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A high-throughput, image-based assay to assess drug sensitivity of Acanthamoeba castellanii cysts
Abstract:
Pathogenic free-living amoebae (FLA) such as Acanthamoeba castellanii are present in soil and water worldwide. A. castellanii causes systemic infections with very high mortality rates, yet drugs specifically targeting this pathogen are not available. Methods to reliably generate and assay cysts, which drive infection recurrence and drug resistance, are unavailable in a high-throughput format suitable for drug screening and testing. In this study, we developed a robust and reproducible protocol for encysting A. castellanii as well as a high-throughput, quantitative cyst viability assay using fluorescent live/dead staining coupled with microscopy and automated image analysis. These methods were used to screen the cysticidal activity of 17 clinically relevant drugs and disinfectants and identified five agents, including caspofungin, as active against cysts.
Insights
New methods enable high-throughput screening of drugs against pathogenic Acanthamoeba castellanii cysts. Five agents, including caspofungin, showed cysticidal activity, offering hope for treating resistant infections.
Area of Science:
- Microbiology
- Infectious Diseases
- Drug Discovery
Background:
- Pathogenic free-living amoebae (FLA), notably Acanthamoeba castellanii, are ubiquitous environmental pathogens.
- A. castellanii causes severe systemic infections with high mortality, and lacks specific anti-amoebic drugs.
- Current methods for generating and assaying infectious Acanthamoeba cysts are not suitable for high-throughput drug screening.
Purpose of the Study:
- To develop a reproducible, high-throughput protocol for Acanthamoeba encystment.
- To establish a quantitative cyst viability assay for drug screening.
- To screen clinically relevant drugs and disinfectants for cysticidal activity.
Main Methods:
- Developed a robust protocol for inducing Acanthamoeba castellanii encystment.
- Implemented a high-throughput, quantitative assay using fluorescent live/dead staining.
- Utilized automated image analysis for cyst viability assessment.
- Screened 17 drugs and disinfectants for activity against Acanthamoeba cysts.
Main Results:
- Successfully established a reproducible encystment protocol and a high-throughput cyst viability assay.
- Identified five agents with significant cysticidal activity against Acanthamoeba cysts.
- Caspofungin was among the identified agents effective against Acanthamoeba cysts.
Conclusions:
- The developed methods provide a powerful tool for screening anti-amoebic compounds.
- The identified cysticidal agents, including caspofungin, warrant further investigation for treating Acanthamoeba infections.
- This work addresses a critical need for effective treatments against drug-resistant Acanthamoeba cysts.
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