Lipopolysaccharide outer core is a ligand for corneal cell binding and ingestion of Pseudomonas aeruginosa

Purpose. Pseudomonas aeruginosa has been observed to be adherent to and inside epithelial cells during experimental corneal infection. The authors identified bacterial ligands involved in adherence and entry of P. aeruginosa into corneal epithelial cells. Methods. In vitro gentamicin survival assays were used to determine the intracellular survival of a panel of P. aeruginosa mutants. Strains (10(6) to 10(7) colony-forming units) were added to primary cultures of rabbit corneal epithelial cells (similar to 10(5)/well) for 3 hours, nonadherent bacteria were washed away, and extracellular bacteria were killed with gentamicin. The antibiotic was then washed away, and epithelial cells were lysed with 0.5% Triton X-100 to release internalized bacteria. Bacterial association (sum of bound and internalized bacteria) was measured by the omission of gentamicin. Similar assays were carried out with whole mouse eyes in situ. Results. A lipopolysaccharide core with an exposed terminal glucose residue was found to be necessary for maximal association and entry of P. aeruginosa into corneal cells. Bacterial pill and flagella were not involved. Mutants of P. aeruginosa strains that do not produce an LPS core with a terminal glucose residue had a significantly lower level of association with (similar to 50%) and ingestion by (>90%, P < 0.01) corneal cells than did strains with this characteristic. Complementation of the LPS production defect by plasmid-borne DNA returned association and ingestion to near parental levels. Lipopolysaccharides and delipidated oligosaccharides with a terminal glucose residue in the core inhibited bacterial association and entry into corneal cells. Experiments using P. aeruginosa LPS mutants and corneal cells on whole mouse eyes confirmed the role of the LPS core in cellular entry. Conclusions. Corneal epithelial cells bind and internalize P. aeruginosa by the exposed LPS core.