title: Lipopolysaccharide outer core is a ligand for corneal cell binding and ingestion of Pseudomonas aeruginosa.
abstract: OBJECTIVE
  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 (approximately 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 pili 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 (approximately 50%) and ingestion by ( > 90%, P < 0.01) corneal cells than did strains with this characteristic. Complementation of the LPS productions 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 internalized P. aeruginosa by the exposed LPS core.
authors.affiliation: Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: T S
authors.initials: TS
authors.lastname: Zaidi
authors.forename: S M
authors.initials: SM
authors.lastname: Fleiszig
authors.forename: M J
authors.initials: MJ
authors.lastname: Preston
authors.forename: J B
authors.initials: JB
authors.lastname: Goldberg
authors.forename: G B
authors.initials: GB
authors.lastname: Pier
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001422:Bacterial Adhesion; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002876:Chromosomes, Bacterial; D015169:Colony Count, Microbial; D003315:Cornea / Q000166:cytology / Q000378:metabolism* / Q000382:microbiology*; D003320:Corneal Ulcer / Q000382:microbiology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism / Q000382:microbiology; D005839:Gentamicins / Q000494:pharmacology; D008024:Ligands; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011817:Rabbits; D014774:Virulence
chemical_list: D000900:Anti-Bacterial Agents; D005839:Gentamicins; D008024:Ligands; D008070:Lipopolysaccharides; D009844:Oligosaccharides