Falciparum Ortholog Research Articles

Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

Genomics of Plasmodium vivax in Colombia reveals evidence of local bottle-necking and inter-country connectivity in the Americas.

Colombia aims to eliminate malaria by 2030 but remains one of the highest burden countries in the Americas. Plasmodium vivax contributes half of all malaria cases, with its control challenged by relapsing parasitaemia, drug resistance and cross-border spread. Using 64 Colombian P. vivax genomes collected between 2013 and 2017, we explored diversity and selection in two major foci of transmission: Chocó and Córdoba. Open-access data from other countries were used for comparative assessment of drug resistance candidates and to assess cross-border spread. Across Colombia, polyclonal infections were infrequent (12%), and infection connectivity was relatively high (median IBD = 5%), consistent with low endemicity. Chocó exhibited a higher frequency of polyclonal infections (23%) than Córdoba (7%), although the difference was not significant (P = 0.300). Most Colombian infections carried double pvdhfr (95%) and single pvdhps (71%) mutants, but other drug resistance mutations were less prevalent (< 10%). There was no evidence of selection at the pvaat1 gene, whose P. falciparum orthologue has recently been implicated in chloroquine resistance. Global population comparisons identified other putative adaptations. Within the Americas, low-level connectivity was observed between Colombia and Peru, highlighting potential for cross-border spread. Our findings demonstrate the potential of molecular data to inform on infection spread and adaptation.

Cation ATPase (ATP4) Orthologue Replacement in the Malaria Parasite Plasmodium knowlesi Reveals Species-Specific Responses to ATP4-Targeting Drugs.

ABSTRACTSeveral unrelated classes of antimalarial compounds developed against Plasmodium falciparum target a parasite-specific P-type ATP-dependent Na+ pump, PfATP4. We have previously shown that other malaria parasite species infecting humans are less susceptible to these compounds. Here, we generated a series of transgenic Plasmodium knowlesi orthologue replacement (OR) lines in which the endogenous pkatp4 locus was replaced by a recodonized P. knowlesi atp4 (pkatp4) coding region or the orthologous coding region from P. falciparum, Plasmodium malariae, Plasmodium ovale subsp. curtisi, or Plasmodium vivax. Each OR transgenic line displayed a similar growth pattern to the parental P. knowlesi line. We found significant orthologue-specific differences in parasite susceptibility to three chemically unrelated ATP4 inhibitors, but not to comparator drugs, among the P. knowlesi OR lines. The PfATP4OR transgenic line of P. knowlesi was significantly more susceptible than our control PkATP4OR line to three ATP4 inhibitors: cipargamin, PA21A092, and SJ733. The PvATP4OR and PmATP4OR lines were similarly susceptible to the control PkATP4OR line, but the PocATP4OR line was significantly less susceptible to all ATP4 inhibitors than the PkATP4OR line. Cipargamin-induced inhibition of Na+ efflux was also significantly greater with the P. falciparum orthologue of ATP4. This confirms that species-specific susceptibility differences previously observed in ex vivo studies of human isolates are partly or wholly enshrined in the primary amino acid sequences of the respective ATP4 orthologues and highlights the need to monitor efficacy of investigational malaria drugs against multiple species. P. knowlesi is now established as an important in vitro model for studying drug susceptibility in non-falciparum malaria parasites.

Identification and preliminary characterization of Plasmodium falciparum proteins secreted upon gamete formation

Malaria long-term elimination depends on parasite transmission control. Plasmodium sexual stage maturation in the mosquito, including egress from the host erythrocyte, is one of the prime targets for transmission-blocking interventions. This work aims to identify candidate molecules potentially involved in gamete emergence from the host erythrocyte, as novel transmission blocking targets. We analyzed by quantitative mass spectrometry the proteins released/secreted by purified Plasmodium falciparum gametocytes upon induction of gametogenesis. The proteome obtained showed a good overlap (74%) with the one previously characterized in similar conditions from gametocytes of the rodent malaria parasite P. berghei. Four candidates were selected based on comparative analysis of their abundance values in released vs total gametocyte proteome. We also characterized the P. falciparum orthologue of the microgamete surface protein (MiGS), a marker of male gametocyte secretory vesicles in murine models of malaria. The findings of this study reveal that all the selected candidate proteins are expressed in both genders and localize to vesicle-like structures that respond to gametogenesis stimuli. This result, together with the fact that the selected proteins are released during gamete emergence in both Plasmodium species, makes them interesting candidates for future functional studies to investigate their potential role in the gametogenesis process.

