Autophagy-Related Atg8 Localizes to the Apicoplast of the Human Malaria Parasite <em>Plasmodium falciparum</em>

<div><p>Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite <em>Plasmodium falciparum</em> (PfAtg8). PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although <em>Plasmodium</em> parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.</p> </div

PfAtg8 is associated with membranes.

<p>(A) Specificity of the two independently generated anti-PfAtg8 antibodies (#1 and #2). Crude antisera and purified antibodies were used for immunoblotting of lysates of asynchronized <i>P. falciparum</i> parasites. (B) Expression of PfAtg8 increases during the erythrocytic stage of development. Highly synchronized <i>P. falciparum</i> parasites were collected at 0, 12, 24, 32, and 40 h after invasion. The duration of one cycle of the erythrocyte stage was approximately 42 h. Expression levels of PfAtg8 were analyzed by immunoblotting. An antibody against HSP70 was used as a loading control. (C) PfAtg8 exogenously expressed in mammalian cells (lane 1), endogenous PfAtg8 expressed in <i>P. falciparum</i> (lane 2), and the mixture of these two samples were subjected to immunoblot analysis using anti-PfAtg8 antibody. (D) Lysates of asynchronized <i>Plasmodium</i> were separated into low-speed (13,000×<i>g</i>) pellet (LSP), high-speed (100,000×<i>g</i>) pellet (HSP), and high-speed supernatant (HSS) fractions, and analyzed by immunoblotting using anti-PfAtg8 antibody. (E) The LSP fraction prepared in (D) was treated with 2 M urea or 2% Triton-X 100 and separated into 100,000×<i>g</i> pellet (P) and supernatant (S). (F) Infected erythrocytes were cultured in the presence of the indicated concentration of chloroquine and expression of PfAtg8 was analyzed.</p

PfAtg8 localizes to the apicoplast.

<p><i>P. falciparum</i> FCR3 (A–E) and <i>P. falciparum</i> 3D7 transfected with ACP-GFP (F–H) were stained with the indicated organelle markers and visualized by confocal microscopy (because ACP-GFP was not uniformly expressed, some merozoites displayed only faint GFP signals). Anti-PfAtg8 antibody #1 was used in (A–F), and anti-PfAtg8 antibody #2 was used in (G). Apical membrane antigen 1 (AMA1) as a microneme marker (A), rhoptry-associated protein 1 (RAP1) as a rhoptry body marker (B), rhoptry neck protein 2 (RON2) as a rhoptry neck marker (C), the ring-infected erythrocyte surface antigen (RESA) as a dense granule marker (D), MitoTrackerRed CMXRos as a mitochondria marker (E), ACP-GFP (F–H) and the organellar histone-like protein PfHU (H) as an apicoplast marker were used. Scale bar, 1 μm.</p

PfAtg8 is associated with the apicoplast membrane.

<p>(A) <i>P. falciparum</i> FCR3 parasites at the schizont stage were analyzed by immunoelectron microscopy (immunogold and silver enhancement method) with an antibody against PfAtg8 (#1). (a) A schizont in an erythrocyte. (b) A magnified image of the area indicated in (a). (c) Another typical image of a PfAtg8-positive structure. (B) <i>P. falciparum</i> transfectant expressing ACP-GFP was analyzed as in panel (A) with an antibody against GFP. A, apicoplast; Mt, mitochondrion. Scale bars, (A, a) 1 μm, (A, b and c, and B) 200 nm.</p

PfAtg8 localizes to tubular and branched apicoplasts.

<p><i>P. falciparum</i> transfectant expressing ACP-GFP at late trophozoite and early schizont stages was stained with anti-GFP and anti-PfAtg8 antibodies and MitoTrackerRed CMXRos, and visualized by confocal microscopy. Scale bar, 1 μm.</p

PfAtg8 localization is not affected by chloroquine or wortmannin treatment.

<p><i>P. falciparum</i> transfectant expressing ACP-GFP was treated with chloroquine (100 or 300 nM) (A), or wortmannin (10 μM) (B) for 2 h. Scale bar, 1 μm.</p

Western blots of the membrane preparations from C41 <i>E. coli</i> strains expressing microsporidian AOX proteins.

<p>Lane 1 shows <i>A. locustae</i> rAOX and Lane 2 shows <i>T. hominis</i> rAOX. Lane 3 shows purified alternative oxidase protein from <i>Sauromatum guttatum</i>.</p

Alignment of <i>A. locustae, T. hominis</i>, <i>S. guttatum</i> and <i>T. brucei</i> AOX sequences.

<p>The four-helix bundles are underlined with a solid line. The putative quinone binding site is underlined with a broken line. Conserved amino acid sites are marked with a star and semi conserved sites are marked with dots.</p

MOESM4 of Suppression of experimental cerebral malaria by disruption of malate:quinone oxidoreductase

Additional file 4. Deficiency of FH and MQO has no effect on gametocyte production. Blood was obtained from infected mice showing 3% parasitaemia and cultured for 22 h under standardized in vitro culture conditions. Then, mature gametocytes and schizonts were collected by Nycodenz density-gradient centrifugation. (A and B) Expression of gametocyte-specific genes. mdv-1/peg3 [21] and g377 [22] were subjected to semi-quantitative RT–PCR using specific primers (see Additional files 2, 3). The hsp70 was used as a positive control. Samples treated with DNase-treated RNA template (hsp70 (-)) were used as a negative control that is the control of eventual DNA contamination of the RNA preparations. Experiments were performed in duplicate and representative data are shown. (C) Control and Δfh parasites-infected erythrocytes cultured for 22 h. (D) Control and Δmqo parasites-infected erythrocytes cultured for 22 h. White arrows indicate representative mature gametocytes. The scale bars indicate 20 μm. Note that sex-specific features such as nuclear enlargement, the distribution of pigment granules throughout the cytoplasm and enlargement of the cells are observed in both Δfh- and Δmqo-parasite cultures just the same as reported by Mons [23]

Phylogenetic analyses of microsporidian AOX sequences.

<p><b>A.</b> Global MrBayes AOX phylogeny, posterior probabilities and PhyML bootstraps from an analysis of 500 bootstrapped datasets are shown above and below respectively, key and well supported clades (>70% Bootstrap). <b>B.</b> Short alignment PhyML phylogeny including the translated amplified sequences from all four microsporidia. Bootstrap support from 100 datasets is shown next to microporidian nodes. <b>C.</b> Microsporidian distribution of the alternative oxidase gene plotted onto a phylogeny of microsporidian SSU rDNA sequences. Scale bars in all trees indicate substitutions per site.</p