Pathogenesis of Fungal Keratitis

真菌性角膜炎的发病机制

• 批准号: 8018103
• 负责人: Eric Pearlman
• 金额: $ 47.68万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8018103
• 关键词: Accidents; Agriculture; Air; Antifungal Agents; Apoptosis; Apoptotic; Area; Awareness; Biochemical Pathway; C-Type Lectins; Caring; Cells; Cleaved cell; Collaborations; Collagen Fibril; Contact Lenses; Cornea; Corneal Stroma; Dendritic Cells; Descemet' s membrane; Diagnosis; Diagnostic; Disease; Disease Outbreaks; Dust; Epithelial Cells; Epithelium; Fusarium; Hydrophilic Contact Lenses; Hyphae; Immune; Infection; Inflammation; Invaded; Keratitis; Mediating; Mediator of activation protein; Methods; Microbial Biofilms; Molds; Molecular; Mus; Mutagenesis; Names; Neutrophil Infiltration; Organism; Pathogenesis; Penetration; Peptide Hydrolases; Peptides; Prevalence; Principal Investigator; Proteins; Proteomics; Reproduction spores; Research; Role; Rural; Soil; Southeastern Asia; Trauma; Ulcer; Vegetables; anterior chamber; corneal epithelium; dectin 1; fungus; improved; lens; microbial; mouse dectin-2; mouse model; neutrophil; novel; programs; prolyl-glycyl-proline; prolyl-prolyl-glycine; research study; response

DESCRIPTION (provided by applicant): Fusarium solani is a filamentous fungus that was the causative organism in an outbreak of keratitis in the USA in 2005/2006 that was traced to a lens care product. This outbreak caused an increased awareness of, and improved diagnostics for Fusarium, indicating that the prevalence of Fusarium keratitis is much higher than was previously estimated. Our findings show that Fusarium forms a biofilm on soft contact lenses, which protects the organisms from anti-mycotics, and also induces programmed cell death (apoptosis) in corneal epithelial cells. This mechanism may be important in penetration of hyphae through the epithelium to the stroma. Fusarium is also a major cause of microbial keratitis in rural southern USA, in southeast Asia and in many parts of the developing world, where spores (conidia) are common in the soil and in the air. Keratitis occurs as a result of agricultural accidents or other forms of trauma where dust or vegetable matter containing spores enter the corneal stroma and germinate. We developed a mouse model of Fusarium keratitis where mice develop severe corneal opacification and ulceration within 24h, and organisms are cleared within 48h following an intense neutrophil infiltration to the corneal stroma. In contrast, in immune deficient IL-1R1-/- and MyD88-/- mice, the organisms invade the anterior chamber and continue to replicate. Aim 1 will examine the mechanism of IL-1R1/MyD88 in resident cells in the cornea and on the anti- fungal activity of neutrophils. Experiments will also examine the role of C-type lectins Dectin-1 and Dectin-2, and the role of Fusarium proteases that mediate penetration of hyphae through the corneal stroma and Descemet's membrane, and will examine the effect of these proteases in generating the neutrophil chemotactic peptide Pro-Gly-Pro by cleaving collagen fibrils. Aim 2 will utilize Proteomics and targeted mutagenesis methods to identify biochemical pathways and Fusarium proteins associated with biofilm formation, and generate organisms in which key proteins in biofilm formation are disrupted. These strains will be examined for their ability to form biofilm, and to induce pro-apoptotic and immunomodulatory responses in corneal epithelial cells and Langerhans dendritic cells, and to induce contact lens associated keratitis. Experiments proposed in this aim will also identify key mediators in biofilm-induced apoptosis of corneal epithelial cells. Results of the proposed studies will greatly increase our understanding of the pathogenesis of this disease, and will identify novel targets for therapy and diagnosis. The common soil fungus Fusarium solani is a major cause of agriculture related and contact lens associated corneal infection and inflammation in southern, humid areas of the USA in the developing world, and was the causative organism in an outbreak of in the USA in 2005/2006 that was traced to a lens care product. Experiments outlined in this proposal will examine the molecular mechanisms by which resident and infiltrating cells in the cornea respond to the growing fungal hyphae to eliminate the organism and cause disease. Experiments will also identify Fusarium proteins that are responsible for biofilm formation on contact lenses, and together with the first set of studies will identify targets for interventional therapy for this disease.

描述（由申请人提供）：索拉尼镰刀菌是一种丝状真菌，是2005/2006年美国角膜炎爆发中的病因，可追溯到镜片护理产品。这次爆发引起了对镰刀菌的认识和诊断的提高，表明角膜炎的患病率比以前估计的高得多。我们的发现表明，镰刀菌在软接触透镜上形成生物膜，可保护生物体免受抗肌动物的影响，并在角膜上皮细胞中诱导程序性细胞死亡（凋亡）。这种机制对于通过上皮到基质的菌丝穿透可能很重要。镰刀菌也是美国南部农村，东南亚和发展中国家的许多地区微生物角膜炎的主要原因，那里的孢子（分生孢子）在土壤和空气中很常见。角膜炎是由于农业事故或其他形式的创伤而导致的，其中含有孢子的灰尘或植物物质进入角膜基质并发芽。我们开发了一种小鼠角膜炎的小鼠模型，其中小鼠在24小时内形成严重的角膜糊化和溃疡，并且在严重的中性粒细胞浸润到角膜基质之后，在48小时内清除了生物体。相反，在免疫缺陷的IL-1R1 - / - 和myd88 - / - 小鼠中，生物体侵入前腔并继续复制。 AIM 1将检查角膜中居民细胞和中性粒细胞的抗真菌活性中IL-1R1/MYD88的机制。实验还将检查C型凝集素Dectin-1和dectin-2的作用，以及辅助蛋白酶的作用，这些蛋白酶的作用介导了菌丝通过角膜基质和Descemet膜的渗透，并将检查这些蛋白酶在产生中性粒细胞化学肽蛋白质蛋白质蛋白质蛋白质蛋白质中的作用，从而产生f胶状纤维螺旋螺旋。 AIM 2将利用蛋白质组学和靶向诱变方法来鉴定与生物膜形成相关的生化途径和镰刀菌蛋白质，并产生生物膜形成中关键蛋白的生物体受到破坏。这些菌株的形成生物膜的能力将被检查，并诱导角膜上皮细胞和兰格汉树突状细胞中的促凋亡和免疫调节反应，并诱导与隐形眼镜相关的角膜炎。在此目标中提出的实验还将确定生物膜诱导的角膜上皮细胞凋亡中的关键介体。拟议研究的结果将大大增加我们对该疾病发病机理的理解，并将确定治疗和诊断的新靶标。常见的土壤真菌镰刀菌是与农业相关的主要原因，并且在发展中国家，美国南部，美国潮湿地区，美国潮湿地区的角膜感染和炎症相关的主要原因，是2005/2006年在美国爆发中的病变生物体，可追溯到镜片护理产品。该提案中概述的实验将检查角膜中驻留和浸润细胞对生长的真菌菌丝的反应以消除生物体并引起疾病的分子机制。实验还将鉴定出负责隐形眼镜生物膜形成的镰刀菌蛋白质，以及第一组研究将确定该疾病介入治疗的靶标。