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MUC1 ectodomain is a flagellin-targeting decoy receptor and biomarker operative during Pseudomonas aeruginosa lung infection

ABSTRACT: We previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialidase-mediated MUC1 ectodomain (MUC1-ED) desialylation and MUC1-ED shedding from murine lungs in vivo. Here, we asked whether Pa in the lungs of patients with ventilator-associated pneumonia might also increase MUC1-ED shedding. The levels of MUC1-ED and Pa-expressed flagellin were dramatically elevated in bronchoalveolar lavage fluid (BALF) harvested from Pa-infected patients, and each flagellin level, in turn, predicted MUC1-ED shedding in the same patient. Desialylated MUC1-ED was only detected in BALF of Pa-infected patients. Clinical Pa strains increased MUC1-ED shedding from cultured human alveolar epithelia, and FlaA and FlaB flagellin-expressing strains provoked comparable levels of MUC1-ED shedding. A flagellin-deficient isogenic mutant generated dramatically reduced MUC1-ED shedding compared with the flagellin-expressing wild-type strain, and purified FlaA and FlaB recapitulated the effect of intact bacteria. Pa:MUC1-ED complexes were detected in the supernatants of alveolar epithelia exposed to wild-type Pa, but not to the flagellin-deficient Pa strain. Finally, human recombinant MUC1-ED dose-dependently disrupted multiple flagellin-driven processes, including Pa motility, Pa biofilm formation, and Pa adhesion to human alveolar epithelia, while enhancing human neutrophil-mediated Pa phagocytosis. Therefore, shed desialylated MUC1-ED functions as a novel flagellin-targeting, Pa-responsive decoy receptor that participates in the host response to Pa at the airway epithelial surface.

SUBMITTER: Verceles A

PROVIDER: S-EPMC8608881 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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.

| S-EPMC6333888 | biostudies-literature

Topical flagellin protects the injured corneas from Pseudomonas aeruginosa infection.

Project description:Among bacterial pathogens, Pseudomonas (P.) aeruginosa infection is the most sight threatening. The corneal innate immune responses are key mediators of the host's defense to P. aeruginosa. Using a mouse model of Pseudomonas keratitis, we evaluated the protective effects of topical application of flagellin, a ligand for Toll-Like receptor 5 (TLR5), on the development of Pseudomonas keratitis and elucidated the underlying mechanisms. Topical application of purified flagellin 6 and 24 h prior to P. aeruginosa inoculation on injured mouse corneas significantly attenuated clinical symptoms of P. aeruginosa keratitis, decreased bacterial burden, and suppressed infection induced inflammation in the B6 mouse cornea. Topical application of flagellin on wounded cornea induced PMN infiltration and markedly upregulated cathelicidin-related antimicrobial peptide (CRAMP) expression. In PMN depleted mice, flagellin promoted bacterial clearance in the cornea compared to that of the PBS treated mice, but was unable to prevent corneal perforation and systemic bacterial dissemination and sepses. Deletion of CRAMP increased corneal susceptibility to P. aeruginosa and abolished flagellin-induced protection in B6 mice. Our findings illustrate the profound protective effect of flagellin on the cornea innate defense, a response that can be exploited for prophylactic purposes to prevent contact lens associated Pseudomonas keratitis.

| S-EPMC3191947 | biostudies-literature

Pseudomonas aeruginosa keratitis: passive immunotherapy with antibodies raised against divalent flagellin.

