Novel insights into the potential role of ion transport in sensory perception in Acanthamoeba

Background Acanthamoeba is well known to produce a blinding keratitis and serious brain infection known as encephalitis. Effective treatment is problematic, and can continue up to a year, and even then, recurrence can ensue. Partly, this is due to the capability of vegetative amoebae to convert into resistant cysts. Cysts can persist in an inactive form for decades while retaining their pathogenicity. It is not clear how Acanthamoeba cysts monitor environmental changes, and determine favourable conditions leading to their emergence as viable trophozoites. Methods The role of ion transporters in the encystation and excystation of Acanthamoeba remains unclear. Here, we investigated the role of sodium, potassium and calcium ion transporters as well as proton pump inhibitors on A. castellanii encystation and excystation and their effects on trophozoites. Results Remarkably 3′,4′-dichlorobenzamil hydrochloride a sodium–calcium exchange inhibitor, completely abolished excystation of Acanthamoeba. Furthermore, lanthanum oxide and stevioside hydrate, both potassium transport inhibitors, resulted in the partial inhibition of Acanthamoeba excystation. Conversely, none of the ion transport inhibitors affected encystation or had any effects on Acanthamoeba trophozoites viability. Conclusions The present study indicates that ion transporters are involved in sensory perception of A. castellanii suggesting their value as potential therapeutic targets to block cellular differentiation that presents a significant challenge in the successful prognosis of Acanthamoeba infections.


Background
In the course of the past few decades Acanthamoeba have acquired much attention as important human pathogens producing vision-threatening keratitis and a rare but fatal encephalitis known as granulomatous amoebic encephalitis (GAE) [1,2]. The most disturbing characteristic is that the mortality concomitant with GAE because of pathogenic Acanthamoeba has endured significance (more than 90%) in spite of developments in antimicrobial chemotherapy and supportive care. Likewise, present diagnosis of Acanthamoeba keratitis is difficult [1,3], and the existing treatments are lengthy and not entirely effective against all strains [4], in part this is owing to the ability of amoebae to convert into resistant cysts [5,6], occasioning in infection recurrence. Additionally, cysts can endure up to several years while maintaining their pathogenicity, and this poses a major problem in chemotherapeutic treatment [5][6][7]. Cysts are double-walled, comprising of an outer ectocyst and an inner endocyst. Both walls meet at points known as arms or rays. Moreover, the cysts comprise pores identified as "ostioles", these are acknowledged by the presence of an operculum bounded by a circular ridge that is apparent on the surface of mature cysts [8]. Ostioles are most likely used to observe environmental changes. As long as conditions are harsh, amoebae remain in the cyst form. Under favourable conditions, amoebae emerge from the cyst form and reproduce, resulting in infection recurrence [9].
To date, much of the research in Acanthamoeba has been concentrated on the infective trophozoite form and its pathogenic mechanisms. For example, in a recent study an ATP-sensitive potassium channel in the mitochondria of A. castellanii trophozoites was described [10]. However, the cellular differentiation processes, as well as how the cysts monitor the environment remains incompletely understood. The main components of cyst wall are acid-resistant proteins and cellulose that provide a physical barrier and making it resistant to biocides, desiccation and drugs [11]. These findings suggested further that redox balance reactions and membrane functions are potential target for the rational development of therapeutic interactions [11]. For cellulose, glycogen phosphorylase is the key enzyme that breaks down glycogen to provide glucose for the synthesis of cellulose [11,12]. Garajová et al. [13] detected clustering of intramembranous particles during encystation. It was suggested that during endocyst formation, intramembranous particle clusters represent cellulose microfibril terminal complexes involved in cellulose synthesis that are reduced after cyst wall completion. It was proposed that disruption of this pathway would affect the synthesis of cyst wall and cyst resistance to chemotherapeutic agents. Given lack of specific drug to target Acanthamoeba infections, in addition to cellulose, polysaccharides composed of galactose are also highlighted as potential targets [14]. This could prove to be an important target given that there is a lack of established drug targets for a pharmaceutical intervention at the cyst stage.
In the present study, for the first time we investigated the role of ion transporters in sensory perception in the cyst stage of A. castellanii belonging to the T4 genotype. Based on the genome information, the presence of potassium ion transporters has been suggested [15]. However, their functional role in phenotypic switching is missing. Furthermore, identification of the ion transport pathway(s) in the cyst stage may guide in the detection of new anti-amoebic targets, as it is the cyst stage which presents a key challenge in treatment. Here, we investigated the effects of potassium, sodium and calcium transporter and proton pump inhibitors in cellular differentiation of A. castellanii of the T4 genotype.

