The effects of phosphanegold(I) thiolates on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype

Background Gold compounds have shown promise in the treatment of non-communicable diseases such as rheumatoid arthritis and cancer, and are considered of value as anti-microbial agents against Gram-negative and Gram-positive bacteria, and have anti-parasitic properties against Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, Leishmania infantinum, Giardia lamblia, and Entamoeba histolytica. They are known to affect enzymatic activities that are required for the cellular respiration processes. Methods Anti-amoebic effects of phosphanegold(I) thiolates were tested against clinical isolate of A. castellanii belonging to the T4 genotype by employing viability assays, growth inhibition assays, encystation assays, excystation assays, and zymographic assays. Results The treatment of A. castellanii with the phosphanegold(I) thiolates tested (i) had no effect on the viability of A. castellanii as determined by Trypan blue exclusion test, (ii) did not affect amoebae growth using PYG growth medium, (iii) did not inhibit cellular differentiation, and (iv) had no effect on the extracellular proteolytic activities of A. castellanii. Conclusion Being free-living amoeba, A. castellanii is a versatile respirator and possesses respiratory mechanisms that adapt to various aerobic and anaerobic environments to avoid toxic threats and adverse conditions. For the first time, our findings showed that A. castellanii exhibits resistance to the toxic effects of gold compounds and could prove to be an attractive model to study mechanisms of metal resistance in eukaryotic cells.


Background
Acanthamoeba is a free living pathogenic protist that can cause cutaneous lesions, a vision-threatening keratitis, and a rare but fatal infection of the brain, identified as granulomatous amoebic encephalitis [1][2][3][4]. Acanthamoeba keratitis infection is of explicit concern given the rise in the number of wearers of contact lenses worldwide, a population susceptible to this infection. Treatment involves hourly topical application of a mixture of drugs comprising of polyhexamethylene biguanide or chlorhexidine digluconate together with propamidine isethionate or hexamidine. Moreover, chloramphenicol or neomycin is also given to prevent mixed bacterial infection [5]. Treatment lasts for several months [5,6]. Furthermore, the treatment is problematic and cumbersome, in part due to the ability of this facultative parasite to go through phenotypic interchanging into a double-walled cyst form, which is impervious to many anti-microbial drugs and harsh conditions, and an active vegetative trophozoite stage that is more vulnerable to anti-microbials, often leading to recurrence of infection [7][8][9]. Consequently, there is a crucial need to develop anti-microbials targeting both the cyst stage and the trophozoite stage of Acanthamoeba.
Gold compounds have been well recognised for their putative properties and potential medical applications [10,11]. For example, the assessment of the potential anti-cancer activity and the determination of signalling pathways for apoptosis of phosphane gold(I) carbonimidothioates, Ph 3 PAu[SC(OR) = NPh], R = Me, Et and iPr, and related species have been carried out recently [12][13][14], see Fig. 1 for chemical structures. Moreover, closely related compounds have shown potential as anti-microbial agents against Gram-positive bacteria [15]. Gold(I) compounds have potential medical applications and shown to possess anti-tumour activities [16,17], anti-parasitic [18] and anti-microbial activities [19][20][21] via a variety of mechanisms including respiration. In this study, for the first time, we determined the effects of phosphanegold(I) thiolates, AAu1-AAu3, Fig. 1, on a keratitis-causing isolate of A. castellanii belonging to the T4 genotype. Furthermore, the effects on viability, growth, encystation and excystation are examined.

Chemicals
All chemicals were purchased from Sigma Labs (Poole, Dorset, England), unless otherwise stated. The phosphanegold(I) thiolates, AAu1-AAu3, were prepared and characterised using methodology as previously described [14]. The molecular structures and weights of AAu1-AAu3 are given in Fig. 1. A stock solution (10 mM) was prepared and stored at −20°C until used. Control cultures contained the same volume of respective solvents.

Cultures of A. castellanii
A. castellanii belonging to the T4 genotype (ATCC 50492) is a clinical isolate that was initially isolated from a keratitis patient and grown in 75 cm 2 tissue culture flasks in 10 mL at a cell density of 5×10 5 cells per mL in PYG medium [proteose peptone 0.75% (w/v), yeast extract 0.75% (w/v) and glucose 1.5% (w/v)] without shaking at 30°C as described previously [22,23]. At this cell density, parasites reach confluency within 48 h. Active trophozoites are attached to the bottom of the flasks while any dormant cells are non-adherent in the supernatant. To obtain trophozoites, supernatant was aspirated and 10 mL of RPMI-1640 was added. Next, flasks were placed on ice for 20 min to detach bound amoebae followed by gentle tapping and observed under the inverted microscope to ensure amoebae detachment had occurred. Finally, the parasites were collected in 50 mL tubes, followed by centrifugation at 1500×g for 5 min, resuspended in one mL of RPMI-1640 and used in experiments.

