Bacteria-Mucin Interactions at the Ocular Surface

眼表面的细菌-粘蛋白相互作用

• 批准号: 7754383
• 负责人: ILENE K. GIPSON
• 金额: $ 48.39万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7754383
• 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; 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; public health relevance; receptor; response

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）：在眼表面，感染通过表面粘蛋白屏障或粘蛋白屏障发生。第一个的例子是金黄色葡萄球菌（SA）或肺炎链球菌（SPE）（封装）角膜炎，这是由于创伤引起的上皮表面损伤而导致的。其他类型的感染，例如，肺炎链球菌（SPN）发生的细菌结膜炎爆发，没有眼表面损伤的证据。大多数对宿主病原体相互作用的研究都使用了涉及在感染前物理诱导上皮损伤的模型，从而使病原体越过顶端粘膜粘蛋白屏障。相反，人们对如何通过流行病病原体直接操纵或损害粘蛋白屏障以促进感染的理解有限。我们建议比较SA，SPE（机会主义生物）和不可塑造的，非封装的SPN（在没有已知或怀疑的眼表面创伤的情况下能够引起流行性结膜炎的生物），与膜相关的粘液相互作用并影响膜相关的粘蛋白进入上皮细胞。研究将使用角膜和结膜上皮细胞系，在天然上皮（MUC1、4和16）（MUC1、4和16）和3个临床分离株-SA，SPE，SPE，SPE和SPN均来自Ocular Surface Inctections中发现的糖基化膜相关的粘蛋白。 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：确定非封装SP驱虫诱导膜粘蛋白MUC16释放的机制。目的III：通过比较（1）SA和SP菌株的细胞病变效应和内在化速率，确定膜粘蛋白在多大程度上呈现细菌或它们的摄取的障碍，以达到上皮表面膜的障碍，并在对照人的角膜 - 膜上皮细胞（HCLE）中，并将MAM固定在MAM中，并在其上被MMAM所用的MAM所用的siRN和sir sir sir serm NAM所用，或（2）通过比较SA，SPE或SPN的效果或它们对应激诱导的细胞因子IL-6，IL-8和TNF-的表达的效果？通过HCLE细胞和MAM表达被废除的HCLE细胞或粘蛋白的糖基化改变。 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在全球范围内，大多数感染源于粘膜边界 - 呼吸道，胃肠道，泌尿生殖道或眼睛的粘膜表面。假设湿表面上的粘膜上的粘蛋白为不连续暴露于数万亿微生物的持续障碍，而我们实验室的初步数据表明，与这些上皮的两种类型的粘蛋白相关的粘蛋白相关的两种类型的粘蛋白相关，是膜相关的粘液粘合蛋白与表面上的粘液膜相关的粘液蛋白。本应用程序中提出的研究将提供有关如何使流行病结膜炎的细菌与机会性病原体相比，如何跨越膜粘蛋白屏障的能力越过膜粘蛋白屏障，并且会为治疗替代方案提供线索。