Parasitology Research (2019) 118:641–651 https://doi.org/10.1007/s00436-018-6192-0 PROTOZOOLOGY - ORIGINAL PAPER G418 induces programmed cell death in Acanthamoeba through the elevation of intracellular calcium and cytochrome c translocation Zisis Koutsogiannis1 & Ewan T. MacLeod2 & Sutherland K. Maciver1 Received: 22 October 2018 /Accepted: 19 December 2018 /Published online: 7 January 2019 # The Author(s) 2019 Abstract Acanthamoeba is a widely distributed opportunistic parasite which causes a vision-threatening keratitis and a life-threatening encephalitis. The cyst stage of this amoeba is especially resistant to currently used therapeutics and so alternative agents are urgently required. Growing evidence supports the existence of a programmed cell death system (PCD) in Acanthamoeba and while some features are shared by higher eukaryote cells, others differ. It is hoped that by understanding these differences we can exploit them as targets for novel drug intervention to activate PCD pathways in the amoebae but not the invaded human tissue. Here, we use the aminoglycoside G418 to activate PCD in Acanthamoeba. This drug caused a shape change in the treated amoebae. Cells rounded up and contracted, and after 6 h fragments of cells resembling the ‘apoptotic bodies’ of vertebrate cells were observed. G418 causes an increase in intracellular calcium from a resting level of 24 nM to 60 nM after 6 h of treatment. Mitochondrial function as assayed by the ΔΨm reporting dye JC-1 and CTC a redox dye becomes inhibited during treatment and we have found that cytochrome c is released from the mitochondria. Cells stained with Hoechst showed first an alteration in chromatin structure and then a vesiculation of the nucleus with G418 treatment, although we found no obvious breakdown in genomic DNA in the early stages of PCD. Keywords Acanthamoeba . Amoebozoa . Programmed cell death . Apoptosis . G418 . Cytochrome c Introduction et al. 2002; Corsaro and Venditti 2018) based on 18S rRNA gene sequences which equates approximately to classification Acanthamoeba is a eukaryotic, opportunistic parasitic proto- at the species level (Hewett et al. 2003; Fuerst 2014). While zoan, which is widely distributed in the natural environment some named species accord with this system, others do not. (Geisen et al. 2014). Although species classification has not The most commonly encountered genotype is T4, and while it been completely formalized, the genus has been divided into is the most frequently isolated from the environment, it is also 21 different genotypes, T1-T21 (Stothard et al. 1998; Booton disproportionally associated with pathology (Maciver et al. 2013). Acanthamoeba (especially T4) is the causative agent of Acanthamoeba keratitis (Lorenzo-Morales et al. 2015)a Handling Editor: Julia Walochnik painful, vision-threatening infection and worse, a granuloma- tous amoebic encephalitis (GAE), an infection of the central Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00436-018-6192-0) contains supplementary nervous system which is usually fatal (Duggal et al. 2017). material, which is available to authorized users. Many drugs have been used to combat Acanthamoeba infec- tions (Siddiqui and Khan 2012;Martín-Navarroetal.2013; * Sutherland K. Maciver Lorenzo-Morales et al. 2016), but they tend not to be as effec- [email protected] tive due to a lack Acanthamoeba specificity and some com- 1 monly used drugs are known to cause harm to the patient at Centre for Discovery Brain Sciences, Edinburgh, UK currently used dosages (Ehlers and Hjortdal 2004;Moonetal. 2 Division of Infection and Pathway Medicine, Biomedical Sciences, 2018). Part of the problem is that Acanthamoeba alternates Edinburgh Medical School, University of Edinburgh, Hugh Robson between the active and infective trophozoite, and a dormant, Building, George Square, Edinburgh, Scotland EH8 9XD, UK 642 Parasitol Res (2019) 118:641–651 double walled cyst. Treatment of the infections caused by 2.0% agar plate onto which Eschericia coli bacteria have been Acanthamoeba is made difficult by the presence of these cysts spread. This was repeated until a pure population of because they are resistant to many disinfectants and other Acanthamoeba was established. Clonal strains were then treatments (Lorenzo-Morales et al. 2015). New drugs are ur- adapted to axenic media (see below) and their T-type deter- gently needed, but Acanthamoeba is a eukaryote which means mined by PCR by the method of Lorenzo-Morales (Lorenzo- that its biochemical pathways are unfortunately similar to that Morales et al. 2005). One particular T4 strain, GS-336, that of their human hosts, limiting the availability of specific drug was closely related to the reference Acanthamoeba castellanii targets. It is clear that we need to identify and exploit differ- Neff strain (ATTC30010, CCAP1501/1B) and was used in ences between Acanthamoeba and ourselves to find better this study and routinely cultured in axenic media (7.15 g/l drug targets, and