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Sample records for apicoplasts

  1. Anti-malarial Drug Design by Targeting Apicoplasts: New Perspectives

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    Avinaba Mukherjee

    2016-03-01

    Full Text Available Objectives: Malaria has been a major global health problem in recent times with increasing mortality. Current treatment methods include parasiticidal drugs and vaccinations. However, resistance among malarial parasites to the existing drugs has emerged as a significant area of concern in anti-malarial drug design. Researchers are now desperately looking for new targets to develop anti-malarials drug which is more target specific. Malarial parasites harbor a plastid-like organelle known as the ‘apicoplast’, which is thought to provide an exciting new outlook for the development of drugs to be used against the parasite. This review elaborates on the current state of development of novel compounds targeted againstemerging malaria parasites. Methods: The apicoplast, originates by an endosymbiotic process, contains a range of metabolic pathways and housekeeping processes that differ from the host body and thereby presents ideal strategies for anti-malarial drug therapy. Drugs are designed by targeting the unique mechanism of the apicoplasts genetic machinery. Several anabolic and catabolic processes, like fatty acid, isopenetyl diphosphate and heme synthess in this organelle, have also been targeted by drugs. Results: Apicoplasts offer exciting opportunities for the development of malarial treatment specific drugs have been found to act by disrupting this organelle’s function, which wouldimpede the survival of the parasite. Conclusion: Recent advanced drugs, their modes of action, and their advantages in the treatment of malaria by using apicoplasts as a target are discussed in this review which thought to be very useful in desigining anti-malarial drugs. Targetting the genetic machinery of apicoplast shows a great advantange regarding anti-malarial drug design. Critical knowledge of these new drugs would give a healthier understanding for deciphering the mechanism of action of anti-malarial drugs when targeting apicoplasts to overcome drug

  2. Understanding the biology of the Plasmodium falciparum apicoplast; an excellent target for antimalarial drug development.

    Science.gov (United States)

    Chakraborty, Arnish

    2016-08-01

    Malaria is a life-threatening tropical disease, caused by the intracellular parasite Plasmodium falciparum. The World Health Organization counts malaria as one of the top ten causes of worldwide death. The unavailability of a successful malaria vaccine and the ever-increasing instances of drug resistance in the malaria parasite demand the discovery of new targets within P. falciparum for the development of next generation antimalarials. Fortunately, all apicomplexan parasites, including P. falciparum harbor a relict, non-photosynthetic plastid known as the apicoplast. The apicoplast is a semi-autonomous organelle within P. falciparum containing a 35kb circular genome. Despite a genome of its own, majority of the apicoplast proteins are encoded by the parasite nucleus and imported into the apicoplast. The organelle has been shown to be essential to P. falciparum survival and the loss the apicoplast manifests as a 'delayed death' response in the parasite. The apicoplast has evolved out of cyanobacteria in a complex, two step endosymbiotic event. As a result the architecture and the gene expression machinery of the apicoplast is quite bacteria-like and is susceptible to a wide range of antibiotics such as fosmidomycin, tetracycline, azithromycin, clindamycin and triclosan. The biosynthetic pathways for isoprenoids, fatty acids and heme operate within the malaria apicoplast, making the organelle an excellent target for drug development. The review focuses on the evolution, biology and the essentiality of the apicoplast within the malaria parasite and discusses some of the recent achievements towards the design and discovery of apicoplast targeted antimalarial compounds.

  3. Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum

    Science.gov (United States)

    Goodman, Christopher D.; McFadden, Geoffrey I.

    2016-01-01

    Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane. PMID:27442138

  4. Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum.

    Science.gov (United States)

    Lim, Liting; Sayers, Claire P; Goodman, Christopher D; McFadden, Geoffrey I

    2016-01-01

    Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane. PMID:27442138

  5. Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Liting Lim

    Full Text Available Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane.

  6. Selective inhibition of apicoplast tryptophanyl-tRNA synthetase causes delayed death in Plasmodium falciparum.

    Science.gov (United States)

    Pasaje, Charisse Flerida A; Cheung, Vanessa; Kennedy, Kit; Lim, Erin E; Baell, Jonathan B; Griffin, Michael D W; Ralph, Stuart A