Ion transporter inhibitors
Ion transport inhibitors were purchased from Sigma-Aldrich (Subang Jaya, Malaysia and dissolved in the solvents dimethyl sulfoxide (DMSO), distilled water, or 95% ethanol. Table 1 depicts the chemical nomenclature, class, empirical formula, molar mass and solvent solubility of the ion transport inhibitors. Stock solutions were prepared at 5 mM concentration.

Cultivation of Acanthamoeba castellanii
Acanthamoeba castellanii was acquired from American Type Culture Collection (ATCC) 50492 of the T4 genotype and grown axenically in 75 mm 3 culture flasks at 30 °C in proteose peptone yeast glucose (PYG) medium (proteose peptone 0.75%, yeast extract 0.75%, glucose 1.5%) as described previously [16]. Growth media were refreshed every 20 h to obtain vegetative trophozoites. Flasks were examined under phase contrast microscope on a daily basis until confluent A. castellanii were observed prior to experimentation.

Amoebicidal assays
To study the role of ion transport inhibitors on the viability of A. castellanii trophozoites, 5 × 10 5 amoebae/0.5 ml/ well were dispersed in Roswell Park Memorial Institute medium (RPMI-1640) in the absence or presence of various inhibitors at different concentrations as described previously [17]. Plates were incubated at 37 °C for 24 h. Subsequently, viability of the amoebae was determined by addition of 0.1% Trypan blue and the number of living (non-stained) and dead (stained) A. castellanii counted by means of a haemocytometer. Control experiments were conducted in RPMI-1640 alone and RPMI-1640 containing respective amount of solvents. The data are representative of three independent experiments and are given as the mean ± standard error.

Amoebistatic assays
An amoebistatic assay was employed to establish the role of ion transport inhibitors on A. castellanii growth [1]. Specifically, 5 × 10 5 trophozoites were incubated with varying concentrations of the inhibitors in growth medium, i.e. PYG in 24-well plates. Plates were reserved at 30 °C for 48 h. As controls, 5 × 10 5 trophozoites were inoculated in 100% PYG medium, 100% non-nutritive phosphate-buffered saline (PBS) solution and the corresponding amounts of solvents plus PYG medium, and kept in the aforementioned conditions. Subsequently, the number of amoebae was quantified via haemocytometer enumeration. All experimental data are representative of the mean ± standard error of three experiments conducted in duplicate.

Provision of A. castellanii cysts and excystation assays
To acquire A. castellanii cysts, encystation was motivated by dispersing 5 × 10 6 A. castellanii trophozoites on nonnutrient agar plates. Plates were kept at 30 °C for 14 days [16,18]. Food deprivation ensued in conversion from the trophozoite stage into the cyst form. Afterward, 10 ml of PBS was incorporated to each plate. Cysts were scratched off the agar surface with a cell scraper and counted using a haemocytometer. To study effects of ion channel inhibitors on excystation, assays were carried out by placing A. castellanii cysts in the presence or absence of different concentrations of inhibitors in PYG medium (2 × 10 4 cysts per ml per well of 24-well plates). Plates were kept at 30 °C and observed every 24 h for the appearance of viable trophozoites for up to 72 h.

