Inhibition of HSP90 in Trypanosoma cruzi Induces a Stress Response but No Stage Differentiation

Parasites. T. cruzi Tulahuen (World Health Organization reference strain M/HOM/CH/00/Tulahuen C2) epimastigotes were maintained in logarithmic growth in LIT medium (3) containing 10% fetal calf serum and subcultivated by weekly dilutions. Blood form trypomastigotes were maintained by weekly inoculations of semiconfluent HG39 cells and drawn from the supernatants for experimental procedures. The concentration of parasites was determined by counting viable cells, i.e., cells that did not stain with trypan blue, in a Neubauer chamber. Parasite cells were applied to glass slides, fixed, and stained with Giemsa's stain for light microscopy.

Chemicals.GA was purchased from Sigma-Aldrich Chemicals, Taufkirchen, Germany, or from A.G. Scientific, San Diego, Calif. A stock solution of 1 mg ml⁻¹ was prepared in dimethyl sulfoxide (DMSO). Hydroxyurea (HU; stock, 500 mM in DMSO), paclitaxel (Taxol [TX], stock, 10 mM in DMSO), and DMSO were all purchased from Sigma-Aldrich Chemicals.

Indirect immunofluorescence microscopy.Approximately 10⁵T. cruzi cells were applied to poly-l-lysine-coated glass slides. Cells were fixed (5 min in methanol), blocked (0.2% iBlock, 0.02% Tween 20, 0.1% Triton X-100 in phosphate-buffered saline [PBS]), and incubated for 1 h at 35°C with a monoclonal anti-alpha-tubulin antibody (1:2,000 in blocking buffer; clone B-5-2-1; Sigma). Detection was achieved using a 5-(4,6-dichlorotriazinyl)-amino fluorescein-labeled goat anti-mouse antibody (Dianova). Samples were analyzed on a Leica DM RB microscope with a confocal TCS NT system at a wavelength of 492 nm.

Scanning electron microscopy. T. cruzi blood form trypomastigotes were washed twice in PBS, fixed in 2% glutaraldehyde in sodium cacodylate buffer, and postfixed with 1% osmium. Samples were dehydrated at increasing ethanol concentrations (30 to 100%). After critical-point drying, samples were treated with gold and analyzed with a Philips SEM500 scanning electron microscope.

Flow cytometry. T. cruzi epimastigotes were harvested by centrifugation (800 × g, 5 min), washed twice in PBS, and fixed for 1 h in 70% methanol at 4°C, followed by treatment with 20 μg of RNase A/ml (20 min, 37°C). Cells were resuspended in citrate buffer (45 mM MgCl₂, 30 mM sodium citrate, 20 mM morpholinepropanesulfonic acid [MOPS; pH 7.0], 0.1% Triton X-100) and labeled for 20 min with 1 μM SYTOX Green nucleic acid stain (Molecular Probes). Following staining, cells were washed twice in PBS supplemented with 5% fetal calf serum and 0.01% sodium azide. Samples were stored in the dark at 4°C until analysis. The fluorescence of 10⁴ cells was analyzed on a fluorescence-activated cell sorter (Becton Dickinson, Heidelberg, Germany).

Immunoblot analysis.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western transfer were performed as described previously (8). Briefly, membranes were treated with blocking buffer (5% milk powder and 0.1% Tween 20 in Tris-buffered saline), with chicken polyclonal immunoglobulin Y (diluted 1:2,500 to 1:5,000 in blocking buffer) raised against Leishmania HSP, and with anti-chicken immunoglobulin G-alkaline phosphatase conjugate (diluted 1:2,500 in blocking buffer; Dianova). Blots were stained with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate.