Amoebicidal assays
To determine amoebicidal activity of AAu1-AAu3, A. castellanii trophozoites (5 × 10 5 amoebae/0.5 mL/well) were incubated in RPMI-1640 with various concentrations of AAu1-AAu3 in 24-well plates as described previously [20][21][22][23][24]. Plates were incubated at 37°C for 24 h. Following this incubation, amoebae viability was determined by adding 0.1% Trypan blue and number of live (non-stained) and dead (stained) A. castellanii were enumerated using a haemocytometer. The counts from A. castellanii incubated with RPMI-1640 alone, and the solvent alone (chloroform) were used as controls. Data are represented as the mean ± standard error of at least three independent experiments. To determine whether the effects of AAu1-AAu3 are irreversible, A. castellanii, 5 × 10 5 trophozoites, were incubated with AAu1-AAu3 for 24 h as described above. After this incubation, amoebae were centrifuged for 10 min at 1,000xg and supernatant was aspirated, followed by the addition of 0.5 mL of RPMI-1640. This process was repeated 3X to remove extracellular AAu1-AAu3. Finally, A. castellanii were re-suspended in PYG as a food source and inoculated in 24-well plates. Plates were incubated at 37°C for up to 72 h and re-emergence of trophozoites was considered as viable amoebae, and absence of trophozoites was considered as non-viable amoebae. In some experiments, plates were incubated for up to a week to observe the emergence of viable trophozoites.

Amoebistatic assays
To determine the effects of AAu1-AAu3 on the growth of A. castellanii, assays were performed by exposing 5 × 10 5 trophozoites to different concentrations of AAu1-AAu3 in growth medium, i.e., PYG in 24-well plates. Next, the plates were incubated at 30°C for 48 h. For controls, 5 × 10 5 trophozoites were inoculated in 100% PYG medium, 100% non-nutritive PBS and respective amounts of solvents plus PYG medium and incubated in the above-mentioned conditions. After this incubation, the number of amoebae was Fig. 1 Chemical diagrams, abbreviations and molecular weights for AAu1-AAu3 determined by haemocytometer counting. All experiments were performed at least three times in duplicate.

Preparation of A. castellanii cysts and excystation assays
To prepare A. castellanii cysts, encystation was induced by inoculating 5 × 10 6 A. castellanii trophozoites onto non-nutrient agar plates [prepared using 3% (w/v) bacteriological agar] and incubating at 30°C for up to 14 days [25]. Food deprivation resulted in trophozoite transformation into the cyst form. Next, 10 mL of PBS was added to each plate. Cysts were then gently scraped off the agar surface using a cell scraper. PBS containing cysts was collected in 15 mL tube and centrifuged at 3000 × g for 10 min to pellet cysts. The supernatant was aspirated and cysts resuspended in RPMI-1640, enumerated using a haemocytometer and used in experiments.
To determine the effects of AAu1-AAu3 on excystation, assays were performed by inoculating A. castellanii cysts (5 × 10 4 cysts per mL PYG per well of 24-well plates) in the presence or absence of different concentrations of AAu1-AAu3. Plates were incubated at 30°C and observed every 24 h under the inverted microscope for the emergence of viable trophozoites for up to 72 h.

Encystation assays
Encystation assays were performed as described previously [25]. Briefly, 2 × 10 6 amoebae were incubated in 0.5 mL of PBS containing 50 mM MgCl 2 and 10% glucose (i.e., encystation trigger) per well of 24-well plates. The plates were incubated at 30°C for 72 h without shaking. After this incubation, amoebae viability was quantified using a haemocytometer via Trypan blue exclusion assay. Next, SDS (0.5% final conc.) was added for 10 min. At this concentration, SDS solubilizes amoebae trophozoites but not cysts. Finally cysts were enumerated using a haemocytometer and used in experiments. To determine the effects of AAu1-AAu3 on encystation, assays were performed in the presence of different concentrations of drugs. Briefly 2 × 10 6 amoebae were incubated in PBS with various concentrations of drugs and incubated at room temperature for 20 min. Following this, 50 mM MgCl 2 and 10% glucose was added as a trigger for encystation and plates were incubated at 30°C for 72 h. Finally, parasites counts were determined using a haemocytometer. Amoebae incubated without inhibitors and encystation trigger were used as controls. The respective amounts of solvents were used as solvent controls.

Zymographic assays
The extracellular proteolytic activities of Acanthamoeba were determined using zymographic assays as previously described [26]. Briefly, A. castellanii were incubated in the presence or absence of various concentrations of AAu1-AAu3 for 24 h. Next day, cell-free supernatants (CM, conditioned medium) were collected by centrifugation. The CM were electrophoresed on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) containing gelatin (2 mg/mL) as a protease substrate as previously described [26]. Following electrophoresis, gels were washed in 2.5% Triton X-100 (w/v) for 60 min, then incubated in developing buffer (50 mM Tris-HCl, pH 7.5, containing 10 mM CaCl 2 ) at 37°C overnight. Next day, gels were stained with Coomassie Brilliant Blue. Areas of gelatin digestion were visualised as non-staining regions in the gel.