Optimization of an in vivo model to study immunity to Plasmodium falciparum pre-erythrocytic stages

BackgroundThe circumsporozoite protein (CSP) of Plasmodium is a key surface antigen that induces antibodies and T-cells, conferring immune protection in animal models and humans. However, much of the work on CSP and immunity has been developed based on studies using rodent or non-human primate CSP antigens, which may not be entirely translatable to CSP expressed by human malaria parasites, especially considering the host specificity of the different species.MethodsUsing a genetically engineered strain of Plasmodium berghei that expresses luciferase, GFP and the Plasmodium falciparum orthologue of CSP, the effect of laboratory preparation, mosquito treatment and mouse factors on sporozoite infectivity was assessed using an in vivo bioluminescence assay on mice. This assay was compared with a PCR-based protection assay using an already described monoclonal antibody that can provide sterile protection against sporozoite challenge.ResultsBioluminescence assay demonstrated similar detection levels of the quantity and kinetics of liver-stage infection, compared to PCR-based detection. This assay was used to evaluate treatment of sporozoite and delivery method on mouse infectivity, as well as the effects of age, sex and strain of mice. Finally, this assay was used to test the protective capacity of monoclonal antibody AB317; results strongly recapitulate the findings of previous work on this antibody.ConclusionsThe PbGFP-Luc line and in vivo bioluminescence imaging provide highly sensitive read-outs of liver-stage infection in mice, and this method can be useful to reliably evaluate potency of pre-erythrocytic interventions.

Naturally Acquired Antibody Response to Malaria Transmission Blocking Vaccine Candidate Pvs230 Domain 1

Plasmodium vivax malaria incidence has increased in Latin America and Asia and is responsible for nearly 74.1% of malaria cases in Latin America. Immune responses to P. vivax are less well characterized than those to P. falciparum, partly because P. vivax is more difficult to cultivate in the laboratory. While antibodies are known to play an important role in P. vivax disease control, few studies have evaluated responses to P. vivax sexual stage antigens. We collected sera or plasma samples from P. vivax-infected subjects from Brazil (n = 70) and Cambodia (n = 79) to assess antibody responses to domain 1 of the gametocyte/gamete stage protein Pvs230 (Pvs230D1M). We found that 27.1% (19/70) and 26.6% (21/79) of subjects from Brazil and Cambodia, respectively, presented with detectable antibody responses to Pvs230D1M antigen. The most frequent subclasses elicited in response to Pvs230D1M were IgG1 and IgG3. Although age did not correlate significantly with Pvs230D1M antibody levels overall, we observed significant differences between age strata. Hemoglobin concentration inversely correlated with Pvs230D1M antibody levels in Brazil, but not in Cambodia. Additionally, we analyzed the antibody response against Pfs230D1M, the P. falciparum ortholog of Pvs230D1M. We detected antibodies to Pfs230D1M in 7.2 and 16.5% of Brazilian and Cambodian P. vivax-infected subjects. Depletion of Pvs230D1M IgG did not impair the response to Pfs230D1M, suggesting pre-exposure to P. falciparum, or co-infection. We also analyzed IgG responses to sporozoite protein PvCSP (11.4 and 41.8% in Brazil and Cambodia, respectively) and to merozoite protein PvDBP-RII (67.1 and 48.1% in Brazil and Cambodia, respectively), whose titers also inversely correlated with hemoglobin concentration only in Brazil. These data establish patterns of seroreactivity to sexual stage Pvs230D1M and show similar antibody responses among P. vivax-infected subjects from regions of differing transmission intensity in Brazil and Cambodia.

