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）：在眼睛表面，感染是通过表面粘蛋白屏障的破裂或穿过粘蛋白屏障而发生的。第一个例子是金黄色葡萄球菌 (SA) 或肺炎链球菌 (SPE)（包膜）角膜炎，这是由于外伤导致上皮表面损伤而发生的。其他类型的感染，例如非包膜肺炎链球菌（SPN）引起的细菌性结膜炎的爆发，没有眼表损伤的证据。大多数宿主与病原体相互作用的研究都使用涉及感染前物理诱导上皮损伤的模型，从而使病原体能够穿过顶粘膜粘蛋白屏障。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（机会性微生物）和不可分型、非封装的SPN（在没有已知或疑似眼表创伤的情况下能够引起流行性结膜炎的微生物）如何与膜相关粘蛋白相互作用并影响其进入上皮细胞。研究将使用角膜和结膜上皮细胞系，这些细胞系顶部表达天然上皮细胞（MUC1、4和16）中发现的糖基化膜相关粘蛋白以及3种临床分离株（SA、SPE和SPN），所有分离株均源自眼表感染。我们的具体目标是： 目标 I：比较人角膜和结膜上皮细胞上 MAM MUC1、MUC4 和 MUC16 的胞外域释放、细胞表面密度和糖基化特征，以响应两种机会性细菌 SA 和封装 SP 以及引起流行的非封装 SP 菌株及其胞外产物的培养。目标 II：确定非封装 SP 外产物诱导膜粘蛋白 MUC16 释放的机制。目标 III：通过比较 (1) SA 和 SP 菌株进入对照人角膜缘上皮 (HCLE) 细胞和进入 HCLE 细胞（其中 MAM 表达被 siRNA 消除或粘蛋白糖基化被阻断）的细胞病变效应和内化率，确定膜粘蛋白对细菌或其外产物到达上皮表面膜的障碍程度。 (2)通过比较SA、SPE或SPN或其外产物对应激诱导的细胞因子IL-6、IL-8和TNF-α表达的影响。 HCLE 细胞和​​ MAM 表达被消除或粘蛋白糖基化被改变的 HCLE 细胞中。目标 IV：确定三种类型的细菌是否与泪膜中存在的特定释放的粘蛋白、MUC 1、4 和 16 和/或分泌型杯状细胞粘蛋白 MUC5AC 结合，以及这种结合是否起到保护机制的作用，防止在对照、膜相关粘蛋白敲低或去糖基化粘蛋白条件下与 HCLE 细胞粘附。 公共卫生相关性 感染仍然是发病和死亡的主要原因 在世界范围内，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。据推测，湿表面粘膜上的粘蛋白可以提供屏障，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，在这些上皮细胞上的两种类型的粘蛋白（分泌型或膜相关型）中，膜相关粘蛋白是形成这种细胞屏障的表面膜的组成部分。本申请中提出的研究将提供关于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替代方案提供线索。