The unfolded protein response as a therapeutic target for fungal keratitis

未折叠蛋白反应作为真菌性角膜炎的治疗靶点

• 批准号: 10624339
• 负责人: Kevin K. Fuller
• 金额: $ 35.36万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10624339
• 关键词: 3-Dimensional; Address; Affect; Animals; Anti-Inflammatory Agents; Antifungal Agents; Antigen Presentation; Aspergillus fumigatus; Attenuated; Automobile Driving; Biomass; Blindness; Catabolism; Cell Wall; Cell Wall Alteration; Cells; Clinical; Collagen; Cornea; Corneal Diseases; Corneal Stroma; DNA Microarray Chip; Defect; Development; Disease; Disease Progression; Doxycycline; Drug Targeting; Endoplasmic Reticulum; Environment; Enzymes; Exhibits; Flow Cytometry; Fungal Antigens; Fungal Proteins; Future; Genes; Genetic; Glucose; Golgi Apparatus; Growth; Harvest; Histopathology; Hypersensitivity; Hyphae; In Vitro; Incubated; Infection; Inflammation; Inflammatory; Inflammatory Response; Investigation; Keratitis; Keratoplasty; Leucocytic infiltrate; Macrophage; Membrane; Modeling; Molds; Monitor; Morbidity - disease rate; Mus; NF-kappa B; Nutrient; Operative Surgical Procedures; Orthologous Gene; Outcome; Pathway interactions; Peptide Hydrolases; Perforation; Persons; Pharmaceutical Preparations; Physiological; Play; Proliferating; Protein Secretion; Proteins; RNA; Repression; Research Priority; Resolution; Role; Sampling; Severity of illness; Signal Transduction; Site; Source; Strategic Planning; Stress; Structure; Testing; Tetracyclines; Topical application; Virulence; Virulence Factors; Visual; Work; chromatin immunoprecipitation; clinically relevant; collagenase; cytokine; drug repurposing; feasibility testing; follow-up; fungus; genome-wide; improved; in vitro activity; in vivo; inhibitor; insight; interest; knock-down; melting; microbial; misfolded protein; mouse model; mutant; neutrophil; new therapeutic target; next generation sequencing; novel; pathogen; pharmacologic; promoter; protein folding; receptor; response; targeted treatment; therapeutic target; transcription factor; transcriptome sequencing; uptake

PROJECT SUMMARY Fungal keratitis (FK) has emerged as a leading source of ocular morbidity and unilateral blindness worldwide. Though better antifungals are urgently needed, their development first requires an understanding of fungal proteins/enzymes that could serve as drug targets. The site of fungal growth during FK is the corneal stroma, which is rich in collagen and other proteins, but ostensibly poor in glucose or freely diffusible nutrients. We therefore predicted that (1) fungi breakdown these proteins as a primary nutrient source during infection, and (2) fungal pathways that support protein catabolism (e.g. protease secretion) represent important virulence factors and putative drug targets. Using a predominant agent of FK, Aspergillus fumigatus, we have confirmed both parts. First, fungal protease expression was up-regulated in A. fumigatus isolated from infected mouse corneas, suggesting the fungus is indeed trying to catabolize stromal protein. Second, an A. fumigatus mutant defective in protease secretion, ∆hacA, was unable to establish corneal infection in the model. The hacA gene encodes a transcription factor that plays a critical role in the unfolded protein response (UPR), a pathway that detects and resolves the accumulation of misfolded proteins in the endoplasmic reticulum and promotes traffic through the ER-Golgi pathway. This project seeks to follow up these foundational observations and potentially elevate the fungal UPR as a novel target for FK treatment. In Aim 1, we will evaluate the role of HacA within the infected cornea. First, we will determine if cell wall alterations associated with the hacA mutant influence host-fungal interactions and pro-inflammatory signaling. Second, we will determine the impact of repressing hacA expression on disease progression/resolution. Specifically, we will infect mice with a strain of A. fumigatus in which hacA expression can be repressed through the addition of doxycycline. In this way, infected corneas will be ‘treated’ with doxycycline and the effect on fungal growth, inflammation, and corneal damage will be monitored. In Aim 2, we will test the feasibility of repurposing a mammalian UPR inhibitor for treating FK. The compound of interest, 4µ8C, inhibits Ire1, which signals upstream of the HacA ortholog in the UPR pathway. As the UPR plays a critical role in cytokine secretion, it follows that 4µ8C dampens the inflammatory response. We have further established that the Ire1 ortholog in A. fumigatus is essential for growth, and the 4µ8C displays antifungal effects in vitro. Accordingly, we will test whether 4µ8C can be used as a dual-edged treatment to block both fungal growth and damaging inflammation in our mouse FK model. Finally, it is clear that the A. fumigatus UPR regulates downstream genes/proteins that are critical for corneal virulence, but these targets remain largely uncharacterized. In Aim 3, will employ both chromatin immunoprecipitation (ChIP-seq) on the HacA protein as well as RNA-seq (WT vs. ∆hacA) following growth in a 3D corneal model. In doing so, we will identify genes under direct and indirect control of the UPR. The characterization of these genes and their role in corneal virulence will serve as the basis for future inquiry.

