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

基本信息

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
项目类别：
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

项目摘要

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）已成为全世界眼部发病率和单侧失明的主要来源。尽管迫切需要更好的抗真菌剂，但他们的发展首先需要了解真菌可以用作药物靶标的蛋白质/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，但表面上的葡萄糖或可自由扩散的营养较差。我们因此，（1）真菌分解这些蛋白质是感染过程中主要营养源的主要营养源，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病毒因素和推定的药物靶标。我们使用FK的主要主体，曲霉烟曲霉，我们已经确认这两个部分。首先，真菌蛋白酶表达在从感染的小鼠中分离出的烟曲霉上上调角膜，表明真菌确实在试图分解基质蛋白。第二，烟曲霉突变体蛋白酶分泌有缺陷的∆HACA无法在模型中建立角膜感染。 HACA基因编码转录因子在展开的蛋白质反应（UPR）中起关键作用，这是一种途径检测并解决内质网中错误折叠蛋白的积累并促进交通通过ER-Golgi途径。该项目旨在跟进这些基本观察，并可能将真菌UPR提升为FK治疗的新目标。在AIM 1中，我们将评估HACA内部的作用感染的角膜。首先，我们将确定与HACA突变体影响相关的细胞壁改变宿主 - 企业相互作用和促炎信号传导。其次，我们将确定反思的影响 HACA表达疾病进展/解决方案。具体而言，我们将用A菌株感染小鼠。可以通过添加强力霉素来反射HACA表达的富马图斯。这样，受感染的角膜将以强力霉素的“治疗”以及对真菌生长，感染和角膜的影响损坏将被监视。在AIM 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抑制了IRE1，它在HACA直系同源的上游信号 UPR路径。由于UPR在细胞因子分泌中起着关键作用，因此4µC抑制了炎症反应。我们进一步确定，烟曲霉中的IRE1直系同源物对于生长，4µ8C在体外显示抗真菌作用。彼此之间，我们将测试是否可以使用4µ8C 作为双刃治疗，可阻止我们的小鼠FK模型中真菌生长和破坏感染。最后，很明显，烟曲霉UPR调节对角膜至关重要的下游基因/蛋白病毒，但这些靶标在很大程度上仍然没有特征。在AIM 3中，将使用两种染色质 HACA蛋白上的免疫沉淀（CHIP-SEQ）以及A的RNA-Seq（WT与∆HACA）在A中生长后 3D角膜模型。在此过程中，我们将在直接和间接控制UPR的控制下识别基因。这这些基因的表征及其在角膜病毒中的作用将成为未来探究的基础。