Tear Protein Microbial Regulation

泪液蛋白微生物调节

• 批准号: 9010167
• 负责人: Gordon William Laurie
• 金额: $ 39.5万
• 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 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9010167
• 关键词: Amino Acids; Antibodies; Apoptosis; Asses; Bacteria; Blepharitis; Blindness; Buffers; C-terminal; Carrier Proteins; Cathepsin G; Cell Death; Cessation of life; Characteristics; Cleaved cell; Clinical; Coagulase; Coenzyme A; Complex; Cytolysis; Development; Diphosphates; Electrons; Endophthalmitis; Escherichia coli; Eukaryota; Excision; Eye; Eye Infections; Eyelid structure; Foundations; Goals; Growth; Hemolysis; Human; Individual; Infection; Inflammation; Keratitis; Lead; MMP9 gene; Membrane; Metabolic; Monitor; N-terminal; Nisin; Nuclear Magnetic Resonance; Pathogenicity; Peptide Antibiotics; Peptides; Phosphatidylethanolamine; Phospholipids; Pseudomonas aeruginosa; Putrescine; Reactive Oxygen Species; Reflex action; Regulation; Resistance; Risk; Risk Factors; Role; Serine Protease; Source; Spermidine; Staphylococcus aureus; Staphylococcus epidermidis; Sterility; Stress; Supplementation; Surface; Testing; Therapeutic; Toxic effect; Type III Secretion System Pathway; Ulcer; Validation; Viral; Virulence; Western Blotting; Work; antimicrobial; bacterial resistance; bactericide; cofactor; commensal microbes; comparative efficacy; corneal epithelium; in vivo; killings; metabolomics; microbial; microbicide; ocular surface; pathogen; protein function; public health relevance; research study; synthetic peptide; tear proteins

 DESCRIPTION (provided by applicant): Tear microbicidal activity protects the surface of the eye from environmental pathogens and may regulate levels of commensal bacteria. Human basal and reflex tears are enriched in lacritin. Lacritin is 10-fold less in tears from individuals suffering from fungal keratitis and selectively downregulated in blepharitis an inflammation of the eyelid associated with overgrowth of commensal bacteria. A cleavage potentiated C- terminal fragment of lacritin is bactericidal for gram negative and positive bacteria including S. epidermidis, S. aureus, P.aeruginosa and E. coli. Removal of lacritin C-terminal fragments from human tears by repeated passage over anti-lacritin C- (but not N-) terminal antibody columns depleted all antimicrobial activity. One possible implication is that lacritin via C-terminal fragment(s) might be the primary source of antimicrobial activity in tears, and that eyes with insufficient tear lacritin may have significantly increased risk of microbial pathogenicity. Furthe, topical lacritin C-terminal fragment may be therapeutic. The fragment is releasable by a serine protease and is active in buffers as high as 380 mOsm/l. Only 2 - 4 µM fragment is required, in keeping with 18 - 27 µM lacritin in normal basal tears that effectively serves as a lacritin reservoir. Lacritin deletion and synthetic peptide analysis has narrowed the activity to a 15 amino acid region, but with the largest active fragment 54 amino acids long. Although this latent activity promotes bacterial pore formation (but not hemolysis), a second death mechanism is involved. Lacritin bactericidal activity widely compromises bacterial metabolic capacity. It also promotes an accumulation of several amino acids and pyrophosphate. Pyrophosphate is beneficial to bacterial growth. Thus, disrupts bacterial membranes. It also promotes regulated cell death. Our working hypothesis is that lacritin's latent bactericidal activity may be the main regulator of ocular surface sterility, and that lack of lacritin's C-terminal fragment may be a ris factor for pathogenic infection. Our immediate goal is to explore mechanisms regulating this activity. Our long-term goal is to harness this activity as an ocular therapeutic. Our first aim ass how cleaved C-terminus targets bacteria and their comparative efficacy against a broad spectrum of clinical isolates and in vivo. Our second aim explores the mechanisms of bacterial killing. Our third aim asks how cleavage is regulated.

 描述(由申请人提供)：泪液杀菌活性保护眼睛表面免受环境病原体的伤害，并可能调节共生菌的水平。人类的基础泪水和反射性泪水富含催乳素。Lacritin的个人泪水减少了10倍 患有真菌性角膜炎，并在眼睑炎症中选择性下调 与共生细菌过度生长有关的眼皮。裂解增强的拉丁C末端片段对革兰氏阴性和阳性细菌包括表皮葡萄球菌、金黄色葡萄球菌、铜绿假单胞菌和大肠杆菌都有杀菌作用。通过反复通过抗泪液素C-端(但不是N-)端抗体柱从人泪液中去除泪液素C-末端片段会耗尽所有的抗菌活性。一种可能的暗示是，通过C-末端片段(S)的催乳素可能是泪液中抗菌活性的主要来源，泪液催乳素不足的眼睛可能会显著增加微生物致病的风险。此外，局部使用Lacritin C-末端片段可能具有治疗作用。该片段可被丝氨酸蛋白酶释放，在高达380 mOsm/L的缓冲液中具有活性。只需要2-4微米的片段，与正常基础泪液中18-27微米的泪蛋白保持一致，后者有效地充当了泪液的储存库。Lacritin缺失和合成肽分析将活性缩小到15个氨基酸区域，但最大的活性片段长54个氨基酸。虽然这种潜在的活性促进了细菌毛孔的形成(但不促进溶血)，但涉及到第二种死亡机制。乳酸菌素的杀菌活性广泛影响细菌的代谢能力。它还促进了几种氨基酸和焦磷酸的积累。焦磷酸盐有利于细菌生长。因此，会破坏细菌的细胞膜。它还促进了受调控的细胞死亡。我们的工作假设是，Lacritin潜在的杀菌活性可能是眼表不孕症的主要调节因素，而缺乏Lacritin的C-末端片段可能是致病感染的RIS因素。我们的近期目标是探索规范这一活动的机制。我们的长期目标是将这种活动作为一种眼部治疗手段来利用。我们的第一个目标是C端如何针对细菌，以及它们对广泛的临床分离株和体内的比较有效性。我们的第二个目标是探索细菌杀死的机制。我们的第三个目标是问乳沟是如何受到监管的。