A homolog of cyclophilin D is expressed in Trypanosoma cruzi and is involved in the oxidative stress-damage response

Abstract

Mitochondria have an important role in energy production, homeostasis and cell death. The opening of the mitochondrial permeability transition pore (mPTP) is considered one of the key events in apoptosis and necrosis, modulated by cyclophilin D (CyPD), a crucial component of this protein complex. In Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, we have previously described that mitochondrial permeability transition occurs after oxidative stress induction in a cyclosporin A-dependent manner, a well-known cyclophilin inhibitor. In the present work, a mitochondrial parasite cyclophilin, named TcCyP22, which is homolog to the mammalian CyPD was identified. TcCyP22-overexpressing parasites showed an enhanced loss of mitochondrial membrane potential and loss of cell viability when exposed to a hydrogen peroxide stimulus compared with control parasites. Our results describe for the first time in a protozoan parasite that a mitochondrial cyclophilin is a component of the permeability transition pore and is involved in regulated cell death induced by oxidative stress.

Figure 1

Expression and subcellular localization of TcCyP22 in transgenic epimastigotes. (a) Monoclonal antibodies against GFP were used to detect TcCyP22 (green). Co-localization with MitoTracker (red) was observed. Merge images show the co-localization in yellow. DAPI was used to stain nucleus and kinetoplast (blue). DIC, differential interference contrast microscopy. (b) Antibodies against GFP detected unique bands of the expected sizes in parasite lysates. Antibodies against α1-tubulin were used to confirm equal loading. 22, epimastigotes carrying the pTREX-TcCyP22-GFP; T, epimastigotes carrying the pTREX-GFP empty vector, as a control; wt, wild-type epimastigotes.

Figure 2

Loss of mitochondrial membrane potential (ΔΨmm) in oxidative stress conditions. Parasites were loaded with the ΔΨmm fluorescent indicator, MitoTracker. Oxidative stress conditions were generated with 5 mM H₂O₂. At 15 and 60 min, parasites were analyzed by flow cytometry. In (a), dot plots of a representative experiment are shown. (b) Quantification of three independent experiments. TREX, Control parasites carrying the empty pTREX-eGFP vector; 22-OE, transgenic parasites overexpressing TcCyP22-GFP (means±s.d., n=3, ***P<0.001 (22-OE versus TREX at 15 min), two-way – RM – ANOVA).

Figure 3

DNA damage in oxidative stress conditions. Oxidative stress conditions were generated with 5 mM H₂O₂. At 60 and 180 min, parasites were fixed and stained for TUNEL, according to manufacturers’ specifications. (a) Fluorescence images of a representative experiment. TUNEL⁺ parasites present nuclei with red staining. (b) Quantification of TUNEL⁺ parasites, expressed as percentage over total parasites for each condition. TREX, Control parasites carrying the empty pTREX-eGFP vector; 22-OE, transgenic parasites overexpressing TcCyP22-GFP (means±s.d., n=3, ***P<0.001 (22-OE versus TREX at 60 min), Student’s t-test).

Figure 4

Loss of plasmatic membrane integrity during oxidative stress conditions. Parasites were incubated with 5 mM H₂O₂. At 60 and 180 min, parasites were stained with propidium iodide and analyzed by flow cytometry. In (a), dot plots of a representative experiment are shown. (b) Quantification of three independent experiments. TREX, Control parasites carrying the empty pTREX-eGFP vector; 22-OE, transgenic parasites overexpressing TcCyP22-GFP (means±s.d., n=3, **P<0.01 (22-OE versus TREX at 60 min); ***P<0.001 (22-OE versus TREX at 180 min), Two-way – RM – ANOVA).

Figure 5

Phosphatidylserine exposure during oxidative stress. Parasites were incubated with 5 mM H₂O₂. At 60 and 180 minutes, parasites were stained with annexin V-phicoeritrin and 7AAD and analyzed by flow cytometry. Heat-shock treated parasites were used as positive control. In (a), dot plots of a representative experiment are shown. (b) Quantification of three independent experiments. TREX, Control parasites carrying the empty pTREX-eGFP vector; 22-OE, transgenic parasites overexpressing TcCyP22-GFP ***P<0.001 (22-OE versus TREX at 180 min), two-way – RM – ANOVA).