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Connie Tam, James J. Mun, David J. Evans, Suzanne M. Fleiszig; Novel Small (<20 amino acid) Antimicrobial Peptides in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5822.
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We previously found that exposure to human tear fluid increased corneal epithelial cell resistance to P. aeruginosa invasion, cytotoxicity and translocation in vitro. Correlating with this, size-fractionated cell lysates containing molecules of 3-10 kDa or <3 kDa were more bactericidal and more protective against P. aeruginosa virulence if cells were exposed to tear fluid before lysis. Here we examined lysates of tear fluid-treated corneal epithelial cells to mine for novel, small-molecule host defense peptides.
Telomerase-immortalized human corneal epithelial cells (TiHCE) were exposed to human tear fluid or cell culture media for 16 h then lysed and fractionated. Fractions of 3-10 kDa and <3 kDa were analyzed by mass spectrometry, and detected peptide fragments analyzed with web-based proteomic tools (ExPASy). Peptides of interest were synthesized and evaluated for antimicrobial activity against P. aeruginosa clinical and laboratory isolates. Protection against bacterial invasion (quantified by gentamicin survival) and cytotoxicity (trypan blue staining) were evaluated using fresh TiHCE cells inoculated with P. aeruginosa clinical isolate 6294 (invasive, ~106 cfu for 2 h) or 6206 (cytotoxic, ~104 cfu for 3 h).
Detected peptides belonged to 12 and 18 proteins respectively in the <3 kDa and 3-10 kDa lysate fractions of tear-treated cells, or 1 and 16 proteins in corresponding fractions of media-treated cells. A 19-amino-acid peptide of a family of fragments was detected in all fractions and had predicted antimicrobial activity based on bioinformatic analyses. It was confirmed to be bactericidal (3 h, 200 ug/ml) for clinical isolates 6206 (99.9% killed), 6294 (97.1%); lab isolates PAO1, (96.1%), PA14 (98.7%), and PAC1R and its LPS mutants (93.9% and 93.7-98.9%); and cystic fibrosis isolate 2192 (96.9%) (all P<0.001). The peptide also reduced bacterial invasion (by 89.1%) and cytotoxicity (by 85.1%) (both P<0.001). Video microscopy showed loss of bacterial motility. TAMRA-tagged peptide and SYTOX blue staining indicated that the peptide binds bacteria and has pore forming activity.
A corneal epithelial cell expressed 19 amino-acid peptide fragment belonging to a constitutively expressed protein with no known anti-infective function was found to have antimicrobial and protective activity. These findings contribute to our understanding of corneal epithelial defense against microbes, and could have therapeutic value in the treatment and prevention of infectious diseases.
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