title: Role of Pseudomonas aeruginosa ExsA in penetration through corneal epithelium in a novel in vivo model.
abstract: OBJECTIVE
  The scarified cornea keratitis model was modified to study Pseudomonas aeruginosa infection of healing corneal epithelium. The new model was then used to study the role of ExsA, a transcriptional activator of P. aeruginosa, in bacterial penetration through injured and healing corneal epithelia.
  
  
  METHODS
  Scratch-injured corneas of C57BL/6 mice were allowed to heal for 0, 6, 9, or 12 hours before inoculation with a cytotoxic (6206) or invasive (PAO1) P. aeruginosa strain. Disease progression was monitored for 14 days. The integrity of the healing epithelium was studied in uninfected eyes by fluorescein staining and by histologic examination. In other experiments, the effect of bacterial exsA mutation was studied after 0, 6, or 12 hours of healing. Three hours after infection, these eyes were used to quantify early bacterial colonization levels by viable counts, or they were sectioned to study bacterial penetration through the epithelium by microscopy.
  
  
  RESULTS
  Corneas remained susceptible to infection 6 but not 12 hours after scratch injury. By 6 hours, the previously exposed stroma was already completely covered by several layers of epithelial cells. Fluorescein staining unexpectedly occurred even after 12 hours of healing time, showing that resistance to infection preceded full restoration of epithelial barrier function. Mutation of exsA reduced both bacterial colonization levels and penetration through the epithelium 3 hours after bacterial inoculation, but only in the 6-hour healing situation, and only for the cytotoxic strain (PA103). Mutation of exsA in the invasive strain (PAO1) had no effect on 3-hour colonization or penetration levels under any circumstances.
  
  
  CONCLUSIONS
  The 6-hour healing infection model showed a role for ExsA in early interactions with the corneal epithelium that was not detectable with the conventional (0-hour) scratch model. Comparison of the 6- and 12-hour healing models, which showed that factors additional to barrier function contribute to defense against infection, could be used to gain new insights into corneal defense mechanisms, and the methods used by bacteria to circumvent them.
authors.affiliation: Morton D. Sarver Laboratory for Cornea and Contact Lens Research, School of Optometry, University of California at Berkeley, Berkeley, CA 94720-2020, USA.
authors.forename: Ellen J
authors.initials: EJ
authors.lastname: Lee
authors.forename: David J
authors.initials: DJ
authors.lastname: Evans
authors.forename: Suzanne M J
authors.initials: SM
authors.lastname: Fleiszig
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000502:physiology; D015169:Colony Count, Microbial; D003320:Corneal Ulcer / Q000382:microbiology* / Q000473:pathology; D004268:DNA-Binding Proteins / Q000502:physiology*; D004195:Disease Models, Animal; D019573:Epithelium, Corneal / Q000293:injuries / Q000382:microbiology* / Q000473:pathology; D015818:Eye Infections, Bacterial / Q000382:microbiology* / Q000473:pathology; D005131:Eye Injuries / Q000382:microbiology; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D011552:Pseudomonas Infections / Q000382:microbiology* / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000302:isolation & purification / Q000502:physiology*; D013997:Time Factors; D015534:Trans-Activators / Q000502:physiology*; D014945:Wound Healing / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C095092:ExsA protein, bacteria; D015534:Trans-Activators