Overproduction of the CFTR R Domain Leads to Increased Levels of AsialoGM1 and Increased Pseudomonas aeruginosa Binding by Epithelial Cells

Abstract

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), particularly the common DeltaF508 mutation, have been associated with alterations in glycolipid sialylation and the availability of receptors for Pseudomonas aeruginosa binding. The surface properties of 9HTEo- tracheal epithelial cell lines transfected with plasmids that overproduce the regulatory (R) domain of CFTR (pCEP-R) and lack cyclic adenosine monophosphate-stimulated Cl- conductance were compared with control cell lines with normal CFTR function. There was increased bacterial adherence to the mutant cell lines with abnormal CFTR activity. Cell lines with overexpression of the R domain had surface properties similar to cells expressing the common DeltaF508 mutation in CF. P. aeruginosa adherence correlated with the increased numbers of asialoGM1 residues available on the surface of the epithelial cells with altered CFTR function; and antibody to asialoGM1, a P. aeruginosa pilin receptor, was able to compete with piliated bacteria for epithelial binding sites. The pCEP-R cell lines with increased bacterial binding were also associated with increased production of interleukin-8 in response to adherent P. aeruginosa compared with cells transfected with the empty vector pCEP. P. aeruginosa pil mutants that lack the adhesin specific for the asialoGM1 receptor did not discriminate between epithelial cells with normal or deficient CFTR function. These results confirm a direct relationship between aberrant CFTR function and increased levels of apical asialoGM1, and support the role of these asialylated glycolipids as P. aeruginosa receptors that initiate an epithelial proinflammatory response in response to bacterial ligands.

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.…”

“…It is surprising, in light of the many studies reporting Gg 3 and Gg 4 binding by Pseudomonas (Bryan et al, 1998; (or its T4P) and other bacterial pathogens (Deal & Krivan, 1990;Krivan et al, 1991;Strömberg et al, 1988), that the effect of depletion of cellular GSLs on bacterial binding has not been reported.…”

“…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.…”

Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patients compared with those of isolates from other origins

“…The nature of the protein encoded by this gene in P. aeruginosa PAO1 is unknown (Stover et al, 2000). Several arguments suggest that nan1 encodes a sialidase, an enzyme theoretically able to release sialic acid from sialylated gangliosides, thus increasing the amount of asialoGM1, a major receptor for adherence to the respiratory tract (Bryan et al, 1998;de Bentzmann et al, 1996;Imundo et al, 1995;Saiman et al, 1992;Saiman & Prince, 1993). The first argument is that the deduced bacterial protein encoded by nan1 possesses four Asp-boxes, a characteristic of bacterial sialidases (Taylor, 1996;Vimr, 1994).…”

Transgenic Cystic Fibrosis Mice Exhibit Reduced Early Clearance of Pseudomonas aeruginosa from the Respiratory Tract

“…These include decreased bacterial killing due to ineffective antimicrobial peptides (21,23,24), increased adherence of P. aeruginosa to respiratory epithelial cells of CF patients (25,26), and decreased bacterial clearance from the periciliary layer above the epithelial cells due to dehydration (27,28). Although all of these hypotheses include factors that could contribute to decreased removal of P. aeruginosa from the lungs of CF patients, none adequately explain the high-level association of P. aeruginosa infection with CF.…”