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Bacteria-Mucin Interactions at the Ocular Surface

眼表面的细菌-粘蛋白相互作用

基本信息

批准号：
8209123
负责人：
ILENE K. GIPSON
金额：
$ 46.09万
依托单位：
SCHEPENS EYE RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-01 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8209123
关键词：
Adhesions Affect Apical Bacteria Bacterial conjunctivitis Binding Biological Assay Cell Line Cell surface Cells Characteristics Clinical Conjunctivitis Cornea Data Disease Outbreaks Encapsulated Epidemic Epithelial Epithelial Cells Epithelium Exposure to Eye Eye Infections Film Gastrointestinal tract structure Genes Genitourinary system Goblet Cells Health Human IL8 gene In Vitro Infection Injury Interleukin-6 Keratitis Laboratories MUC5AC gene Membrane Microbe Modeling Morbidity - disease rate Mucin-1 Staining Method Mucins Mucous Membrane Organism Pathogenesis Pattern Pneumococcal Infections Positioning Attribute Research Respiratory System Respiratory tract structure Small Interfering RNA Staphylococcus aureus Streptococcus pneumoniae Stress Surface Systems Analysis TNF gene Testing Tissue membrane Tissues Trauma alternative treatment cytokine density glycosylation insight limbal mortality novel strategies ocular surface pathogen prevent receptor response

项目摘要

DESCRIPTION (provided by applicant): At the surface of the eye, infections occur via breaks in the surface mucin barrier or through the mucin barrier. Examples of the first are Staphylococcus aureus (SA) or Streptococcus pneumoniae (SPE) (encapsulated) keratitis that occurs as the result of epithelial surface damage from trauma. Other types of infection, e.g., outbreaks of bacterial conjunctivitis from non-encapsulated Streptococcus pneumoniae (SPN), occur without evidence of ocular surface damage. Most studies of host-pathogen interactions have used models that involve the physical induction of epithelial damage prior to infection, allowing a pathogen to cross the apical mucosal mucin barrier. In contrast, there is limited understanding of how the mucin barrier can be manipulated or compromised directly by epidemic-causing pathogens to facilitate infection. We propose to compare how SA, SPE (opportunistic organisms), and nontypeable, non-encapsulated SPN (organisms capable of causing epidemic conjunctivitis in the absence of known or suspected ocular surface trauma), interact with and affect membrane-associated mucins to gain access to epithelial cells. Studies will use corneal and conjunctival epithelial cell lines expressing apically the glycosylated membrane-associated mucins found in native epithelia (MUC1, 4 and 16) and 3 clinical isolates-SA, SPE, and SPN-all derived from ocular surface infections. Our Specific Aims are: Aim I: Compare ectodomain release, cell surface density and glycosylation characteristics of MAMs MUC1, MUC4 and MUC16 on human corneal and conjunctival epithelial cells, in response to culture with the two opportunistic bacteria, SA and encapsulated SP, and the epidemic-causing non-encapsulated strain of SP, and their exoproducts. Aim II: Determine the mechanism by which non-encapsulated SP exoproducts induce release of the membrane mucin MUC16. Aim III: Determine the extent to which membrane mucins present an obstacle to bacteria or their exoproducts to reach the epithelial surface membrane by comparing (1) cytopathic effects and internalization rates for SA and the SP strains into control human corneal-limbal epithelial (HCLE) cells, and into HCLE cells in which MAM expression is abrogated by siRNA, or in which glycosylation of mucins is blocked, and (2) by comparing effects of SA, SPE, or SPN, or their exoproducts on expression of stress-induced cytokines IL-6, IL-8 and TNF-? by HCLE cells and in HCLE cells in which MAM expression is abrogated or in which glycosylation of the mucins is altered. Aim IV: Determine if the three types of bacteria bind to specific released mucins, MUCs 1, 4 and 16, and/or secreted goblet cell mucin MUC5AC present in the tear film, and whether such binding acts as a protective mechanism, preventing adhesion to HCLE cells in control, membrane-associated mucin knockdown or deglycosylated mucin conditions PUBLIC HEALTH RELEVANCE Infection remains a leading cause of morbidity and mortality worldwide, and most infections originate at a mucosal boundary-the mucosal surface of the respiratory tract, gastrointestinal tract, urogenital tract or the eye. Mucins on the wet-surfaced mucosa are hypothesized to provide a barrier against continuous exposure to trillions of microbes, and preliminary data from our laboratory indicate that, of the two types of mucins on these epithelia, secreted or membrane associated, it is the membrane-associated mucins integral to surface membranes that form this cellular barrier. The research proposed in this application will provide new information about how bacteria that cause epidemic conjunctivitis are more capable of crossing the membrane mucin barrier than are the opportunistic pathogens and, in doing so, will provide clues for treatment alternatives.

