Tear microbicidal activity protects the surface of the eye from environmental pathogens and may regulate levels of commensal bacteria. Human basal and reflex tears are enriched in lacritin. Lacritin is 10-fold less in tears from individuals suffering from fungal keratitis and selectively downregulated in blepharitis an inflammation of the eyelid associated with overgrowth of commensal bacteria. A cleavage potentiated C- terminal fragment of lacritin is bactericidal for gram negative and positive bacteria including S. epidermidis, S. aureus, P.aeruginosa and E. coli. Removal of lacritin C-terminal fragments from human tears by repeated passage over anti-lacritin C- (but not N-) terminal antibody columns depleted all antimicrobial activity. One possible implication is that lacritin via C-terminal fragment(s) might be the primary source of antimicrobial activity in tears, and that eyes with insufficient tear lacritin may have significantly increased risk of microbial pathogenicity. Furthe, topical lacritin C-terminal fragment may be therapeutic. The fragment is releasable by a serine protease and is active in buffers as high as 380 mOsm/l. Only 2 - 4 M fragment is required, in keeping with 18 - 27 M lacritin in normal basal tears that effectively serves as a lacritin reservoir. Lacritin deletion and synthetic peptide analysis has narrowed the activity to a 15 amino acid region, but with the largest active fragment 54 amino acids long. Although this latent activity promotes bacterial pore formation (but not hemolysis), a second death mechanism is involved. Lacritin bactericidal activity widely compromises bacterial metabolic capacity. It also promotes an accumulation of several amino acids and pyrophosphate. Pyrophosphate is beneficial to bacterial growth. Thus, disrupts bacterial membranes. It also promotes regulated cell death. Our working hypothesis is that lacritin's latent bactericidal activity may be the main regulator of ocular surface sterility, and that lack of lacritin's C-terminal fragment may be a ris factor for pathogenic infection. Our immediate goal is to explore mechanisms regulating this activity. Our long-term goal is to harness this activity as an ocular therapeutic.
Our first aim ass how cleaved C-terminus targets bacteria and their comparative efficacy against a broad spectrum of clinical isolates and in vivo.
Our second aim explores the mechanisms of bacterial killing.
Our third aim asks how cleavage is regulated.
Microbial infection of the eye can lead to ulceration, a precursor to blindness. This project works out the mechanism(s) by which cleavage of a natural human tear protein releases a potent bactericidal peptide that helps protects the eye from microbial infection.
| Willcox, Mark D P; Argüeso, Pablo; Georgiev, Georgi A et al. (2017) TFOS DEWS II Tear Film Report. Ocul Surf 15:366-403 |
