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: 10554363
• 负责人: Kevin K. Fuller
• 金额: $ 26.34万
• 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/10554363
• 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; Epithelium; 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; 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; virulence gene

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.

项目总结/文摘