The unfolded protein response as a therapeutic target for fungal keratitis

项目总结 真菌性角膜炎(FK)已成为世界范围内眼部疾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，但它们的开发首先需要对真菌的了解 可以作为药物靶标的蛋白质/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)真菌在感染期间分解这些蛋白质，作为主要的营养来源，并且 (2)支持蛋白质分解代谢的真菌途径(例如，蛋白酶分泌)代表重要的毒力 因素和可能的药物靶点。使用FK的优势制剂烟曲霉，我们已经证实 两部分都有。首先，真菌蛋白水解酶在感染小鼠体内的表达上调 这表明这种真菌确实在试图分解基质蛋白。第二，烟曲霉变异体 蛋白酶分泌缺陷的∆HACA无法在该模型中建立角膜感染。HACA基因 编码一种转录因子，在未折叠蛋白反应(UPR)中发挥关键作用，UPR是一条 检测并解决错误折叠的蛋白质在内质网中的堆积，促进运输 通过ER-高尔基体途径。该项目寻求跟踪这些基本观察结果，并潜在地 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 被感染的角膜。首先，我们将确定与HACA突变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。第二，我们将确定镇压的影响 HACA在疾病进展/解决中的表达。具体地说，我们将用一种A菌株感染老鼠。 可通过添加强力霉素抑制HACA表达的烟曲霉菌。就这样， 感染的角膜将被多西环素“治疗”，以及对真菌生长、炎症和角膜的影响 将对损害进行监测。在目标2中，我们将测试将哺乳动物的UPR抑制剂重新用于 治疗FK。目标化合物4µ8C抑制IRE1，IRE1信号位于HACA直系同源基因的上游 UPR途径。由于UPR在细胞因子的分泌中起着关键作用，因此4µ8C抑制了 炎症反应。我们进一步证实了烟曲霉菌中的IRE1同源基因对于 4µ8C在体外表现出抗真菌作用。因此，我们将测试是否可以使用4µ8C 作为一种双刃式治疗方法，在我们的小鼠FK模型中既能阻止真菌生长，又能抑制破坏性炎症。 最后，很明显，烟曲霉UPR调节下游对角膜至关重要的基因/蛋白。 毒力，但这些目标在很大程度上仍未确定。在AIM 3中，将使用两种染色质 对HacA蛋白的免疫沉淀(ChIP-seq)以及rna-seq(WT与∆HacA的比较) 三维角膜模型。在这样做的过程中，我们将识别UPR直接和间接控制下的基因。这个 这些基因的特征及其在角膜毒力中的作用将成为未来研究的基础。

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.