The recent advances of chemical biology in artemisinin target and mechanism

Artemisinin is a natural compound that is extracted from the plant Artemisia annua. While it was used to treat fever and pain in the past, it is currently approved by World Health Organization (WHO) to be used in combating malaria. It is an effective anti-malarial drug because of its high efficacy even towards multi-drug resistant Plasmodium strains and exhibits no significant side effects in humans. However, artemisinin has poor solubility in water or oil, poor bioavailability and a short half-life in vivo . Therefore, derivatives of artemisinin consisting of semi-synthetic and fully synthetic compounds with improved properties have been synthesized. Semi-synthetic derivatives include artesunate, artemether and dihydroartemisinin. Artemisinin and its derivatives are sesquiterpene lactones that contain a unique endoperoxide bond in its trioxane ring, which is essential for their anti-malarial activity. The importance of this bond is shown by the findings of studies that the abrogation of the endoperoxide bond led to a significant reduction in toxicity of the drugs. For example, the removal of one O-atom in the endoperoxide moiety was associated with a complete loss of anti-malarial activity. The activation mechanism of artemisinin is hypothesized to involve the reduction of the endoperoxide bond by Fe2+, which is a catalyst that can generate free radicals from peroxidic structures. The selective toxicity of the drug towards parasite-infected cells as compared to non-infected cells is attributed to the higher heme level in parasite-infected cells. In malarial parasite-infected red blood cells, the parasites break down hemoglobin in the food vacuole to gain amino acids for synthesis of their parasite-specific proteins. As a result, heme is released, which can be further degraded into iron in the vacuole. Therefore, hemoglobin degradation gives rise in an increase in both heme and free iron level in infected erythrocytes. As a result, whether heme or iron is the crucial activator of the drug is yet to be elucidated. One of the pleiotropic effects of the drug is that the reduced endoperoxide bond forms cytotoxic carbon-centered radicals that can alkylate target proteins and affect their functions. Other effects include the alkylation of heme, generation of reactive oxygen species (ROS) and inhibition of PfATP6, which is the P. falciparum ortholog of the endoplasmic reticulum Ca2+ pump. Some studies have also found that artemisinin builds up within neutral lipids and damage the parasite membrane. The combination of these toxic effects of artemisinin and its derivatives result in parasitic death. Artemisinin is used to relieve pain and fever in the older days. Today, artemisinin is widely used as an anti-malarial drug as our first line of defense to combat the emergence of drug resistance malaria parasite. Besides its anti-malarial properties, artemisinin is being investigated in other diseases. It is found to possess a wide spectrum of pharmacological activities, including anticancer, anti-asthma etc. However, its mechanism of action (MOA) is still not fully understood. Recently, “omics” approach has been used in identify the targets and unravel the mechanism of action of artemisinin. This review will summarize the recent progress in artemisinin target and mechanism study.

Receptor for Activated C-Kinase 1 (PfRACK1) is required for Plasmodium falciparum intra-erythrocytic proliferation

Emerging resistance to current anti-malarials necessitates a more detailed understanding of the biological processes of Plasmodium falciparum proliferation, thus allowing identification of new drug targets. The well-conserved protein Receptor for Activated C-Kinase 1 (RACK1) was originally identified in mammalian cells as an anchoring protein for protein kinase C (PKC) and has since been shown to be important for cell migration, cytokinesis, transcription, epigenetics, and protein translation. The P. falciparum ortholog, PfRACK1, is expressed in blood stages of the parasite and is diffusely localized in the parasite cytoplasm. Using a destabilizing domain to allow inducible knockdown of the endogenous protein level, we evaluated the requirement for PfRACK1 during blood-stage replication. Following destabilization, the parasites demonstrate a nearly complete growth arrest at the trophozoite stage. The essential nature of PfRACK1 suggests that the protein itself or the pathways regulated by the protein are potential targets for novel anti-malarial therapeutics.

Characterization of a Plasmodium falciparum Orthologue of the Yeast Ubiquinone-Binding Protein, Coq10p.

Coenzyme Q (CoQ, ubiquinone) is a central electron carrier in mitochondrial respiration. CoQ is synthesized through multiple steps involving a number of different enzymes. The prevailing view that the CoQ used in respiration exists as a free pool that diffuses throughout the mitochondrial inner membrane bilayer has recently been challenged. In the yeast Saccharomyces cerevisiae, deletion of the gene encoding Coq10p results in respiration deficiency without inhibiting the synthesis of CoQ, suggesting that the Coq10 protein is critical for the delivery of CoQ to the site(s) of respiration. The precise mechanism by which this is achieved remains unknown at present. We have identified a Plasmodium orthologue of Coq10 (PfCoq10), which is predominantly expressed in trophozoite-stage parasites, and localizes to the parasite mitochondrion. Expression of PfCoq10 in the S. cerevisiae coq10 deletion strain restored the capability of the yeast to grow on respiratory substrates, suggesting a remarkable functional conservation of this protein over a vast evolutionary distance, and despite a relatively low level of amino acid sequence identity. As the antimalarial drug atovaquone acts as a competitive inhibitor of CoQ, we assessed whether over-expression of PfCoq10 altered the atovaquone sensitivity in parasites and in yeast mitochondria, but found no alteration of its activity.

Molecular modeling of the Plasmodium falciparum pre-mRNA splicing and nuclear export factor PfU52.

UAP56/SUB2 is a DExD/H-box RNA helicase that is critically involved in pre-mRNA splicing and mRNA nuclear export. This helicase is broadly conserved and essential in many eukaryotic lineages, including protozoan and metazoan parasites. Previous research suggests that helicases from parasites could be promising drug targets for treating parasitic diseases. Accordingly, characterizing the structure and function of these proteins is of interest for structure-based, de novo design of new lead compounds. Here, we used homology modeling to construct a three-dimensional structure of PfU52 (PMDB ID: PM0079288), the Plasmodium falciparum ortholog of UAP56/SUB2, and explored the detailed architecture of its functional sites. Comparative in silico analysis revealed that although PfU52 shared many physicochemical, structural and dynamic similarities with its human homolog, it also displayed some unique features that could be exploited for drug design.

