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Acanthamoeba is a genus of small free-living amoebas characterized by a life cycle of active trophozoites and dormant cysts (21, 28). Human infection due to Acanthamoeba spp. involving the brain, eyes, lungs, and skin has increased significantly during the last 10 years (10, 13, 16). Numerous in vitro studies, carried out to elucidate the virulence factors responsible for Acanthamoeba infections, have shown both contact-dependent cell killing and cell-free cytopathogenicity due to metabolites released by trophozoites (14, 15, 22), which confirms that amoebic exotoxins, enzymes, or other unidentified molecules may be involved in the interactions between the amoebas and host cells. The role played by soluble molecules that may participate in acanthamoebal cytopathogenicity has yet to be completely elucidated.

Alizadeh et al. (2, 22) have reported that corneal epithelial cells, melanoma cells, and murine neuroblastoma cells exposed to aqueous extracts of Acanthamoeba trophozoites undergo lysis by a process involving apoptosis.

We have previously shown (17) that heat-resistant and nonproteinic molecules with a low molecular weight (<10 kDa), released by viable A. castellanii trophozoites, produce a serious cytopathic effect in human epithelial Wish cells in vitro, causing a cytosolic-free-calcium ([Ca²⁺]_i) increase, morphological changes, cytoskeletal alterations, a decrease in cell viability, and cell death. Since the increase in cytosolic free-calcium concentration was immediate in Wish cells, in this connection we also hypothesized that the [Ca²⁺]_i rise may be the prime cause of cell death, but the identity of the molecules inducing calcium overload and the mechanisms of cell death were not characterized.

The present investigation was undertaken to identify the chemical nature of these amoebic toxic compounds and to further characterize their cytotoxic action on human epithelial Wish cells.

Our study was performed using trophozoites of A. castellanii, isolated from a case of amoebic keratitis (in Ancona, Italy), axenically grown at 25°C in PYG medium (11). The species identification of this isolate was based on cyst morphology and indirect immunofluorescence microscopy. Our previous observations have shown that this A. castellanii isolate, at 37°C, exerted contact-dependent cell killing on Wish cells.

Preparation of amoebic cell-free supernatants. Amoebic cell-free supernatants were prepared as described previously (17). Amoebas were washed twice in phosphate-buffered saline solution (PBS) without Ca²⁺ and Mg²⁺ at pH 7.4 were resuspended (6 × 10⁶ cells/ml) in the same buffer or in RPMI medium containing 20 mM HEPES and incubated for 2 h at 25°C. Supernatants of these cultures, obtained by centrifugation at 500 × g for 15 min, were treated at 95°C for 10 min, ultrafiltered through Centriprep-10 microconcentrators (Amicon), which have a molecular cutoff of 10 kDa, and used immediately after processing as cPBS and cRPMI, respectively. In particular, cPBS was used only in [Ca²⁺]_i measurements, since some aromatic RPMI medium components could interfere with this assay.