Statistical analysis
Statistical significance for differences was evaluated using 2 sample t-test; two-tailed distribution, comparing the mean of two independent groups in Excel. A critical value of P < 0.05 was used for all analysis. For graphical representation of the data, y-axis error basis indicate the standard error of the data for each point on the figure.
Phosphanegold(I) thiolates, AAu1-AAu3, did not affect excystation in A. castellanii When incubated in growth medium, the number of amoebae increased from 5 × 10 4 to 3.91 × 10 5 ± 1.63 × 10 4 as compared to 5 × 10 4 to 1.24 × 10 5 ± 1.38 × 10 4 in RPMI medium, which is a non-nutritive medium (Fig. 4a). However, for AAu1-AAu3, the number of amoebae increased from 5 × 10 4 to 3.50 × 10 5 ± 1.63 × 10 4 , 3.73 × 10 5 ± 2.50 × 10 4 and 3.21 × 10 5 ± 2.81 × 10 4 , respectively at 300 μM (Fig. 4a). Nonetheless, this was not significant when compared to the respective growth  Fig. 2 a The effects of AAu1-AAu3 on the viability of A. castellanii belonging to T4 genotype. Briefly A. castellanii (5 × 10 5 trophozoites) were incubated with gold thiolates at 37°C for 24 h. Next day, Trypan blue exclusion assays were performed and amoebae were counted using haemocytometer. Note that none of the compounds shows significant effect on the viability of A. castellanii as compared to control. The results represent the mean ± standard error of three different experiments performed in duplicates. b Representative effects of AAu1-AAu3 on survival of A. castellanii. Briefly, A. castellanii (5 × 10 5 trophozoites) were incubated with AAu1-AAu3 at 37°C for 24 h and were counted using a heamocytometer. Next, drugs-treated amoeba were washed and re-inoculated in fresh PYG at 37°C for up to 24 h and observed under a microscope. The results are representative of three independent experiments. B1 is Amoeba alone; B2 is solvent alone (chloroform 15 μL); B3 is AAu1 (300 μM); B4 is AAu2 (300 μM); B5 is AAu3 (300 μM); B6 is chlorhexidine (300 μM) medium control and the results revealed that none of the compounds tested had any effects on excystation, and amoebae were able to excyst at rates comparable to controls (Fig. 4b).
Phosphanegold(I) thiolates, AAu1-AAu3, did not effect A. castellanii extracellular proteolytic activity To determine the effect of AAu1-AAu3 on the extracellular proteases of A. castellanii, zymographic assays were performed using gelatin as substrate as described in materials and methods. In the absence of any trial compound, A. castellanii exhibited proteolytic activities and a visible band of 140 kDa was observed (Fig. 6). Similarly, both, A. castellanii treated in the presence of different concentrations of AAu1-AAu3 and in RPMI alone exhibited extracellular proteases at similar levels ( Fig. 6).

