Abstract
Purpose :
For internal organs, such as the lung, fungal infection drives the development of tissue hypoxia due to inflammation, vascular occlusion and necrosis. It is unknown however whether the cornea –a thin tissue with ostensibly good access to atmospheric oxygen– similarly becomes hypoxic during fungal keratitis (FK). In this study, we tested development of corneal hypoxia, as well as the role of fungal hypoxia adaptation, in the pathogenesis of Aspergillus fumigatus keratitis.
Methods :
C57BL/6J mice were immunosuppressed with methylprednisolone on the day preceding inoculation. The next day, corneas were abraded with an algerbrush and topically inoculated with germinated A. fumigatus spores. To measure tissue hypoxia, animals were injected with pimonidazole (Hypoxyprobe) 90 min prior to tissue harvest. Ocular sections were imaged by fluorescence microscopy following staining with anti-hypoxyrprobe or anti-Aspergillus antibodies. The srbA gene of A. fumigatus was deleted via a CRISPR-Cas9 approach. For the virulence studies, animals were inoculated with wild-type (WT) or the ΔsrbA mutant and eyes were imaged daily by slit-lamp and optical coherence tomography (OCT). At 24 and 48 h post-inoculation (p.i.), corneas were isolated for histopathology (PASH stain), fungal burden (CFUs) or flow cytometry.
Results :
In corneas infected with A. fumigatus, positive hypoxyprobe staining was observed as early as 12 h p.i. and this signal intensity increased 24 and 48 h p.i. Given these results, we hypothesized that a key regulator of the hypoxic response in A. fumigatus, SrbA, would regulate growth within the infected cornea. We first deleted the srbA gene and, consistent with previous reports, found the mutant was unable to grow in vitro at oxygen levels below 3%. In contrast to corneas infected with WT A. fumigatus, those infected with ΔsrbA failed to develop signs of disease (opacification or ulceration), displayed normal corneal structure by OCT, and did not harbor viable fungus. This corresponded with histopathology and flow cytometry, which revealed a lack of inflammatory cells in ΔsrbA-infected corneas.
Conclusions :
Taken together, these results support a model in which fungal antigen rapidly drives the development of corneal hypoxia, thereby rendering SrbA essential for fungal growth and virulence. Consequently, proteins essential for fungal hypoxia adaptation could serve as targets for novel FK therapy.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.