G418 induces programmed cell death in Acanthamoeba through the elevation of intracellular calcium and cytochrome c translocation
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.
KeywordsAcanthamoeba Amoebozoa Programmed cell death Apoptosis G418 Cytochrome c
Acanthamoeba is a eukaryotic, opportunistic parasitic protozoan, which is widely distributed in the natural environment (Geisen et al. 2014). Although species classification has not been completely formalized, the genus has been divided into 21 different genotypes, T1-T21 (Stothard et al. 1998; Booton et al. 2002; Corsaro and Venditti 2018) based on 18S rRNA gene sequences which equates approximately to classification at the species level (Hewett et al. 2003; Fuerst 2014). While some named species accord with this system, others do not. The most commonly encountered genotype is T4, and while it is the most frequently isolated from the environment, it is also disproportionally associated with pathology (Maciver et al. 2013). Acanthamoeba (especially T4) is the causative agent of Acanthamoeba keratitis (Lorenzo-Morales et al. 2015) a painful, vision-threatening infection and worse, a granulomatous amoebic encephalitis (GAE), an infection of the central nervous system which is usually fatal (Duggal et al. 2017). Many drugs have been used to combat Acanthamoeba infections (Siddiqui and Khan 2012; Martín-Navarro et al. 2013; Lorenzo-Morales et al. 2016), but they tend not to be as effective due to a lack Acanthamoeba specificity and some commonly used drugs are known to cause harm to the patient at currently used dosages (Ehlers and Hjortdal 2004; Moon et al. 2018). Part of the problem is that Acanthamoeba alternates between the active and infective trophozoite, and a dormant, double walled cyst. Treatment of the infections caused by Acanthamoeba is made difficult by the presence of these cysts because they are resistant to many disinfectants and other treatments (Lorenzo-Morales et al. 2015). New drugs are urgently needed, but Acanthamoeba is a eukaryote which means that its biochemical pathways are unfortunately similar to that of their human hosts, limiting the availability of specific drug targets. It is clear that we need to identify and exploit differences between Acanthamoeba and ourselves to find better drug targets, and we argue that such differences may be found within the programmed cell death (PCD) system.
It was something of a surprise to find that vertebrate cells possess the deliberate means of self-destruction (Kerr et al. 1972) as this seems so blatantly anti-Darwinian, but we now know that cells are required to eliminate themselves in a variety of ways in several different situations within the vertebrate body especially during development. The suggestion that single celled organisms can also kill themselves is even more unexpected, but evidence is accumulating to support this. PCD processes have been identified and described in numerous unicellular organisms including yeast such as Saccharomyces (Ludovico et al. 2001; Madeo et al. 1999) slime molds such as Dictyostelium (Cornillon et al. 1994), parasitic protozoans including Trypanosoma (Welburn et al. 1996; Nguewa et al. 2004; Duszenko et al. 2006; Jiménez-Ruiz et al. 2010), Leishmania (Lee et al. 2002), Entamoeba (Villalba et al. 2007) and Plasmodium (Al-Olayan et al. 2002). Although details on the actors of PCD are scant for protists as they generally lack the caspase enzymes that are commonly seen in vertebrates. For example, caspases are absent in Dictyostelium discoideum (Olie et al. 1998), Acanthamoeba castellanii (Clarke et al. 2013) and Naegleria gruberi (Fritz-Laylin et al. 2010). It is hoped that a fuller understanding of these PCD pathways will reveal targets that may act as efficient ways of killing these parasites through drugs designed to activate parasite but not host PCD pathways. The aim of this study is to investigate the role of calcium influx and mitochondrial misfunction in the early stages of the process of G418-induced programmed cell death in Acanthamoeba.