Characterization of heat-resistant compounds. To characterize the chemical nature of the heat-resistant compounds with low molecular weight released by Acanthamoeba trophozoites we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis techniques. Several peptides, such as staphylococcal aplha-toxin and delta-toxin (5), dissolving in the lipid bilayer have been shown to increase the membrane permeability to specific inorganic ions and to produce cytolysis. Tricine SDS-PAGE experiments, performed according to the method of Schagger and von Jagow (26), nevertheless excluded the presence of small peptides in both cPBS and cRPMI (data not shown). Capillary electrophoresis analysis of amoebic-cell-free supernatants was performed according to the method of Banditelli et al. (4), with a P/ACE 2100 System (Beckman Instruments) at 20 kV, using capillary tubing 50 µm in diameter and 50 cm long and a buffer of 40 mM glycine and 50 mM sodium dihydrogen phosphate (pH 9). These experiments have shown the presence of ADP in significant micromolar concentrations (Fig. 1), and the ADP concentration in both cPBS and cRPMI was calculated to be about 20 µM. Similar results were obtained when we incubated amoebae for 2 h at 37°C. Extracellular purine nucleosides and nucleotides are ubiquitous, phylogenetically ancient intercellular signals that can interact with specific cell surface receptors to mediate a variety of biological responses (20, 23). In the past decade, the cytotoxic properties of extracellular nucleotides have received a lot of attention. It has been shown, in fact, that ATP and ADP can affect the plasma membrane permeability of cultured cells (9, 27) and cause elevation of the [Ca²⁺]_i concentration and apoptosis in certain types of cell (1, 7, 19, 25, 30). In order to establish the role of amoebic released-ADP in A. castellanii cell-free cytopathogenicity, therefore, we have compared the cytopathic effect exerted on human epithelial Wish cells, by both ultrafiltered Acanthamoeba cell-free supernatants and external ADP (ADP₀).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Electropherograms of 100 µM purified ADP solution (A), heat-treated filtered cell-free RPMI (cRPMI) conditioned for 2 h by A. castellanii (6 × 106 trophozoites/ml) (B), or heat-treated filtered cell-free PBS (cPBS) conditioned for 2 h by A. castellanii (6 × 106 trophozoites/ml) (C).

[Ca²⁺]_i measurement. The Wish cell line was routinely maintained in RPMI 1640 medium (GIBCO BRL/Life Technologies Italy) supplemented with 10% heat-inactivated fetal calf serum (GIBCO BRL/Life Technologies Italy), 100 U of penicillin G, and 100 µg of streptomycin sulfate per ml and grown in 25-cm² plastic flasks at 37°C in a 5% CO₂ atmosphere. Intracellular calcium in Wish cells was monitored by using the fluorescent calcium probe FURA 2-AM (Sigma-Aldrich S.r.l., Milano, Italy). Dye loading was standardized by incubating cells, suspended in HEPES buffer (137 mM NaCl, 1.2 mM MgSO₄, 1.5 mM CaCl₂, 5 mM KCl, 15 mM glucose; pH 7.4); with 3 µM Fura 2-AM for 10 min at room temperature. Loaded cells were washed twice with the same buffer, and the assay was performed on a stirred aliquot (0.5 ml), at 37°C, with the use of a Hitachi F-2000 spectrophotometer. The excitation and emission wavelengths were 340 to 380 nm and 510 nm, respectively, detected every 500 ms, and stored in separate memories of the F-2000 spectrophotometer. A data Menager was used to monitor the fluorescence signal of Fura-2 AM-loaded cells. Basal and stimulated cytosolic calcium were quantified according to the method of Grynkiewicz et al. (12) by using the ratio technique and a K_d of 224 nM as the dissociation constant of Fura-2 AM; maximal and minimal values of fluorescence were evaluated after the addition of of 0.006% Triton X-100 and 10 mM EGTA, respectively. Hitachi F-2000 software was used for calculation.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Time courses of the [Ca2+]i increase evoked in Wish cells, loaded with Fura 2-AM (3 µM) by 80 µl of heat-treated filtered cell-free PBS (cPBS), conditioned for 2 h by (6 × 106/ml) A. castellanii trophozoites (A) by the same volume of PBS containing 20 µM ADP0 (B); by cPBS (C) or 20 µM ADP0 (D) after chelation of extracellular Ca2+ with 5 mM EGTA; and by cPBS (E) and 20 µM ADP0 (F) after 20 min of loaded Wish cell exposure to 10 µM ryanodine.

