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Background <p>Acanthamoeba keratitis (AK) is a severe ocular infection. The primary treatment is polyhexamethylene biguanide (PHMB), but its efficacy is limited due to cyst resistance. This study investigated the role of DNA methylation in <i>Acanthamoeba</i> encystation and the potential efficacy of combining 5-azacytidine (5-AzaC; a methyltransferase inhibitor) with PHMB treatment.</p> Methods <p>Encystation was induced in <i>Acanthamoeba castellanii</i> using encystation buffer, followed by calcofluor white staining. Gene expression levels of virulence genes and the methyltransferase gene were assessed by quantitative PCR. The cell viability assay of <i>A. castellanii</i> on Statens Seruminstitut Rabbit Cornea (SIRC) cells was evaluated to determine functional virulence. Overexpression of methyltransferase via electroporation was established to further investigate its function.</p> Results <p>qPCR results revealed upregulated methyltransferase expression during encystation. Calcofluor white staining showed significantly reduced cyst formation in <i>A. castellanii</i> treated with 5-AzaC. This treatment also induced significant transcriptional reduction in virulence genes. Consistently, 5-AzaC-treated <i>A. castellanii</i> did not exhibit increased cytotoxicity against SIRC cells. Overexpression of the methyltransferase increases the encystation ratio. Combination treatment with PHMB and 5-AzaC significantly decreased <i>A. castellanii</i> survival.</p> Conclusions <p>Inhibiting DNA methylation enhances the antiamoebic effects of PHMB, offering a potential AK treatment strategy. DNA methylation may serve as a critical regulatory switch in <i>A. castellanii</i>, governing both morphological transformation and virulence genes expression. This work expands our understanding of epigenetic regulation in protozoa.</p> Graphical Abstract <p></p>

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Enhanced antiamoebic efficacy of PHMB via methyltransferase inhibition to maintain drug sensitivity and block encystation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月4日

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  • Jui-Chi Wu,
  • Chun-Hsien Chen,
  • Jian-Ming Huang

摘要

Background

Acanthamoeba keratitis (AK) is a severe ocular infection. The primary treatment is polyhexamethylene biguanide (PHMB), but its efficacy is limited due to cyst resistance. This study investigated the role of DNA methylation in Acanthamoeba encystation and the potential efficacy of combining 5-azacytidine (5-AzaC; a methyltransferase inhibitor) with PHMB treatment.

Methods

Encystation was induced in Acanthamoeba castellanii using encystation buffer, followed by calcofluor white staining. Gene expression levels of virulence genes and the methyltransferase gene were assessed by quantitative PCR. The cell viability assay of A. castellanii on Statens Seruminstitut Rabbit Cornea (SIRC) cells was evaluated to determine functional virulence. Overexpression of methyltransferase via electroporation was established to further investigate its function.

Results

qPCR results revealed upregulated methyltransferase expression during encystation. Calcofluor white staining showed significantly reduced cyst formation in A. castellanii treated with 5-AzaC. This treatment also induced significant transcriptional reduction in virulence genes. Consistently, 5-AzaC-treated A. castellanii did not exhibit increased cytotoxicity against SIRC cells. Overexpression of the methyltransferase increases the encystation ratio. Combination treatment with PHMB and 5-AzaC significantly decreased A. castellanii survival.

Conclusions

Inhibiting DNA methylation enhances the antiamoebic effects of PHMB, offering a potential AK treatment strategy. DNA methylation may serve as a critical regulatory switch in A. castellanii, governing both morphological transformation and virulence genes expression. This work expands our understanding of epigenetic regulation in protozoa.

Graphical Abstract