Materials and methods
Amoebae isolation, culture and microscopy
A new strain of Acanthamoeba, GS-336, was isolated from a soil sample taken from George Square gardens, Edinburgh, using the modified (De Obeso Fernandez Del Valle et al. 2017) ‘walkout’ method of Neff (Neff 1958). Soil was dissolved in Neff’s saline buffer NSB see below), placed on a non-nutrition 2.0% agar plate (2% agar NSB) and left overnight at room temperature. Small sections of agar were excised, inverted and placed on a second-fresh non-nutrition 2.0% agar plate onto which Eschericia coli bacteria have been spread. This was repeated until a pure population of Acanthamoeba was established. Clonal strains were then adapted to axenic media (see below) and their T-type determined by PCR by the method of Lorenzo-Morales (Lorenzo-Morales et al. 2005). One particular T4 strain, GS-336, that was closely related to the reference Acanthamoeba castellanii Neff strain (ATTC30010, CCAP1501/1B) and was used in this study and routinely cultured in axenic media (7.15 g/l yeast extract, 14.3 g/l peptone, 15.4 g/l glucose, 0.486 g/l KH2PO4, 0.51 g/l Na2HPO4, pH 6.5). Experiments were conducted in Neff’s saline buffer (NSB) (NaCl 0.12 g/L, MgSO47H2O 4 mg/L, CaCl2 4 mg/L, Na2HPO4 0.142 g/L, KH2PO4 0.136 g/L). Light microscopic observations were made on living amoebae using an inverted Leica DMIRB microscope equipped with Hoffman modulation contrast optics. Imaging/image analysis was performed in the IMPACT Imaging Facility, Centre for Discovery Brain Sciences, University of Edinburgh. Confocal microscopy was carried out using a Nikon A1R microscope with various filters. NIS Elements software was used for all collected images which were then further processed with Imaris and ImageJ software.
Sequences were compiled with others from Genbank and aligned using the ‘muscle’ algorithm (Edgar 2004) implemented using ‘Seaview’ version 4 (Gouy et al. 2010). The alignments were then manually trimmed using ‘BioEdit’ (Hall 1999) and maximum likelihood phylogenetic trees produced by PhlyML algorithm with the GTR model (Guindon and Gascuel 2003). The non-parametric analysis was performed with 1000 bootstrap pseudo-replicates, using the 18S sequence from Acanthamoeba pyriformis (Tice et al. 2016) as the outgroup.
Cells were counted manually using a haemocytometer and an inverted microscope (× 20 objective).
Intracellular Ca2+ concentration
R = sample ratio fluorescence, Rmin = calcium-zero conditions (EGTA 10 μΜ), Rmax = calcium-saturated conditions (CaCl2 4 mM + 10 μM ionomycin) and β = ratio of Rmin/Rmax at 380 nm, Kd = 224 nM at 30 °C (Grynkiewicz et al. 1985).
Hoechst 33342 staining protocol and Hoechst fluorescence intensity
Hoechst 33342 is a cell-permanent nucleic acid stain which we have used to study chromatin structure in Acanthamoeba. Briefly, trophozoites treated with IC90 G-418 in NSB and incubated at 37 °C for preschedule period (0 to 8 h). Amoebae were then centrifuged at 500×g for 10 min, washed once with NSB and fixed with fresh 4% paraformaldehyde (PFA) for 20 min at room temperature. Cells washed again with NSB and centrifuged at 500×g for 10 min. Hoechst 33342 was applied at a final concentration of 10 μM. Cells incubated at 37 °C in the dark for 30 min and finally washed twice with 1 mL NSB to remove dye excess. Stained trophozoites were observed by confocal microscopy, under an epifluorescence microscope (Nikon A1R). DAPI filters were applied in order to detect fluorescence. Analysis was later made using Image analysis Software Imaris and ImageJ. Emission signal intensity of Hoechst 33342 was monitored and measured by a LS55 PerkinElmer luminesce spectrometer when excited at 510 nm.
CTC staining and fluorescence intensity
CTC (5-cyano-2,3-ditolyl tetrazolium chloride) is a redox dye used here to report the respiratory activity of Acanthamoeba mitochondria. Respiring Acanthamoeba trophozoites loaded with CTC solution reduce the dye to produce a visible insoluble formazan, conversely, dead or mitochondrial dysfunctional trophozoites show lower or no such accumulation. Acanthamoeba were treated with IC90 G418 in NSB and incubated at 37 °C for various periods (1–3–6 h). Cells were centrifuged at 500×g for 10 min. Cells washed twice with NSB and centrifuged as before. 5-Cyano-2,3-ditolyl tetrazolium chloride at a final concentration of 5 mM in NSB were applied and cells incubated at 37 °C, in dark for 30 min before washed twice with 1 mL NSB to rinse dye excess. Cells were fixed with 4% paraformaldehyde for 20 min, washed with 1 mL NSB and centrifuged at 500×g for 10 min before applied on a micro slide and observed by confocal microscopy, Epi-Filter rhodamine red; excitation/emission (nm): 480/630 using NIS elements software. Fluorescence was quantified using a LS55 Perkin Elmer luminesce spectrometer.
