Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: cornea: tears/tear film/dry eye • cornea: epithelium • cornea: surface mucins