Tear Protein Microbial Regulation

泪液蛋白微生物调节

• 批准号: 10398176
• 负责人: Gordon William Laurie
• 金额: $ 45.81万
• 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/10398176
• 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; 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

泪液的杀微生物活性保护眼睛表面免受环境病原体的侵害，并可能调节水平 的细菌。Lacritin是一种富含于人类基础泪液和反射泪液中的促分泌有丝分裂原， C-末端蛋白形式的裂解增强释放，包括对E.大肠杆菌、铜绿假单胞菌 （Migula和PA 14）、L. monctyogenes、单孢S.金黄色葡萄球菌和表皮C-末端蛋白形式的去除 人泪液通过反复通过抗催泪蛋白C-（而不是N-）末端抗体柱耗尽所有泪液 抗微生物活性，一种封装在合成肽AQKLLKKFSLLKPWA“N-104”中的活性，也是一种 蛋白质型作为一种阳离子，主要是α-螺旋两亲性肽，膜破裂导致的死亡将是可能的。 预期-这一假设在很大程度上被模型细菌膜的表面等离子体共振排除， 用亲本“N-65”片段进行代谢组学分析，表明受调节的细胞死亡机制。非常 筛选出N-104抗性突变株。大肠杆菌庆应义塾收集了3,985个非必需的 基因敲除敲除feoB、potH、ybaE、yhfZ或ybdM足以赋予抗性，但不足以 等摩尔量的氨苄青霉素。同样的情况也发生在feoB和potH转座子插入突变体中。 条件致病菌铜绿假单胞菌PA 14。YbaE和YbdM在E.大肠杆菌和铜绿假单胞菌 其中功能可能不同。YhfZ不存在于铜绿假单胞菌中。FeoB是一种众所周知的致病因子， 呼吸道铜绿假单胞菌、F. tularensis和L. pneumophila、gut C. jejuni和H. pylori和尿路致病性E. 大肠杆菌，但以前没有与眼部感染。FeoB和PotH有助于或形成 相应的亚铁和腐胺和摄取通道，YbaE（建议名称为“细菌细胞外 可溶性结合蛋白“）似乎是一种ABC转运蛋白亚基，与外膜有遗传相互作用 蛋白A，YbdM是含有ParB结构域的核酸酶。亚铁和腐胺都是必不可少的 细菌细胞生长，但也需要宿主细胞，特别是无血管角膜。媒体 补充10倍摩尔过量的腐胺，但不补充多胺亚精胺， 消除了N-104依赖性细菌死亡（泪液腐胺是N-104蛋白形式的1/10）。 potH-E的互补。而potH+ cDNA的表达则相反。既往代谢组学研究（未 检测亚铁）显示N-104亲本“N-65”快速抑制细胞内E.大肠杆菌腐胺。我们 当前的焦点是FeoB和PotH。我们的工作假设是FeoB和/或PotH是毒力 眼表病原体的机制，其中N-104是泪液杀菌活性的主要来源。我们眼前的 目的是阐明N-104如何通过FeoB或PotH触发杀伤。 University of Virginia弗吉尼亚大学 哈佛大学波士顿马萨诸塞州