Elucidating pathways that regulate fungal keratitis pathogenesis

阐明调节真菌性角膜炎发病机制的途径

• 批准号: 10341206
• 负责人: Kevin K. Fuller
• 金额: $ 27.51万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10341206
• 关键词: Address; Amoeba genus; Animals; Antifungal Agents; Attenuated; Bacteria; Blindness; Bovine Serum Albumin; Carbon; Catabolism; Cell Death; Collagen; Complex; Contact Lenses; Cornea; Corneal Stroma; Data; Data Set; Defect; Development; Disease Outbreaks; Down-Regulation; Drug Targeting; Endoplasmic Reticulum; Environment; Enzymes; Epithelial; Exposure to; Extracellular Matrix; Eye; Fungal Proteins; Fusarium; Gene Expression; Gene Expression Profile; Genes; Genome; Glucose; Golgi Apparatus; Growth; Homeostasis; Hydrolase; Hypoxia; Immune response; Immunity; In Vitro; Infection; Inflammatory Response; Introns; Keratitis; Keratoplasty; Lead; Lesion; Medical; Membrane; Messenger RNA; Metabolic; Metabolic Pathway; Microbe; Micronutrients; Milk; Modality; Modeling; Molecular Chaperones; Morbidity - disease rate; Mus; Mutation; Needles; Nutrient; Nutritional; Oklahoma; Organism; Orthologous Gene; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Play; Proteins; RNA Splicing; Reproduction spores; Ribonucleases; Role; Signal Pathway; Signal Transduction; Source; Stress; System; Testing; Tissues; Translating; Tunicamycin; United States; Up-Regulation; Virulence; Virulence Factors; Virus; Work; Yeasts; amino acid metabolism; endoplasmic reticulum stress; fungus; improved outcome; in vivo; inhibitor; insight; microbial; mouse model; mutant; novel; protein folding; protein misfolding; response; sensor; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

Project Summary/Abstract Fungal keratitis is an important source of ocular morbidity and unilateral blindness worldwide. Current antifungal regimes fails in up to 60% of patients, resulting in the need for at least one and sometimes repeated corneal transplants. Novel antifungals are therefore required, but their development requires a better understanding of fungal proteins/enzymes that could serve as drug targets. As the corneal stroma is effectively an extracellular matrix comprised of collagen and other proteins, we hypothesize that pathways that support fungal protein catabolism are essential for fungal growth in the eye and, by extension, virulence factors that might be targeted in treatment. In order for fungi to utilize proteins as a nutritional source, they must first secrete copious amounts of proteases into the environment/host tissue. This secretory burden leads to an accumulation of unfolded proteins within the endoplasmic reticulum (ER) that, if not resolved, leads to a “clogging” of the secretion pathway that will severely inhibit fungal growth. The unfolded protein response (UPR) plays a critical role in this regard by first sensing unfolded proteins and subsequently regulating genes that that promote the protein folding capacity within the ER lumen (e.g., chaperones). In Aim 1, we will test the hypothesis that fungal UPR promotes the corneal pathogenesis of a common agent of keratitis, Fusarium solani. We will first generate UPR-deficient mutants of F. solani and then test whether the mutants are defective for growth on protein substrates as we predict. We will then assess the virulence of the mutants in a mouse model of fungal keratitis. The observation that the UPR-deficient strains are hypovirulent would suggest that inhibitors of the UPR could be used as novel antifungals. The transcriptional profile of a fungus varies largely as a function of the nutrient source. The transition from glucose-rich to glucose-limiting media, for example, leads to a down-regulation of glycolytic genes, upregulation of secreted hydrolases, and an upregulation of metabolic enzymes involved in amino acid metabolism. Therefore, in Aim 2, we will test the hypothesis that the F. solani utilizes proteins in the cornea by comparing the transcriptome of the fungus in vivo (from infected eyes) against the transcriptome of the fungus grown under defined nutrient conditions (collagen v. glucose) in vitro. We predict that the most highly expressed genes in vivo will mirror the most highly expressed genes on collagen. However, we do not expect a one-to-one correspondence between the two datasets due to environmental conditions that are unique to the eye, such as stresses imparted by the inflammatory response. In this way, we stand to gain novel insight into the fungal adaptive response during keratitis infection, which will lead to the identification of novel virulence genes and putative drug targets.

项目摘要/摘要 真菌角膜炎是全球眼部发病率和单侧失明的重要来源。当前的 抗真菌政权最多失败了60％的患者，导致至少需要一个，有时需要 重复的角膜移植。因此，需要新颖的抗真菌剂，但是它们的发展需要 更好地了解可以用作药物靶标的真菌蛋白/酶。作为角膜 基质实际上是胶原蛋白和其他蛋白质的细胞外基质，我们假设 支持真菌蛋白分解代谢的途径对于眼睛的真菌生长至关重要 扩展，可能针对治疗的病毒因素。 为了使真菌利用蛋白质作为营养来源，它们必须首先秘密大量 蛋白酶进入环境/宿主组织。这个秘密的伯恩导致了 内质网（ER）中展开的蛋白质，如果无法解决，则会导致“堵塞” 分泌途径将严重抑制真菌的生长。展开的蛋白质反应（UPR）扮演 在这方面的关键作用是首先感测未折叠的蛋白质和随后调节的基因 促进ER管腔内的蛋白质折叠能力（例如伴侣）。在AIM 1中，我们将测试 真菌UPR促进角膜炎的角膜发病机理的假设， 镰刀菌。我们将首先生成F. solani的UPR缺陷突变体，然后测试是否是否 正如我们预期的那样，突变体的蛋白质底物生长有缺陷。然后，我们将评估病毒 真菌性角膜炎小鼠模型中突变体的突变体。观察到UPR缺陷菌株是 静态病毒剂会表明，UPR的抑制剂可以用作新型抗真菌性。 真菌品种的转录曲线在很大程度上是养分来源的函数。过渡 例如，从富含葡萄糖到葡萄糖限制培养基，例如，糖酵解基因的下调， 分泌水解酶的上调，以及参与氨基酸的代谢酶的上调 代谢。因此，在AIM 2中，我们将检验以下假设：F。solani利用蛋白质 通过比较体内真菌的转录组（来自感染的眼睛）与角膜 在体外定义的营养条件（胶原蛋白案）下生长的真菌的转录组。我们 预测体内最高表达的基因将反映在 胶原。但是，由于 眼睛独有的环境条件，例如炎症赋予的压力 回复。这样，我们将获得有关角膜炎期间真菌自适应反应的新颖洞察力 感染，这将导致鉴定新的病毒基因和推定的药物靶标。