Increased HSP expression in T. cruzi following treatment with GA.The inhibition of HSP90 by GA can induce elevated HSP levels in the related protozoan L. donovani (31, 32). In order to analyze the effect of HSP90 inhibition on T. cruzi, we incubated epimastigotes at a concentration of 10⁶ cells ml⁻¹ with various concentrations of GA and analyzed the effects by SDS-PAGE and immunoblotting. As shown in Fig. 1A, distinct protein bands of 90 and 70 kDa were induced in a GA dose-dependent manner. These bands with increased intensity were identified as the proteins HSP70 and HSP90 by immunoblotting (Fig. 1B). HSP100 is barely detectable by immunoblotting under standard culture conditions. However, GA treatment increased HSP100 abundance severalfold. We conclude that the inhibition of HSP90 in T. cruzi induces the expression of at least three HSP, a hallmark of the cellular stress response.

• Open in new tab
• Download powerpoint

FIG. 1.

GA induces a dose-dependent increase of HSP expression and blocks the proliferation of T. cruzi epimastigotes. Cells were axenically cultured with increasing concentrations of GA for 48 h. After lysis of cells, the proteins of 10⁶ cells were separated by SDS-PAGE and visualized with Coomassie blue (A). A replica gel was blotted and probed with anti-HSP polyclonal antiserum (B). Cells were counted with a Neubauer chamber (C). The experiment was performed in quadruplicate with identical outcomes.

Growth arrest due to HSP90 inhibition.In order to analyze the effect of GA on epimastigote proliferation, cells were seeded at 10⁶ cells ml⁻¹ and incubated with GA at various concentrations. After 48 h, the cell density was determined and plotted against GA concentration (Fig. 1C). The proliferation of T. cruzi epimastigotes was completely blocked at 200 ng ml⁻¹, with a 50% inhibitory concentration between 50 and 100 ng ml⁻¹. Removal of GA reestablished the proliferation of epimastigotes after approximately 7 days (data not shown). Blood form trypomastigotes cultivated xenically showed inhibition of proliferation at a GA concentration of 50 ng ml⁻¹ (data not shown). However, feeder cell proliferation was also abolished in these cultures at the same GA concentration.

Under axenic conditions, blood form trypomastigotes do not proliferate but transform into epimastigotes via intermediate stages. These intermediate stages resume cell division at a low rate. In the presence of GA, the formation of intermediate stages in the trypomastigote-to-epimastigote transition was blocked (see Fig. 3) and no proliferation was observed (data not shown).

GA arrests cell cycle progression of trypanosomes in G₁ phase.Epimastigotes and blood form trypomastigotes were incubated axenically in the presence or absence of 200 ng of GA ml⁻¹ for 72 h. Control cultures were incubated with either an equivalent amount of the solvent (DMSO); 1 mM HU, which blocks cell cycle progression in G₁; or 25 μM TX, which blocks cell cycle progression in G₂/M. While the controls incubated with DMSO displayed continuous logarithmic growth after 72 h, with a typical biphasic staining representative of cells in G₁ and G₂/M phases, cells incubated with GA were arrested in the G₁ phase of the cell cycle (Fig. 2). As expected, growth of epimastigotes that were incubated with HU and TX was arrested in G₁ phase and G₂/M phase, respectively. We also observed an upshift of fluorescence intensity in the samples that were treated with the growth inhibitors. This is a phenomenon we and others observe frequently (see Discussion).

• Open in new tab
• Download powerpoint

FIG. 2.

GA arrests the growth of T. cruzi epimastigotes in G₁ phase of the cell cycle. Cells (10⁷) growing logarithmically were incubated with either 200 ng of GA ml⁻¹, the solvent DMSO at an equivalent concentration, 5 mM HU, or 25 μM TX for 72 h. Subsequently, cells were stained with SYTOX Green and analyzed by flow cytometry. G₁ and G₂/M designate cell populations with a DNA content representative of the corresponding cycle stages. The experiment was performed in triplicate with identical outcomes. FL1-H, relative fluorescence intensity.

