Tear Protein Microbial Regulation

泪液蛋白微生物调节

基本信息

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
项目类别：
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

项目摘要

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（适用提供）：撕裂微生物活性可保护眼表面免受环境病原体的影响，并可能调节共生细菌的水平。人的基础和反射眼泪富含透眼素。流人的眼泪少10倍患有真菌性角膜炎，在睑缘炎中有选择地下调与共生细菌过度生长有关的眼睑。裂解势c-末端的c-末端片段是革兰氏阴性和阳性细菌的细菌，包括表皮链球菌，金黄色葡萄球菌，p.aeruginosa和大肠杆菌。通过反复通过抗透眼蛋白C-（但不是N-）末端抗体柱从人眼泪中清除透眼蛋白C末端片段，从而消耗了所有抗菌活性。一种可能的含义是，通过C末端片段（S）通过泪液中的抗菌活性的主要来源，而眼泪不足的眼睛可能会显着增加微生物致病性的风险。从事近表质素C末端片段可能是治疗性的。该片段由串行蛋白释放，并在高达380 MOSM/L的缓冲液中活跃。在正常的基本泪液中，仅需要2-4 µm片段，与18-27 µm的曲息蛋白保持一致，这有效地用作透眼蛋白储存剂。细胞蛋白缺失和合成肽分析已将活性范围缩小到15个氨基酸区域，但最大的活性片段为54氨基酸。尽管这种潜在活性促进了细菌孔的形成（但不是溶血），但涉及第二种死亡机制。细菌活性广泛损害细菌代谢能力。它还促进了几种氨基酸和焦磷酸盐的积累。焦磷酸对细菌的生长有益。那破坏了细菌机制。它还促进受调节的细胞死亡。我们的工作假设是，酸性蛋白的潜在细菌活性可能是眼表不育的主要调节剂，缺乏细胞蛋白的C末端片段可能是致病感染的RIS因素。我们的直接目标是探索机制调节此活动。我们的长期目标是利用这项活动作为眼部疗法。我们的第一个瞄准方法如何将细菌靶向细菌及其比较有效性，以靠广泛的临床分离株和体内。我们的第二个目标探讨了细菌杀死的机制。我们的第三个目标询问如何调节分裂。