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Published on: July 22, 2021
Cytopathic effect of Acanthamoeba on human corneal fibroblasts
Noriko Takaoka-Sugihara1, Satoru Yamagami, Seiichi Yokoo
1Department of Ophthalmology, Tokyo Women's Medical University Medical Center East, Tokyo Japan.
Purpose:
Acanthamoeba keratitis is associated with keratocyte depletion in humans. We investigated how Acanthamoebae isolated from corneas affected by Acanthamoeba keratitis interacted with human corneal stromal cells in vitro.
Methods:
Acanthamoebae were isolated from 6 patients with Acanthamoeba keratitis and genotyping was done. Whether the isolated Acanthamoebae could invade the corneal stroma was assessed with denuded corneal stroma ex vivo. The cytopathic effect of Acanthamoeba on cultured corneal fibroblasts from donor corneas was quantitatively evaluated by the MTT assay after culture under various conditions. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Annexin V staining were employed to detect apoptotic cells among the corneal fibroblasts co-cultured with Acanthamoebae.
Results:
All 6 Acanthamoebae isolated from the patients with Acanthamoeba keratitis were shown to have the T4 genotype by 18S rDNA sequence analysis. Acanthamoebae invaded the denuded corneal stroma in the ex vivo experiments and had a cytopathic effect on human corneal fibroblasts after direct adhesion, but not via chemical mediators. A cytopathic effect was detected with all 6 Acanthamoebae and corneal fibroblasts mainly died by apoptosis, as evidenced by Annexin V staining.
Conclusions:
Acanthamoebae isolated from patients with Acanthamoeba keratitis had a cytopathic effect on human corneal fibroblasts, mainly via induction of apoptosis after direct adhesion. Our findings may provide some clues to the pathophysiology of corneal keratocyte depletion in patients with Acanthamoeba keratitis.
Insights
Acanthamoeba keratitis (AK) causes human corneal cell death. Researchers found that Acanthamoebae from AK patients directly adhere to and induce apoptosis in corneal fibroblasts, explaining keratocyte depletion.
Area of Science:
- Ophthalmology
- Microbiology
- Cell Biology
Background:
- Acanthamoeba keratitis (AK) is a severe corneal infection.
- Keratocyte depletion is a hallmark of human AK.
- The precise mechanism of keratocyte loss in AK remains unclear.
Purpose of the Study:
- To investigate the in vitro interaction between Acanthamoebae from AK patients and human corneal stromal cells.
- To determine the mechanism by which Acanthamoebae affect corneal fibroblasts.
Main Methods:
- Acanthamoebae were isolated from 6 AK patients and genotyped (T4 genotype).
- Ex vivo corneal stroma invasion assays were performed.
- Cytopathic effects on cultured corneal fibroblasts were assessed using MTT assays.
- Apoptosis was detected via TUNEL and Annexin V staining.
Main Results:
- All isolated Acanthamoebae (T4 genotype) invaded corneal stroma ex vivo.
- Acanthamoebae caused direct cytopathic effects on corneal fibroblasts upon adhesion.
- Corneal fibroblasts primarily underwent apoptosis when co-cultured with Acanthamoebae.
Conclusions:
- Acanthamoebae from AK patients induce corneal fibroblast apoptosis through direct adhesion.
- This mechanism likely contributes to keratocyte depletion in Acanthamoeba keratitis.
- Findings offer insights into the pathophysiology of AK.
