Aspergillus fumigatushypoxia adaptation is critical for the establishment of fungal keratitis

Abstract

Purpose: The poor visual outcomes associated with fungal keratitis (FK) underscore a need to identify fungal pathways that can serve as novel antifungal targets. In this report, we investigated whether hypoxia develops in the FK cornea and, by extension, if fungal hypoxia adaptation is essential for virulence in this setting. Methods: C57BL/6j mice were inoculated withAspergillus fumigatusandFusarium solanivarpetroliphilumvia topical overlay or instrastromal injection. At various time points post-inoculation (p.i.), animals we were injected with pimonidazole for the detection of tissue hypoxia through immunofluorescence imaging. TheA. fumigatus srbAgene was deleted through Cas9-mediated homologous recombination and its virulence was assessed in the topical infection model using slit-lamp microscopy and optical coherence tomography (OCT). Results: Topical inoculation withA. fumigatusresulted in diffuse pimonidazole staining across the epithelial and endothelial layers within 6 h. Stromal hypoxia was evident by 48 h p.i., which corresponded to leukocytic infiltration. Instrastromal inoculation with eitherA. fumigatusorF. solanisimilarly led to diffuse staining patterns across all corneal cell layers. TheA. fumigatus srbAdeletion mutant was unable to grow at oxygen levels below 3%in vitro,and corneas inoculated with the mutant failed to develop signs of corneal opacification, inflammation or fungal burden. Conclusions: These results suggest that fungal antigen rapidly drives the development of corneal hypoxia, thus rendering fungal SrbA or related pathways essential for the establishment of infection. Such pathways may therefore serve as targets for novel antifungal intervention.