Pathogenesis of Fungal Keratitis

真菌性角膜炎的发病机制

• 批准号: 7561719
• 负责人: Eric Pearlman
• 金额: $ 47.29万
• 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/7561719
• 关键词: Accidents; Agriculture; Air; Antifungal Agents; Apoptosis; Apoptotic; Area; Awareness; Beds; Biochemical Pathway; C-Type Lectins; C57BL/6 Mouse; Caring; Cells; Cicatrix; Cleaved cell; Collagen; Collagen Fibril; Contact Lenses; Cornea; Corneal Diseases; Corneal Stroma; Data; Dendritic Cells; Descemet' s membrane; Diagnosis; Diagnostic; Disease; Disease Outbreaks; Dust; Endophthalmitis; Epithelial Cells; Epithelium; Eye; Failure; Frequencies; Fusarium; Genes; Germination; Glucans; Human; Hydrophilic Contact Lenses; Hyphae; Immune; Immune response; Infection; Inflammation; Inflammatory Response; Injection of therapeutic agent; Invaded; Keratitis; Keratoplasty; Knockout Mice; Mediating; Mediator of activation protein; Methods; Microbial Biofilms; Modeling; Molds; Molecular; Mus; Mutagenesis; Neutrophil Antimicrobial Peptides; Neutrophil Infiltration; Organism; Oryctolagus cuniculus; Pathogenesis; Pathway interactions; Penetration; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Prevalence; Proteins; Proteomics; Recombinants; Reproduction spores; Rest; Role; Rural; Severities; Soil; Solubility; Southeastern Asia; Technology; Testing; Transplantation; Trauma; Ulcer; Vegetables; Visual; anterior chamber; corneal epithelium; dectin 1; fungus; high risk; improved; lens; macrophage; microbial; microbial host; mouse dectin-2; mouse model; mutant; neutrophil; novel; prolyl-glycyl-proline; 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）：茄病镰刀菌是一种丝状真菌，是 2005/2006 年美国爆发的角膜炎的致病微生物，可追溯到镜片护理产品。此次疫情的爆发提高了人们对镰刀菌的认识并改进了诊断方法，表明镰刀菌角膜炎的患病率远高于之前的估计。我们的研究结果表明，镰刀菌在软性隐形眼镜上形成生物膜，保护生物体免受抗真菌药物的侵害，并诱导角膜上皮细胞的程序性细胞死亡（细胞凋亡）。这种机制对于菌丝穿过上皮到达基质的渗透可能很重要。镰刀菌也是美国南部农村、东南亚和发展中国家许多地区微生物性角膜炎的主要原因，这些地方孢子（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造成的，其中含有孢子的灰尘或植物物质进入角膜基质并发芽。我们开发了镰刀菌角膜炎小鼠模型，小鼠在 24 小时内出现严重的角膜混浊和溃疡，在中性粒细胞强烈浸润角膜基质后，微生物在 48 小时内被清除。相反，在免疫缺陷的 IL-1R1-/- 和 MyD88-/- 小鼠中，微生物侵入前房并继续复制。目标 1 将检查角膜驻留细胞中 IL-1R1/MyD88 的机制以及中性粒细胞的抗真菌活性。实验还将检查 C 型凝集素 Dectin-1 和 Dectin-2 的作用，以及介导菌丝穿透角膜基质和后弹力层的镰刀菌蛋白酶的作用，并将检查这些蛋白酶在通过裂解胶原纤维生成中性粒细胞趋化肽 Pro-Gly-Pro 方面的作用。目标 2 将利用蛋白质组学和定向诱变方法来识别与生物膜形成相关的生化途径和镰刀菌蛋白，并产生生物膜形成中的关键蛋白被破坏的生物体。将检查这些菌株形成生物膜的能力，以及在角膜上皮细胞和朗格汉斯树突细胞中诱导促凋亡和免疫调节反应的能力，以及诱导隐形眼镜相关角膜炎的能力。为此目的提出的实验还将确定生物膜诱导的角膜上皮细胞凋亡的关键介质。拟议研究的结果将极大地增加我们对这种疾病发病机制的了解，并将确定治疗和诊断的新靶点。常见的土壤真菌茄病镰刀菌 (Fusarium solani) 是美国南部潮湿地区发展中国家农业相关和隐形眼镜相关角膜感染和炎症的主要原因，也是 2005/2006 年美国暴发的致病微生物，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的常驻细胞和浸润细胞对生长的真菌菌丝做出反应以消灭微生物并引起疾病的分子机制。实验还将鉴定负责隐形眼镜上生物膜形成的镰刀菌蛋白，并与第一组研究一起确定该疾病介入治疗的靶点。