Abstract
Purpose: :
Lacritin is a 119 amino acid prosecretory mitogen found in tears that stimulates basal tearing in rabbits and promotes human corneal epithelial cell proliferation with a biphasic 1-10 nM dose optimum. Lacritin shows slight sequence alignment with the C-terminus of dermcidin, an important sweat antibiotic. Deletion mutants of lacritin containing C-terminal sequences are antimicrobial against E. coli in the uM range. The human antimicrobial proteins dermcidin and cathelicidin (LL-37) are cleavage-activated by host proteases. Here we ask whether lacritin also displays cleavage dependent antibiotic activity and by what mechanism.
Methods: :
Lacritin deletion mutants were generated, expressed in E. coli, and then purified by chitin and DEAE chromatography. A colony forming unit assay was used to assess antimicrobial activity. Bacterial membrane permeabilization was analyzed with the impermeable dye Sytox Green and mammalian membrane permeabilization with the hemolysis assay. Lacritin with supernatants from bacterial cultures were monitored by Western bloting with lacritin C-terminal or N-terminal specific antibodies.
Results: :
The deletion mutant N-65, but not intact lacritin is bactericidal against Staphylococcus epidermidis, and Pseudomonas aeruginosa at physiological conditions. The mechanism of bactericidal activity involves rapid membrane permeabilization. No lysis of red blood cells is detected under equivalent conditions, suggesting the involvement of a bacterial surface protein. Incubation of lacritin with bacterial supernatants generated a fragment that is recognized by the C-terminal specific antibody but not the N-terminal specific antibody. A synergistic bactericidal effect is observed when N-65 is combined with human lysozyme.
Conclusions: :
We propose that bacteria-dependent cleavage of lacritin releases a potent gram negative and positive bactericidal activity that functions under physiological conditions in a synergistic mechanism with other antimicrobial factors found in human tear film.
Keywords: cornea: tears/tear film/dry eye • lacrimal gland • antibiotics/antifungals/antiparasitics