Molecular basis for defective glycosylation and Pseudomonas pathogenesis in cystic fibrosis lung

Abstract

The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P. aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments.

Laboratory and clinical Pseudomonas aeruginosa strains do not bind glycosphingolipids in vitro or during type IV pili-mediated initial host cell attachment

“…An accumulation of Gg 4 in CF cells (Bryan et al, 1998;Saiman & Prince, 1993), resulting from hyposialylation (Poschet et al, 2001) due to an altered Golgi pH in CFTR mutant cells, has been questioned (Jiang et al, 1997), but was verified in our work. IB3-1 cells contain at least twice the levels of Gg 4 of S9 cells; intracellular Gg 4 , in structures consistent with Golgi/ER, was observed for both cell types.…”

“…Anti-Gg 4 antibodies have been shown to react directly with the bacterium (Schroeder et al, 2001), providing an alternative explanation for earlier findings. Gg 4 has been reported to be increased in cells containing non-functional CFTR (Bryan et al, 1998;Saiman & Prince, 1993) due to reduced sialylation (Poschet et al, 2001), providing an attractive hypothesis for the increased colonization of respiratory epithelium of CF patients.…”

Interleukin-1β Differentially Regulates β2 Adrenoreceptor and Prostaglandin E2-mediated cAMP Accumulation and Chloride Efflux from Calu-3 Bronchial Epithelial Cells

“…Alterations in ion flux in cystic fibrosis lungs is thought to be central in the pathogenesis of lung disease, although the mechanism is debated. Several hypotheses have been suggested with the biochemical defect being linked to impaired antibacterial activity of ␤-defensins (7), mucociliary clearance (8), and altered sialisation of bacterial receptors (9).…”

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Mediated Residual Chloride Secretion Does Not Protect against Early Chronic Pseudomonas aeruginosa Infection in F508del Homozygous Cystic Fibrosis Patients

“…In this study, we addressed the issue of whether the manifestation of the CF basic defect, as assessed by the absence or presence of CFTR-mediated residual Cl Ϫ secretion, is predictive for the onset of PA colonization, as CFTR has been shown to play a key role for the acquisition of PA (14,(17)(18)(19). Among patients who are homozygous for F508del, a considerable percentage show some residual Cl Ϫ secretion (9), indicating that the CFTR mutation genotype does not exclusively predict the manifestation of the CF basic defect.…”

“…It has been reported that CFTR, apart from its well known role as a Cl Ϫ channel, is also a receptor for PA in the airways and, consequently, unimpaired CFTR function must be vital for the clearance of the bacteria from the lungs (16 -18). Recently, it has been suggested that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network in CF lung epithelial cells, being the molecular basis for defective glycosylation and increased PA adherence (19). In summary, the molecular mechanism that links CFTR Cl Ϫ channel dysfunction to the adhesion of PA to epithelial cells and to the persistence of respiratory colonization with mucoid PA in CF patients has not been satisfactorily resolved so far.…”