Pathogenesis of Fungal Keratitis

描述（由申请人提供）：梭兰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生物，可追溯到一种镜片护理产品。这次暴发提高了对镰刀菌的认识，并改进了对镰刀菌的诊断，表明镰刀菌角膜炎的流行率远高于以前的估计。我们的研究结果表明，镰刀菌在软性隐形眼镜上形成一层生物膜，保护生物体免受抗真菌药的侵害，并诱导角膜上皮细胞的程序性细胞死亡（凋亡）。这一机制在菌丝通过上皮向间质渗透过程中可能是重要的。镰刀菌也是美国南部农村、东南亚和发展中国家许多地方的微生物角膜炎的主要原因，孢子（分生孢子）在土壤和空气中很常见。角膜炎的发生是由于农业事故或其他形式的创伤，其中含有孢子的灰尘或植物物质进入角膜基质并发芽。我们建立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，在角膜基质中大量中性粒细胞浸润后，生物体在48小时内被清除。相反，在免疫缺陷的IL-1R1-/-和MyD88-/-小鼠中，生物体侵入前房并继续复制。目的1将探讨IL-1R1/MyD88在角膜驻留细胞中的作用机制以及中性粒细胞的抗真菌活性。实验还将研究c型凝集素Dectin-1和Dectin-2的作用，以及镰刀菌蛋白酶介导菌丝穿过角膜基质和Descemet膜的作用，并将研究这些蛋白酶通过切割胶原原纤维产生中性粒细胞趋化肽Pro-Gly-Pro的作用。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，并产生生物膜形成中关键蛋白被破坏的生物体。这些菌株将被检测其形成生物膜的能力，并在角膜上皮细胞和朗格汉斯树突状细胞中诱导促凋亡和免疫调节反应，以及诱导隐形眼镜相关性角膜炎。本实验还将确定生物膜诱导角膜上皮细胞凋亡的关键介质。所提出的研究结果将大大增加我们对该疾病发病机制的理解，并将确定新的治疗和诊断靶点。在发展中国家的美国南部潮湿地区，常见的土壤真菌镰刀菌是农业相关和与隐形眼镜相关的角膜感染和炎症的主要原因，并且是2005/2006年美国爆发的致病生物，可追溯到一种镜片护理产品。本提案中概述的实验将检查角膜中常驻和浸润细胞对生长的真菌菌丝作出反应以消除生物体并引起疾病的分子机制。实验还将确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这种疾病的介入治疗目标。

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.