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（由申请人提供）：眼泪的杀菌活性可保护眼睛表面免受环境病原体的侵害，并可调节共生细菌的水平。人的基础泪液和反射泪液中的泪泌素含量比个体泪液中的低 10 倍。患有真菌性角膜炎，并在睑缘炎（睑缘炎）中选择性下调与共生细菌过度生长相关的眼睑，增强的乳泌素 C 末端片段对革兰氏阴性和阳性细菌具有杀菌作用，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。一种可能的暗示是，人类眼泪反复通过抗乳泌素 C-（而非 N-）末端抗体柱，会耗尽所有抗菌活性。的观点是，通过C-末端片段的泪泌素可能是泪液中抗菌活性的主要来源，并且泪液泌素不足的眼睛可能显着增加微生物致病性的风险。此外，局部泪泌素C-末端片段可能具有治疗作用。该片段可通过丝氨酸蛋白酶释放，并且在高达 380 mOsm/l 的缓冲液中具有活性，与此一致，仅需要 2 - 4 µM 片段。正常基底泪液中的 18 - 27 µM 泪泌素可有效地充当泪泌素储存库，并且合成肽分析已将活性缩小到 15 个氨基酸区域，但最大的活性片段长为 54 个氨基酸。细菌孔的形成（但不是溶血），涉及第二种死亡机制，它还广泛损害细菌的代谢能力。焦磷酸盐有利于细菌生长。因此，它还能促进受调节的细胞死亡。我们的工作假设是，lacritin 的潜在杀菌活性可能是眼表面无菌性的主要调节剂。我们的近期目标是探索调节这种活性的机制，我们的首要目标是利用这种活性作为眼部治疗方法。我们的第二个目标是探索细菌杀伤机制，我们的第三个目标是如何调节切割。