Discussion
Gold(I) complexes have potential medical applications [10,11]. Thus, gold(I) derivatives have been explored for anti-tumour activity [16,17] as well as anti-parasitic [18] and anti-microbial agents [19][20][21]. Gold has properties such as high thermal/chemical stability and resistant to oxidation, yet is mechanically soft with high electric conductivity enabling its applications in several disciplines ranging from healthcare to engineering. For example, gold compounds have been successfully used in the treatment of rheumatoid arthritis and are shown to slow down the progression of rheumatic disorder [27,28]. Many of the biologically active gold(I) compounds contain thiolates and/or phosphane as ligands [10,11,16,17,21] and inhibit thioredoxin reductase [29,30]. More recently, it is shown that the gold(I) compounds exhibit anti-parasitic activities such as targeting Schistosoma mansoni [31], Trypanosoma brucei [32], Echinococcus granulosus [33], Plasmodium falciparum [34], Leishmania infantinum [35] Giardia lamblia [36], and Entamoeba histolytica [37]. Furthermore, it was shown that  Fig. 3 The effects of AAu1-AAu3 on the growth of A. castellanii belonging to T4 genotype. Briefly A. castellanii (5 × 10 5 trophozoites) were incubated with AAu1-AAu3 in growth medium, PYG at 37°C for 24 h. After this period, amoebae were counted using haemocytometer. Note that none of the trial compounds shows significant effect on the growth of A. castellanii as compared to control. The results represent the mean ± standard error of three different experiments performed in duplicates gold(I) compounds target E. histolytica by inhibiting thioredoxin reductase activity [37]. The anti-bacterial activities of gold(I) compounds showed that these compounds affect Clostridium difficile and Treponema denticola by disrupting the selenium metabolism by targeting selenoproteins required for energy [38,39], while Staphylococcus aureus growth is inhibited by gold(I) compounds [40]. Other studies proposed targets including the inhibition of mitochondrial enzymes and of the proteasome compounds [41,42] and the inhibition of the zinc finger protein poly (adenosine diphosphate (ADP) ribose) polymerase 1 (PARP-1) [43,44]. Notably, PARP's are crucial proteins that are important in drug resistance in cancer as they play an essential role in DNA repair by detecting DNA strand breaks and catalyzing poly (ADP-ribosylation) [45]. Other biological targets of gold(I) compounds with prokaryotic and eukaryotic cells are yet to be discovered. Based on these findings, it was logical to test the antiamoebic effects of phosphanegold(I) thiolates, AAu1-AAu3, on the biological properties of A. castellanii belonging to the T4 genotype. The results revealed that AAu1-AAu3 did not show any effects on the biological properties of the parasite. This was determined by  Fig. 4 a The effects of AAu1-AAu3 on excystation of A. castellanii belonging to T4 genotype. Briefly A. castellanii (5 × 10 4 cyst) were incubated with AAu1-AAu3 in growth medium, PYG at 37°C for 48 h. After this period, amoebae were counted using a haeamocytometer. Note that AAu1-AAu3 were unable to inhibit excystation. The dotted line represents the original inoculum. The results represent the mean ± standard error of two different experiments performed in duplicates. b Representative effects of AAu1-AAu3 on excystation of A. castellanii. The results are representative of three independent experiments. B1 is Amoeba alone; B2 is solvent alone (chloroform 15 μL); B3 is AAu1 (300 μM); B4 is AAu2 (300 μM); B5 is AAu3 300 (μM) performing (i) viability assays using Trypan blue exclusion test, (ii) amoebae growth using PYG growth medium, (iii) cellular differentiation using encystation and excystation assays and (iv) enzymatic activities by determining extracellular proteases profiles. The reported results are highly reproducible and consistently showed that AAu1-AAu3 do not affect the biological properties of A. castellanii. There could be several explanations for the findings observed in this study. For example, the mode of action of gold requires it to enter the cell, via the hydrophobic cell membrane, to produce damage, most likely through transmembrane proteins that may be different in A. castellanii. Notably, gold(I) compounds are well known to affect enzymatic activities that are required for the cellular respiration processes. Being one of the most ubiquitous protists, the natural  Fig. 6 The effects of AAu1-AAu3 on extracellular proteolytic activity of A. castellanii belonging to T4 genotype. Zymographic assays were performed using gelatin as a substrate to determine the effects of AAu1-AAu3 on extracellular proteases of A. castellanii using 100, 200 and 300 μM concentrations. The results revealed that none of AAu1-AAu3 inhibited A. castellanii proteases when compared with amoeba in RPMI alone. The results are representative of three independent experiments habitat of Acanthamoeba is the environment with diverse respiratory mechanisms and wide exposure to metals, thus Acanthamoeba is likely to possesses mechanisms to inhibit the toxic effects exerted by metals. A. castellanii is well known as a versatile respirator and possesses several mitochondria per cell and respiratory mechanisms that adapt to various aerobic and anaerobic environments to dodge toxic threat and adverse conditions. It is possible that the toxic effects of metals are compensated by switching the type of respiration or the use of an efflux system to rid toxic metals. Future studies are needed to test higher concentration of phosphanegold(I) thiolates compounds and/or in combining phosphanegold(I) thiolates with current anti-amoeba drugs, such as chlorhexidine to determine their improved efficacy against pathogenic Acanthamoeba. Overall, these findings suggest that Acanthamoeba exhibits resistance to toxic effects of gold(I) compounds and could prove to be an attractive model to study mechanisms of metal resistance in eukaryotic cells.

Conclusions
Although gold compounds have shown promise in the treatment of non-communicable diseases such as rheumatoid arthritis, anti-tumour activities, as well as antibacterial properties, and anti-parasitic properties against protozoan pathogens, T. brucei, P. falciparum, L. infantinum, G. lamblia, and E. histolytica, often by targeting respiration pathways, our studies demonstrated that A. castellanii exhibited resistance against their toxic effects. The gold derivatives tested had no effect on the viability of A. castellanii, did not inhibit amoebae growth, or cellular differentiation processes or extracellular proteolytic activities. As Acanthamoeba is a versatile respirator, it can adapt to various aerobic and anaerobic environments to avoid toxic threats. Our studies suggest that Acanthamoeba could prove to be a useful model to study mechanisms of metal resistance in eukaryotic cells.