Tear Protein Microbial Regulation

泪液蛋白微生物调节

基本信息

批准号：
10615707
负责人：
Gordon William Laurie
金额：
$ 47.22万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
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/10615707
关键词：
ATP-Binding Cassette Transporters Affinity Affinity Chromatography Ampicillin Antibodies Bacterial Model Binding Proteins Biochemical Biotinylation Blindness Boston C-terminal Caenorhabditis elegans Campylobacter jejuni Cell Death Cells Cessation of life Chelating Agents Clinical Collection Complement Complementary DNA Cornea Development Encapsulated Escherichia coli Excision Eye Eye Infections Francisella tularensis Genetic Genetic Screening Goals Helicobacter pylori Human Individual Infection Iron Knock-out Legionella pneumophila Mass Spectrum Analysis Massachusetts Mediating Mediator 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 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

项目摘要

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

撕裂杀生活性可保护眼表面免受环境病原体的侵害，并可能调节水平共生细菌。富集在人类基底和反射泪中的起诉促促促促促促丝线元素 C末端蛋白基型的切割式释放释放，包括大肠杆菌的杀菌性，铜绿假单胞菌（Migula和Pa14），L。单胞菌，金黄色葡萄球菌和表皮链球菌。从中去除C末端蛋白基通过反复通过抗透眼蛋白C-（但不是n-）末端抗体柱耗尽的人眼泪抗菌活性，一种封装在合成肽Aqkllkkfsllkpwa'N-104'中的活性，也是蛋白质成型。作为一种阳离子，很大程度上是一个螺旋的两亲性肽，膜破坏的死亡将是预期 - 一个假设在很大程度上被表面等离子体与模型细菌膜共鸣的假设，并且代谢组学分析的“ N-65”片段表明受调节的细胞死亡机制。非常揭示是从3,985个非必需的全大肠杆菌收藏中的N-104抗性突变体的筛选基因淘汰。 FEOB，POTH，YBAE，YHFZ或YBDM的淘汰赛足以赋予抵抗力，但不适合等摩尔量的氨苄青霉素。对于FeOB和Poth Transposon插入突变体也是如此机会性病原体P.铜绿PA14。 YBAE和YBDM在大肠杆菌和铜绿假单胞菌中未表征功能可能有所不同。铜绿假单胞菌没有YHFZ。 FEOB是众所周知的毒力因子呼吸道P.铜绿假单胞菌，F。tularensis和L.肺炎，肠道C. jejuni和H. Pylori以及尿道病E. 大肠杆菌，但以前尚未与眼部感染有关。 Feob和Poth贡献或形成 Ybae（带有细菌细胞外的名称' 溶质结合蛋白'）似乎是ABC转运蛋白亚基，具有与外膜的遗传相互作用蛋白A和YBDM是含有核酸酶的PARB结构域。亚铁和腐烂都是必不可少的细菌细胞生长，但也需要宿主细胞，尤其是血管角膜。媒体补充10倍过量的腐烂，但不添加多胺精子，完全是废除N-104依赖性细菌死亡（撕裂腐烂是N-104蛋白质成型的1/10）。 Poth+ cDNA对Poth-E. cosi的互补并恰恰相反。先前的代谢组学研究（没有检测铁铁）表明N-104母体“ N-65”迅速抑制了细胞内大肠杆菌腐烂。我们的直接的重点是Feob和Poth。我们的工作假设是FEOB和/或POTH是毒力 N-104的眼表病原体机制是泪液活性的主要来源。我们的直接目标是通过FEOB或POTH触发杀戮阐明N-104。弗吉尼亚大学夏洛茨维尔弗吉尼亚大学哈佛大学马萨诸塞州波士顿