Fungal infections of ophthalmic (or ocular) tissue are an important cause of morbidity and can result in blindness, and most common manifestations of ocular infections affect the cornea, resulting in keratitis. Fungal keratitis is predominantly caused by filamentous fungi Fusarium, but yeasts (e.g. Candida) can also cause this disease. Risk factors for mycotic keratitis include trauma, ocular and systemic defects and prior application of corticosteroids. Additionally, contact lens wear is also associated with Fusarium keratitis. Fusarium keratitis is not responsive to most antifungal agents, necessitating penetrating keratoplasty, with a high risk of failure and endophthalmitis. The ability of Fusarium to form biofilms on contact lenses/lens cases was suggested to be one of the factors that contributed to the recent Fusarium keratitis outbreak. These findings underscore the need for detailed investigations into the biology and pathogenesis of Fusarium keratitis. In preliminary studies, we showed that: (1) Fusarium forms biofilms on soft contact lenses, which were resistant to lens cleaning solutions, (2) Fusarium biofilms formed on silicone hydrogel contact lenses induce keratitis on injured corneas in our murine model of contact lens associated Fusarium keratitis, (3) outbreak isolates of Fusarium form robust biofilms that are resistant to antifungal agents, and (4) a direct correlation was observed between the ability of outbreak isolates to form biofilms and disease severity in the murine keratitis model, (5) proteomics analysis identified 29 proteins that were differentially expressed in Fusarium biofilms compared to planktonically grown cells, (6) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the most overexpressed among these proteins (by 9-fold) in Fusarium biofilms, (7) inhibition of GAPDH resulted in attenuated biofilms, (8) biofilms contained higher number of apoptotic cells, and (9) GAPDH is translocated from the cytosol to the nucleus in fungal biofilms. In this application, we decided to investigate the role of GAPDH in biofilm formation because: (a) it is the most over-expressed protein in both Fusarium and Candida biofilms, suggesting that it may be a common regulator of fungal biofilms, (b) GAPDH was overexpressed in ocular tissues obtained from patients with fungal keratitis, and (c) in addition to its enzymatic activity, GAPDH also exhibits non-enzymatic activities that modulate key cellular processes (e.g. apoptosis, intracellular signaling). Based on these observations, we hypothesize that GAPDH acts as a modulator of Fusarium biofilms. We propose the following specific aims:
Aim I. Use a biochemical approach to determine the role of enzymatic and non-enzymatic activities of GAPDH in Fusarium biofilm formation.
Aim II. Use a molecular approach to (a) construct isogenic mutant strains of Fusarium lacking GAPDH, and (b) determine the effect of targeted gene disruption and overexpression on Fusarium biofilms.
Fusarium keratitis is an important cause of morbidity and can result in blindness, and was associated with a recent global outbreak. The ability of Fusarium to form biofilms is believed to be one of the factors that contributed to this outbreak, and we found that these biofilms overproduce the glycolytic proteins GAPDH. Studies proposed in the current application will reveal the role of GAPDH in Fusarium biofilms, and may facilitate the use of this protein as a potential biomarker for biofilm infections.
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