asialo-gm1-ganglioside and Pseudomonas-Infections

Pseudomonas aeruginosa is an opportunistic organism, which frequently colonizes the respiratory tract of patients with impaired host defence. In cystic fibrosis (CF) patients, this pathogen causes a progressive destructive bronchitis and bronchiolitis and is responsible for high mortality. Normal respiratory epithelium is protected against bacteria via mucus and mucociliary clearance. Alteration of mucociliary clearance and of glycosylation of mucins in CF facilitates the access of bacteria to the underlying airway epithelial cells. Intact respiratory epithelium does not bind P. aeruginosa, whereas injured respiratory epithelium is highly susceptible to P. aeruginosa adherence. We found that the high affinity of respiratory epithelium, from CF and non-CF sources, for P. aeruginosa, during the wound repair process is related to the apical expression of asialo ganglioside M1 (aGM1). The affinity of repairing respiratory epithelium for P. aeruginosa is time-dependent, and is related to transient apical expression of aGM1 at the surface of repairing respiratory epithelial cells. CF respiratory epithelial cells apically express more aGM1 residues with relation to an increased affinity for P. aeruginosa than non CF cells. High epithelial damage followed by repair represents a major cause of P. aeruginosa adherence to airway epithelium in cystic fibrosis. However, P. aerurignosa adherence and colonization are not restricted to cystic fibrosis disease and P. aeruginosa pneumonia may also occur in severely immunocompromised patients, suggesting that epithelial injury and decreased host-response favour the colonization of the airways by P. aeruginosa.

Topics: Bacterial Adhesion; Bronchiolitis; Bronchitis; Cystic Fibrosis; Disease Susceptibility; Epithelium; G(M1) Ganglioside; Gene Expression; Humans; Immunocompromised Host; Mucociliary Clearance; Mucus; Opportunistic Infections; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory System; Wound Healing

Pseudomonas aeruginosa causes severe life-threatening airway infections that are a frequent cause for hospitalization of cystic fibrosis (CF) patients. These Gram-negative pathogens possess flagella that contain the protein flagellin as a major structural component. Flagellin binds to the host cell glycolipid asialoGM1 (ASGM1), which appears enriched in luminal membranes of respiratory epithelial cells. We demonstrate that in mouse airways, luminal exposure to flagellin leads to inhibition of Na+ absorption by the epithelial Na+ channel ENaC, but does not directly induce a secretory response. Inhibition of ENaC was observed in tracheas of wild-type mice and was attenuated in mice homozygous for the frequent cystic fibrosis conductance regulator (CFTR) mutation G551D. Similar to flagellin, anti-ASGM1 antibody also inhibited ENaC. The inhibitory effects of flagellin on ENaC were attenuated by blockers of the purinergic signaling pathway, although an increase in the intracellular Ca2+ concentration by recombinant or purified flagellin or whole flagella was not observed. Because an inhibitor of the mitogen-activated protein kinase (MAPK) pathway also attenuated the effects of flagellin on Na+ absorption, we conclude that flagellin exclusively inhibits ENaC, probably due to release of ATP and activation of purinergic receptors of the P2Y subtype. Stimulation of these receptors activates the MAPK pathway, thereby leading to inhibition of ENaC. Thus, P. aeruginosa reduces Na+ absorption, which could enhance local mucociliary clearance, a mechanism that seem to be attenuated in CF.

Topics: Adenosine Triphosphate; Amiloride; Animals; Bronchi; Butadienes; Calcium Signaling; Cells, Cultured; Cystic Fibrosis; Egtazic Acid; Epithelial Cells; Epithelial Sodium Channels; Estrenes; Flagellin; G(M1) Ganglioside; Humans; Ion Transport; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred CFTR; Nitriles; Patch-Clamp Techniques; Phosphatidylinositol 4,5-Diphosphate; Pseudomonas aeruginosa; Pseudomonas Infections; Pyrrolidinones; Receptors, Purinergic P2; Receptors, Purinergic P2Y12; Respiratory Tract Infections; Sodium; Sodium Channels; Trachea

Pulmonary infection with Pseudomonas aeruginosa in patients with cystic fibrosis (CF) causes a chronic destructive bronchitis. A xenograft model was used to study the susceptibility of the CF respiratory epithelium to P. aeruginosa strain PAK and the virulence of certain mutants. Despite an early trend toward increased susceptibility, colonization of CF xenografts (ID(95), 62 colony-forming units [cfu]) was not statistically different (P=.5) than in xenografts with normal respiratory cells (ID(95), 1.2x10(3) cfu). Infection severity in 12 CF xenografts (mean polymorphonuclear leukocyte [PMNL] density, 1.88x10(6)+/-1.75x10(6)/xenograft) was similar to that in 16 non-CF xenografts (3.19x10(6)+/-2.45x10(6) PMNL/xenograft; P=.38), despite slightly greater bacterial density in the CF xenografts (mean, 1.57+/-2.73x10(6) cfu/xenograft) versus xenografts with normal epithelium (mean, 1.03+/-1.3x10(6) cfu/xenograft). P. aeruginosa mutants pilA and fliF, but not rpoN, colonized normal respiratory xenografts, indicating that colonization and infection in this model depend on an uncharacterized RpoN-controlled gene. This model appears to be suitable for genetic study of P. aeruginosa virulence but not of the CF respiratory tract's unique susceptibility.

