Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays

Abstract

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.

Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis

“…The cell integrity of the Acanthamoeba castellanii strains 1BU and SIN20 was monitored under various conditions using serum-free corneal epithelial cell medium (CEpiCM), CEpiCM with serum, and peptone yeast extract glucose (PYG) medium, as described in our previous study [ 23 ]. In brief, the amoeba count remained constant over 24 h, indicating that they stopped dividing in serum-free CEpiCM.…”

“…To investigate whether serine proteases play an important role in the induction of host cell death by Acanthamoeba , HCECs were treated with PMSF—a cell-permeable, irreversible inhibitor of serine proteases—before they were inoculated with the strains 1BUH3x and SIN20H3x. At 6 h post inoculation, the viability of HCECs was evaluated using the CellTiter 96 ® AQueous One Solution Reagent, which contains a tetrazolium salt, which is reduced to formazan by viable mammalian cells and not by Acanthamoeba [ 23 ]. The viability of untreated and treated HCECs co-cultured with Acanthamoeba was assessed by measuring the quantity of formazan produced by the mammalian host cells (Fig.…”

“…The transient nature of the observed proteolytic activities is particularly interesting. In our previous study, we showed that the Acanthamoeba strains 1BU and SIN20 were able to reduce cell viability by 32.31% and 21.40% within 4 h and 6.36% and 4.6% within 8 h of co-culture with HCECs, respectively [ 23 ]. Thus, the comparably short time of activity of the novel proteases found in the current study may be attributed to the reduced viability of the HCECs during co-culture with amoebae, again leading to a decrease in protease secretion by the amoebae.…”

“…The pathogenicity of untreated Acanthamoeba and Acanthamoeba treated with serine protease inhibitors and their effect on the viability of HCECs were evaluated using the CellTiter 96 ® AQueous One Solution Reagent as previously described [ 23 ]. Phase contrast microscopy was used to observe the effect of this inhibition; a Nikon Eclipse E800 microscope with a Nikon DS-Fi2 camera (Nikon Optoteam, Vienna, Austria) was used for microphotography.…”