Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Nov 12:14:1477463.
doi: 10.3389/fcimb.2024.1477463. eCollection 2024.

The mammalian Ire1 inhibitor, 4µ8C, exhibits broad anti- Aspergillus activity in vitro and in a treatment model of fungal keratitis

Manali M Kamath  1 , Emily M Adams  2 , Jorge D Lightfoot  2 , Becca L Wells  1 , Kevin K Fuller  1   2   3
Affiliations

The mammalian Ire1 inhibitor, 4µ8C, exhibits broad anti- Aspergillus activity in vitro and in a treatment model of fungal keratitis

Manali M Kamath et al. Front Cell Infect Microbiol. .

Abstract

Objective: The fungal unfolded protein response consists of a two-component relay in which the ER-bound sensor, IreA, splices and activates the mRNA of the transcription factor, HacA. Previously, we demonstrated that hacA is essential for Aspergillus fumigatus virulence in a murine model of fungal keratitis (FK), suggesting the pathway could serve as a therapeutic target. Here we investigate the antifungal properties of known inhibitors of the mammalian Ire1 protein both in vitro and in a treatment model of FK.

Methods: The antifungal activity of Ire1 inhibitors was tested against conidia of several A. fumigatus isolates by a broth microdilution assay and against fungal biofilm by XTT reduction. The influence of 4μ8C on hacA mRNA splicing in A. fumigatus was assessed through gel electrophoresis and qRT-PCR of UPR regulatory genes. The toxicity and antifungal profile of 4μ8C in the cornea was assessed by applying drops to uninfected or A. fumigatus-infected corneas 3 times daily starting 4 hours post-inoculation. Corneas were evaluated daily through slit-lamp imaging and optical coherence tomography, or at endpoint through histology or fungal burden quantification via colony forming units.

Results: Among six Ire1 inhibitors screened, the endonuclease inhibitor 4μ8C displayed the strongest antifungal profile with an apparent fungicidal action. The compound both blocked conidial germination and hyphal metabolism of A. fumigatus Af293 in the same concentration range that blocked hacA splicing and UPR gene induction (60-120 µM). Topical treatment of sham-inoculated corneas with 0.5 and 2.5 mM 4μ8C did not impact corneal clarity, but did transiently inhibit epithelialization of corneal ulcers. Relative to vehicle-treated Af293-infected corneas, treatment with 0.5 and 2.5 mM drug resulted in a 50% and >90% reduction in fungal load, respectively, the latter of which corresponded to an absence of clinical signs of infection or corneal pathology.

Conclusion: The in vitro data suggest that 4μ8C displays antifungal activity against A. fumigatus through the specific inhibition of IreA. Topical application of the compound to the murine cornea can furthermore block the establishment of infection, suggesting this class of drugs can be developed as novel antifungals that improve visual outcomes in FK patients.