项目概要 真菌性角膜炎（FK）已成为全球眼部发病和单眼失明的主要原因。 尽管迫切需要更好的抗真菌药物，但其开发首先需要了解真菌 可以作为药物靶点的蛋白质/酶。 FK 期间真菌生长的部位是角膜基质， 它富含胶原蛋白和其他蛋白质，但表面上缺乏葡萄糖或可自由扩散的营养物质。我们 因此预测（1）真菌在感染过程中分解这些蛋白质作为主要营养源，并且 (2) 支持蛋白质分解代谢（例如蛋白酶分泌）的真菌途径代表重要的毒力 因素和假定的药物靶点。使用 FK 的主要药剂烟曲霉，我们已经证实 两个部分。首先，从受感染小鼠中分离出的烟曲霉中真菌蛋白酶的表达上调 角膜，表明真菌确实试图分解基质蛋白。二、烟曲霉突变体 蛋白酶分泌缺陷，ΔhacA，无法在模型中建立角膜感染。 hacA基因 编码在未折叠蛋白反应 (UPR) 中发挥关键作用的转录因子，该途径 检测并解决内质网中错误折叠蛋白质的积累并促进运输 通过 ER-高尔基体途径。该项目旨在跟进这些基本观察结果并可能 将真菌 UPR 提升为 FK 治疗的新靶点。在目标 1 中，我们将评估 HacA 在以下方面的作用： 受感染的角膜。首先，我们将确定细胞壁改变是否与 hacA 突变体影响相关 宿主-真菌相互作用和促炎症信号传导。其次，我们将确定镇压的影响 hacA 表达对疾病进展/缓解的影响。具体来说，我们将用 A 菌株感染小鼠。 fumigatus 中 hacA 的表达可以通过添加强力霉素来抑制。这样， 受感染的角膜将用多西环素进行“治疗”，并对真菌生长、炎症和角膜产生影响 损害将受到监控。在目标 2 中，我们将测试重新利用哺乳动物 UPR 抑制剂的可行性 治疗FK。感兴趣的化合物 4μ8C 抑制 Ire1，Ire1 向 HacA 直向同源物上游发出信号 UPR途径。由于 UPR 在细胞因子分泌中起着至关重要的作用，因此 4μ8C 会抑制 炎症反应。我们进一步确定烟曲霉中的 Ire1 直向同源物对于 生长，4μ8C 在体外表现出抗真菌作用。因此，我们将测试4μ8C是否可以使用 在我们的小鼠 FK 模型中，它是一种双刃剑，可以阻止真菌生长和破坏性炎症。 最后，很明显烟曲霉 UPR 调节对角膜至关重要的下游基因/蛋白质 毒力，但这些目标在很大程度上仍然未知。在目标 3 中，将同时使用染色质 对 HacA 蛋白进行免疫沉淀 (ChIP-seq) 以及在 a 中生长后的 RNA-seq（WT 与 ΔhacA） 3D 角膜模型。在此过程中，我们将鉴定受 UPR 直接和间接控制的基因。这 这些基因的特征及其在角膜毒力中的作用将作为未来研究的基础。