Mitochondrial membrane potential measurement
Mitochondrial membrane potential was measured using the florescent reported, JC-1. This membrane permeant dye accumulates in mitochondria at a rate determined by the potential difference across the mitochondrial membrane. The monomer emits around 530 nm and as it concentrates in mitochondria it forms fluorescent aggregates which emit a longer wavelength centred at 590 nm. Consequently, a reduction in the J-aggregate fluorescence indicates depolarization whereas a rise means hyperpolarization. Trophozoites were treated with G418 IC90 for predefined periods (1–3–6 h), harvested and washed twice with NSB. After centrifuging at 500×g cells were suspended in Neff’s saline buffer containing 6 μM JC-1 and left for 20 min at 37 °C, in dark. Trophozoites were then washed twice with 1 mL NSB and centrifuged. Cells were examined immediately by fluorescence microscopy using excitation FITC (490-540 nm) and emission JC-1 (580–610 nm) filters. Simultaneously, JC-1 fluorescence was measured spectrophotometrically at 488 nm using a LS55 Perkin Elmer luminesce spectrometer.
Release of cytochrome c from mitochondria
Cytochrome c release was assayed using a commercial kit (Abcam’s cytochrome c releasing apoptosis assay kit (ab65311)). Acanthamoeba trophozoites were incubated with IC90 G418 in NSB at 37 °C for predetermined period (0, 1, 3, 6 h). Cells were collected in Eppendorf tubes and centrifuged at 500g for 5 min, washed once with NSB and supernatant was removed. One millilitre of 1x cytosol extraction buffer mix containing DTT and protease inhibitors was used to re-suspend the pellet while sample was incubated in ice for 10 min. Approximately 4–6 cycles of freeze–thaw were conducted using dry ice to freeze Acanthamoeba suspension (~ 4 min) and a tube thermal incubator set at 37 °C (~ 3 min). The homogenization process was monitored under a microscope by applying 5–6 μL of cell suspension onto a coverslip. The homogenate was centrifuged at 10,000g to separate cytosolic from mitochondrial fraction for 30 min at 4 °C. Supernatant was collected in a new Eppendorf tube and labeled as cytosolic fraction. Pellet was re-suspended in 100 μL mitochondrial extraction buffer mix that contained DTT and protease inhibitors, while was vortexed for 10 to 15 s and labeled as mitochondrial fraction. Protein concentrations were determined using a Pierce BCA protein assay kit. Samples (10 μg) of each cytosolic and mitochondrial fractions isolated from treated and untreated Acanthamoeba were separated by 16% SDS-PAGE and then Western Blotted using a Bio-Rad ‘mini protean’ system and probed with a polyclonal rabbit anti-cytochrome c antibody (Abcam ab90529) at a final dilution of 1:200 in skimmed dry milk 3.5% w/v, in tris-buffered saline (TBS). 0.4 mm PVDF membranes were incubated at 4 °C overnight with slight agitation and washed twice for 5 min with TBS-T (TBS-0.1% Tween-20). A secondary fluorescent antibody was used to label rabbit’s anti-cytochrome c antibody. Odyssey goat anti-rabbit IgG IR dye was diluted in skimmed dry milk 3.5% w/v, in tris-buffered saline to 1 μg/mL and applied to PVDF protein membranes for 1 h at room temperature, in the dark with gentle agitation. Membrane were washed twice for 5 min with TBS-T at room temperature, visualized with a LI-COR Odyssey Classic imager scanner and analyzed by win Image studio 5.2 analysis software.
The strain of Acanthamoeba used for this study GS-336 was chosen as it was similar to the well-characterised T4 Neff strain (Acanthamoeba castellanii ATCC 30010, CCAP1501/1A) whose genome has been sequenced (Clarke et al. 2013). It was important to use a fresh strain since the original Neff strain has become altered by its prolonged period in culture (Köhsler et al. 2008). Phylogenetic analysis shows that GS-336 is a T4 strain and groups with the Neff strain (Supplementary Fig. 1).
G418 effect on Acanthamoeba trophozoites viability and morphology
G418 increases intracellular calcium concentration
G418 induces nuclear morphological changes
Mitochondria in Acanthamoeba PCD
It is well known that various types of programmed cell deaths are necessary part of embryogenesis and development in higher multicellular eukaryotes. For example, cells are required to die in the limb-bud to define digits in the developing limb (Zaleske 1985) and cells of the immune system that react to self must die during the process of thymic education (Murphy et al. 1990). Programmed cell death has also been described in many unicellular organisms including yeast (Madeo et al. 1999), several protozoans (Cornillon et al. 1994; Welburn et al. 1996; Olie et al. 1998; Verdi et al. 1999; Al-Olayan et al. 2002) and even bacteria (Yarmolinsky 1995). Whereas PCD makes sense in metazoans, it is less obvious why a single cell organism such as Acanthamoeba might express proteins to destroy itself. It is suggested that PCD may be in the interest of parasite populations within a host to limit the parasite load thereby allowing reproduction and spread of more parasites in the longer term (Al-Olayan et al. 2002; Nguewa et al. 2004), but in free living amoebae, PCD may be a mechanism to prevent the spread of pathogenic bacteria and viruses through local populations.