Encystation assays
To determine the effects of inhibitors on cyst formation, amoebae inoculation on agar plates is required. For this purpose, encystation assay using liquid medium was used as described previously [19]. In brief, 2 × 10 6 amoebae were dispersed in PBS and 50 mM MgCl 2 and 10% glucose (i.e. encystation trigger) in a 24-well tissue culture plates at 30 °C for 72 h (without shaking). Following incubation, SDS (0.5% final concentration) was added for 10 min to solubilize trophozoites and the cysts were  counted using a haemocytometer. To study effects of ion channel inhibitors on encystation, assays (PBS plus 50 mM MgCl 2 and 10% glucose) were conducted in the presence of varying concentrations of the inhibitors. In brief, 2 × 10 6 amoebae were dispersed in PBS containing varying concentrations of inhibitors and in the presence of 50 mM MgCl 2 and kept at room temperature for 20 min. Next, 10% glucose was added as a trigger for encystation and plates were incubated at 30 °C for 72 h. Encystation in wells without inhibitors was used as positive controls and wells without the inhibitors as well as encystation trigger were used as negative controls. The corresponding amounts of solvents were incubated in wells plus A. castellanii and used as solvent controls. Amoebae were counted using a haemocytometer [20]. All experimental data are representative of the mean ± standard error of at least three independent experiments completed in duplicate.

Statistical analysis
Statistical significance for differences was evaluated using a 2-sample t-test; two-tailed distribution, comparing the mean and resulting P-values < 0.05 were considered significant. For graphical representation of the data, y-axis error indicates the standard error of the data.

3′,4′-Dichlorobenzamil hydrochloride, stevioside hydrate and CLP257 inhibited excystation of A. castellanii
To examine the effects of ion transporter inhibitors on the excystation of A. castellanii cysts, excystation in amoebae incubated without inhibitors was considered as 100% and the effects of inhibitors/solvent are presented as the relative change. Of note, 100 μM 3′,4′-dichlorobenzamil hydrochloride, a sodium-calcium exchange inhibitor, abolished the excystation of A. castellanii as depicted in Fig. 1

Ion transport inhibitors did not inhibit the encystation of A. castellanii
To determine the effects of ion transport inhibitors on the encystation of A. castellanii, encystation assays were carried out. Untreated amoebae showed 0% encystation.  Fig. 2. However, these results were not different when compared with the respective solvent controls.

Ion transport inhibitors did not exhibit amoebicidal activity against A. castellanii trophozoites
To assess the effects of ion transport inhibitors, amoebicidal assays were accomplished. Viability in untreated amoebae was considered as 100% and the effects of inhibitors/solvent are presented as the relative change.
The results showed that ion transport inhibitors had no amoebicidal activity against A. castellanii trophozoites (Fig. 3). In the presence of 100 µM 3′,4′-dichlorobenzamil hydrochloride, and gadolinium (III) chloride, the percentage cell viability was 70% and 61.92%, respectively. However, these results were not different when compared with the respective solvent controls. The use of 100 μM of stevioside hydrate, tenatoprazole, cariporide, PF-03716556, lanthanum oxide, CLP257 and strontium chloride did not reveal any statistically significant amoebicidal activity as they resulted in respective cell viability of 81.62%, 81.73%, 90.3%, 92.03%, 92.05%, 100% and 100%. Overall, the results revealed that the ion transport inhibitors had limited or no effects on the viability of A. castellanii trophozoites.

Ion transport inhibitors did not inhibit the growth of A. castellanii trophozoites
Amoebistatic assays were completed in the presence and absence of ion transport inhibitors to study the effects of the inhibitors on the growth of A. castellanii trophozoites. Growth in untreated amoebae was considered as 100% and the effects of inhibitors/solvent are presented as the relative change. The number of untreated amoebae increased from 5 × 10 5 to 1.17 × 10 6 when incubated in PYG growth medium. Addition of 100 μM of strontium   Fig. 4. However, none of the growth inhibition was statistically significant when compared with the corresponding solvent controls.

