# Tear Protein Microbial Regulation

> **NIH NIH R01** · UNIVERSITY OF VIRGINIA · 2021 · $495,316

## Abstract

Tear microbicidal activity protects the surface of the eye from environmental pathogens and may regulate levels
of commensal bacteria. Lacritin, a prosecretory mitogen enriched in human basal and reflex tears, is subject to
cleavage-potentiated release of C-terminal proteoforms, including those bactericidal for E. coli, P. aeruginosa
(Migula and PA14), L. monoctyogenes, S. aureus and S. epidermidis. Removal of C-terminal proteoforms from
human tears by repeated passage over anti-lacritin C- (but not N-) terminal antibody columns depletes all tear
antimicrobial activity, an activity encapsulated in the synthetic peptide AQKLLKKFSLLKPWA 'N-104', also a
proteoform. As a cationic, largely a-helical amphipathic peptide, death by membrane disruption would be
expected - a hypothesis largely ruled out by surface plasmon resonance with model bacterial membranes, and
metabolomic analysis with parent 'N-65' fragment that suggests a regulated cell death mechanism. Very
revealing were screens for N-104 resistant mutants out of the full E. coli Keio collection of 3,985 nonessential
gene knockouts. Knockout of feoB, potH, ybaE, yhfZ or ybdM was sufficient to confer resistance, but not to
equimolar amounts of ampicillin. The same is true for feoB and potH transposon insertion mutants of
opportunistic pathogen P. aeruginosa PA14. YbaE and YbdM are uncharacterized in E. coli and P. aeruginosa
where functions may differ. YhfZ is absent from P. aeruginosa. FeoB is a well-known virulence factor of
respiratory P. aeruginosa, F. tularensis and L. pneumophila, gut C. jejuni and H. pylori, and uropathogenic E.
coli, but has not previously been associated with ocular infections. FeoB and PotH contribute to or form
respective ferrous iron and putrescine and uptake channels, YbaE (with proposed name 'bacterial extracellular
solute-binding protein') appears to be an ABC transporter subunit with a genetic interaction to outer membrane
protein A, and YbdM is a ParB domain containing nuclease. Ferrous iron and putrescine are each essential for
bacterial cell growth, and yet are also required by host cells, especially the avascular cornea. Media
supplementation with a 10-fold molar excess of putrescine, but not fellow polyamine spermidine, completely
abrogates N-104 dependent bacterial death (tear putrescine is 1/10th that of N-104 proteoform).
Complementation of potH- E. coli by potH+ cDNA does the opposite. Prior metabolomic studies (that did not
detect ferrous iron) revealed that N-104 parent 'N-65' rapidly suppresses intracellular E. coli putrescine. Our
immediate focus is on FeoB and PotH. Our working hypothesis is that FeoB and/or PotH are virulence
mechanisms for ocular surface pathogens with N-104 a main source of tear bactericidal activity. Our immediate
goal is to elucidate how N-104, through FeoB or PotH triggers killing.
University of Virginia Charlottesville Virginia
Harvard University Boston Massachusetts

## Key facts

- **NIH application ID:** 10211706
- **Project number:** 2R01EY026171-04
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** Gordon William Laurie
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $495,316
- **Award type:** 2
- **Project period:** 2016-09-30 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10211706

## Citation

> US National Institutes of Health, RePORTER application 10211706, Tear Protein Microbial Regulation (2R01EY026171-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10211706. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*