View this table: [in this window] [in a new window]   TABLE 1.   Effect of two purinergic antagonists on the cytosolic free-calcium peak increase ([Ca2+]i) induced on Wish cells stimulated with heat-treated filtered cell-free PBS-20 mM HEPES conditioned for 2 h by A. castellanii trophozoites (6 × 106/ml) (cPBS) or PBS-20 mM HEPES containing 20 µM ADP0a

Phase-contrast microscopy, actin microfilament analysis, and assessment of apoptosis. It is known that [Ca²⁺]_i deregulation is an important link and signaling event in cells following a variety of injuries. The [Ca²⁺]_i increase, by activation of proteases, endonucleases and phospholipase, can cause cellular prelethal changes which can occur by two principal patterns: oncosis or apoptosis (29). It has also been demonstrated that changes in cytoskeletal organization can be relevant for the cell destiny (6). Hence, to characterize the cytopathic effect induced by amoebic-cell-free supernatant and ADP₀, Wish cell monolayers growing on glass coverslips, in a CO₂ incubator at 37°C, were exposed for 1, 3, 6, 9, and 24 h to 0.5 ml of cRPMI (obtained as described above), 0.5 ml of RPMI containing 20 µM ADP₀, or the same volume of fresh medium (cell controls). At the selected time intervals, the cell morphology was observed with a Zeiss (Tilaval 31) microscope equipped with a 40 × lens. After incubation with cRPMI or RPMI containing 20 µM ADP₀, Wish cells underwent morphological changes which are typical of classical apoptosis. In both cases they developed extensive cell membrane blebs, nuclear condensation, and overall cell shrinkage (Fig. 3A, C, and E). Cell damage, on the contrary, was not observed in samples incubated for up to 9 h, with cRPMI or RPMI containing 20 µM ADP₀, in the presence of 20 µM suramin (Fig. 3B, D, and F).

View larger version (129K): [in this window] [in a new window]   FIG. 3.   Phase-contrast microscopy of Wish cells incubated for 3 h at 37°C in 5% CO2 atmosphere with RPMI medium in the absence (A) or in the presence (B) of 20 µM suramin; with heat-treated filtered cRPMI, conditioned for 2 h by 6 × 106 A. castellanii trophozoites, in the absence (C) or in the presence (D) of 20 µM suramin; or with RPMI containing 20 µM ADP0 in the absence (E) or in the presence (F) of 20 µM suramin. Magnification, × 400.

View larger version (85K): [in this window] [in a new window]   FIG. 4.   Actin microfilaments of Wish cells incubated for 3 h at 37°C in 5% CO2 atmosphere with RPMI medium (A), with cRPMI for 3 h (C) and 6 h (D), or with RPMI containing 20 µM ADP0 for 3 h (F) and 6 h (G) and nuclei of Wish cells incubated for 3 h at 37°C in a 5% CO2 atmosphere with RPMI medium (B), with cRPMI (E), or with RPMI containing 20 µM ADP0 (H). Magnifications: A, C, D, F, and G, ×420; B, H, and F, ×350.

Conclusions. The present findings show that A. castellanii trophozoites constitutively release ADP in the medium and suggest that this compound might play an important role in cell-free cytopathogenicity due to A. castellanii. Our data, albeit indirectly, demonstrate that ADP is very probably the heat-resistant low-molecular-weight component of the amoebic-cell-free supernatant that causes increase in cytosolic free calcium, morphological changes, cytoskeletal damage, and death in Wish cells. In fact, purified ADP and A. castellanii culture supernatants cause a similar pattern of calcium fluxes and apoptotic cell death that are blocked by the P_2y2 inhibitor suramin. It has been shown that ATP can also interact with P_2y2 cell surface receptors to induce elevation of cytosolic free calcium and apoptosis in mammalian cells. In our experimental conditions, however, only ADP has been detected in the conditioned medium. We still do not know whether apoptotic cell death caused by both cRPMI and purified ADP depends directly on ADP molecules or also on ADP-metabolite generates, for example, by cleavage of the phosphate groups. Further studies are necessary to conclusively demonstrate this and to determine whether the ADP released from A. castellanii trophozoites is derived from exocytotic granules and/or vesicles or from the cytosolic ADP pool via intrinsic plasma membrane channels or pores.