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Title: Compartmentation of Redox Metabolism in Malaria Parasites}
Article:
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.
Introduction:
Malaria threatens more than 40% of the world's population. Current estimations point to 200–300 million clinical episodes and about 1 million human deaths each year[1]. The unicellular parasite Plasmodium falciparum causes the most dangerous form of this tropical disease including the development of cerebral malaria. Malaria parasites are continuously exposed to high fluxes of toxic reactive oxygen species( ROS)[2]. This is due to their life style in different intra- and extracellular environments, the high metabolic rate of the rapidly multiplying parasite, the intraparasitic haemoglobin digestion, and the ROS produced by the host's immune system[3]. In recent years Plasmodium has been shown to possess two major NADPH-dependent redox systems with a broad range of antioxidant defence mechanisms. This includes a complete glutathione system[2] comprising glutathione reductase[4]( GR), glutathione, glutaredoxin, and different glutaredoxin-like proteins[5],[6], glutathione S-transferase[7], and a functional glutathione dependent glyoxalase system[8]. Additionally, a complete thioredoxin system comprising thioredoxin reductase( TrxR), different thioredoxins and thioredoxin-like proteins, and thioredoxin-dependent peroxidases( TPx) has been characterised[2],[9],[10],[11]. Furthermore, two functional superoxide dismutases[12],[13], as well as two dihydrolipoamide dehydrogenase-like proteins[14], are present in the parasite. It has been demonstrated that malaria parasites are vulnerable to disruption of this redox equilibrium during their erythrocytic life stages[15]. This vulnerability is impressively underscored by the malaria-protective effects of glucose-6-phosphate dehydrogenase deficiency, one of the most frequent human gene defects worldwide, which leads to a lack of reducing equivalents provided by NADPH[16]. Two major antioxidant enzymes, catalase and a genuine glutathione peroxidase, do not occur in the parasite[2]. Taken together, this constellation offers great potential for the development of chemotherapeutic agents that act by perturbing the redox equilibrium of Plasmodium[3],[17],[18]. Many of the redox-active enzymes described above are predicted to contain protein targeting sequences. However, their localization has not yet been experimentally demonstrated. Although prediction algorithms have significantly improved over the years, experimental verification of protein localization is still essential. Predictions can often not be verified with regard to their significance and reliability, and multiple targeting of proteins has only rarely been considered by prediction methods[19]. In this study we systematically analyzed the subcellular compartmentation of the major redox-related proteins in Plasmodium falciparum( Access. No. provided in Table S1). Our data, together with published information, result in a comprehensive redox-map of the parasite.
Discussion:
Our study provides the first comprehensive picture of the subcellular compartmentation of cellular redox metabolism in malaria parasites. An overview of the data is given in Figure 5. The apicoplast, a non-photosynthetic plastid of Plasmodium, homologous to chloroplasts of plants, harbours indispensable plant-like metabolic pathways including fatty-acid and haem biosynthesis[24]. Until today no extensive data existed about the antioxidative capacity of the apicoplast. Based on our data, five directly redox-related enzymes are targeted to this apicomplexan-specific organelle( see Fig. 5). AOP[9],[21] and TPxGl[2],[22] belong to the family of peroxiredoxins, which are important components of the cellular antioxidative and redox-regulatory systems( for TPxGl see also cytosol). PfAOP is one of the few AOPs reported in a non-plant organism and likely to be the only Prx in P. falciparum that turns over lipid hydroperoxides[9],[21]. We could also show that, apart from a cytosolic glyoxalase couple, two other glyoxalase related proteins, GILP[8] and tGloII[8],[30], are transported to the apicoplast. It is known that the apicoplast imports triosephosphate-isomerase[24],[41] and its substrates and that one of the sources of toxic methylglyoxal is an incomplete triosephosphate-isomerase reaction[8],[24]. Therefore, an apicoplast-glyoxalase-system to detoxify methylglyoxal and other 2-oxoaldehydes would have a functional importance. The existence of a functional GSH-dependent glyoxalase system in the apicoplast is further supported by our finding that a previously unidentified isoform of PfGR is targeted to the apicoplast( Fig. 2). GR is a single copy gene within the Plasmodium genome, and the apicoplast isoform is generated by alternative-translation-initiation( ATI) at a start-codon upstream of the canonical start( schematically depicted in Fig. 3). The expression of the cytosolic GR is strictly dependent on the integrity of the downstream AUG triplet as shown by in vitro and in vivo functional analysis of mutants. This indicates that the phenomenon is a direct consequence of the translational process, supporting the leaky scanning model for ATI[37],[38]. In conclusion, the discovery of an antioxidative and redox-regulatory system within the apicoplast fills an important gap in our knowledge about this apicomplexan-specific organelle and has an impact on the interpretation of other studies[24],[42]. For example, the plastidic-type ferredoxin and ferredoxin-NADP+ reductase( FNR) of P. falciparum provide electrons for the synthesis of isoprenoid precursors in the apicoplast[42]. FNR activity is controlled through monomer-dimer interconversion by oxidizing and reducing agents, such as H2O2 and glutathione[42]. Furthermore, our data represent a solid basis for detailed functional analyzes. The mitochondrial antioxidant and redox-regulatory proteins of Plasmodium proposed so far comprise Trx2[20] and TPx2[20] as well as a mitochondrial SOD[13] and a dihydrolipoamide dehydrogenase[14]. The assumption that mitochondrial TrxR does not exist in malarial parasites led to the postulation that the low-Mr thiol lipoic acid, an essential cofactor of mitochondrial α-ketoacid dehydrogenase complexes, acts as a reductant for PfTrx2 in vivo[20]. Here we describe a previously unidentified isoform of PfTrxR localized to the mitochondrion( Fig. 4), proposing a disulfide-reductase based reduction system. As with GR, TrxR is a single copy gene within the Plasmodium genome and the mitochondrion targeted isoform is generated by an ATI-site upstream of the canonical start. We did not detect mitochondrial targeting for Trx2[20], however its localization to the parasitophorous vacuole could be confirmed[25]. Furthermore, as described in detail in the results section, we found Trx2 localizing to a yet unidentified, non-dividing organelle of the parasite( Fig. S3, Fig. S4). For Tlp2, targeting to the mitochondrion could be shown unambiguously( Fig. 1D), and mitochondrial targeting of TPx2 could be confirmed( Fig. S1B). Therefore, the existence of a mitochondrial thioredoxin-dependent antioxidative network in P. falciparum can be postulated. Additionally, we demonstrated that GLP3 is a mitochondrial protein, despite a lack of predictable protein targeting signals( Table S2; Fig. 1E). The function of Trx2 in the parasitophorous vacuole was recently unraveled[25]. Trx2 appears to be part of a newly discovered export machinery of the malaria parasite in the parasitophorous vacuolar membrane and is potentially involved in unfolding of target proteins for their passage through the export channel[25]. Within the frame of this study we identified a thioredoxin targeted to the ER( Fig. 1C). The localization of Trx3 to the ER might indicate a specialized function with an involvement in protein folding through the formation and isomerization of disulfide bonds[43]. The cytosol harbors the major part of the redox-capacity and antioxidative potential of the parasite( Fig. 5). Apart from the previously known cytosolic components, our study unravels further important details of this system. Within the glutathione-system of P. falciparum, GLP 1 and 2 were shown to be cytosolic proteins, although GLP1 was predicted to possess an N-terminal mitochondrial targeting sequence[5],[6]( Fig. S5A, B). Especially intriguing in this light is a recent report by McFadden and coworkers, who could demonstrate that exon 1 of GLP1 perfectly fulfils the criteria of a transit peptide for apicoplast targeting. When placed between a signal peptide and GFP, exon 1 of GLP1 mediates accurate plastid targeting[35]. The targeting mechanism of TPxGl, a Prx, both to the cytosol and to the apicoplast has not yet been analyzed in further detail( Fig. 1B). Considering that the gene sequence contains two additional ATG-codons within the target sequence, we propose that TPxGl is targeted to both compartments due to ATI[37],[38], as we have shown here for GR and TrxR. In the present study we mapped the cellular redox system of malaria parasites, providing a tool to better understand the cellular functions and interactions of this essential network. Furthermore, we demonstrated that ATI exists in apicomplexan parasites and is used to translate protein isoforms with differing subcellular localization from one gene. The differentiated control of ATI-sites may furthermore provide a mechanism for the fine tuning of the ratio between the synthesized protein isoforms. In fact, reports of dual/multiple targeting[39],[44] in apicomplexan parasites exist where ATI could be a possible explanation for the described results. Identification of the genes that might have evolved dual targeting capability because of ATI-sites is likely to change our in silico view of organelle function.
The unicellular parasite Plasmodium falciparum is the causative agent of tropical malaria , which represents a global health burden . In order to survive in its human host and the malaria vector Anopheles , malaria parasites depend on adequate antioxidant defense and efficient redox regulation . Furthermore , as shown by glucose-6 phosphate dehydrogenase deficiency , a genetic variation protecting from malaria , redox equilibrium plays a vital role in parasite pathogenicity . Using a green fluorescent protein reporter gene , we systematically studied the subcellular compartmentation of redox networks in Plasmodium falciparum . Based on our results and data from literature , we provide the first thorough map of redox compartmentation . Most interestingly , for the two major antioxidant redox-enzymes – glutathione reductase ( GR ) and thioredoxin reductase ( TrxR ) – Plasmodium falciparum makes use of alternative translation initiation to translate protein isoforms with differing subcellular localization from one gene . Dual localization of proteins due to alternative translation initiation might occur frequently in Apicomplexa and identification of further genes that have evolved alternative translation initiation is likely to offer new therapeutic strategies against this devastating disease .