Abstract
Purpose :
The poor visual outcomes associated with fungal keratitis (FK) is attributable, in part, to the ineffectiveness of current antifungals. The development of novel therapy requires the identification of putative targets in the fungus, which we reasoned include pathways involved in the assimilation of corneal collagen (protein) as a nutrient source. The fungal unfolded protein response (UPR) is critical in this regard as it maintains the fidelity of the secretion pathway and, consequently, the release of collagenases into the cornea. Accordingly, we tested the hypothesis that the UPR is essential for corneal virulence in a predominant agent of FK, Aspergillus fumigatus.
Methods :
The gene encoding HacA, a transcription factor essential for the UPR, was deleted in A. fumigatus using a CRISPR/Cas9 method, and the resulting mutant (hacA KO) was confirmed by PCR. Collagenase secretion was measured in culture supernatants using azocollagen hydrolysis (Millipore Sigma). Stress sensitivity assays were performed in rich (YPD) or minimal media as indicated. For the virulence studies, C57BL/6J mice were immunosuppressed with methylprednisolone (100mg/kg) on the days preceding and following infection. On the day of infection, corneas were abraded with an algerbrush and topically inoculated with germinated spores of the WT or hacA KO strains (n= 20/group). At 48 or 72 h post-inoculation, eyes were clinically scored and corneas were resected for histological analysis (PASH staining) or fungal burden assessment using colony-forming units (CFUs). WT and hacA KO data sets were compared by student’s T-test.
Results :
The A. fumigatus hacA KO mutant displayed hypersensitivity to stresses that induce protein misfolding, including increased temperature, DTT, and tunicamycin. The mutant was also deficient in secreted collagenase activity and could not grow on biological tissue ex vivo. Compared to mice infected with WT A. fumigatus, hacA KO-infected corneas displayed decreased clinical scores and fungal burden; this corresponded with the histopathology, which revealed reduced neutrophil infiltration and essentially no fungal invasion into the stroma.
Conclusions :
Our results confirm our hypothesis that the UPR is essential for A. fumigatus nutrient acquisition on protein-rich substrates as well as virulence in a murine model of FK. These data suggest that the fungal UPR could serve as a target for antifungal intervention.
This is a 2021 ARVO Annual Meeting abstract.