    2016-01-01

    The malaria parasite Plasmodium falciparum relies on efficient protein translation. An essential component of translation is the tryptophanyl-tRNA synthetase (TrpRS) that charges tRNA(trp). Here we characterise two isoforms of TrpRS in Plasmodium; one eukaryotic type localises to the cytosol and a bacterial type localises to the remnant plastid (apicoplast). We show that the apicoplast TrpRS aminoacylates bacterial tRNA(trp) while the cytosolic TrpRS charges eukaryotic tRNA(trp). An inhibitor of bacterial TrpRSs, indolmycin, specifically inhibits aminoacylation by the apicoplast TrpRS in vitro, and inhibits ex vivo Plasmodium parasite growth, killing parasites with a delayed death effect characteristic of apicoplast inhibitors. Indolmycin treatment ablates apicoplast inheritance and is rescuable by addition of the apicoplast metabolite isopentenyl pyrophosphate (IPP). These data establish that inhibition of an apicoplast housekeeping enzyme leads to loss of the apicoplast and this is sufficient for delayed death. Apicoplast TrpRS is essential for protein translation and is a promising, specific antimalarial target. PMID:27277538

  7. Apicoplast Biosynthetic Pathways as Possible Targetsfor Combination Therapy of Malaria

    Institute of Scientific and Technical Information of China (English)

    Solomon Tesfaye; Bhanu Prakash; Prati Pal Singh

    2015-01-01

    The emergence of malaria parasite strains resistant to practically all the antimalarial drugs in clinical use is now making itnecessary to discover and develop both new antimalarial drugs and treatments. Recent advances in molecular techniques along withthe availability of genome sequence ofPlasmodiumfalciparum may provide a wide range of novel targets in metabolic pathways likeisoprenoid biosynthesis, fatty acid biosynthesis and heme biosynthesis in the apicoplast of Plasmodiurn. On the other hand, thecombination therapy approach (currently used to retard the selection of parasite strains resistant to individual components of acombination of drugs) has proved to be a success in the combination of sulphadoxine and pyrimethamine, which targets two differentsteps in the folate pathway of malaria parasite. However, after the success of this therapeutic combination, the efficacy of othercombinations of drugs which target different enzymes in a particular metabolic pathway has, apparently, not been reported. Therefore,herein, we review various drug targets so far discovered in apicoplast-related anabolic pathways, especially, with a sharper focus onthe possibility to target more than one enzyme at a time in a particular metabolic pathway of malaria parasites.

  8. Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences

    OpenAIRE

    Michelle Klein Sercundes; Samantha Yuri Oshiro Branco Valadas; Lara Borges Keid; Tricia Maria Ferreira Souza Oliveira; Helena Lage Ferreira; Ricardo Wagner Almeida Vitor; Fábio Gregori; Rodrigo Martins Soares

    2016-01-01

    Abstract Phylogenies within Toxoplasmatinae have been widely investigated with different molecular markers. Here, we studied molecular phylogenies of the Toxoplasmatinae subfamily based on apicoplast and mitochondrial genes. Partial sequences of apicoplast genes coding for caseinolytic protease (clpC) and beta subunit of RNA polymerase (rpoB), and mitochondrial gene coding for cytochrome B (cytB) were analyzed. Laboratory-adapted strains of the closely related parasites Sarcocystis falcatula ...

  9. Role of Calcium Signaling in the Transcriptional Regulation of the Apicoplast Genome of Plasmodium falciparum

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    Sabna Cheemadan

    2014-01-01

    Full Text Available Calcium is a universal second messenger that plays an important role in regulatory processes in eukaryotic cells. To understand calcium-dependent signaling in malaria parasites, we analyzed transcriptional responses of Plasmodium falciparum to two calcium ionophores (A23187 and ionomycin that cause redistribution of intracellular calcium within the cytoplasm. While ionomycin induced a specific transcriptional response defined by up- or downregulation of a narrow set of genes, A23187 caused a developmental arrest in the schizont stage. In addition, we observed a dramatic decrease of mRNA levels of the transcripts encoded by the apicoplast genome during the exposure of P. falciparum to both calcium ionophores. Neither of the ionophores caused any disruptions to the DNA replication or the overall apicoplast morphology. This suggests that the mRNA downregulation reflects direct inhibition of the apicoplast gene transcription. Next, we identify a nuclear encoded protein with a calcium binding domain (EF-hand that is localized to the apicoplast. Overexpression of this protein (termed PfACBP1 in P. falciparum cells mediates an increased resistance to the ionophores which suggests its role in calcium-dependent signaling within the apicoplast. Our data indicate that the P. falciparum apicoplast requires calcium-dependent signaling that involves a novel protein PfACBP1.