Topics: Animals; Colony Count, Microbial; Cystic Fibrosis; Disease Models, Animal; Epitopes; Female; G(M1) Ganglioside; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; N-Acetylneuraminic Acid; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Transplantation, Heterologous

Pseudomonas aeruginosa strains are opportunistic pathogens associated with infections in immunocompromised hosts and patients with cystic fibrosis. Like many other mucosal pathogens, P. aeruginosa cells express flagella which provide motility and chemotaxis toward preferred substrates but also provide a ligand for clearance by phagocytic cells. We tested the role of flagella in the initial stages of respiratory tract infection by comparing the virulence of fliC mutants in a neonatal mouse model of pneumonia. In the absence of fliC, there was no mortality, compared with 30% mortality attributed to the parental strain PAK or 15% mortality associated with infection due to a pilA mutant PAK/NP (P < 0.0001). The fliC mutants caused pneumonia in only 25% of the mice inoculated, regardless of whether there was expression of the pilus, whereas the parental strain was associated with an 80% rate of pneumonia. Histopathological studies demonstrated that the fliC mutants caused very focal inflammation and that the organisms did not spread through the lungs as seen in infection due to either PAK or PAK/NP. Purified flagellin elicited an intense inflammatory response in the mouse lung. 125I-labeled flagellin bound to the glycolipids GM1 and GD1a and to asialoGM1 in an in vitro binding assay. However, flagellin-mediated binding to epithelial gangliosides was a relatively unusual event, as quantified by binding assays of wild-type or fliC mutant organisms to CHO Lec-2 cells with membrane-incorporated GM1. Fla+ organisms but not fliC mutants were efficiently taken up by murine macrophages. P. aeruginosa flagella are important in the establishment of respiratory tract infection and may act as a tether in initial interactions with epithelial membranes. This function is offset by the contribution of flagella to host clearance mechanisms facilitating phagocytic clearance and the role of flagellar genes in mucin binding and clearance.

Topics: Animals; Bacterial Adhesion; Bacterial Proteins; CHO Cells; Cricetinae; DNA-Binding Proteins; Fimbriae Proteins; Flagella; Flagellin; Flow Cytometry; G(M1) Ganglioside; Gangliosides; Inflammation; Macrophages; Mice; Mice, Inbred BALB C; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Tract Infections; Tumor Cells, Cultured; Virulence

We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients. Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model. At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury. P aeruginosa adherence to regenerating respiratory epithelial cells was found to be significantly greater in the delta F 508 homozygous CF group than in the non-CF group (P < 0.001). In vitro competitive binding inhibition assays performed with rabbit polyclonal antibody against asialo GM1 demonstrated that blocking asialo GM1 reduces P. aeruginosa adherence to regenerating respiratory epithelial cells in delta F 508 homozygous cultures (P < 0.001) as well as in non-CF cultures (P < 0.001). Blocking of asialo GM1 was significantly more efficient in CF patients than in non-CF subjects (P < 0.05). Distribution of asialo GM1 as determined by preembedding labelling and immunoelectron microscopy clearly demonstrated the specific apical membrane expression of asialo GM1 by regenerating respiratory epithelial cells, whereas other cell phenotypes did not apically express asialo GM1. These results demonstrate that (i) asialo GM1 is an apical membrane receptor for P. aeruginosa expressed at the surface of CF and non-CF regenerating respiratory epithelial cells and (ii) asialo GM1 is specifically recovered in regenerating respiratory epithelium. These results suggest that in CF, epithelial repair represents the major event which exposes asialo GM1 for P. aeruginosa adherence.

Topics: Adolescent; Adult; Animals; Bacterial Adhesion; Cells, Cultured; Child; Cystic Fibrosis; Epithelium; Female; G(M1) Ganglioside; Humans; Male; Microscopy, Electron, Scanning; Microscopy, Immunoelectron; Opportunistic Infections; Pseudomonas aeruginosa; Pseudomonas Infections; Rabbits; Regeneration; Respiratory Physiological Phenomena; Respiratory System; Respiratory Tract Infections

Extensive immunohistochemical and thin-layer chromatogram-immunostain analyses were carried out to establish whether asialo GM1, a glycolipid which contains binding sites for Pseudomonas aeruginosa, is present in corneal epithelium. The data suggest that rabbit corneal epithelium does not contain detectable levels of asialo GM1 even after corneas are scarified and incubated with trypsin, P. aeruginosa, or P. aeruginosa exoproducts to expose potential cryptic sites. Preliminary immunohistochemical analyses indicated that asialo GM1 is also not found in human corneas.

Topics: Animals; Cornea; Corneal Injuries; Eye Infections, Bacterial; G(M1) Ganglioside; Immunohistochemistry; Organ Culture Techniques; Pseudomonas Infections; Rabbits