Project description:Objectives:Pseudomonas aeruginosa infections such as keratitis are considered among the major health problems worldwide due to the complexity of pathogenesis and antibiotic resistance crisis, thus, finding new effective approaches for prevention and treatment of the infections seem to be still vital. In this report, we aimed to investigate the therapeutic effects of topical administration of the antibodies against type a and b-flagellin (FLA and FLB) in Pseudomonas keratitis model of infection in mice. Materials and Methods:Scratched corneas of mice were treated with approximately 107 CFUs/eye of PAK and/or PAO1 strains of P. aeruginosa. Specific IgG to FLA, FLB or divalent flagellin were topically applied to the infected corneas for 20 min, 24, and 36 hr post-infection. The bacterial burden and myeloperoxidase activity (as a marker for polymorphonuclears (PMNs) infiltration) were determined in the corneas. The biological activity of the anti-FLA and FLB IgG was evaluated in vitro by opsonophagocytosis test. Results:Compared to other treated corneas, divalent anti-flagellin IgG treatment showed a significant decrease in the bacterial CFUs and myeloperoxidase activity in the infected corneas (P<0.05). Results of opsonophagocytosis revealed that the specific antibodies raised against FLA and FLB had more potent opsonic killing activity on their homologous strains as compared with control group (P<0.05). Conclusion:It appears that in P. aeruginosa keratitis, topical administration of the combined antibodies likely via decreasing the bacterial load, and PMNs infiltration as well as increasing opsonophagocytosis could lead to dramatic improvement of the infected corneas.

| S-EPMC6437458 | biostudies-literature

Comparison of flagellin genes from clinical and environmental Pseudomonas aeruginosa isolates.

Project description:Pseudomonas aeruginosa, an important opportunistic pathogen, was isolated from environmental samples and compared to clinically derived strains. While P. aeruginosa was isolated readily from an experimental mushroom-growing unit, it was found only rarely in other environmental samples. A flagellin gene PCR-restriction fragment length polymorphism analysis of the isolates revealed that environmental and clinical P. aeruginosa strains are not readily distinguishable. The variation in the central regions of the flagellin genes of seven of the isolates was investigated further. The strains used included two strains with type a genes (998 bp), four strains with type b genes (1,258 bp), and one strain, K979, with a novel flagellin gene (2,199 bp). The route by which flagellin gene variation has occurred in P. aeruginosa is discussed.

| S-EPMC91160 | biostudies-literature

A genomic island in Pseudomonas aeruginosa carries the determinants of flagellin glycosylation.

Project description:Protein glycosylation has been long recognized as an important posttranslational modification process in eukaryotic cells. Glycoproteins, predominantly secreted or surface localized, have also been identified in bacteria. We have identified a cluster of 14 genes, encoding the determinants of the flagellin glycosylation machinery in Pseudomonas aeruginosa PAK, which we called the flagellin glycosylation island. Flagellin glycosylation can be detected only in bacteria expressing the a-type flagellin sequence variants, and the survey of 30 P. aeruginosa isolates revealed coinheritance of the a-type flagellin genes with at least one of the flagellin glycosylation island genes. Expression of the b-type flagellin in PAK, an a-type strain carrying the glycosylation island, did not lead to glycosylation of the b-type flagellin of PAO1, suggesting that flagellins expressed by b-type bacteria not only lack the glycosylation island, they cannot serve as substrates for glycosylation. Providing the entire glycosylation island of PAK, including its a-type flagellin in a flagellin mutant of a b-type strain, results in glycosylation of the heterologous flagellin. These results suggest that some or all of the 14 genes on the glycosylation island are the genes that are missing from strain PAO1 to allow glycosylation of an appropriate flagellin. Inactivation of either one of the two flanking genes present on this island abolished flagellin glycosylation. Based on the limited homologies of these gene products with enzymes involved in glycosylation, we propose that the island encodes similar proteins involved in synthesis, activation, or polymerization of sugars that are necessary for flagellin glycosylation.

| S-EPMC55422 | biostudies-literature

Pseudomonas aeruginosa increases MUC1 expression in macrophages through the TLR4-p38 pathway.

