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A. Kumar, J. Zhang, F.–S. Yu; Signal transduction pathways activated in human corneal epithelial cells in response to Staphylococcus aureus challenge . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4985.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the signal transduction induced by Staphylococcus aureus (live and heat killed), exoproteins and cell wall components ( Peptidoglycan and LTA ) in cultured human corneal epithelial cells. Methods: S.aureus strain 8325 was grown in TSB in mid exponential growth phase and used to challenge epithelial cells in ratio of ∼ 30 bacteria/ cell. The heat–killed was made by boiling bacterial suspension for 10 min. Bacterial exoproteins were prepared by culturing the bacteria in chemically defined medium till it reached stationary phase, the resulting supernatant was filter sterlized and used at ratio of 1:10 to treat HUCL, a telomerase–immortalized human corneal epithelial cell line. Bacterial cell wall components PGN and LTA were obtained commercially and used at 10 and 25µg/ml respectively. Phosphorylation and degradation of IkB–α and activation of MAP–kinase signaling pathways (p38,JNK–1/2,ERK–1/2) was evaluated by western blotting using phospho–specific antibodies. Results: Exposure of HUCL cells to live, but not heated killed, bacteria resulted in IkB–α phosphorylation and degradation and activation of ERK and JNK in time dependent manner. Phosphorylated IkB–α was detected at 2h and reached a peak at 6h after exposure, while ERK and JNK were activated at 1h and 6h respectively. Bacterial exoproteins stimulated a rapid response as phosphorylated IkB–α start appearing after 10 min of stimulation and reached maximum at 60 min, and the activation of p38 within 10 min. Cells challenged with peptidoglycan also showed rapid IkB–α phosphorylation after 40 min of stimulation and activation of ERK in 10 min. No response was detected in cells exposed to lipoteichoic acid. Conclusions: Bacterial exoproteins, live bacteria and peptidoglycan, but not the LTA, might be an important components of S.aureus in triggering epithelial inflammatory response during keratitis.
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