ED signal (n = 3). *, increased BALF MUC1-ED signal compared with PBS controls at p < 0.05. (A and F–H), molecular masses in kDa are indicated on the left. IP, immunoprecipitate; IB, immunoblot; IB*, immunoblot after stripping. PD, pulldown. The results are representative of three independent experiments.
Figure 4.
Figure 4.
NEU1 is required for maximal Pa-induced MUC1-ED desialylation and shedding. A–D, BALB/c mice were administered intraperitoneally with the indicated amounts of C9-BA-DANA, DANA, or the PBS vehicle. At 24 h postadministration, mice were infected i.n. with 1.0 × 105 CFUs/mouse of PAK. At 24 h postinfection, BALF was collected. A, BALFs were incubated with PNA-agarose and the PNA-binding proteins processed for MUC1-ED immunoblotting. B, densitometric analyses of the blots in (A). B and C, error bars represent mean ± S.E. BALF desialylated MUC1-ED signal or shed MUC1-ED levels (n = 3). C, BALF MUC1-ED levels were quantified by ELISA and normalized to BALF protein. D, BALFs were processed for MUC1-ED immunoblotting. **, decreased desialylated MUC1-ED signal or MUC1-ED level compared with PBS control at p < 0.05. †, decreased desialylated MUC1-ED signal or MUC1-ED level in mice administered DANA compared with mice administered C9-BA-DANA at p < 0.05. E–G, BALB/c mice were administered i.t. with Ad-NEU1, Ad-NEU1-G68V, or Ad-Null. At 3 days postinfection, BALF was collected. E, BALFs were incubated with PNA-agarose and the PNA-binding proteins processed for MUC1-ED immunoblotting. F, densitometric analyses of the blots in (E). G, BALF MUC1-ED levels were quantified by ELISA and normalized to BALF protein. F and G, error bars represent mean ± S.E. BALF desialylated MUC1-ED signal or MUC1-ED levels (n = 5). *, increased desialylated MUC1-ED signal or MUC1-ED level compared with Ad-Null or Ad-NEU1-G68V controls at p < 0.05. A, D, and E, Molecular masses in kDa are indicated on the left. H, BALB/c mice were infected i.n. with 1.0 × 105 CFUs of WT NanPs-expressing PAO1 or the NanPs-deficient PAO1/NanPs− isogenic mutant. At 24 h postinfection, MUC1-ED levels in BALF were quantified and normalized to BALF protein (n = 6). I, HEK293 cells were transfected with plasmids encoding for WT MUC1 or the MUC1-ED S317A protease recognition site mutant and incubated for 24 h. The ECs were infected with increasing m.o.i.s of Ad-GFP or Ad-NEU1. After 48 h, shed MUC1-ED levels in cell culture supernatants were quantified by ELISA and normalized to total supernatant protein. Error bars represent mean ± S.E. shed MUC1-ED levels (n = 3). *, increased MUC1-ED levels versus Ad-GFP-infected MUC1-HEK293 cells at p < 0.05. **, decreased MUC1-ED levels versus Ad-NEU1-infected MUC1-HEK293 cells at p < 0.05. The results are representative of three independent experiments. IB, immunoblot; PD, pulldown.
Figure 5.
Figure 5.
MUC1-ED–enriched BALF inhibits flagellin binding and Pa adhesion to airway ECs and Pa motility. BALB/c mice were administered i.n. with 1.0 × 105 CFUs/mouse of PAK or the PBS vehicle. At increasing times postadministration, BALF was collected. A and B, A549 cells were infected with Ad-NEU1 (m.o.i. = 100) and cultured for 48 h. A, Alexa Fluor 594–labeled flagellin was incubated for 30 min at 4 °C with BALF from uninfected or PAK-infected mice and assayed for binding to Ad-NEU1–infected A549 cells. B, PAK was incubated for 30 min at 4 °C with BALF from uninfected or PAK-infected mice and assayed for adhesion to Ad-NEU1–infected A549 cells. C, PAK was incubated for 30 min at 4 °C with BALF from uninfected or PAK-infected mice and assayed for bacterial motility. A–C, error bars represent mean ± S.E. flagellin binding or Pa adhesion or motility (n = 3). D, BALFs incubated with anti-MUC1-ED or control Abs were processed for MUC1-ED immunoblotting. To control for equal protein and loading, the blot was stripped and reprobed for β-tubulin. Molecular masses in kDa are indicated on the left. IB, immunoblot; IB*, immunoblot after stripping. A–C, in selected experiments, BALF from PAK-infected mice collected at 48 h postinfection was incubated with anti-MUC1-ED Ab or a species- and isotype-matched nonimmune IgG control. Igs were immobilized on protein G–agarose and removed by centrifugation. The MUC1-ED–immunodepleted or control IgG-treated BALFs were incubated with Alexa Fluor 594–flagellin or Pa, and processed for (A) flagellin binding or (B) bacterial adhesion to Ad-NEU1–infected A549 cells or (C) bacterial motility. *, increased flagellin binding, or Pa adhesion or motility, following incubation with MUC1-ED–immunodepleted BALFs compared with nonimmune IgG-treated BALFs at p < 0.05. **, decreased flagellin binding or Pa adhesion or motility following incubation with BALFs from Pa-infected mice compared with incubation with BALF from uninfected controls at p < 0.05. The results are representative of three independent experiments.
