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Mohammad Sharifian Gh., Fatemeh Norouzi, Mihaela G Gadjeva, Gordon W Laurie; Putrescine Transporter Subunit 'PotH' is Essential for Lacritin Peptide 'N-104' Bactericidal Activity. Invest. Ophthalmol. Vis. Sci. 2020;61(7):404.
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Antimicrobial proteins and peptides on the eye surface are essential for innate defense and tear sterility. 'Lacritin', a prosecretory and pro-homeostatic factor, is subject to C-terminal processing giving rise to ~12 different predicted or demonstrated bactericidal fragments including the 15 amino acid, largely amphipathic alpha-helical 'N-104'. Since N-104's primary mechanism of action does not seem to involve substantial membrane disruption, we screened the 3,985 single-gene knockout Keio E. coli K-12 collection for resistant mutants.
A cell viability screen was designed using 106 cfu/ml suspensions of wild-type or individual Keio E. coli K-12 mutant strains that were incubated for 5 hours at 34°C (surface temperature of the eye) in 96-well plates prior to addition of, and subsequent 25-hour incubation with, 100 µM N-104 or inactive lacritin peptide N-80/C-25 (negative control) or ampicillin (positive control). Data were expressed as alamarBlue-monitored viability at 30 hrs normalized to viability prior to the addition of peptide or ampicillin, thereby controlling for differential proliferation capacity of mutants. Resistant mutants from two independent screens were rescreened twice for subsequent confirmation using CFU assays. For validation, the mutant potH strain was transfected with potH plasmid for testing with N-104. The effect of exogenous putrescine on the bactericidal efficacy of N-104 toward the wild-type strain was studied.
Five different gene mutants were resistant at the screening threshold of 0.75 (treated to untreated) including potH, feoB, yhfZ, ybdM, and ybaE. The potH knockout was of particular interest with N-acetylputrescine substantially depleted in E. coli treated for 15 min with N-104-containing lacritin fragment 'N-65' (McKown et al, JBC '14). potH plasmid transfection restored N-104 sensitivity. Moreover, exogenous putrescine reduced the bactericidal efficacy of N-104 in a concentration-dependent manner - in keeping with PotH’s role as a putrescine ABC transporter.
PotH is essential for N-104 bactericidal activity, but exactly how it does so remains to be determined. P. aeruginosa PotH is 65% identical. Whether PotH or one of the other four gene products mediate N-104's capacity to block P. aeruginosa biofilm formation, and if so how, are also questions of interest.
This is a 2020 ARVO Annual Meeting abstract.
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