June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Exploring the unfolded protein response as a novel therapeutic target for fungal keratitis.
Author Affiliations & Notes
  • Kevin K Fuller
    Ophthalmology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
    Microbiology & Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
  • Manali M Kamath
    Microbiology & Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
  • Emily M Adams
    Ophthalmology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
  • Footnotes
    Commercial Relationships   Kevin Fuller None; Manali Kamath None; Emily Adams None
  • Footnotes
    Support  1R01EY033866-01
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3794. doi:
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      Kevin K Fuller, Manali M Kamath, Emily M Adams; Exploring the unfolded protein response as a novel therapeutic target for fungal keratitis.. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3794.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The fungal unfolded protein response (UPR) is a two-component relay initiated by ER-bound IreA, which splices and activates the mRNA of the transcription factor HacA. We have shown that the hacA deletion mutant in the mold Aspergillus fumigatus is unable to establish infection in a murine model of fungal keratitis (FK). Here, we test the activity of a known inhibitor of the mammalian IreA ortholog against the fungus and explore its utility as a novel therapeutic for FK in mice.

Methods : The antifungal activity of 4μ8C against various Aspergillus and was tested by a micro broth dilution assay and against fungal biofilm by XTT reduction. The influence of 4μ8C on fungal gene expression was determined through aRT-PCR and on hacA mRNA splicing by gel electrophoresis and sequencing of the cloned hacA cDNA. Toxicity of 4μ8C tested on cultured human epithelial cells through lactate dehydrogenase release, and in murine corneas through histology, ocular coherence tomography (OCT), and fluorescein staining. The antifungal activity of 4μ8C in vivo was evaluated by treating murine eyes infected with WT A. fumigatus topically 3 times/day starting 1 day post-infection with either PBS or 4μ8C (n= 10/group). At 72 h post-inoculation, eyes were clinically scored and corneas were resected for fungal burden assessment using colony forming units (CFUs).

Results : The compound 4μ8C inhibited A. fumigauts growth and killed fungal biofilms at the same concentrations that blocked the ribonuclease activity of IreA, suggesting the protein is essential for growth. Indeed, attempts to isolate an ireA knockout were unsuccessful and repression of ireA expression with a tetracycine-repressible promoter almost completely blocked hyphal proliferation. The compound furthermore led to a significan reduction in fungal burden when applied topically to FK mice several times a day. 4μ8C was not toxic to corneal epithelial cells in culture and did not impact corneal clarity in healthy controls, although re-epithelization of corneal ulcers was transiently inhibited.

Conclusions : Taken together, our results confirm that the UPR is essential for A. fumigatus to establish infection and its inhibition with the Ire1 inhibitor, 4μ8C, can significantly reduce fungal growth in the murine model of FK. This suggests that such inhibitors can be developed for the treatment of FK.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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