Abstract
Purpose :
Lacritin non-lytic peptides comprising the protease-released 'N-104' domain are a primary source of natural basal tear bactericidal activity that target Gram-negative inner-membrane proteins FeoB and SpuG (PotH) - in part via the outer-membrane lipoprotein YaiW. While N-104 is highly potent (MIC < 2 μM) against ocular surface pathogen P. aeruginosa (PA14), its efficacy is significantly reduced at physiological conditions - as per many cationic antimicrobial peptides. Here, we searched for peptides or proteins in human normal basal tears that might ligate N-104 thereby synergistically enhancing its bactericidal efficacy.
Methods :
Normal human basal tears from 0.5% proparacaine-anesthetized eyes were collected onto Schirmer strips for 5 min. Schirmer strips with ≥ 12 mm of wicked tears were eluted in PBS at 4°C for a pooled tear equivalent of 100 mm, and diluted 5-fold in PBS containing protease inhibitor cocktails (cOmplete Mini, 11836153001). Tears were precleared for 5 hours while rocking at 4°C with 100 μl of Pierce™ Control Agarose beads, and then were added in equal amounts to SulfoLink® immobilized Cys-N-104 or to negative control Cys-C-95 beads (each 100 μl) for overnight incubation at 4°C. C-95 is the inactive N-terminus of lacritin. After collection of flow-through, beads were washed with 100 bead volumes of PBS, eluted via a step gradient of 0.3, 0.5 and 1.0 M KCl for SDS-PAGE/silver staining, and then subjected to MS/MS focusing on the 0.5 M KCl eluate. Mass spectra were analyzed using the Sequest against UniProt Human.
Results :
Captured by N-104 and absent from C-95 beads were fifty-eight different proteins above a threshold. Threshold of ≥ 0.24% was defined based on a histogram of the percentage over total values of each hit in the N-104 column. Nine are reported to be antimicrobial including: PIGR, F2, GSN, DMBT1, CST4, IGKC, PRR4, SCGB2A1, and LYZ. Also detected was IGLC2 that although also on C95 beads was 2.6x enriched on N-104.
Conclusions :
N-104 appears to target numerous antimicrobial proteins/peptides in normal basal tears under physiological conditions. Whether the relationship might be functionally synergistic has yet to be determined. Given that N-104 is non-lytic, potential synergism may occur via enhancement of N-104’s electrostatic interaction with the bacterial surface and/or with YaiW.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.