Heat shock protein 90 homeostasis controls stage differentiation in Leishmania donovani

Abstract

The differentiation of Leishmania parasites from the insect stage, the promastigote, toward the pathogenic mammalian stage, the amastigote, is triggered primarily by the rise in ambient temperature encountered during the insect-to-mammal transmission. We show here that inactivation of heat shock protein (Hsp) 90, with the use of the drugs geldanamycin or radicicol, mimics transmission and induces the differentiation from the promastigote to the amastigote stage. Geldanamycin also induces a growth arrest of cultured promastigotes that can be forestalled by overexpression of the cytoplasmic Hsp90. Moreover, we demonstrate that Hsp90 serves as a feedback inhibitor of the cellular heat shock response in Leishmania. Our results are consistent with Hsp90 homeostasis serving as cellular thermometer for these primitive eukaryotes, controlling both the heat shock response and morphological differentiation.

Figure 1

Geldanamycin-induced growth arrest. (A) Cells of three strains, wild-type (Ld wt), vector control (Ld [pcosTL]), and Hsp90-overexpressing cells (Ld [pcos90]) were seeded at 5 × 10⁶ cells/ml and incubated for 24 h at the indicated concentrations of geldanamycin (GA). Cell density was measured, and the mean values from six independent experiments were calculated and plotted against drug concentration. The error bars represent the SD. (B) Leishmania promastigotes from logarithmically growing (25°C - lg) or from stationary (25°C - st) culture as well as cells heat shocked at 37°C or treated with GA (200 μg/ml) were analyzed by flow cytometry. Culture aliquots were removed after 24 h and stained with SYTOX. G1, S, and G2/M refer to the corresponding cell cycle stages. Percentages of cells in respective cell cycle stages are given.

Figure 2

Amplification of Hsp90 gene copies attributable to geldanamycin selection. (A) SDS-PAGE analysis of wild-type promastigotes (WT) and of two escape strains, I and II, selected at 150 and 200 ng/ml GA, respectively. An equivalent of 10⁶ cells per lane was dissolved in sample buffer and run on a 7.5% gel. The gel was stained with Coomassie blue. The sizes of selected marker proteins (10-kDa ladder; Life Technologies, Gaithersburg, MD) are shown on the left. (B) Immunoblot analysis of the samples described in A. After Western blot, the membrane was probed with a mixture of antibodies directed against Hsp70, Hsp90 and Hsp100. (C) Karyotyping. Chromosomes of L. donovani wild-type and escape strains I and II were separated by pulsed-field gel electrophoresis and stained with ethidium bromide. Saccharomyces cerevisiae chromosomes (strain YPH80) were used as size markers (M). (D) The gel shown in C was subjected to Southern blot, and the membrane was hybridized with a digoxigenin-labeled hsp90 gene probe. The arrows point at bands positive for Hsp90 hybridization. (E) L. donovani wild-type (wt) or escape mutant (e1) cells were seeded at 2 × 10⁶ and incubated for 24 h at increasing GA concentrations. Cell density was measured and plotted against GA concentration.

Figure 3

Effect of geldanamycin on Hsp levels in Leishmania. L. donovani promastigotes were incubated at increasing geldanamycin concentrations. After 24 h, cells were lysed, and the equivalent of 10⁶ cells per lane was separated by SDS-PAGE. Proteins were blotted and probed with a mixture of antibodies directed against Hsp70, Hsp90, and Hsp100. Marker proteins (10-kDa ladder; Life Technologies) are shown on the left.

Figure 4

Geldanamycin-induced synthesis of amastigote-specific A2 proteins. (A) Immunoblot analysis. Samples were collected daily during an in vitro promastigote-to-amastigote differentiation of L. donovani and analyzed by SDS-PAGE and immunoblot with the use of anti-A2 mAb (lanes 1–7). Cells were treated with 200 ng/ml GA at 25°C (lane 8) for 24 h at neutral pH. The pH was shifted to 5.5, and incubation was continued at 25°C with 200 ng/ml GA (lanes 9–13). Note that a family of closely related proteins of different sizes is recognized by the antibody. (B) Immunoblot analysis of wild-type (lane 1–10) and escape strain I (lane 12–19) parasites, incubated for 48 h at 37°C (lanes 1 and 11), with the solvent DMSO (lanes 2 and 12), at the indicated GA concentrations (lanes 3–8 and 13–19), or at the indicated RAD concentrations (lanes 9 and 10). The equivalent of 10⁶ cells per lane was subjected to SDS-PAGE and Western blot and probed with anti-A2 monoclonal antibodies. The positions of marker proteins (10-kDa ladder; Life Technologies) are shown on the left.

Figure 5

Taxol-induced synthesis of A2 protein family. (A) Leishmania promastigotes were incubated for 48 h at various concentrations (nanograms per milliliter) of geldanamycin (GA; lanes 2–5) or radicicol (RAD; lane 6). Alternatively, taxol (TX; lanes 7–8) and hydroxy urea (HU; lanes 9–10) were added at the indicated concentrations. Control cells were cultivated with the solvent DMSO (lane 1), until they were stationary (lane 11) or at 37°C (lane 12). (B) Wild-type (wt; lanes 1–6) and escape strain I (e1; lanes 7–11) promastigotes were incubated at the indicated concentrations of taxol (TX; lanes 4–6 and 9–11) or with the solvent DMSO (lanes 2 and 8). To exclude effects attributable to DMSO, in lane 1 and 7, lysates of untreated cells are shown. After 48 h the cells were lysed, and an equivalent of 10⁶ cells per lane was subjected to SDS-PAGE and Western blot and probed with anti-A2 monoclonal antibodies.

Figure 6

Morphological differentiation of Leishmania cells after Hsp90 inhibition. Promastigote parasites were cultivated for 48 h under the indicated conditions. Cells were then stained with anti–alpha-tubulin antibodies and anti-chicken IgG/DTAF and visualized by fluorescence microscopy.

Figure 7

Morphological differentiation of GA-treated Leishmania cells. Promastigote L. donovani cells at 25°C, parasites incubated at 37°C for 24 h, axenic amastigotes after 5 d differentiation at 37°C and pH 5.5, and parasites treated at 25°C, 100 ng/ml GA for 24 h were imaged by scanning electron microscopy. Bar, 10 μm.