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 an d inside epithelial cells during experimental corneal infection. The a uthors identified bacterial ligands involved in adherence and entry of P. aeruginosa into corneal epithelial cells. Methods. In vitro gentam icin survival assays were used to determine the intracellular survival of a panel of P. aeruginosa mutants. Strains (10(6) to 10(7) colony-f orming units) were added to primary cultures of rabbit corneal epithel ial cells (similar to 10(5)/well) for 3 hours, nonadherent bacteria we re 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 as sociation (sum of bound and internalized bacteria) was measured by the omission of gentamicin. Similar assays were carried out with whole mo use eyes in situ. Results. A lipopolysaccharide core with an exposed t erminal glucose residue was found to be necessary for maximal associat ion and entry of P. aeruginosa into corneal cells. Bacterial pill and flagella were not involved. Mutants of P. aeruginosa strains that do n ot produce an LPS core with a terminal glucose residue had a significa ntly lower level of association with (similar to 50%) and ingestion by (>90%, P < 0.01) corneal cells than did strains with this characteris tic. Complementation of the LPS production defect by plasmid-borne DNA returned association and ingestion to near parental levels. Lipopolys accharides and delipidated oligosaccharides with a terminal glucose re sidue in the core inhibited bacterial association and entry into corne al cells. Experiments using P. aeruginosa LPS mutants and corneal cell s on whole mouse eyes confirmed the role of the LPS core in cellular e ntry. Conclusions. Corneal epithelial cells bind and internalize P. ae ruginosa by the exposed LPS core.