Figure 6.
Figure 6.
Deglycosylated protein backbone of human MUC1-ED inhibits Pa adhesion to human airway ECs. A, PAK was incubated with human MUC1-ED isolated from supernatants of A549 airway ECs infected with Ad-NEU1, Ad-NEU1–G68V, or Ad-GFP (m.o.i. = 100), washed, and assayed for adhesion to fresh, unmanipulated A549 cells (n = 3). B, Alexa Fluor 594–labeled PAK flagellin was incubated for 30 min at 37 °C with 25 mm of Gal or GalNAc, 25 mm Gal plus 25 mm GalNAc, or the PBS vehicle control. The ECs were washed and processed for flagellin binding by fluorometry (n = 3). C, PAK was incubated with human MUC1-ED isolated from the supernatants of A549 cells cultured in the presence of 1.0 μg/ml tunicamycin, 5.0 μm GalNAc-O-bn, or the PBS vehicle control, washed, and assayed for adhesion to fresh, unmanipulated A549 cells (n = 3). D, PAK was incubated with human rMUC1-ED prepared from E. coli transformed with a MUC1 expression plasmid or the empty vector control, washed, and assayed for adhesion to A549 cells (n = 3). Error bars represent mean ± S.E. Pa adhesion (A, C, D) or flagellin binding (B). **, significantly decreased Pa adhesion versus Ad-GFP, PBS, or empty vector controls at p < 0.05. E, human MUC1-ED isolated from A549 cells cultured in the presence of 1.0 μg/ml tunicamycin, 5.0 μm GalNAc-O-bn, or the PBS vehicle control, and E. coli-expressed human rMUC1-ED were processed for MUC1-ED immunoblotting. Molecular masses in kDa are indicated on the left. IB, immunoblot. The results are representative of three independent experiments.
Figure 7.
Figure 7.
Human rMUC1-ED inhibits flagellin binding and Pa adhesion to human airway ECs and Pa motility. A, PAK was incubated with 25 μm of human rMUC1-ED for 1 h at 4 °C. The bacteria were incubated with mouse anti-MUC1-ED Ab (panel i) or nonimmune mouse IgG (panel ii), each at 1:5000 dilution, for 1 h at 4 °C, followed by gold-labeled goat anti-mouse IgG secondary Ab at 1:10,000 dilution for 1 h at 4 °C, and examined by transmission immunoelectron microscopy. Each section was photographed at 11,000× or 44,000× (insert). Arrowheads indicate immunogold labeling. Scale bar, 100 nm. B and C, Human rMUC1-ED at the indicated concentrations or the PBS vehicle control were incubated with (B) Alexa Fluor 594–labeled Pa flagellin (n = 3) or (C) WT PAK or the flagellin-deficient PAK/fliC− mutant (n = 3) and assayed for (B) flagellin binding or (C) Pa adhesion to Ad-NEU1–infected A549 cells. D, the indicated concentrations of human rMUC1-ED or the PBS vehicle control were incubated with WT PAK or the PAK/fliC− mutant, and the bacteria were stab-inoculated into 0.3% agar plates and incubated overnight. Pa colony diameter was measured as an indicator of bacterial motility (n = 3). Error bars represent mean ± S.E. flagellin binding (B), Pa adhesion (C), or Pa motility (D). **, significantly decreased flagellin binding or Pa adhesion or motility versus the PBS control at p < 0.05. The results are representative of three independent experiments.
Figure 8.
Figure 8.
Human rMUC1-ED protects against lethal Pa lung infection. WT PAK or the PAK/fliC− mutant (1.0 × 105 CFUs/mouse) were co-administered i.n. with PBS or 2.5, 5.0, 12.5, or 25 μg/kg body weight (BW) of human rMUC1-ED to BALB/c mice. A–E, lung (A) and BALF CFUs (B), TNFα (C) and KC (D) levels in BALF, and lung MPO (E) levels were measured at 24 h postinfection. Error bars represent the mean ± S.E. Pa CFUs/g lung protein or CFUs/mg BALF protein, TNFα ng/ml and KC ng/ml BALF, or MPO activity/g lung protein (n = 3). **, reduced WT PAK CFUs, TNFα, KC, or MPO following Pa co-administered with human rMUC1-ED versus the PBS controls at p < 0.05. F, WT PAK (1.0 × 105 CFUs/mouse) was co-administered i.n. with PBS (panel i) or 25 μg/kg BW of human rMUC1-ED (panel ii) to BALB/c mice. At 24 h postinfection, lungs were harvested and sections were stained with hematoxylin and eosin, and examined by microscopy. Each section was photographed at 400×. Scale bar, 50 μm. The photomicrographs are representative of three mouse lung sections. G, WT PAK (2.0 × 107 CFUs/mouse) was co-administered i.n. with PBS, or 125 or 250 μg/kg BW of human rMUC1-ED to BALB/c mice and survival was measured daily by Kaplan-Meier analysis (n = 7/group). The results are representative of three independent experiments.
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