Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays
BackgroundIn vitro models for studying interactions between Acanthamoeba and host cells are crucial for understanding the pathomechanism of Acanthamoeba and assessing differences between strains and cell types. The virulence of Acanthamoeba strains is usually assessed and monitored by using cell cyt...
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| Format: | Article |
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1175469/full |
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| author | Alvie Loufouma Mbouaka Iwona Lesiak-Markowicz Irene Heredero-Bermejo Rounik Mazumdar Julia Walochnik Tania Martín-Pérez |
| author_facet | Alvie Loufouma Mbouaka Iwona Lesiak-Markowicz Irene Heredero-Bermejo Rounik Mazumdar Julia Walochnik Tania Martín-Pérez |
| author_sort | Alvie Loufouma Mbouaka |
| collection | DOAJ |
| description | BackgroundIn vitro models for studying interactions between Acanthamoeba and host cells are crucial for understanding the pathomechanism of Acanthamoeba and assessing differences between strains and cell types. The virulence of Acanthamoeba strains is usually assessed and monitored by using cell cytotoxicity assays. The aim of the present study was to evaluate and compare the most widely used cytotoxicity assays for their suitability to assess Acanthamoeba cytopathogenicity.MethodsThe viability of human corneal epithelial cells (HCECs) after co-culture with Acanthamoeba was evaluated in phase contrast microscopy.ResultsIt was shown that Acanthamoeba is unable to considerably reduce the tetrazolium salt and the NanoLuc® Luciferase prosubstrate to formazan and the luciferase substrate, respectively. This incapacity helped to generate a cell density-dependent signal allowing to accurately quantify Acanthamoeba cytotoxicity. The lactate dehydrogenase (LDH) assay led to an underestimation of the cytotoxic effect of Acanthamoeba on HCECs since their co-incubation negatively affected the lactate dehydrogenase activity.DiscussionOur findings demonstrate that cell-based assays using the aqueous soluble tetrazolium-formazan, and the NanoLuc® Luciferase prosubstrate products, in contrast to LDH, are excellent markers to monitor the interaction of Acanthamoeba with human cell lines and to determine and quantify effectively the cytotoxic effect induced by the amoebae. Furthermore, our data indicate that protease activity may have an impact on the outcome and thus the reliability of these tests. |
| first_indexed | 2024-04-09T16:06:33Z |
| format | Article |
| id | doaj.art-9b93c7c3eb69460982ae3679710e8740 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-09T16:06:33Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-9b93c7c3eb69460982ae3679710e87402023-04-25T05:23:39ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-04-011410.3389/fmicb.2023.11754691175469Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assaysAlvie Loufouma Mbouaka0Iwona Lesiak-Markowicz1Irene Heredero-Bermejo2Rounik Mazumdar3Julia Walochnik4Tania Martín-Pérez5Center for Pathophysiology, Infectiology and Immunology, Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, AustriaCenter for Pathophysiology, Infectiology and Immunology, Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, AustriaDepartment of Biomedicine and Biotechnology, Faculty of Pharmacy, University of Alcalá, Madrid, SpainMax Perutz Labs Vienna, Department of Medical Biochemistry, Medical University of Vienna, Vienna, AustriaCenter for Pathophysiology, Infectiology and Immunology, Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, AustriaCenter for Pathophysiology, Infectiology and Immunology, Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, AustriaBackgroundIn vitro models for studying interactions between Acanthamoeba and host cells are crucial for understanding the pathomechanism of Acanthamoeba and assessing differences between strains and cell types. The virulence of Acanthamoeba strains is usually assessed and monitored by using cell cytotoxicity assays. The aim of the present study was to evaluate and compare the most widely used cytotoxicity assays for their suitability to assess Acanthamoeba cytopathogenicity.MethodsThe viability of human corneal epithelial cells (HCECs) after co-culture with Acanthamoeba was evaluated in phase contrast microscopy.ResultsIt was shown that Acanthamoeba is unable to considerably reduce the tetrazolium salt and the NanoLuc® Luciferase prosubstrate to formazan and the luciferase substrate, respectively. This incapacity helped to generate a cell density-dependent signal allowing to accurately quantify Acanthamoeba cytotoxicity. The lactate dehydrogenase (LDH) assay led to an underestimation of the cytotoxic effect of Acanthamoeba on HCECs since their co-incubation negatively affected the lactate dehydrogenase activity.DiscussionOur findings demonstrate that cell-based assays using the aqueous soluble tetrazolium-formazan, and the NanoLuc® Luciferase prosubstrate products, in contrast to LDH, are excellent markers to monitor the interaction of Acanthamoeba with human cell lines and to determine and quantify effectively the cytotoxic effect induced by the amoebae. Furthermore, our data indicate that protease activity may have an impact on the outcome and thus the reliability of these tests.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1175469/fullAcanthamoebahuman corneal epithelial cellspathogenesisamoeba-host cell interactionviabilitycytotoxicity |
| spellingShingle | Alvie Loufouma Mbouaka Iwona Lesiak-Markowicz Irene Heredero-Bermejo Rounik Mazumdar Julia Walochnik Tania Martín-Pérez Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays Frontiers in Microbiology Acanthamoeba human corneal epithelial cells pathogenesis amoeba-host cell interaction viability cytotoxicity |
| title | Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays |
| title_full | Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays |
| title_fullStr | Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays |
| title_full_unstemmed | Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays |
| title_short | Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays |
| title_sort | assessing acanthamoeba cytotoxicity comparison of common cell viability assays |
| topic | Acanthamoeba human corneal epithelial cells pathogenesis amoeba-host cell interaction viability cytotoxicity |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1175469/full |
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