Fungal keratitis is an important source of ocular morbidity and unilateral blindness worldwide. Current antifungal regimes fails in up to 60% of patients, resulting in the need for at least one and sometimes repeated corneal transplants. Novel antifungals are therefore required, but their development requires a better understanding of fungal proteins/enzymes that could serve as drug targets. As the corneal stroma is effectively an extracellular matrix comprised of collagen and other proteins, we hypothesize that pathways that support fungal protein catabolism are essential for fungal growth in the eye and, by extension, virulence factors that might be targeted in treatment. In order for fungi to utilize proteins as a nutritional source, they must first secrete copious amounts of proteases into the environment/host tissue. This secretory burden leads to an accumulation of unfolded proteins within the endoplasmic reticulum (ER) that, if not resolved, leads to a ?clogging? of the secretion pathway that will severely inhibit fungal growth. The unfolded protein response (UPR) plays a critical role in this regard by first sensing unfolded proteins and subsequently regulating genes that that promote the protein folding capacity within the ER lumen (e.g., chaperones).
In Aim 1, we will test the hypothesis that fungal UPR promotes the corneal pathogenesis of a common agent of keratitis, Fusarium solani. We will first generate UPR-deficient mutants of F. solani and then test whether the mutants are defective for growth on protein substrates as we predict. We will then assess the virulence of the mutants in a mouse model of fungal keratitis. The observation that the UPR-deficient strains are hypovirulent would suggest that inhibitors of the UPR could be used as novel antifungals. The transcriptional profile of a fungus varies largely as a function of the nutrient source. The transition from glucose-rich to glucose-limiting media, for example, leads to a down-regulation of glycolytic genes, upregulation of secreted hydrolases, and an upregulation of metabolic enzymes involved in amino acid metabolism. Therefore, in Aim 2, we will test the hypothesis that the F. solani utilizes proteins in the cornea by comparing the transcriptome of the fungus in vivo (from infected eyes) against the transcriptome of the fungus grown under defined nutrient conditions (collagen v. glucose) in vitro. We predict that the most highly expressed genes in vivo will mirror the most highly expressed genes on collagen. However, we do not expect a one-to-one correspondence between the two datasets due to environmental conditions that are unique to the eye, such as stresses imparted by the inflammatory response. In this way, we stand to gain novel insight into the fungal adaptive response during keratitis infection, which will lead to the identification of novel virulence genes and putative drug targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Exploratory Grants (P20)
Project #
5P20GM134973-02
Application #
10114313
Study Section
Special Emphasis Panel (ZGM1)
Project Start
2020-03-01
Project End
2025-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
2
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Type
DUNS #
878648294
City
Oklahoma City
State
OK
Country
United States
Zip Code
73104