The multifunctional autophagy pathway in the human malaria parasite,Plasmodium falciparum

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 A₁ 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.     

Characterization of the Autophagy Marker Protein Atg8 Reveals Atypical Features of Autophagy in Plasmodium falciparum

Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast biogenesis. Collectively, our results reveal several atypical features of autophagy in malaria parasites, which may be largely associated with non-degradative processes.     

Plasmodium falciparum Erythrocytic Stage Parasites Require the Putative Autophagy Protein PfAtg7 for Normal Growth

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite’s erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.     

The Suf Iron-Sulfur Cluster Synthesis Pathway Is Required for Apicoplast Maintenance in Malaria Parasites

The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.     

Characterization of the Plasmodium falciparum and P. berghei glycerol 3‐phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti‐malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3‐phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N‐terminal targeting sequence to GFP and 3′ tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site‐directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3‐phosphate acyltransferase in malaria parasites.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> apicoplast and its transcriptional regulation through calcium signaling

Malaria has been present since ancient time and remains a major global health problem in developing countries. Plasmodium falciparum belongs to the phylum Apicomplexan, largely contain disease-causing parasites and characterized by the presence of apicoplast. It is a very essential organelle of P. falciparum responsible for the synthesis of key molecules required for the growth of the parasite. Indispensable nature of apicoplast makes it a potential drug target. Calcium signaling is important in the establishment of malaria parasite inside the host. It has been involved in invasion and egress of merozoites during the asexual life cycle of the parasite. Calcium signaling also regulates apicoplast metabolism. Therefore, in this review, we will focus on the role of apicoplast in malaria biology and its metabolic regulation through Ca⁺⁺ signaling.     

Autophagy-related protein PfATG18 participates in food vacuole dynamics and autophagy-like pathway in Plasmodium falciparum

Plasmodium falciparum has a limited repertoire of autophagy-related genes (ATGs), and the functions of various proteins of the autophagy-like pathway are not fully established in this protozoan parasite. Studies suggest that some of the autophagy proteins are crucial for parasite growth. PfATG18, for example, is essential for parasite replication and has a noncanonical role in apicoplast biogenesis. In this study, we demonstrate the conserved functions of PfATG18 in food vacuole (FV) dynamics and autophagy. Intriguingly, the P. falciparum FV is found to undergo fission and fusion and PfATG18 gets enriched at the interfaces of the newly generated multilobed FV during the process. In addition, expression of PfATG18 is induced upon starvation, both at the mRNA and protein level indicating its participation in the autophagy-like pathway, which is independent of its role in apicoplast biogenesis. The study also shows that PfATG18 is transported to the FV via the haemoglobin trafficking pathway. Overall, this study establishes the conserved functions of Atg18 in this important apicomplexan.     

A Nondiscriminating Glutamyl-tRNA Synthetase in the Plasmodium Apicoplast: THE FIRST ENZYME IN AN INDIRECT AMINOACYLATION PATHWAY*

The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNA^Gln biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNA^Glu and tRNA^Gln, determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNA^Gln biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.     

Independent losses and duplications of autophagy-related genes in fungal tree of life

Autophagy is important for growth, development and pathogenesis in fungi. Although autophagic process is generally considered to be conserved, the conservation and evolution of ATG genes at kingdom-wide remains to be conducted. Here we systematically identified 41 known ATG genes in 331 species and analyzed their distribution across the fungal kingdom. In general, only 20 ATG genes are highly conserved, including most but not all the yeast core-autophagy-machinery genes. Four functional protein groups involved in autophagosome formation had conserved and non-conserved components, suggesting plasticity in autophagosome formation in fungi. All or majority of the key ATG genes were lost in several fungal groups with unique lifestyles and niches, such as Microsporidia, Pneumocystis and Malassezia. Moreover, majority of ATG genes had A-to-I RNA editing during sexual reproduction in two ascomycetes and deletion of FgATG11, the ATG gene with the most editing sites in Fusarium affected ascospore releasing. Duplication and divergence also was observed to several core ATG genes, such as highly divergent ATG8 paralogs in dermatophytes and multiple ATG15 duplications in mushrooms. Taken together, independent losses and duplications of ATG genes have occurred throughout the fungal kingdom and variations in autophagy exist among different lineages and possibly different developmental stages.     

ATG8 localization in apicomplexan parasites: Apicoplast and more?

The ATG genes are highly conserved in eukaryotes including yeasts, plants, and mammals. However, these genes appear to be only partially present in most protists. Recent studies demonstrated that, in the apicomplexan parasites Plasmodium (malaria parasites) and Toxoplasma, ATG8 localizes to the apicoplast, a unique nonphotosynthetic plastid with 4 limiting membranes. In contrast to this established localization, it remains unclear whether these parasites can induce canonical macroautophagy and if ATG8 localizes to autophagosomes. Furthermore, the molecular function of ATG8 in its novel workplace, the apicoplast, is totally unknown. Here, we review recent studies on ATG8 in Plasmodium and Toxoplasma, summarize both consensus and controversial findings, and discuss its potential role in these parasites.     

