Aspergillus fumigatus Hypoxia 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 with Aspergillus fumigatus and Fusarium solani var. petroliphilum via topical overlay or intrastromal injection. At various time points post-inoculation (p.i.), animals were injected with pimonidazole for the detection of tissue hypoxia through immunofluorescence imaging. The A. fumigatus srbA gene 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 with A. fumigatus resulted in diffuse pimonidazole staining across the epithelial and endothelial layers within 6 hours. Stromal hypoxia was evident by 48 hours p.i., which corresponded to leukocytic infiltration. Intrastromal inoculation with either A. fumigatus or F. solani similarly led to diffuse staining patterns across all corneal cell layers. The A. fumigatus srbA deletion 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.

Immune Mechanisms of Filamentous Fungal Keratitis

“…They also observed that during the progression of the infection, as the corneal edema progressed, the degree of hypoxia increased, and the endothelium decreased. Later, in the following stages, the accumulation of fungal metabolites together with leukocyte ROS/cytokines caused further loss of endothelial integrity and subsequent entry of fluid from the anterior chamber [66].…”