描述（由申请人提供）：在眼睛表面，感染通过表面粘蛋白屏障的破裂或通过粘蛋白屏障发生。第一种是金黄色葡萄球菌（SA）或肺炎链球菌（SPE）（囊性）角膜炎，它们是外伤引起的上皮表面损伤的结果。其他类型的感染，例如，由非包裹性肺炎链球菌（SPN）引起的细菌性结膜炎的爆发，没有眼表损伤的证据。大多数宿主-病原体相互作用的研究使用的模型涉及感染前上皮损伤的物理诱导，允许病原体穿过根尖粘膜粘蛋白屏障。相比之下，人们对粘蛋白屏障如何被引起流行病的病原体直接操纵或破坏以促进感染的了解有限。我们建议比较SA、SPE（机会性生物）和不可分型、非包封的SPN（在没有已知或疑似眼表外伤的情况下能够引起流行病结膜炎的生物）如何与膜相关粘蛋白相互作用并影响其进入上皮细胞。研究将使用天然上皮细胞（MUC1、4和16）和3种临床分离物（sa、SPE和spn）中表达糖基化膜相关粘蛋白的角膜和结膜上皮细胞系，这些细胞均来源于眼表感染。我们的具体目标是：目的1：比较MAMs MUC1、MUC4和MUC16在人角膜和结膜上皮细胞上的外结构域释放、细胞表面密度和糖基化特性，以响应SA和包封SP两种机会菌以及引起流行的未包封SP菌株及其外产物的培养。目的二：确定非包封SP外产物诱导膜粘蛋白MUC16释放的机制。目标3:通过比较(1)SA和SP菌株在对照人角膜缘上皮（HCLE）细胞中的细胞病变效应和内化率，以及在MAM表达被siRNA消除或粘蛋白糖基化被阻断的HCLE细胞中的内化率，以及(2)通过比较SA、SPE或SPN的作用，确定膜粘蛋白对细菌或其外产物到达上皮表面膜的阻碍程度。或其外产物对应激诱导细胞因子IL-6、IL-8和TNF-表达的影响？在HCLE细胞中，MAM表达被取消或粘蛋白的糖基化被改变。目标4:确定这三种类型的细菌是否与泪膜中特定释放的黏液蛋白MUCs 1、4和16和/或分泌的杯状细胞黏液蛋白MUC5AC结合，以及这种结合是否作为一种保护机制，在对照、膜相关黏液蛋白敲低或去糖基化的黏液条件下阻止对HCLE细胞的粘附。而且大多数感染起源于粘膜边界——呼吸道、胃肠道、泌尿生殖道或眼睛的粘膜表面。湿表面粘膜上的粘蛋白被假设为提供一种屏障，防止持续暴露于数万亿微生物，我们实验室的初步数据表明，在这些上皮上的两种粘蛋白中，分泌型或膜相关型，膜相关型粘蛋白是表面膜的组成部分，形成了这种细胞屏障。本应用程序中提出的研究将提供有关引起流行性结膜炎的细菌如何比机会性病原体更有能力穿过膜粘蛋白屏障的新信息，并在此过程中为治疗方案提供线索。