Development of a transgenic Plasmodium berghei line (Pb pfpkg) expressing the P. falciparum cGMP-dependent protein kinase, a novel antimalarial drug target.

With the inevitable selection of resistance to antimalarial drugs in treated populations, there is a need for new medicines to enter the clinic and new targets to progress through the drug discovery pipeline. In this study we set out to develop a transgenic rodent model for testing inhibitors of the Plasmodium falciparum cyclic GMP-dependent kinase in vivo. A model was needed that would allow us to investigate whether differences in amino acid sequence of this enzyme between species influences in vivo efficacy. Here we report the successful development of a transgenic P. berghei line in which the cyclic GMP-dependent protein kinase (PKG) was replaced by the P. falciparum orthologue. We demonstrate that the P. falciparum orthologue was able to functionally complement the endogenous P. berghei pkg gene throughout blood stage development and early sexual development. However, subsequent development in the mosquito was severely compromised. We show that this is due to a defect in the female lineage of the transgenic by using genetic crosses with both male and female deficient P. berghei lines. This defect could be due to expression of a female-specific target in the mosquito stages of P. berghei that cannot be phosphorylated by the P. falciparum kinase. Using a previously reported anti-coccidial inhibitor of the cyclic GMP-dependent protein kinase, we show no difference in in vivo efficacy between the transgenic and control P. berghei lines. This in vivo model will be useful for screening future generations of cyclic GMP-dependent protein kinase inhibitors and allowing us to overcome any species-specific differences in the enzyme primary sequence that would influence in vivo efficacy in the rodent model. The approach will also be applicable to in vivo testing of other antimalarial compounds where the target is known.

Virtual Screening and Optimization Yield Low-Nanomolar Inhibitors of the Tautomerase Activity of Plasmodium falciparum Macrophage Migration Inhibitory Factor

The Plasmodium falciparum orthologue of the human cytokine, macrophage migratory inhibitory factor (PfMIF), is produced by the parasite during malaria infection and modulates the host's immune response. As for other MIF orthologues, PfMIF has tautomerase activity, whose inhibition may influence the cytokine activity. To identify small-molecule inhibitors of the tautomerase activity of PfMIF, virtual screening has been performed by docking 2.1 million compounds into the enzymatic site. Assaying of 17 compounds identified four as active. Substructure search for the most potent of these compounds, a 4-phenoxypyridine analogue, identified four additional compounds that were purchased and also shown to be active. Thirty-one additional analogues were then designed, synthesized, and assayed. Three were found to be potent PfMIF tautomerase inhibitors with K(i) of ∼40 nM; they are also highly selective with K(i) &gt; 100 μM for human MIF.

Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

Parasite Replication Trigger

Apicomplexan parasites, including Toxoplasma gondii and the malaria parasite Plasmodium falciparum , actively invade host cells. Little is known about the signals that govern initiation of replication once the parasite is intracellular. Apical membrane antigen 1 (AMA1) is released onto the parasite surface during invasion and cleaved by intramembrane proteolysis, mediated by a rhomboid-like serine protease, ROM4. Santos et al. (p. [473][1], published online 23 December; see the Perspective by [ Cowman and Tonkin ][2]) used conditional expression of Toxoplasma ROM4 to show that ROM4 activity is not essential for invasion, but instead is required for subsequent replication of the intracellular parasite. Furthermore, transgenic expression of the cleaved cytoplasmic tail of AMA1 alone—either from the Toxoplasma AMA1 or from its P. falciparum ortholog—completely restored the replicative capacity of the intracellular parasites. Thus, intramembrane cleavage of AMA1 is required to trigger parasite replication within the host cell. [1]: /lookup/doi/10.1126/science.1199284 [2]: /lookup/doi/10.1126/science.1201692

Investigations into the role of the Plasmodium falciparum SERCA (PfATP6) L263E mutation in artemisinin action and resistance.

