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

泪液杀菌活性可保护眼睛表面免受环境病原体的侵害，并可调节水平 共生细菌。泪素是一种富含人类基础泪液和反射泪液的促分泌性有丝分裂原， C 末端蛋白质形式的裂解增强释放，包括对大肠杆菌、铜绿假单胞菌具有杀菌作用的蛋白质形式 （Migula 和 PA14）、单核乳杆菌、金黄色葡萄球菌和表皮葡萄球菌。去除 C 末端蛋白形式 通过反复通过抗乳泌素 C-（而非 N-）末端抗体柱，人类泪液会耗尽所有泪液 抗菌活性，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、大部分为α螺旋的两亲肽，膜破坏导致的死亡将是 预期 - 这一假设很大程度上被模型细菌膜的表面等离振子共振所排除，并且 使用母体“N-65”片段进行的代谢组学分析表明存在受调节的细胞死亡机制。非常 从包含 3,985 个非必需品的完整大肠杆菌 Keio 集合中筛选出 N-104 抗性突变体，结果揭示了这一点 基因敲除。 feoB、potH、ybaE、yhfZ 或 ybdM 的敲除足以赋予抗性，但不足以赋予抗性 等摩尔量的氨苄西林。 feoB 和 potH 转座子插入突变体也是如此 机会致病菌铜绿假单胞菌 PA14。 YbaE 和 YbdM 在大肠杆菌和铜绿假单胞菌中没有特征 其中功能可能有所不同。铜绿假单胞菌中不存在 YhfZ。 FeoB 是众所周知的毒力因子 呼吸道铜绿假单胞菌、土拉杆菌和嗜肺军团菌、肠道空肠弯曲菌和幽门螺杆菌，以及泌尿道致病性大肠杆菌。 大肠杆菌，但之前并未发现与眼部感染有关。 FeoB 和 PotH 有助于或形成 各自的二价铁和腐胺以及摄取通道，YbaE（建议名称“细菌细胞外 溶质结合蛋白'）似乎是与外膜具有遗传相互作用的 ABC 转运蛋白亚基 蛋白 A，YbdM 是包含核酸酶的 ParB 结构域。二价铁和腐胺对于 细菌细胞生长，但也是宿主细胞，特别是无血管角膜所必需的。媒体 完全补充 10 倍摩尔过量的腐胺，但不完全补充聚胺亚精胺 消除 N-104 依赖性细菌死亡（泪液腐胺是 N-104 蛋白质形式的 1/10）。 potH+ cDNA 对 potH- 大肠杆菌的补充则起到相反的作用。先前的代谢组学研究（没有 检测二价铁）表明 N-104 母体“N-65”可快速抑制细胞内大肠杆菌腐胺。我们的 目前的焦点是 FeoB 和 PotH。我们的工作假设是 FeoB 和/或 PotH 具有毒力 N-104 是泪液杀菌活性的主要来源，对眼表病原体的作用机制。我们的即时 目标是阐明 N-104 如何通过 FeoB 或 PotH 触发杀伤。 弗吉尼亚大学弗吉尼亚州夏洛茨维尔分校 哈佛大学 马萨诸塞州波士顿分校