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Kimberly Brothers, Nicholas A Stella, Kristin M Hunt, Regis P Kowalski, Jes Klarlund, Robert M Q Shanks, Charles T. Campbell Laboratory, UPMC Eye Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA.; Bacterial Impediment of Corneal Cell Migration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4058.
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© ARVO (1962-2015); The Authors (2016-present)
The loss of corneal epithelium in patients with microbial keratitis is well known but the underlying mechanisms are poorly understood. This study set out to determine the impact of bacterial secreted factors on ocular wound healing.
Bacterial secretomes from ocular keratitis isolates were prepared from overnight cultures and filtered to remove bacteria. Using a plate based cell migration assay, secretomes were added to stratified human corneal limbal epithelial (HCLE) cells and incubated. Calcein AM viability stained cell layers were imaged by confocal microscopy. Porcine corneal organ culture was used to assess epithelial wound healing phenotypes ex vivo. Transposon mutagenesis of the Serratia marcescens genome was conducted to identify bacterial genes responsible for corneal cell migration inhibition. LPS was purified by the hot phenol method. The waaG lipopolysaccharide (LPS) gene was cloned using yeast homologous recombination into vector pMQ131.
Corneal cells treated with bacterial secretomes from 4/5 Pseudomonas aeruginosa, 26/27 S. marcescens, and 2/14 Staphylococcus aureus isolates showed dose dependent inhibition of HCLE migration. No inhibition was seen with secretomes from 4 other ocular pathogens. Using an ex vivo porcine corneal wound model, we have recapitulated S. marcescens inhibition of wound healing. A transposon mutation in the S. marcescens LPS biosynthetic locus that prevented bacterial inhibition of epithelial cell migration was identified and complemented with the wild-type gene on a plasmid. Supporting the importance of LPS in the cell migration phenotype, depletion of LPS with polymyxin B agarose inactivated the inhibitory ability of the bacterial secretomes. Purified S. marcescens LPS, but not E. coli LPS was able to inhibit corneal cell migration.
Together these data support that multiple ocular pathogens secrete factors able to inhibit corneal epithelial wound healing. Genetic and biochemical data using S. marcescens as a model organism indicate that S. marcescens LPS is sufficient to prevent corneal epithelial cell migration and wound healing. This study presents a novel host-pathogen interaction with implications for corneal ulcers and other medical problems where bacteria impact wound healing, such as chronic wounds and provides evidence that LPS may be a key factor in the inhibitory mechanism.
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