Project description:Alveolar macrophages (AMs) play a critical role in the clearance of Pseudomonas aeruginosa (Pa) from the airways. However, hyper-activation of macrophages can impair bacterial clearance and contribute to morbidity and mortality. MUC1 mucin is a membrane-tethered, high molecular mass glycoprotein expressed on the apical surface of mucosal epithelial cells and some hematopoietic cells, including macrophages, where it counter-regulates inflammation. We recently reported that Pa up-regulates the expression of MUC1 in primary human AMs and THP-1 macrophages, and that increased MUC1 expression in these cells prevents hyper-activation of macrophages that appears to be important for host defense against severe pathology of Pa lung infection. The aims of this study were to elucidate the mechanism by which Pa increases MUC1 expression in macrophages. The results showed that: (a) Pa stimulation of THP-1 macrophages increased MUC1 expression both at transcriptional and protein levels in a dose-dependent manner; (b) Both Pa- and LPS-induced MUC1 expression in THP-1 cells were significantly diminished by an inhibitory peptide of TLR4; and (c) LPS-stimulated MUC1 expression was diminished at both the mRNA and protein levels by an inhibitor of the p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2, JNK, or IKK. We conclude that Pa-stimulated MUC1 expression in THP-1 macrophages is regulated mainly through the TLR4-p38 signaling pathway.

| S-EPMC5599168 | biostudies-literature

Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK.

Project description:Flagella in procaryotes are complex structures requiring the coordinate expression of over 50 genes, including flagellin, the major repeating structural protein. We have previously shown that a functional RpoN gene product is required for expression of flagellin in Pseudomonas aeruginosa PAK (P. A. Totten and S. Lory, J. Bacteriol. 172:389-396, 1990) and have now cloned, sequenced, and determined the transcriptional start site of the structural gene for this flagellin. The clones containing this gene produced a protein that reacted on Western immunoblots with polyclonal and four different monoclonal antibodies to purified flagella. However, this flagellin protein in Escherichia coli was slightly smaller (41 kDa) than flagellin protein produced in P. aeruginosa PAK (45 kDa), indicating degradation in E. coli or modification in P. aeruginosa. Comparison of the deduced amino acid sequence of this gene with the amino acid sequences of other flagellins revealed a conservation in the N- and C-terminal domains, suggesting conservation of secretion or assembly signals between these organisms. The sequence 5' of the structural gene contained potential RpoN-specific promoters as well as a promoter sequence recognized by RpoF (sigma 28), the alternative sigma factor required for expression of flagellin genes in E. coli (and Bacillus subtilis). Deletion analysis of the promoter region as well as transcriptional start site mapping implicated the RpoF, and not the RpoN, consensus sequences as the functional promoter for the flagellin gene. Models for the involvement of both RpoN and RpoF in the expression of flagellin in P. aeruginosa are presented.

| S-EPMC210844 | biostudies-other

Structural and genetic characterization of glycosylation of type a flagellin in Pseudomonas aeruginosa.

Project description:Type a flagellins from two strains of Pseudomonas aeruginosa, strains PAK and JJ692, were found to be glycosylated with unique glycan structures. In both cases, two sites of O-linked glycosylation were identified on each monomer, and these sites were localized to the central, surface-exposed domain of the monomer in the assembled filament. The PAK flagellin was modified with a heterogeneous glycan comprising up to 11 monosaccharide units that were O linked through a rhamnose residue to the protein backbone. The flagellin of JJ692 was less complex and had a single rhamnose substitution at each site. The role of the glycosylation island gene cluster in the production of each of these glycosyl moieties was investigated. These studies revealed that the orfA and orfN genes were required for attachment of the heterologous glycan and the proximal rhamnose residue, respectively.

| S-EPMC387798 | biostudies-literature

Flagellin glycosylation in Pseudomonas aeruginosa PAK requires the O-antigen biosynthesis enzyme WbpO.

Project description:Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

| S-EPMC2910752 | biostudies-literature

Airway administration of flagellin regulates the inflammatory response to Pseudomonas aeruginosa

Project description:Here, we aim to understand the role of P38 in the airway epithelial cells response to flagellin. We report the gene expression profile of pig airway epithelial cells cultured under air-liquid conditions, pre-incubated or not with the P38 inhibitor SB203580 (20 µM, Tocris Bioscience) for 1 h and then stimulated with 100 ng/ml of a mutated flagellin (FliCΔ174-400) for 2h or 24h. Our data show that flagellin stimulation induce the expression of pro-inflammatory cytokines mainly through the NFkB pathway, with little impact of P38 inhibition in the cells response.

2022-05-21 | GSE173768 | GEO

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