Figure 1.
Canonical pathways of macroautophagy.
(A) Autophagosome biogenesis and fate. The successive events during the generation of autophagosmes are depicted, from induction to breakdown. The involvement of ATG5, ATG12 and ATG8 and the two conjugation pathways, and the sequence in which they act, are depicted. (B) The two conjugation pathways involved in autophagosome biogenesis. The ATG12-ATG5-ATG16 complex formed in the first pathway is involved in the attachment of ATG8 to phosphatidylethanolamine (PE) during the second pathway. These processes in Leishmania differ from those of mammals in that the Leishmania ATG12 has an extended C-terminal domain beyond the glycine residue required for conjugation to ATG5, suggesting cleavage is required. In addition, Leishmania possess two ATG4s, which may act at different stages of autophagosome formation. (C) Cleavage of ATG8 from the surface of mature autophagosomes before they fuse with the lysosomal network, showing the second step involving ATG4.
Figure 2.
Reconstitution of the L. major ATG5-ATG12 conjugation system in vitro.
(A) Recombinant proteins were mixed, as indicated, in the presence (lane 5) or absence (lane 4) of ATP and incubated for 1 h at 30°C before being separated by SDS-PAGE and analyzed by western blotting using the α-His antibody. Components present (+) or absent (−) from the assay are indicated. (B) Recombinant proteins were mixed, as indicated, and incubated as above. The reactions were stopped, subjected to SDS-PAGE and stained with Coomassie Blue. (C) Recombinant proteins were mixed, as indicated, incubated as above and analyzed with α-His antibody.
Figure 3.
ATG5 puncta in L. major promastigotes.
(A) The occurrence of mC-ATG5 puncta in WT L. major promastigotes expressing mC-ATG5 incubated in nutrient-rich medium at log phase at 26°C. (B) The multiplicity of puncta in promastigotes expressing mC-ATG5 after starvation for up to 1 h in PBS at 26°C. The time ranges indicated reflect the starvation period together with the 30 min period during which the observations on the microscope were undertaken. (C) Co-labelling of puncta with mC-ATG5 and GFP-ATG12 co-expressed in promastigotes incubated in PBS for 2 h at 26°C. (D) In nutrient-rich medium, GFP-ATG8 punctum without mC-ATG5 staining is arrowed. (E) Incubated in PBS for 30 min at 26°C. (F) In nutrient-rich medium at 26°C. (G) Incubated in PBS for 30 min at 26°C, the small panels are enlargements of the merged panels. (H–I) Promastigotes in nutrient-rich medium. (J–K) Promastigotes at late log phase in nutrient-rich conditions. (L) Promastigotes at stationary phase in nutrient-rich conditions. Scale bar throughout, 10 µm.
Figure 4.
Phenotypic characterisation of Δatg5.
(A) Growth curve of Leishmania promastigotes in HOMEM medium at 26°C. *, Δatg5 differed significantly from WT (p<0.05). (B) The occurrence of GFP-ATG8 puncta in promastigotes after incubation in nutrient-deprived (PBS, ND) and nutrient-rich (HOMEM medium, NR) conditions for 2 h at 26°C. Scale bar, 10 µm. (C) Occurrence of GFP-ATG8 puncta in promastigotes when incubated in the conditions detailed in (B). Means ± SD from four independent experiments. * and **, occurrence of GFP-ATG8 puncta in Δatg5 were significantly different from in WT in nutrient-deprived and nutrient-rich conditions (p<0.05). (D) Western blot analysis of extracts of promastigotes expressing GFP-ATG8 at logarithmic growth under standard conditions and probed with α-GFP antibody. The faster migrating, lipidated band is labelled GFP-ATG8-II while the un-lipidated band migrating more slowly is labelled GFP-ATG8-I.
Figure 5.
L. major Δatg5 promastigotes have a dysfunctional mitochondrion.
(A) Enlarged (EM) and swollen (WM) mitochondria seen by transmission electron microscopy (TEM) in Δatg5 promastigotes under standard growth conditions. WT is shown at bottom right. Scale bar, 500 nm. (B) Fluorescent intensity from MitoTracker Red (MTR, 0.1 µM) and MitoTracker Green (MTG, 0.2 µM) in 2×106 promastigotes after 30 min incubation at 26°C. Values shown are the means ± SD from three independent experiments. * and **, fluorescence was significantly different between WT and Δatg5 (p<0.05). (C) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP-GFP. Scale bar, 10 µm. (D) Differential staining of promastigotes with both MTR (0.1 µM) and MTG (0.2 µM). Scale bar, 10 µm. (E) Viability, as measured by Alamar Blue reduction, of promastigotes. All data are means ± SD from three independent experiments. *, Alamar blue reduction was significantly different (p<0.05). (F) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotes at 2×106/ml with H2DCFDA at 0.1 mM for 2 h at 26°C. Values shown are the means ± SD from three independent experiments. *, DCF fluorescence was significantly different (p<0.05).
Figure 6.
Phospholipid accumulation in Δatg5 promastigotes.
Negative ion survey scans (650–900 m/z) of WT (A) and Δatg5 (B) promastigotes extracted for lipids and analysed by ES-MS, as described in Materials and Methods. a = (alkylacyl).
Figure 7.
Promastigote differentiation and infectivity.
(A) Proportion of metacyclic promastigotes in stationary phase cultures, assessed using the PNA assay. Values shown are the means ± SD from three independent experiments. *, differed significantly (p<0.05). (B) Western blot analysis of extracts of 107 promastigotes at stationary phase of growth probed with α-HASPB. α-Cysteine Synthase was used as a loading control [56]. (C) Infectivity and survival of promastigotes in peritoneal macrophages in vitro, infected at a ratio 5∶1, with the infection rates being assessed after 1 and 5 days. *, differed significantly (p<0.01). (D) Lesion progression in BALB/c mice inoculated with 5×105 stationary phase promastigotes. Values shown are the means ± SD from 5 mice. *, infection level of Δatg5 differed significantly from WT (p<0.01) and Δatg5::ATG5 (p<0.05). (E) Morphologies of cells isolated from mouse lesions and analysed by SEM. Scale bar: 2 µm.
Figure 8.
Morphology of Δatg5 promastigotes.
(A) SEM analysis of promastigote culture initiated with Δatg5 isolated from a mouse lesion and cultured in nutrient-rich medium. Shown are ovoid and amastigote-like form (left); spindled-shaped form without an external flagellum (centre left panel) and with an external flagellum of varying lengths (centre right and right panels). Scale bar, 10 µm. (B) Distribution of flagella lengths and body lengths of stationary phase promastigotes. Data represent measurements from ∼200 cells from each promastigote population.