we argue that such differences may be found yeast extract, 14.3 g/l peptone, 15.4 g/l glucose, 0.486 g/l within the programmed cell death (PCD) system. KH2PO4, 0.51 g/l Na2HPO4, pH 6.5). Experiments were con- It was something of a surprise to find that vertebrate cells ducted in Neff’s saline buffer (NSB) (NaCl 0.12 g/L, possess the deliberate means of self-destruction (Kerr et al. MgSO47H2O 4 mg/L, CaCl2 4mg/L,Na2HPO4 0.142 g/L, 1972) as this seems so blatantly anti-Darwinian, but we now KH2PO4 0.136 g/L). Light microscopic observations were know that cells are required to eliminate themselves in a vari- made on living amoebae using an inverted Leica DMIRB ety of ways in several different situations within the vertebrate microscope equipped with Hoffman modulation contrast op- body especially during development. The suggestion that sin- tics. Imaging/image analysis was performed in the IMPACT gle celled organisms can also kill themselves is even more Imaging Facility, Centre for Discovery Brain Sciences, unexpected, but evidence is accumulating to support this. University of Edinburgh. Confocal microscopy was carried PCD processes have been identified and described in numer- out using a Nikon A1R microscope with various filters. NIS ous unicellular organisms including yeast such as Elements software was used for all collected images which Saccharomyces (Ludovico et al. 2001; Madeo et al. 1999) were then further processed with Imaris and ImageJ software. slime molds such as Dictyostelium (Cornillon et al. 1994), parasitic protozoans including Trypanosoma (Welburn et al. 1996;Nguewaetal.2004; Duszenko et al. 2006; Jiménez- Phylogenetic analysis Ruiz et al. 2010), Leishmania (Lee et al. 2002), Entamoeba (Villalba et al. 2007)andPlasmodium (Al-Olayan et al. 2002). Sequences were compiled with others from Genbank Although details on the actors of PCD are scant for protists as and aligned using the ‘muscle’ algorithm (Edgar 2004) they generally lack the caspase enzymes that are commonly implemented using ‘Seaview’ version 4 (Gouy et al. seen in vertebrates. For example, caspases are absent in 2010). The alignments were then manually trimmed Dictyostelium discoideum (Olie et al. 1998), Acanthamoeba using ‘BioEdit’ (Hall 1999) and maximum likelihood castellanii (Clarke et al. 2013)andNaegleria gruberi (Fritz- phylogenetic trees produced by PhlyML algorithm with Laylin et al. 2010). It is hoped that a fuller understanding of the GTR model (Guindon and Gascuel 2003). The non- these PCD pathways will reveal targets that may act as effi- parametric analysis was performed with 1000 bootstrap cient ways of killing these parasites through drugs designed to pseudo-replicates, using the 18S sequence from activate parasite but not host PCD pathways. The aim of this Acanthamoeba pyriformis (Tice et al. 2016)asthe study is to investigate the role of calcium influx and mitochon- outgroup. drial misfunction in the early stages of the process of G418- induced programmed cell death in Acanthamoeba. Viability assays Materials and methods Viability of trophozoites was determined in the presence or absence of the aminoglycoside G418 for different periods of Amoebae isolation, culture and microscopy time by trypan blue exclusion assay. Acanthamoeba cells were suspended in 0.4% trypan blue dye in NSB and left for 5 min A new strain of Acanthamoeba, GS-336, was isolated from a at room temperature. The percentage of cell viability was soil sample taken from George Square gardens, Edinburgh, measured as: using the modified (De Obeso Fernandez Del Valle et al. Total viable cells 2017) ‘walkout’ method of Neff (Neff 1958). Soil was dis- Â 100% Total number of cells solved in Neff’s saline buffer NSB see below), placed on a non-nutrition 2.0% agar plate (2% agar NSB) and left over- night at room temperature. Small sections of agar were ex- Cells were counted manually using a haemocytometer and cised, inverted and placed on a second-fresh non-nutrition an inverted microscope (× 20 objective). Parasitol Res (2019) 118:641–651 643 Intracellular Ca2+ concentration insoluble formazan, conversely, dead or mitochondrial dys- functional trophozoites show lower or no such accumulation. Intracellular levels of calcium were monitored by the fluores- Acanthamoeba were treated with IC90 G418 in NSB and in- cent probe Fura-2/AM. After treatment for various periods cubated at 37 °C for various periods (1–3–6h).Cellswere with G418 (IC90 concentration) Acanthamoeba trophozoites centrifuged at 500×g for 10 min. Cells washed twice with were harvested and washed twice in Neff’s saline buffer at NSB and centrifuged as before. 5-Cyano-2,3-ditolyl tetrazoli- 500×g for 5 min at room temperature. Trophozoites were re- um chloride at a final concentration of 5 mM in NSB were suspended in Fura 2/AM loading buffer containing: 6 μM applied and cells incubated at 37 °C, in dark for 30 min before Fura 2 AM, 116 mM NaCl, 54 mM KCl, 0.8 mM MgSO4, washed twice with 1 mL NSB to rinse dye excess.