Tear Protein Microbial Regulation

泪液蛋白微生物调节

• 批准号: 10211706
• 负责人: Gordon William Laurie
• 金额: $ 49.53万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211706
• 关键词: ATP-Binding Cassette Transporters; Affinity; Affinity Chromatography; Ampicillin; Antibodies; Bacterial Model; Binding Proteins; Biochemical; Blindness; Boston; C-terminal; Caenorhabditis elegans; Campylobacter jejuni; Cations; Cell Death; Cells; Cessation of life; Chelating Agents; Clinical; Collection; Complement; Complementary DNA; Cornea; Development; Encapsulated; Escherichia coli; Excision; Eye; Eye Infections; Francisella tularensis; GAG Gene; Genetic; Genetic Screening; Goals; Helicobacter pylori; Human; Individual; Infection; Iron; Knock-out; Lead; Legionella pneumophila; Mass Spectrum Analysis; Massachusetts; Mediating; Mediator of activation protein; Membrane; Membrane Proteins; Mitogens; Mutate; Names; Outcome; Parents; Peptides; Polyamines; Pseudomonas aeruginosa; Putrescine; Reflex action; Regulation; Resistance; Role; Source; Spermidine; Staphylococcus aureus; Staphylococcus epidermidis; Supplementation; Surface; Surface Plasmon Resonance; System; Testing; Ulcer; Universities; Uropathogenic E. coli; Virginia; Virulence; Virulence Factors; Work; antimicrobial; bactericide; cell growth; commensal bacteria; extracellular; inhibitor/antagonist; knockout gene; metabolomics; microbial; microbicide; mouse model; mutant; novel therapeutics; nuclease; ocular surface; opportunistic pathogen; pathogen; periplasm; resistant strain; respiratory; solute; syndecan; synthetic peptide; tear proteins; uptake

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

泪液杀微生物活性保护眼睛表面免受环境病原体的侵害，并可能调节其水平