Blood form trypomastigotes that were drawn from supernatants of infected HG39 cells and further cultivated axenically displayed arrest in G₁ phase irrespective of the presence of GA (data not shown).

Morphological changes due to GA treatment.Epimastigotes and blood form trypomastigotes were incubated axenically with 200 ng of GA ml⁻¹ or with an equivalent amount of the solvent DMSO. After 3 days, cells were adhered onto slides for staining either with Giemsa's stain or by indirect immune fluorescence with anti-alpha-tubulin antibody (Fig. 3). Epimastigotes (Fig. 3A and B) cultured with GA showed no significant morphological change (Fig. 3E and F), apart from the appearance of small numbers of binucleated forms in the culture. Under control conditions, no binucleated forms were observed (Fig. 3C and D).

• Open in new tab
• Download powerpoint

FIG. 3.

GA induces morphological change of T. cruzi blood form trypomastigotes but not of epimastigotes. Cells were axenically incubated with 200 ng of GA ml⁻¹ for 72 h and subsequently adhered onto slides. They were stained either with Giemsa's stain for light microscopy (B, D, F, H, K, and M) or with anti-tubulin antibody and a 5-(4,6-dichlorotriazinyl)-amino fluorescein-labeled secondary antibody for immunofluorescence (A, C, E, G, I, and L). The figure shows representative results from one out of three independent experiments. d0, day 0; d3, day 3.

Under axenic culture conditions with DMSO, blood form trypomastigotes (Fig. 3G and H) transformed towards pear-shaped intermediate stages (Fig. 3I and K), the first step towards epimastigote differentiation. Under GA treatment, by contrast, the transition of blood form trypomastigotes to intermediate stages and then to epimastigotes was abolished. Instead, rounded forms appeared after 2 to 3 days (Fig. 3L and M).

The rounded forms induced by GA treatment of blood form trypomastigotes were further analyzed by scanning electron microscopy (Fig. 4). The trypomastigotes observed at day 0 (Fig. 4A) showed a normal appearance and developed into intermediate forms within 3 days (Fig. 4B) when they were cultivated under control conditions (with DMSO). By contrast, trypomastigotes treated with GA did not differentiate towards intermediate stages but developed forms reminiscent of spheromastigotes, with distinctly visible, short flagella.

• Open in new tab
• Download powerpoint

FIG. 4.

GA induces the development of spheromastigote-like culture forms from T. cruzi blood form trypomastigotes. Trypomastigotes derived from the supernatants of infected feeder cells were incubated axenically for 72 h with 200 ng of GA ml⁻¹ or with an equivalent amount of the solvent DMSO. Cells were visualized by scanning electron microscopy. The figure shows representative results from one out of three independent experiments. d0, day 0; d3, day 3.

In Fig. 5, a more detailed analysis of blood form trypomastigotes treated with GA is shown. Representative micrographs of trypomastigotes (day 0) and of the culture forms after 1, 2, and 3 days of cultivation with or without GA are shown. In the control culture (DMSO), pear-shaped transition forms appeared after 1 day and became the dominant culture form on days 2 and 3. These transition forms were barely observed in the cultures treated with GA. Here, spheromastigotes appeared on day 1 and were the dominant culture forms on days 2 and 3. A quantitative analysis of the three distinguishable culture forms in the samples is given in Table 1.

• Open in new tab
• Download powerpoint

• Open in new tab
• Download powerpoint

FIG. 5.

Development of blood form trypomastigotes under GA treatment (+ GA) or in a control culture with the solvent DMSO added (+DMSO). Cells were axenically incubated with 200 ng of GA ml⁻¹ for up to 3 days and subsequently adhered onto slides. They were stained with Giemsa's stain for light microscopy. The images show representative results for each day and culture.

The spheromastigote-like forms induced by GA did not differentiate back to trypomastigotes after GA removal, and they did not begin to transform into epimastigotes after GA was removed. No proliferation was observed with these forms, and eventually they died.