Discussion
Membrane ion transporters are well known to be pertinent to the permeability of ions, membrane potential and stability of cell microenvironment ranging from single cells to large complex organisms. Microbes possess a considerable selection of ion transporters in their cell membranes [21]. Cellular functions in microbes require tight regulation and it is well accepted that ion channels have a crucial role, for instance in energy transduction [22]. For example, potassium channels are one of the most varied families of membrane proteins, widely described from bacteria to humans. They are found in all eukaryotic organisms, marking them out as essential biological enzymes. Their known roles comprise voltagemembrane potential maintenance, excitability, organogenesis, cell death, pH and cell volume regulation. They are involved in sensing and responding to environmental changes such as acidification, oxygen pressure, osmolarity and ionic concentration [23]. It is not clear how Acanthamoeba cysts monitor environmental changes and determine favourable conditions leading to their emergence as viable trophozoites. There is limited understanding regarding sensory perception in the cyst stage of Acanthamoeba.
Cyst walls are largely composed of acid-resistant proteins and polysaccharides, among which cellulose is one of the key constituents [13]. Magistrado-Coxen et al. [24] demonstrated that purified cyst walls retained an outer ectocyst layer, an inner endocyst layer, and ostioles that connect them. The cyst wall proteins were mostly represented by three families of lectins and are localized in the ectocyst, the endocyst layer and ostioles of mature walls [24], suggesting their potential role in monitoring the environmental conditions.
In this study, for the first time we postulated that ion transport plays a role in sensory perception in the cyst stage of A. castellanii. Our experiments showed that tenatoprazole, a proton pump inhibitor, partially inhibited the formation of cysts while hampering the excystation process to a greater extent. This suggests that proton pump may be involved in the conversion of cysts into trophozoites. Moreover, previous studies in Dictyostelium discoideum revealed that the plasma membrane proton pump served as an intercellular pH regulator [25]. It is plausible that the proton may regulate the pH so as to detect when there are favourable conditions for the trophozoite to emerge, hence being important for excystation. In addition, cariporide, a sodium-proton exchanger, was found to partially inhibit excystation. Sodium hydrogen ion exchange is likely to be involved in regulating pH, and acting similarly to tenatoprazole for trophozoite formation in Acanthamoeba. Furthermore, both stevioside and lanthanum oxide which are potassium channel modulators of activity completely inhibited the excystation process and yet did not have much effect in the transformation of the trophozoites into cyst, indicating that potassium transport may be involved in the sensory perception of the cyst to emerge as a trophozoite but not vice versa. Similarly, CLP257, a potassiumchloride co-transporter activator, also inhibited the excystation to some extent, again suggesting the role of potassium ion transport in the excystation process of A. castellanii. Notably, 3′,4′-dichlorobenzamil hydrochloride, a sodium-calcium exchanger, completely abolished excystation of A. castellanii. This indicates that the Ca 2+ signalling is disturbed and leads to phenotypic transformation. These findings are interesting and can be utilised as a potential drug target. To underline the importance of ion transport in drug discovery, 13% of all drugs on the market to date are targeting ion transporters; this makes them the second most important target for medical intervention after the G-protein coupled receptors [26].
Comparable to other microbes, Acanthamoeba has been shown to display chemosensory responses and is known to have receptor(s) in its plasma membranes to detect chemo-attractants [27]. It was previously suggested that there may be specific receptors that can be comparable to sensory organs for taste and smell to detect favourable prey and lead to subsequent motor mechanisms and determine Acanthamoeba preferential feeding behaviour towards certain bacterial species and it is plausible that ion channels are utilised in these processes [28].

Conclusions
To our knowledge, we demonstrated for the first time that ion transport plays a role in sensory perception in the cyst stage of A. castellanii and could be valuable targets in the rational development of chemotherapeutic interventions. Subsequent studies will determine optimal ion transport activity and associated molecules, knowledge of which can be exploited for blocking excystment and improved strategies for therapeutic interventions. Moreover, identifying the genes encoding ion channels (and transporters) in Acanthamoeba and expression in a heterologous expression cell system will enable their electrophysiological characterisation using patch clamp techniques that can provide information on the functional aspects of important membrane proteins that maybe used as anti-amoebic targets for treatment of these important pathogens.