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R. L. McKown, E. V. Coleman, E. E. Crawley, C. J. Genero, R. W. Raab, C. A. White, G. W. Laurie; Antimicrobial Activity in Recombinant Variants of Prosecretory Mitogen Lacritin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5287. doi: https://doi.org/.
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Lacritin is a human prosecretory mitogen in tears that is derived for the most part from lacrimal gland. Topical lacritin stimulates prolonged basal tearing in rabbits. Lacritin displays a biphasic dose response that is optimal at 0.1 - 10 nM, with little or no mitogenic or prosecretory activity at higher concentrations. Thus >10 nM, other activities are possible. Lacritin shows slight sequence alignment with the C-terminus of dermcidin. Cleavage of dermcidin generates an important sweat antibiotic. Here we ask whether lacritin is also antimicrobial.
New lacritin deletion mutants were created. Each was tested at different concentrations and for different times in physiological salt against gram negative and positive bacteria using a colony forming unit assay. Inner membrane permeabilization of gram negative bacteria was detected by release of cytoplasmic beta-galactosidase. The sheep red blood cell hemolytic assay served as a control for mammalian cells.
Lacritin is antimicrobial; an activity localized to a domain between amino acids 75 and 114 of mature lacritin. This differs from the mitogenic domain that is longer by 10 N-terminal amino acids. The concentration of lacritin needed to kill 50% of the bacteria (LC50) ranged from 0.5 ug/ml (72.5 nM) to 11 ug/ml (894 nM) for the variant lacritin proteins tested. Both gram negative and gram positive pathogenic bacteria (Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa) were sensitive. Lacritin N-55 permeabilized the inner membrane of gram negative bacteria; however, permeabilization was not observed with lacritin C-25. No lysis was observed in the sheep red blood cell assay indicating lack of mammalian cell lysis at this elevated lacritin concentration.
Lacritin is antimicrobial for gram negative and gram positive pathogenic bacteria at concentrations higher than are required for epithelial secretion and growth, a property made possible by lacritin’s epithelial biphasic dose response. The antimicrobial domain differs in length from the proscretory/mitogenic domain, but shares the C-terminal amphipathic alpha helix. Thus lacritin appears to have multiple roles on the ocular surface, and offers a new approach towards treating and/or preventing microbial diseases and infections.
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