Artemisinin-based combination therapies (ACTs) are highly effective for the treatment of Plasmodium falciparum malaria, yet their sustained efficacy is threatened by the potential spread of parasite resistance. Recent studies have provided evidence that artemisinins can inhibit the function of PfATP6, the P. falciparum ortholog of the ER calcium pump SERCA, when expressed in Xenopus laevis oocytes. Inhibition was significantly reduced in an L263E variant, which introduced the mammalian residue into a putative drug-binding pocket. To test the hypothesis that this single mutation could decrease P. falciparum susceptibility to artemisinins, we implemented an allelic-exchange strategy to replace the wild-type pfatp6 allele by a variant allele encoding L263E. Transfected P. falciparum clones were screened by PCR analysis for disruption of the endogenous locus and introduction of the mutant L263E allele under the transcriptional control of a calmodulin promoter. Expression of the mutant allele was demonstrated by reverse transcriptase (RT) PCR and verified by sequence analysis. Parasite clones expressing wild-type or L263E variant PfATP6 showed no significant difference in 50% inhibitory concentrations (IC(50)s) for artemisinin or its derivatives dihydroartemisinin and artesunate. Nonetheless, hierarchical clustering analysis revealed a trend toward reduced susceptibility that neared significance (artemisinin, P approximately = 0.1; dihydroartemisinin, P = 0.053 and P = 0.085; and artesunate, P = 0.082 and P = 0.162 for the D10 and 7G8 lines, respectively). Notable differences in the distribution of normalized IC(50)s provided evidence of decreased responsiveness to artemisinin and dihydroartemisinin (P = 0.02 for the D10 and 7G8 lines), but not to artesunate in parasites expressing mutant PfATP6.

Egress ofPlasmodium bergheigametes from their host erythrocyte is mediated by the MDV-1/PEG3 protein

Malaria parasites invade erythrocytes of their host both for asexual multiplication and for differentiation to male and female gametocytes - the precursor cells of Plasmodium gametes. For further development the parasite is dependent on efficient release of the asexual daughter cells and of the gametes from the host erythrocyte. How malarial parasites exit their host cells remains largely unknown. We here report the characterization of a Plasmodium berghei protein that is involved in egress of both male and female gametes from the host erythrocyte. Protein MDV-1/PEG3, like its Plasmodium falciparum orthologue, is present in gametocytes of both sexes, but more abundant in the female, where it is associated with dense granular organelles, the osmiophilic bodies. Deltamdv-1/peg3 parasites in which MDV-1/PEG3 production was abolished by gene disruption had a strongly reduced capacity to form zygotes resulting from a reduced capability of both the male and female gametes to disrupt the surrounding parasitophorous vacuole and to egress from the host erythrocyte. These data demonstrate that emergence from the host cell of male and female gametes relies on a common, MDV-1/PEG3-dependent mechanism that is distinct from mechanisms used by asexual parasites.

Selection of yeast strains with enhanced expression of Plasmodium falciparum proteins

The poor expression of Plasmodium falciparum proteins in heterologous systems and the difficulty in obtaining sufficient material directly from the parasite have limited the experimental characterization of many of the approximately 5200 proteins encoded by this organism. To improve the expression of P. falciparum proteins in the yeast Saccharomyces cerevisiae, we selected yeast ura3 mutants that acquired the ability to utilize the P. falciparum orthologue ( PfOMPDC) of URA3 to grow on media lacking uracil. Two of these mutant strains, BY#29 and PJ#17, expressed up to 100-fold more of four P. falciparum proteins as a result of mutations in either HRP1 or KAP104, respectively. These mutations, as well as a temperature-sensitive rna15 mutation, likely decrease the efficiency of mRNA 3′ end formation and produce longer mRNAs of P. falciparum genes. These yeast strains may be useful for the analysis and purification of P. falciparum proteins.

Spatial dissection of the cis‐ and trans‐Golgi compartments in the malaria parasite Plasmodium falciparum

The Golgi apparatus forms the heart of the secretory pathway in eukaryotic cells where proteins are modified, processed and sorted. The transport of proteins from the endoplasmic reticulum (ER) to the cis-side of the Golgi complex takes place at specialized ER sub-domains known as transitional ER (tER). We used the Plasmodium falciparum orthologue of Sec13p to analyse tER organization. We show that the distribution of PfSec13p is restricted to defined areas of the ER membrane. These foci are juxtaposed to the Golgi apparatus and might represent tER sites. To further analyse cis- to trans-Golgi architecture, we generated a double transfectant parasite line that expresses the Golgi marker Golgi reassembly stacking protein (GRASP) as a green fluorescent protein fusion and the trans-Golgi marker Rab6 as a DsRed fusion protein. Our data demonstrate that Golgi multiplication is closely linked to tER multiplication, and that parasite maturation is accompanied by the spatial separation of the cis- and trans- face of this organelle.

Gene encoding a deubiquitinating enzyme is mutated in artesunate‐ and chloroquine‐resistant rodent malaria parasites§

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.