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.

项目概要/摘要 真菌性角膜炎是全世界眼部发病和单眼失明的重要来源。当前的 高达 60% 的患者的抗真菌治疗方案失败，导致需要至少一种，有时甚至需要 反复进行角膜移植。因此需要新型抗真菌药物，但其开发需要 更好地了解可以作为药物靶点的真菌蛋白/酶。正如角膜 我们假设基质实际上是由胶原蛋白和其他蛋白质组成的细胞外基质 支持真菌蛋白质分解代谢的途径对于眼睛中的真菌生长至关重要，并且通过 延伸，可能是治疗目标的毒力因子。 为了使真菌利用蛋白质作为营养来源，它们必须首先分泌大量的 蛋白酶进入环境/宿主组织。这种分泌负担会导致 内质网 (ER) 内未折叠的蛋白质，如果不解决，会导致内质网“堵塞” 分泌途径将严重抑制真菌生长。未折叠蛋白反应 (UPR) 在 通过首先感测未折叠蛋白质并随后调节基因，在这方面发挥着关键作用 促进 ER 腔内的蛋白质折叠能力（例如分子伴侣）。在目标 1 中，我们将测试 假设真菌 UPR 促进角膜炎常见病原体的角膜发病机制， 茄病镰刀菌。我们将首先生成 F. solani 的 UPR 缺陷突变体，然后测试是否 正如我们预测的那样，突变体在蛋白质底物上生长有缺陷。然后我们将评估毒力 真菌性角膜炎小鼠模型中的突变体。观察发现 UPR 缺陷菌株 低毒力表明 UPR 抑制剂可以用作新型抗真菌药物。 真菌的转录谱随营养源的变化而变化很大。过渡 例如，从富含葡萄糖的培养基到限制葡萄糖的培养基，会导致糖酵解基因的下调， 分泌水解酶的上调以及涉及氨基酸的代谢酶的上调 代谢。因此，在目标 2 中，我们将检验茄镰孢利用蛋白质的假设 通过比较体内真菌（来自受感染眼睛）的转录组与角膜 在体外特定营养条件（胶原蛋白与葡萄糖）下生长的真菌的转录组。我们 预测体内表达最高的基因将反映体内表达最高的基因 胶原。然而，我们并不期望两个数据集之间存在一对一的对应关系，因为 眼睛特有的环境条件，例如炎症造成的压力 回复。通过这种方式，我们将获得关于角膜炎期间真菌适应性反应的新见解 感染，这将导致新毒力基因和假定药物靶点的鉴定。