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. 2014 Jan;10(1):80-92.
doi: 10.4161/auto.26743. Epub 2013 Nov 11.

The Multifunctional Autophagy Pathway in the Human Malaria Parasite, Plasmodium Falciparum

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Free PMC article

The Multifunctional Autophagy Pathway in the Human Malaria Parasite, Plasmodium Falciparum

Serena Cervantes et al. Autophagy. .
Free PMC article

Abstract

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A 1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.

Keywords: Plasmodium; apicoplast biogenesis; autophagy; gametocytogenesis; protein traffic.

Figures

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Figure 1. Expression of PfATG8 throughout the erythrocytic cycle. (A) Immunoblot analysis of PfATG8 expression in tightly synchronized parasites harvested at the ring, trophozoite, and schizont stage. Protein concentration was determined by Bradford analysis and 30 μg of parasite lysate was loaded in each lane. Using a custom-generated anti-PfATG8 antibody, it was observed that PfATG8 protein is present throughout the asexual cycle, albeit at lower abundance in the trophozoite stage. (B) Immunoblot analysis showing the expression of endogenous PfATG8 in cytosolic, nuclear, and membrane fractions of parasite lysates. Mixed-stage 3D7 parasite cultures were used for subcellular fractionation. Protein concentration was determined by Bradford assay and 30 μg of protein was loaded for the cytosolic and nuclear fractions, while 10 μg of protein was loaded for the membrane fraction. PfATG8 is detected in all fractions, but is predominantly membrane-bound. (C) Immunoblot analysis of the GFP-PfATG8 transiently-transfected P. falciparum cell line. Mixed-stage 3D7 and GFP-PfATG8 parasites were lysed and 30 μg of lysate was loaded into each lane. Endogenous PfATG8 was detected using anti-PfATG8 at 14 kDa, while the fusion protein GFP-PfATG8 was detected by both anti-GFP and anti-PfATG8 antibodies at the expected molecular weight of 41 kDa. For all panels, relative amounts of protein loaded were assessed by Ponceau-staining the full-length PVDF membrane prior to blotting (Fig. S1). The sizes of molecular mass markers are indicated in kDa.
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Figure 2. PfATG8 immunofluorescence staining throughout the erythrocytic cycle. Tightly synchronized P. falciparum D10-ACP-GFP parasites were immunolabeled with anti-PfATG8. D10 parasites express ACP-GFP, an apicoplast signal and transit peptide fused to GFP that localizes to the apicoplast. Localization of ACP-GFP and PfATG8 can be observed, although PfATG8 appears to surround the apicoplast, indicating a possible association with the outer membrane. Additional PfATG8 vesicles can be observed in the cytosol and host red blood cell from the late ring to late trophozoite stage, when parasites are metabolically active. DAPI is used as a DNA marker in blue. Scale bar: 5 μm.
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Figure 3. PfATG8 immunofluorescent staining in apicoplast-minus malaria parasites. The integrity of the apicoplast in these parasites was lost using treatment with the antibiotic chloramphenicol. GFP-ACP parasite cultures were rescued by supplementing media with isopentenyl pyrophosphate (IPP), the essential product from the non-mevalonate isoprenoid precursor pathway in the apicoplast. The large apicoplast PfATG8-labeled branching, as observed in untreated parasites, is disrupted into multiple foci in apicoplast-minus parasites. These foci show colocalization of GFP-ACP and PfATG8 vesicles, although additional PfATG8 vesicles can be observed in the cytosol. DAPI is used as a DNA marker in blue. Scale bar: 2.5 μm.
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Figure 4. PfATG8 immunofluorescent staining in gametocytes. (A) Validation of PfATG8 localization using the ACP-GFP cell line. A clear localization of GFP-PfATG8 and the apicoplast can be observed. (B) Expression of PfATG8 in GFP-PfATG8 gametocytes. An abundance of PfATG8-labeled autophagosomes is observed during gametocytogenesis. In stage II-IV, large vesicles are observed at the apical poles. (C) Differential PfATG8 pattern in male and female gametocytes. Female NF54 gametocytes at stage IV have rounded poles and PfATG8 vesicles are more uniform in size and distribution in the cytosol, while male gametocytes have larger PfATG8 vesicles at the apical poles (white arrowheads). DAPI is used as a DNA marker in blue. Scale bar: 5 μm.
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Figure 5. Effect of antimalarial treatment on PfATG8 expression and localization. (A) PfATG8 immunofluorescent staining in 3D7 parasites treated with lysosomotropic agents and artemisinin. After 3 h incubation with 75 nM of bafilomycin A1, branching of the apicoplast is disrupted and multiple PfATG8 autophagosomes can be observed. Artemisinin treatment at a high concentration (25 μM) results in cell cycle progression arrest, and chloroquine treatment, at a concentration of 100 nM (IC80), causes disruption of the apicoplast integrity. DAPI is used as a DNA marker in blue. Scale bar: 2.5 μm. (B) Graph of PfATG8 vesicles on trophozoite-stage parasites with or without autophagy inhibitor incubation (baf, 75 nM bafilomycin A1; art, 25 μM artemisinin; cq, 100 nM chloroquine). (C) Western blot analysis of PfATG8 expression levels upon bafilomycin A1, chloroquine and artemisinin treatment.

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