Keywords: 4μ8C; Aspergillus fumigatus; IRE1 inhibitors; antifungals; fungal keratitis.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
4µ8C displays antifungal activity against A. fumigatus conidia and hyphae in vitro. (A) Conidia of the indicated strains were inoculated into GMM broth containing drug and incubated for 72 h at 35°C. The minimal inhibitory concentration (MIC) for at least three independent experiments are plotted and groups were compared by Two-way ANOVA, ***<0.0005. (B) Following 72 h incubation of Af293 in a broth microdilution assay described in panel (A), 100 µL aliquots were spread onto YPD plates and incubated for 24 h at 35°C. The data reflect the mean colony counts from triplicate wells of a single broth microdilution experiment and the groups were compared by Two-way ANOVA, ***<0.0005, ****<0.0001. (C) Af293 biofilms generated in GMM were treated with vehicle (DMSO) or the indicated concentration of 4µ8C for 2 h at 35°C. Metabolic activity was measured by XTT reduction. The data reflect the mean of 490 nm absorbance (± SD) of triplicate wells in single experiment and groups were compared by Two-way ANOVA **** <0.0001. Similar results were recapitulated across three independent experiments.
Figure 2
Figure 2
The antifungal activity of 4µ8C corresponds to an inhibition of IreA activity in Af293. Af293 biofilms generated overnight in GMM were pre-treated with 4µ8C or DMSO for 2 h and subsequently spiked with 10 mM DTT and incubated for an additional 2 h prior to total RNA isolation and cDNA synthesis. (A) The full-length hacA message was amplified and the hacAu (uninduced: 665 bp) and hacAi (induced: 645 bp) products were resolved by gel electrophoresis. The end point hacA PCR products were sequenced and a CLUSTAL multiple sequence alignment was performed in the regions spanning the canonical/spliceosome intron highlighted in blue (B) and the IreA-targeted non-canonical intron highlighted in green (C). (D) qPCR was performed on total hacA as well as two chaperone encoding genes, bipA and pdiA. The qPCR data reflect the mean 2ΔΔCt calculations for triplicate fungal samples from a single experiment and groups were compared by Two-way ANOVA, ****<0.0001. Similar results were observed in an independent experiment.
Figure 3
Figure 3
High dose 4µ8C treatment does not impact corneal clarity but does transiently inhibit re-epithelialization. Sham-inoculated (UI) corneas were treated with topical drops of 2.5 mM 4µ8C or vehicle (DMSO), once on the day on ulceration (4 h p.i.), three times (4 h apart) the following three days. (A) Representative external images taken each day post-ulceration. (B) Corneal thickness was measured daily based on OCT images (n = 6/group). Groups were compared by Two-way ANOVA; (C) Representative histological (H&E) sections taken at 72 h post-ulceration; 400X magnification. The arrows are highlighting the epithelial ‘E’ and the stromal ‘S’ layers. (D) In a separate experiment, ulcerated eyes were treated as described above and on each day the penetration of fluorescein was imaged by a fluorescent slit-lamp (Micron IV). The red arrow indicates that the treatment was stopped, and the eyes were monitored for an additional three days.
Figure 4
Figure 4
Topical treatment with 4µ8C blocks fungal growth and disease establishment in a murine model of FK. Sham (UI) or Af293-inoculated (FK) corneas were treated with 2.5 mM 4μ8C or vehicle for up to 72 h p.i. as described in the Figure 3 legend. The data in all panels reflect a pool of two independent experiments (n=6 per UI group; n=13 per FK group). (A) Representative external images taken each day p.i. (B) Average clinical scores at 72 h p.i.; Groups were compared by Ordinary one-way ANOVA p-value **** <0.0001; (C) Fungal burden at 72 h p.i.; Groups were compared by Ordinary one-way ANOVA p-value ** 0.0061, * 0.0173; (D) Representative OCT scans of UI and FK corneas treated with 2.5 mM 4μ8C or vehicle at 48 and 72 h p.i. (E) Average corneal thickness measured at 72 h p.i.. Groups were compared by Ordinary one-way ANOVA p-value **** <0.0001.
See this image and copyright information in PMC

Update of

Similar articles

See all similar articles

References

    1. Acharya Y., Acharya B., Karki P. (2017). Fungal keratitis: study of increasing trend and common determinants. Nepal J. Epidemiol. 7, 685–693. doi: 10.3126/nje.v7i2.17975 - DOI - PMC - PubMed
    1. Altamirano D., Martinez J., Leviste K. D., Parel J. M., Amescua G. (2020). Photodynamic Therapy for Infectious Keratitis. Curr Ophthalmol Rep. 8, 245–251. doi: 10.1007/s40135-020-00252-y - DOI - PMC - PubMed
    1. Ansari Z., Miller D., Galor A. (2013). Current thoughts in fungal keratitis: Diagnosis and treatment. Curr. Fungal Infect. Rep. 7, 209–218. doi: 10.1007/s12281-013-0150-1 - DOI - PMC - PubMed
    1. Atta S., Perera C., Kowalski R. P., Jhanji V. (2022). Fungal keratitis: clinical features, risk factors, treatment, and outcomes. J. Fungi. 8 (9), 962. doi: 10.3390/jof8090962 - DOI - PMC - PubMed
    1. Awasthi D., Chopra S., Cho B. A., Emmanuelli A., Sandoval T. A., Hwang S.-M., et al. . (2023). Inflammatory ER stress responses dictate the immunopathogenic progression of systemic candidiasis. J. Clin. Invest. 133 (17), e167359. doi: 10.1172/JCI167359 - DOI - PMC - PubMed

MeSH terms

LinkOut - more resources