Figure 1.
Temporal and spatial expression of ISCL in L. major.
(A) Elevated ISCL transcript level in stationary phase promastigotes. L. major WT promastigotes were cultured in M199 medium at a starting density of 1.0×105 cells/ml and total RNA was extracted daily. The relative abundance of ISCL mRNA was determined by quantative RT-PCR using the constitutively expressed rRNA45 gene as an internal control. EL: early log phase culture (1–2×106 cells/ml); LL: late log phase culture (6–10×106 cells/ml); S1–S4: day 1–4 stationary phase culture (2.0–3.0×107 cells/ml). *: p<0.05, **: p<0.01. Error bars represent standard deviations from two independent experiments. (B) Increased expression of ISCL protein in stationary phase promastigotes and lesion amastigotes. Left: whole cell lysates from WT late log phase promastigotes (LL), day 1–4 stationary phase promastigotes (S1–S4), and late log phase promastigotes of iscl− and iscl−/+ISCL were subjected to immunoblot analysis using the affinity purified anti-ISCL antibody (top) or an anti-α-tubulin antibody as loading control (bottom). Right: immunoblot of cell lysates from WT day 3 stationary phase promastigotes (S3 pro.) and lesion amastigotes (ama.). Each lane contained material from 1×106 cells. (C–J) Localization of endogenous ISCL in L. major. WT promastigotes (C–F) and amastigotes (G–J) were fixed and permeabilized with ethanol as described in Materials and Methods. Cells were then labeled with rabbit anti-ISCL antibody, followed by staining with goat-anti-rabbit IgG-FITC (D, H) and Mitotracker Red 580 (E, I). (F) Overlay of D and E. (J) Overlay of H and I. Control cells that were labeled with secondary antibody only did not show any green fluorescence (data not shown). Experiments in C–F were performed three times and results from one representative set are shown here.
Figure 2.
Functional analysis of ISCL by mutagenesis.
(A) Schematic diagram of ISCL open reading frame and the mutations introduced in this study. Asterisks represent the three aspartic acids (D116, D200, and D383) that were mutated; grey bars represent the region recognized by the anti-ISCL peptide antibody; and black bars represent the transmembrane helices. (B) Western-blot to confirm the expression of mutated ISCL. Whole cell lysates from WT, iscl−, iscl−/+ISCL, iscl−/+ISCL D116G, iscl−/+ISCL D200G, iscl−/+ISCL D383G, iscl−/+ISCLΔ and iscl−/+pXG (empty vector) promastigotes were probed with either anti-ISCL (top) or anti-α-tubulin (bottom) antibody. Each lane contained material from 6×105 cells. (C–D) The SMase and IPCase activities in log phase promastigotes were determined as described in Materials and Method. Error bars represent standard deviations from 3 independent experiments.
Figure 3.
Mutated forms of ISCL failed to complement iscl− parasites.
(A–B) Promastigotes were cultured in M199 medium and percentages of round cells (defined as those promastigotes whose long axis is shorter than twice the length of the short axis) (A) and dead cells (B) were recorded in late log (LL) and stationary phase promastigotes (S1–S5) as described [21]. Each group of bars represents cells in the following order (from left to right): WT, iscl−, iscl−/+ISCL, iscl−/+pXG, iscl−/+ISCL D116G, iscl−/+ISCL D200G, iscl−/+ISCL D383G, and iscl−/+ISCLΔ. (C–D) Stationary phase promastigotes (•: WT, ○: iscl−, ▾: iscl−/+ISCL, ▪: iscl−/+ISCL D116G, □: iscl−/+ISCL D200G, ⧫: iscl−/+ISCL D383G, ⋄: iscl−/+ISCLΔ) were used to infect bone-marrow derived macrophages (Mφs) from BALB/c mice at a ratio of 15 parasite per Mφ. Percentages of infected Mφs (C) and parasites per 100 Mφs (D) were determined at 2, 24, 48, and 72 hours post infection as described. As a control, WT parasites were also used to infect Mφs that were activated with 100 ng/ml of LPS and 100 ng/ml of IFN-γ (▵: WT+activated Mφs). Experiments were repeated 3 times and error bars represent standard deviations.
Figure 4.
Introduction of a sole SMase or sole IPCase into iscl−.
Whole cell lysates from late log phase promastigotes of WT, iscl−, iscl−/+HA-ISCL, iscl−/+HA-BcSMase, iscl−/+5′HASPb-BcSMase-HA, and iscl−/+CnISC1 parasites were incubated with NBD-labeled SM (A and C) or NBD-labeled IPC (B and D) as described in Materials and Methods. Each reaction contained ∼40 µg of total protein corresponding to 4×106 cells. (A–B) After incubation, lipids were extracted and separated by thin layer chromatography (TLC). Ceramide (Cer), a product of SMase and IPCase, migrates faster than sphingomyelin (SM in A) or IPC (B). O: origin of migration. Positive control (+): 0.1 unit of purified BcSMase (A) or 0.1 unit of purified BcPI-PLC (B). Negative control (−): boiled WT lysate. (C–D) Activity of SMase (C) or IPCase (D) in Leishmania cell lysates was quantified after TLC analysis based on the amount ceramide produced and the amount of protein in each reaction. Error bars represent standard deviations from 3 independent experiments.
Figure 5.
The IPCase activity was required for acid resistance in stationary phase promastigotes.
Promastigotes were cultured under either neutral pH (pH 7.4, A) or acidic pH (pH 5.0, B). Cell viability in stationary phase (S1–S5) was determined by flow cytometry after staining with propidium iodide. Each group of bars represents cells in the following order (from left to right): WT, iscl−, iscl−/+HA-ISCL, iscl−/+HA-BcSMase, iscl−/+5′HASPb-BcSMase-HA, and iscl−/+CnISC1. Error bars represent standard deviations from 3 independent experiments.
Figure 6.
The SMase activity alone was sufficient to restore virulence in iscl−.
(A–B) Bone marrow-derived Mφs from BALB/c mice were infected by stationary phase promastigotes (•: WT, ○: iscl−, ▾: iscl−/+HA-ISCL, ▪: iscl−/+CnISC1, □: iscl−/+HA-BcSMase, ⧫: iscl−/+5′HASPb-BcSMase-HA, ▵: WT+activated Mφs) at a ratio of 15 parasites per Mφ. Percentages of infected Mφs (A) and the number of parasites in 100 Mφs (B) were recorded. (C–D) BALB/c mice were infected in the footpads with day 3 stationary promastigotes (•: WT, ○: iscl−, ▾: iscl−/+HA-ISCL, ▵: iscl−/+HA-BcSMase, ▪: iscl−/+5′HASPb-BcSMase-HA, □: iscl−/+CnISC1) at 1×106 parasites per mouse (5 mice per group). Lesion sizes were measured with a caliper (C) and parasite numbers in infected footpads were determined by the limiting dilution assay (D). Error bars represent standard deviations from 2 independent experiments.
Figure 7.
Cellular localization of HA-tagged BcSMase.
Late log phase promastigotes of iscl−/+HA-BcSMase (A–C) or iscl−/+5′HASPb-BcSMase-HA (D–F) were labeled with a monoclonal anti-HA antibody followed by anti-mouse IgG-FITC. (A, D) DIC images; (B, E) Hoechst staining of DNA; (C, F) anti-HA staining. Control cells that were labeled with secondary antibody only did not show any green fluorescence (data not shown).