Fig 1.
C14dm‾ promastigotes show high mitochondrial membrane potential (ΔΨm).
(A) Log phase promastigotes were labeled with 100 nM of Mitotracker CMXRos followed by DAPI staining. WT parasites pretreated with CCCp were included as controls. DIC: differential interference contrast. (B) Representative transmission electron micrographs showing mitochondria in log phase promastigotes. Black arrows indicate areas containing kinetoplast (top panel) and tubular mitochondria (bottom panel). (C) To measure ΔΨm, log phase and stationary phase (day 1-day 4) promastigotes were labelled with 100 nM of TMRE for 15 min in PBS and mean fluorescence intensity (MFI) were determined by flow cytometry. Controls include WT and c14dm‾ parasites pretreated with CCCp and WT cells pretreated with 0.2 μM of ITZ. Bar values represent averages from three experiments and error bars represent standard deviations. (D) Live parasites imaging after TMRE labeling.
Fig 2.
Inactivation of C14DM leads to ROS accumulation in the mitochondria.
(A, B and F) Promastigotes were incubated in complete M199, serum-free M199, PBS contain 5.5 mM of glucose, or PBS only and labeled with 100 nM of TMRE (A, for 15 min), 5 μM of MitoSox Red (B, for 25 min) or 5 μM of DHE (F, for 30 min). MFIs were determined by flow cytometry. Effects of CCCp (75 μM, 15 min) were also monitored in A and B. (C) Log or stationary phase (day 1-day 4) promastigotes were labeled with 5 μM of MitoSox Red for 25 min in PBS and MFIs were determined by flow cytometry and normalized to values from log phase WT. WT cells grown in the presence of ITZ (for 48 hours) were also analyzed. (D and E) Live cell imaging of L. major promastigotes (D) or L. mexicana axenic amastigotes (grown in the absence or presence of 0.1 μM of ITZ for 48 hours, E) after labelling with MitoSox Red in PBS. White arrows indicate kDNA binding of MitoSox. (E) Live after labeling with MitoSox Red. In A-C and F, averaged values from five experiments are shown and error bars represent standard deviations.
Fig 3.
Increased superoxide dismutase (SOD) activity in c14dm‾ parasites.
(A) Whole cell lysates from log phase and stationary phase (day 1-day 3) promastigotes were collected as described in Materials and Methods. SOD activity was measured using a SOD assay kit and results were normalized based on the amount of protein in each sample. Error bars represent standard deviations from two independent experiments. (B) Mitochondria enriched membrane fractions (Mem.) and cytosolic fractions (Sup.) were isolated from log phase promastigotes as described. Western blots were performed using antibodies against L. amazonensis SODA (mitochondrial) and SODB (both SODB1 and SODB2, glycosomal).
Fig 4.
C14dm‾ parasites show compromised cellular respiration.
(A-C) Log phase promastigotes were incubated in a mitochondrial respiration buffer (A-B) or DMEM (C) and oxygen consumption rates (OCR) were determined using the MitoXpress probe as described in Materials and Methods. Buffer only and buffer with probe (no cells) were included as blanks (A). In A and C, fluorescence signals (in arbitrary units or A.U.) were measured every 90 s. In B, the relative OCR from the experiments in A was calculated by subtracting the 0 min reading from the 15 min reading. WT parasites treated with antimycin A (AA) were included as controls. (D) Cellular ATP contents in log phase promastigotes were measured after 1 hour incubation in an assay buffer (21 mM of HEPES, 0.7 mM of Na2HPO4, and 137 mM of NaCl, pH 7.2) in the absence or presence of glucose (Glu, 5.5 mM), sodium pyruvate (Pyr, 5.5 mM), 2-deoxy-D-glucose (2DG, 5.5 mM), or sodium azide (NaN3, 20 mM). All experiments were repeated three or four times and error bars represent standard deviations.
Fig 5.
C14dm‾ parasites are hypersensitive to starvation.
Log phase promastigotes were inoculated in HBSS in the absence or presence of 2DG (5.5 mM), Pyr (5.5 mM), aspartate (Asp, 5.5 mM), Glu (5.5 mM), or glycerol (0.1 mM). In A, percentages of cell death for c14dm‾ mutants were determined at the indicated time points. In B, percentages of cell death were determined after 24 hours for WT, c14dm‾, c14dm‾/+C14DM, smt‾, and smt‾/+SMT parasites. Error bars represent standard deviations from three independent experiments.
Fig 6.
C14dm‾ parasites show increased uptake of glucose and glycerol.
Incorporation of radiolabeled glucose (A) or glycerol (B and C) were determined as described under Materials and Methods. Transport assays were performed at room temperature (A and B unless specified) or 4 °C (C). Error bars represent standard deviations from three independent experiments.
Fig 7.
ROS accumulation contributes to heat sensitivity in c14dm‾ mutants.
(A-E) Log phase promastigotes were incubated in PBS at 27 °C or 37 °C for 3 hours. In A, percentages of cell death were determined by flow cytometry after PI staining. In B, ΔΨm was determined after TMRE labeling. Unlabeled WT parasites were included as a negative control (Neg). In C-D, parasites were lysed and cytosolic fractions (Sup.) were separated from mitochondrial fractions (Mem.) as described in Materials and Methods. Distribution of cytochrome c and HSP83 (a cytosolic protein marker) were determined by Western blots (C) and quantified (D). In E, parasites were labelled with the cytosolic ROS indicator DHE. Fluorescence intensities were measured by flow cytometry and relative cytosolic ROS levels were determined. (F) Log phase promastigotes were incubated at 37 °C in the absence or presence of reduced GSH. After 8 hours, mitochondrial ROS levels were measured as described. (G) C14dm‾ parasites were incubated at 37 °C in the absence or presence of reduced GSH and percentages of cell death were determined at 0 and 8 hours. (H) WT parasites were incubated at 27 °C or 37 °C in the absence or presence of AA (10 μM) and percentages of dead cells were determined by flow cytometry at the indicated time points. †: Very few WT+AA cells were detectable after 36 hours at 37 °C. Error bars represent standard deviations from three independent experiments.
Fig 8.
C14dm‾ parasites show increased sensitivity to pentamidine (PEN).
(A) Log phase promastigotes were inoculated in M199 media containing various concentrations of PEN. Culture densities were determined after 48 hours and the PEN concentrations required to inhibit growth by 90%, 50%, and 25% were determined (as EC90, EC50 and EC25 respectively) using cells grown in the absence of PEN as controls. “WT + ITZ” represent WT parasites grown in the presence of 0.5 μM of ITZ. (B-C) Log phase promastigotes were inoculated in M199 media containing 2.5 μM (B) or 25 μM (C) of PEN for one hour, followed two washes with PBS. Cells were then re-inoculated in M199 media and culture densities were determined every 24 hours. Some c14dm¯ mutants were also incubated in the presence of 75 μM of CCCp along with PEN (for one hour). Parasites originally inoculated in the absence of PEN were included as controls. Error bars represent standard deviations from three or four independent experiments.