Characterization of the ATG8-conjugation system in 2 Plasmodium species with special focus on the liver stage

Plasmodium parasites successfully colonize different habitats within mammals and mosquitoes, and adaptation to various environments is accompanied by changes in their organelle composition and size. Previously, we observed that during hepatocyte infection, Plasmodium discards organelles involved in invasion and expands those implicated in biosynthetic pathways. We hypothesized that this process is regulated by autophagy. Plasmodium spp. possess a rudimentary set of known autophagy-related proteins that includes the ortholog of yeast Atg8. In this study, we analyzed the activity of the ATG8-conjugation pathway over the course of the lifecycle of Plasmodium falciparum and during the liver stage of Plasmodium berghei. We engineered a transgenic P. falciparum strain expressing mCherry-PfATG8. These transgenic parasites expressed mCherry-PfATG8 in human hepatocytes and erythrocytes, and in the midgut and salivary glands of Anopheles mosquitoes. In all observed stages, mCherry-PfATG8 was localized to tubular structures. Our EM and colocalization studies done in P. berghei showed the association of PbATG8 on the limiting membranes of the endosymbiont-derived plastid-like organelle known as the apicoplast. Interestingly, during parasite replication in hepatocytes, the association of PbATG8 with the apicoplast increases as this organelle expands in size. PbATG3, PbATG7 and PbATG8 are cotranscribed in all parasitic stages. Molecular analysis of PbATG8 and PbATG3 revealed a novel mechanism of interaction compared with that observed for other orthologs. This is further supported by the inability of Plasmodium ATG8 to functionally complement atg8Δ yeast or localize to autophagosomes in starved mammalian cells. Altogether, these data suggests a unique role for the ATG8-conjugation system in Plasmodium parasites.     

Features of autophagic cell death in Plasmodium liver-stage parasites

Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology.     

Plasmodium falciparum OTU‐like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with noncanonical role of Atg8

The metabolic pathways associated with the mitochondrion and the apicoplast in Plasmodium, 2 parasite organelles of prokaryotic origin, are considered as suitable drug targets. In the present study, we have identified functional role of a novel ovarian tumour unit (OTU) domain‐containing cysteine protease of Plasmodium falciparum (PfOTU). A C‐terminal regulatable fluorescent affinity tag on native protein was utilised for its localization and functional characterization. Detailed studies showed vesicular localization of PfOTU and its association with the apicoplast. Degradation‐tag mediated knockdown of PfOTU resulted in abnormal apicoplast development and blocked development of parasites beyond early‐schizont stages in subsequent cell cycle; downregulation of PfOTU hindered apicoplast protein import. Further, the isoprenoid precursor‐mediated parasite growth‐rescue experiments confirmed that PfOTU knockdown specifically effect development of functional apicoplast. We also provide evidence for a possible biological function of PfOTU in membrane deconjugation of Atg8, which may be linked with the apicoplast protein import. Overall, our results show that the PfOTU is involved in apicoplast homeostasis and associates with the noncanonical function of Atg8 in maintenance of parasite apicoplast.     

Apicoplast acetyl Co-A carboxylase of the human malaria parasite is not targeted by cyclohexanedione herbicides

Malaria parasites retain a relict plastid (apicoplast) from a photosynthetic ancestor. The apicoplast is a useful drug target but the specificity of compounds believed to target apicoplast fatty acid biosynthesis has become uncertain, as this pathway is not essential in blood stages of the parasite. Herbicides that inhibit the plastid acetyl Coenzyme A (Co-A) carboxylase of plants also kill Plasmodium falciparum in vitro, but their mode of action remains undefined. We characterised the gene for acetyl Co-A carboxylase in P. falciparum. The P. falciparum acetyl-CoA carboxylase gene product is expressed in blood stage parasites and accumulates in the apicoplast. Ablation of the gene did not render parasites insensitive to herbicides, suggesting that these compounds are acting off-target in blood stages of P. falciparum.     

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.     

Enzymes involved in plastid‐targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver‐stage development

Malaria parasites scavenge nutrients from their host but also harbour enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast‐targeted type II fatty acid synthesis, which is essential for late liver‐stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast‐targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three‐step reaction utilizing three enzymes: glycerol 3‐phosphate dehydrogenase, glycerol 3‐phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbour genes for both apicoplast‐ and cytosol/endoplasmic reticulum‐targeted phosphatidic acid synthesis. Our research shows that apicoplast‐targeted Plasmodium yoelii glycerol 3‐phosphate dehydrogenase and glycerol 3‐phosphate acyltransferase are expressed only during liver‐stage development and deletion of the encoding genes resulted in late liver‐stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast‐targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite life cycle. Our results suggest that P. yoelii has an incomplete apicoplast‐targeted phosphatidic acid synthesis pathway that is essential for liver‐stage maturation.     

