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2011 OMIG Abstract 27

Corneal cell migration blocked by Serratia marcescens factor in an in vitro wound healing model
N.A. Stella, J.K. Klarlund, R.M.Q. Shanks
The Charles T. Campbell Laboratory, UPMC Eye Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA

Purpose: To test the hypothesis that S. marcescens secreted factors inhibit corneal wound healing.

Methods: Sheets of human corneal limbal epithelial (HCLE) cells were grown to confluence in 12-well plates with agarose strips.  Bacteria-free secreted fractions (BSF) were isolated from laboratory and clinical keratitis isolates of S. marcescens, Esherichia coli and Pseudomonas aeruginosa, grown overnight in LB medium, normalized by optical density, and filter sterilized.  BSFs were used directly or heat treated (10 minutes at 95˚C) and added directly to culture medium.  The effect of epidermal growth factor (EGF) and serum concentration was also assessed.  LB medium alone was used as a negative control.  After 20-24 incubation, cell layers were fixed and stained with gentian violet and the remaining wound was assessed.

Results: Incubation of cell layers with BSF from S. marcescens, but not E. coli or P. aeruginosa, led to a dose-dependent inhibition of cell migration in this in vitro wound healing model.  Mutant bacterial strains were analyzed and a previously uncharacterized bacterial transcription factor was found to be necessary for the inhibition phenotype.  Furthermore, the inhibition effect was unaltered by exogenous EGF, and was only partially prevented by heat treatment of the BSF.

Conclusions: We have partially characterized a novel host-pathogen interaction that may have profound importance in corneal infections, given the common isolation of Serratia species from contact lens cases.

Support:  Research to Prevent Blindness, NIH EY08098

Disclosures: N

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