Phospholipid profile of females positive survey.

<p><b>A–D)</b> Positive ion ES-MS survey scans (600–1000 m/z) of total lipid extracts from female wild type (A, atg7^+/+) and atg7^−/− (B) flies as well as from spermidine-fed female wild type (C, atg7^+/+) and atg7^−/− (D) flies. Arrows indicate significant changes compared to normal flies untreated.</p

Climbing activity.

<p><b>A)</b> Percentage of flies able to climb 8 cm in 10 seconds in wild type and atg7^−/− male flies fed 1 mM spermidine or not. 3 independent replicates were monitored (50 flies in each group and replicate). <b>B)</b> Percentage of flies able to climb 8 cm in 10 seconds in wild type and atg7^−/− female flies fed 1 mM spermidine or not. 3 independent replicates were monitored (50 flies in each group and replicate). *p<0.05; ***p<0.001.</p

Food intake, glycogen and protein contents.

<p><b>A)</b> Mean +/− SEM of food intake in wild type flies of both sexes fed 1 mM spermidine or not as measured by colorimetry (flies fed blue dye and dye intensity measured). 2 independent replicates were monitored (5 or 6 independent samples for each group in each replicate). <b>B, C)</b> Mean +/− SEM of glycogen (B) and protein (C) content in wild type or atg7^−/− flies of both sexes fed 1 mM spermidine or not as measured by colorimetry (anthrone reaction for B and Bradford reaction for C). 3 independent replicates were monitored (4 or 5 independent samples for each group in each replicate). **p<0.01; ***p<0.001.</p

Phospholipid profile of males positive survey.

<p><b>A–D)</b> Positive ion ES-MS survey scans (600–850 m/z) of total lipid extracts from male wild type (A, atg7^+/+) and atg7^−/− (B) flies as well as from spermidine-fed male wild type (C, atg7^+/+) and atg7^−/− (D) flies. Arrows indicate significant changes compared to normal flies untreated.</p

A Class of 5‑Nitro-2-furancarboxylamides with Potent Trypanocidal Activity against Trypanosoma brucei in Vitro

Recently, the World Health Organization approved the nifurtimox–eflornithine combination therapy for the treatment of human African trypanosomiasis, renewing interest in nitroheterocycle therapies for this and associated diseases. In this study, we have synthesized a series of novel 5-nitro-2-furancarboxylamides that show potent trypanocidal activity, ∼1000-fold more potent than nifurtimox against in vitro Trypanosoma brucei with very low cytotoxicity against human HeLa cells. More importantly, the most potent analogue showed very limited cross-resistance to nifurtimox-resistant cells and vice versa. This implies that our novel, relatively easy to synthesize and therefore cheap, 5-nitro-2-furancarboxylamides are targeting a different, but still essential, biochemical process to those targeted by nifurtimox or its metabolites in the parasites. The significant increase in potency (smaller dose probably required) has the potential for greatly reducing unwanted side effects and also reducing the likelihood of drug resistance. Collectively, these findings have important implications for the future therapeutic treatment of African sleeping sickness

Phenotypic characterisation of Δ<i>atg5</i>.

<p>(A) Growth curve of <i>Leishmania</i> promastigotes in HOMEM medium at 26°C. *, Δ<i>atg5</i> 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 Δ<i>atg5</i> 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.</p

Promastigote differentiation and infectivity.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002695#ppat.1002695-Coombs1" target="_blank">[56]</a>. (C) Infectivity and survival of promastigotes in peritoneal macrophages <i>in vitro</i>, 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 Δ<i>atg5</i> differed significantly from WT (p<0.01) and Δ<i>atg5</i>::<i>ATG5</i> (p<0.05). (E) Morphologies of cells isolated from mouse lesions and analysed by SEM. Scale bar: 2 µm.</p

<i>L. major</i> Δ<i>atg5</i> promastigotes have a dysfunctional mitochondrion.

<p>(A) Enlarged (EM) and swollen (WM) mitochondria seen by transmission electron microscopy (TEM) in Δ<i>atg5</i> 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 Δ<i>atg5</i> (p<0.05). (C) Types of mitochondrial morphology observed by fluorescence microscopy of Δ<i>atg5</i> 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).</p

Canonical pathways of macroautophagy.

<p>(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 <i>Leishmania</i> differ from those of mammals in that the <i>Leishmania</i> ATG12 has an extended C-terminal domain beyond the glycine residue required for conjugation to ATG5, suggesting cleavage is required. In addition, <i>Leishmania</i> 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.</p

Morphology of Δ<i>atg5</i> promastigotes.

<p>(A) SEM analysis of promastigote culture initiated with Δ<i>atg5</i> 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.</p