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.

项目摘要 真菌性角膜炎（FK）已成为全球眼部发病率和单侧失明的主要来源。 虽然迫切需要更好的抗真菌药物，但它们的发展首先需要了解真菌 蛋白质/酶可以作为药物靶点。FK期间真菌生长的部位是角膜基质， 其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素。我们 因此预测：（1）真菌在感染期间将这些蛋白质分解为主要营养源， (2)支持蛋白质分解的真菌途径（例如蛋白酶分泌）代表重要的毒力 因素和假定的药物靶点。使用FK的优势因子烟曲霉，我们已经证实 两个部分。首先，真菌蛋白酶在A.从受感染小鼠分离的烟曲霉 角膜，这表明真菌确实试图分解代谢基质蛋白。第二，A。烟曲霉突变体 蛋白酶分泌缺陷型的EscherichacA不能在模型中建立角膜感染。hacA基因 编码在未折叠蛋白反应（UPR）中起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， 通过内质网-高尔基体途径。该项目旨在跟踪这些基本观察结果，并可能 提升真菌UPR作为FK治疗的新靶点。在目标1中，我们将评估HacA在 感染的角膜首先，我们将确定与hacA突变体相关的细胞壁改变是否会影响 宿主-真菌相互作用和促炎信号传导。其次，我们将确定镇压的影响， hacA表达对疾病进展/消退的影响。具体来说，我们将用一种A. 其中hacA表达可以通过加入多西环素来抑制。通过这种方式， 受感染的角膜将用强力霉素进行“治疗”， 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4µ 8 C抑制Ire 1，Ire 1在HacA直系同源物的上游发出信号， UPR途径。由于UPR在细胞因子分泌中起着关键作用，因此4µ 8 C抑制了细胞因子的分泌。 炎症反应。我们进一步确定了A.烟曲霉是必不可少的， 生长，4µ 8 C在体外显示抗真菌作用。因此，我们将测试是否可以使用4µ 8 C 作为一个双刃剑的治疗，以阻止真菌生长和破坏性炎症在我们的小鼠FK模型。 最后，很明显，A.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没有特征。在目标3中，将使用染色质 HacA蛋白上的免疫沉淀（ChIP-seq）以及在细胞中生长后的RNA-seq（WT vs. 3D角膜模型。在这样做的过程中，我们将确定直接和间接控制下的UPR基因。的 这些基因的特性及其在角膜毒性中的作用将作为未来研究的基础。