Compartmentation of redox metabolism in malaria parasites

Abstract

Malaria, caused by the apicomplexan parasite Plasmodium, still represents a major threat to human health and welfare and leads to about one million human deaths annually. Plasmodium is a rapidly multiplying unicellular organism undergoing a complex developmental cycle in man and mosquito - a life style that requires rapid adaptation to various environments. In order to deal with high fluxes of reactive oxygen species and maintain redox regulatory processes and pathogenicity, Plasmodium depends upon an adequate redox balance. By systematically studying the subcellular localization of the major antioxidant and redox regulatory proteins, we obtained the first complete map of redox compartmentation in Plasmodium falciparum. We demonstrate the targeting of two plasmodial peroxiredoxins and a putative glyoxalase system to the apicoplast, a non-photosynthetic plastid. We furthermore obtained a complete picture of the compartmentation of thioredoxin- and glutaredoxin-like proteins. Notably, for the two major antioxidant redox-enzymes--glutathione reductase and thioredoxin reductase--Plasmodium makes use of alternative-translation-initiation (ATI) to achieve differential targeting. Dual localization of proteins effected by ATI is likely to occur also in other Apicomplexa and might open new avenues for therapeutic intervention.

Figure 1. GFP targeting by various P. falciparum redox proteins.

(A) Apicoplast targeting of the AOP N-terminus. (B) Dual localization (cytosol and apicoplast) of the TPx_Gl N-terminal amino acid sequence. (C) ER-targeting of Trx3. (D) Mitochondrial targeting of Tlp2. (E) Mitochondrial targeting of GLP3. (F) Apicoplast targeting of the GILP N-terminal sequence. Colocalization of GFP with the mitochondrial dye MitoTrackerOrange in fixed cells. Colocalization of GFP and the apicoplast marker ACP or the ER marker BiP in fixed, immunodecorated cells.

Figure 2. Dissection of the dual localization of P. falciparum GR.

(A) Dual localization (cytosol and apicoplast) of GR-N71-GFP effected by a newly discovered N-terminal extension/leader. (B) Western blot analysis of parasites stably expressing GR-N71-GFP using anti-GFP antibodies. TP, transit peptide still attached; Pr, transit peptide processed; ★, GFP degradation product. (C) Cytosolic targeting of a construct lacking the signal peptide (GR-TP-GFP). (D) Western blot analysis of parasites stably expressing GR-SP using anti-GFP, anti-Hsp70, and anti-SERP antibodies shows targeting of GR-SP to the parasitophorous vacuole. Lysate, erythrocyte cytosol plus the soluble contents of the parasitophorous vacuole; Parasite, cellular contents of the parasite. (E) Apicoplast targeting of construct (A) after mutation of methionine 47 to alanine (GR-N71-GFP M47A). (F) Thermolysin protection assays on parasites stably expressing GR-N71-GFP M47A confirm complete apicoplast localization of GR-N71 M47A. Parasites permeabilized using the detergents digitonin (plasma membrane) and Triton X-100 (plasma membrane and organellar membranes) were treated with the protease thermolysin. Tubulin is the cytosolic control that is not protected from thermolysin after digitonin permeabilization; sCdc48 is the apicoplast-targeted control protein that is protected from thermolysin after digitonin permeabilization but not after Triton X-100 permeabilization. Degradation could be inhibited with the addition of EDTA, an inhibitor of thermolysin, suggesting that the loss of protein we observed was specifically due to thermolysin degradation. Colocalization of GFP and the apicoplast marker ACP in fixed, immunodecorated cells. Live cell imaging of erythrocytes infected with transgenic parasites for solely cytosolic GFP signals.

Figure 3. A schematic representation of GR distribution in P. falciparum by ATI.

(1) A single gene is transcribed, leading to a single transcript. (2) An upstream translational start site followed by a second AUG-codon, leading to two translational products. (3) GR carrying an N-terminal apicoplast targeting sequence. (4) GR lacking an N-terminal targeting sequence. (5) GR translocates into the apicoplast. (6) GR remains in the cytosol. PV, parasitophorous vacuole.

Figure 4. Dissection of the dual localization of P. falciparum TrxR.

(A) Dual localization (cytosol and mitochondria) of TrxR-GFP effected by a newly discovered N-terminal extension/leader. (B) Mitochondrial targeting of a construct (TrxR-N76-GFP) containing solely the TrxR 5′-extension. (C) Detection of TrxR-GFP fusion constructs in transgenic parasites by Western blot analysis against the GFP-moiety, showing that the dual localization of TrxR-GFP is not due to a weak mitochondrial signal sequence. (D) Western blot analysis of parasites stably expressing TrxR-N76-GFP using anti-GFP antibodies, showing proteolytically processed TrxR. Colocalization of GFP with the mitochondrial dye MitoTrackerOrange in fixed cells.

Figure 5. Compartmentation of the redox metabolism in malaria parasites.

Schematic representation of an intra-erythrocytic trophozoite, highlighting key parasite intracellular compartments. AOP, antioxidant protein; 1-Cys Prx, 1-cysteine peroxiredoxin; ER, endoplasmic reticulum; GILP, glyoxalase-1-like protein; Glo, glyoxalase; GLP, glutaredoxin-like protein; GR, glutathione reductase; Grx, glutaredoxin; GST, glutathione-S-transferase; LipDH, lipoamide dehydrogenase-like protein; Plrx, plasmoredoxin; SOD, superoxide dismutase; Tlp, thioredoxin-like protein; TPx, thioredoxin-dependent peroxidase; Trx, thioredoxin; TrxR, thioredoxin reductase. *, tGloII was found to localize to the cytosol and the apicoplast; ^#, Trx2 was targeted to the parasitophorous vacuole and a yet unidentified organelle of the parasite; ⁺, TPx_Gl was localized both to the cytosol and the apicoplast (targeting mechanism not yet analyzed). Please refer to the main text for further details and references. Endogenous glutathione peroxidases and a catalase are not present in Plasmodium.