In agreement with former studies (Martín-Navarro et al. 2015; Martín-Navarro et al. 2017; Sifaoui et al. 2017; Baig et al. 2017; Lopez-Arencibia et al. 2017; Moon et al. 2018), we have found morphological changes in Acanthamoeba associated with a PCD-linked series of events. These include rounded up, cell shrinkage, intracellular ion fluctuations, mitochondrial dysfunction nuclear and chromatin condensation and finally breakup and release of apoptotic body-like particles. Confocal microscopy revealed chromatin condensation reflected by an increase in the intensity of Hoechst staining while the nucleus began to vesiculate. However, electrophoresis after DNA isolation of the G418 treated trophozoites did not reveal DNA cleavage or the ladder-like characteristic pattern seen in Entamoeba (Villalba et al. 2007).
An elevation of [Ca2+]i has been associated with early and late stages of PCD in a wide variety of cell types and many PCD proteins are calcium sensitive (Orrenius et al. 2003). We have found that [Ca2+]i also rises in Acanthamoeba in the presence of G418, before any sign of cellular death was apparent. A steady rise in [Ca2+]i was observed in the presence of 75 μg/mL G418 in treated Acanthamoeba trophozoites from a resting [Ca2+]i level around 20 nM rising to 60 nM plateau around 90 min. This is similar to the rise in [Ca2+]i of Entamoeba histolytica caused by G418 (10 μg/mL) in (Villalba et al. 2007) where resting levels were 20 nM and increased to around 47 nM after 120 min. The PCD inducer of Dictyostelium, DIF also caused an increase in [Ca2+]i (Schaap et al. 1996).
It is reported that the mitochondria of A. castellanii actively accumulate Ca2+ which leads to a decrease in mitochondrial membrane potential ∆Ψm (Domka-Popek and Michejda 1986; Trocha and Stobienia 2007). We have found that mitochondrial function as measured by CTC and JC-1 decreased as the [Ca2+]i increased. Furthermore, we have also discovered the release of cytochrome c from Acanthamoeba mitochondria by Western Blot analysis (Fig. 8). The well-characterised antibody reacted to a band at around 25 kDa, close to double that of the 12.9 kDa size expected from the encoding sequence of the conserved Acanthamoeba cytochrome c gene; however, the Acanthamoeba protein contains an exposed cysteine residue at position 1213 (see red arrow in Supplementary fig. 2) and as it is known that cytochrome c has a strong propensity to polymerise through domain-pair exchange (Hirota et al. 2010), requiring denaturation conditions to convert to the monomeric state (Margoliash and Lustgarten 1962). We hypothesise that the 25 kDa band that is recognised by the anti-cytochrome c antibody is a homodimer (possibly stabilized/mediated by cysteine disulphide bonds) of Acanthamoeba cytochrome c. Spectrometric analysis of mitochondria from stressed Acanthamoeba also indicate that cytochrome c is released (Trocha and Stobienia 2007). Cytochrome c release is known to be calcium-dependent in vertebrate neurons (Schild et al. 2001) and cytochrome c released from mitochondria is known to accumulate in the nucleus where it is implicated in chromatin remodelling (Nur-E-Kamal et al. 2004). It is interesting to note that the peroxidase activity of dimeric cytochrome c is far greater than the monomer perhaps making it a more potent mediator of PCD (Wang et al. 2011).
Several differences in G418 induced PCD compared to other inducers are evident. When compared directly, different amoebicidal drugs show differences in PCD profile (Moon et al. 2018) perhaps indicating that they are activating different pathways or different targets in a single pathway. In summary, we have found that G418 causes PCD-like changes in Acanthamoeba. In the early stages of the process, there is an increase in intracellular calcium concentration leading to mitochondrial misfunction and the release of cytochrome c.
The biological pathways that lead to PCD in Acanthamoeba are far from being fully understood; however, an understanding of cell death-triggering mechanisms, mediators, and executioner pathways will offer the potential to identify targets for therapy, not only for Acanthamoeba infections but for many other disease-causing protists that share these PCD pathways.
The authors would like to thank Dr. Anisha Kubasik-Thayil for assistance with confocal microscopy.
Compliance with ethical standards
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
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