The unfolded protein response as a therapeutic target for fungal keratitis

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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.