Bacteria-Mucin Interactions at the Ocular Surface

描述（由申请方提供）：在眼表面，感染通过表面粘蛋白屏障的破坏或穿过粘蛋白屏障发生。第一个例子是金黄色葡萄球菌（SA）或肺炎链球菌（SPE）（包囊）角膜炎，发生的结果是上皮表面损伤的创伤。其他类型的感染，例如，来自非包囊肺炎链球菌（SPN）的细菌性结膜炎的爆发在没有眼表面损伤的证据的情况下发生。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，允许病原体穿过顶端粘膜粘蛋白屏障。相比之下，对于粘蛋白屏障如何被致炎病原体直接操纵或损害以促进感染的理解有限。我们建议比较SA，SPE（机会微生物），和nontypeable，非封装SPN（生物体能够引起流行性结膜炎，在没有已知或疑似眼表创伤），相互作用，并影响膜相关粘蛋白获得上皮细胞。研究将使用角膜和结膜上皮细胞系，这些细胞系在顶部表达天然上皮中发现的糖基化膜相关粘蛋白（MUC 1、4和16）和3种临床分离株-SA、SPE和SPN-均来自眼表感染。我们的具体目标是：目标一：比较人角膜和结膜上皮细胞上MAMs MUC 1、MUC 4和MUC 16的胞外结构域释放、细胞表面密度和糖基化特征，以响应与两种条件性细菌SA和包封的SP以及引起条件性细菌的非包封的SP菌株及其胞外产物的培养。目的II：确定非包封的SP外产物诱导膜粘蛋白MUC 16释放的机制。目标三：通过比较（1）SA和SP菌株进入对照人角膜缘上皮（HCLE）细胞和进入其中MAM表达被siRNA消除或其中粘蛋白的糖基化被阻断的HCLE细胞的细胞病变效应和内化速率，（2）比较SA、SPE、SPN及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。通过HCLE细胞和在其中MAM表达被消除或其中粘蛋白的糖基化被改变的HCLE细胞中。目标四：确定这三种类型的细菌是否与泪膜中存在的特异性释放的粘蛋白MUC 1、4和16和/或分泌的杯状细胞粘蛋白MUC 5AC结合，以及这种结合是否作为保护机制，防止在对照、膜相关粘蛋白敲低或去糖基化粘蛋白条件下粘附于HCLE细胞。并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屏障以防止连续暴露于数万亿微生物，并且来自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中，分泌的或膜相关的，是膜相关的粘蛋白与形成这种细胞屏障的表面膜是一体的。本申请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致病菌更能够穿过膜粘蛋白屏障的新信息，并且这样做将为治疗方案提供线索。

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.