Role of ATG3 in the parasite Toxoplasma gondii

Toxoplasma gondii belongs to the phylum Apicomplexa, a diverse group of early branching unicellular eukaryotes related to dinoflagellates and ciliates. Like several other Apicomplexa such as Plasmodium (the causative agent of malaria), T. gondii is a human pathogen responsible for a potentially lethal disease called toxoplasmosis. Most Apicomplexa have complex life cycles, involving intermediate hosts and vectors, which include obligatory intracellular developmental stages. In the case of malaria and toxoplasmosis, it is that replicative process, leading to the ultimate lysis of the host cell, which is causing the symptoms of the disease. For Toxoplasma, the invasive and fast-replicating form of the parasite is called the tachyzoite. While autophagy has been a fast-growing field of research in recent years, not much was known about the relevance of this catabolic process in medically important apicomplexan parasites. Vesicles resembling autophagosomes had been described in drug-treated Plasmodium parasites in the early 1970s and a potential role for autophagy in organelle recycling during differentiation between Plasmodium life stages has also been recently described. Interestingly, recent database searches have identified putative orthologs of the core machinery responsible for the formation of autophagosomes in several protists, including Toxoplasma. In spite of an apparently reduced machinery (only about one-third of the yeast ATG genes appear to be conserved), T. gondii seemed thus able to perform macroautophagy, but the cellular functions of the pathway for this parasite remained to be demonstrated.     

An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins

Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.     

A Process Similar to Autophagy Is Associated with Cytocidal Chloroquine Resistance in Plasmodium falciparum

Resistance to the cytostatic activity of the antimalarial drug chloroquine (CQ) is becoming well understood, however, resistance to cytocidal effects of CQ is largely unexplored. We find that PfCRT mutations that almost fully recapitulate P. falciparum cytostatic CQ resistance (CQR^CS) as quantified by CQ IC₅₀ shift, account for only 10–20% of cytocidal CQR (CQR^CC) as quantified by CQ LD₅₀ shift. Quantitative trait loci (QTL) analysis of the progeny of a chloroquine sensitive (CQS; strain HB3)×chloroquine resistant (CQR; strain Dd2) genetic cross identifies distinct genetic architectures for CQR^CS vs CQR^CC phenotypes, including identification of novel interacting chromosomal loci that influence CQ LD₅₀. Candidate genes in these loci are consistent with a role for autophagy in CQR^CC, leading us to directly examine the autophagy pathway in intraerythrocytic CQR parasites. Indirect immunofluorescence of RBC infected with synchronized CQS vs CQR trophozoite stage parasites reveals differences in the distribution of the autophagy marker protein PfATG8 coinciding with CQR^CC. Taken together, the data show that an unusual autophagy – like process is either activated or inhibited for intraerythrocytic trophozoite parasites at LD₅₀ doses (but not IC₅₀ doses) of CQ, that the pathway is altered in CQR P. falciparum, and that it may contribute along with mutations in PfCRT to confer the CQR^CC phenotype.     

Deciphering apicoplast targeting signals – feature extraction from nuclear-encoded precursors of Plasmodium falciparum apicoplast proteins

The malaria causing protozoan Plasmodium falciparum contains a vestigal, non-photosynthetic plastid, the apicoplast. Numerous proteins encoded by nuclear genes are targeted to the apicoplast courtesy of N-terminal extensions. With the impending sequence completion of an entire genome of the malaria parasite, it is important to have software tools in place for prediction of subcellular locations for all proteins. Apicoplast targeting signals are bipartite; containing a signal peptide and a transit peptide. Nuclear-encoded apicoplast protein precursors were analyzed for characteristic features by statistical methods, principal component analysis, self-organizing maps, and supervised neural networks. The transit peptide contains a net positive charge and is rich in asparagine, lysine, and isoleucine residues. A novel prediction system (PATS, predict apicoplast-targeted sequences) was developed based on various sequence features, yielding a Matthews correlation coefficient of 0.91 (97% correct predictions) in a 40-fold cross-validation study. This system predicted 22% apicoplast proteins of the 205 potential proteins on P. falciparum chromosome 2, and 21% of 243 chromosome 3 proteins. A combination of the PATS results with a signal peptide prediction yields 15% potentially nuclear-encoded apicoplast proteins on chromosomes 2 and 3. The prediction tool will advance P. falciparum genome analysis, and it might help to identify apicoplast proteins as drug targets for the development of novel anti-malaria agents.