  10. Comparative Analysis of Apicoplast-Targeted Protein Extension Lengths in Apicomplexan Parasites.

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    Seliverstov, Alexandr V; Zverkov, Oleg A; Istomina, Svetlana N; Pirogov, Sergey A; Kitsis, Philip S

    2015-01-01

    In general, the mechanism of protein translocation through the apicoplast membrane requires a specific extension of a functionally important region of the apicoplast-targeted proteins. The corresponding signal peptides were detected in many apicomplexans but not in the majority of apicoplast-targeted proteins in Toxoplasma gondii. In T. gondii signal peptides are either much diverged or their extension region is processed, which in either case makes the situation different from other studied apicomplexans. We propose a statistic method to compare extensions of the functionally important regions of apicoplast-targeted proteins. More specifically, we provide a comparison of extension lengths of orthologous apicoplast-targeted proteins in apicomplexan parasites. We focus on results obtained for the model species T. gondii, Neospora caninum, and Plasmodium falciparum. With our method, cross species comparisons demonstrate that, in average, apicoplast-targeted protein extensions in T. gondii are 1.5-fold longer than in N. caninum and 2-fold longer than in P. falciparum. Extensions in P. falciparum less than 87 residues in size are longer than the corresponding extensions in N. caninum and, reversely, are shorter if they exceed 88 residues.

  11. Identification of vital and dispensable sulfur utilization factors in the Plasmodium apicoplast.

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    Joana M Haussig

    Full Text Available Iron-sulfur [Fe-S] clusters are ubiquitous and critical cofactors in diverse biochemical processes. They are assembled by distinct [Fe-S] cluster biosynthesis pathways, typically in organelles of endosymbiotic origin. Apicomplexan parasites, including Plasmodium, the causative agent of malaria, harbor two separate [Fe-S] cluster biosynthesis pathways in the their mitochondrion and apicoplast. In this study, we systematically targeted the five nuclear-encoded sulfur utilization factors (SUF of the apicoplast [Fe-S] cluster biosynthesis pathway by experimental genetics in the murine malaria model parasite Plasmodium berghei. We show that four SUFs, namely SUFC, D, E, and S are refractory to targeted gene deletion, validating them as potential targets for antimalarial drug development. We achieved targeted deletion of SUFA, which encodes a potential [Fe-S] transfer protein, indicative of a dispensable role during asexual blood stage growth in vivo. Furthermore, no abnormalities were observed during Plasmodium life cycle progression in the insect and mammalian hosts. Fusion of a fluorescent tag to the endogenous P. berghei SUFs demonstrated that all loci were accessible to genetic modification and that all five tagged SUFs localize to the apicoplast. Together, our experimental genetics analysis identifies the key components of the SUF [Fe-S] cluster biosynthesis pathway in the apicoplast of a malarial parasite and shows that absence of SUFC, D, E, or S is incompatible with Plasmodium blood infection in vivo.

  12. Sequential processing of the Toxoplasma apicoplast membrane protein FtsH1 in topologically distinct domains during intracellular trafficking.

    Science.gov (United States)

    Karnataki, Anuradha; DeRocher, Amy E; Feagin, Jean E; Parsons, Marilyn

    2009-08-01

    FtsH proteins are hexameric transmembrane proteases found in chloroplasts, mitochondria and bacteria. In the protozoan Toxoplasma gondii, FtsH1 is localized to membranes of the apicoplast, a relict chloroplast present in many apicomplexan parasites. We have shown that although T. gondii FtsH1 lacks the typical bipartite targeting presequence seen on apicoplast luminal proteins, it is targeted to the apicoplast via the endoplasmic reticulum. In this report, we show that FtsH1 undergoes processing events to remove both the N- and C-termini, which are topologically separated by the membrane in which FtsH1 is embedded. Pulse-chase analysis showed that N-terminal cleavage precedes C-terminal cleavage. Unlike the processing of the N-terminal transit peptide of luminal proteins, which occurs in the apicoplast, analysis of ER-retained mutants showed that N-terminal processing of FtsH1 occurs in the endoplasmic reticulum. Two of four FtsH1 mutants bearing internal epitope tags accumulated in structures peripheral to the apicoplast, implying that FtsH1 trafficking is highly sensitive to changes in protein structure. These mutant proteins did not undergo C-terminal processing, suggesting that this processing step occurs after localization to the plastid. Mutation of the peptidase active site demonstrated that neither processing event occurs in cis. These data support a model in which multiple proteases act at different points of the trafficking pathway to form mature FtsH1, making its processing more complex than other FtsHs and unique among apicoplast proteins described thus far. PMID:19450729

  13. Crystallization and preliminary X-ray analysis of the Plasmodium falciparum apicoplast DNA polymerase.

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    Milton, Morgan E; Choe, Jun-yong; Honzatko, Richard B; Nelson, Scott W

    2015-03-01

    Infection by the parasite Plasmodium falciparum is the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment of Escherichia coli DNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystallization of and preliminary X-ray diffraction data from crystals of P. falciparum apPOL are reported. Data complete to 3.5 Å resolution were collected from a single crystal (2 × 2 × 5 µm) using a 5 µm beam. The space group P6522 (unit-cell parameters a = b = 141.8, c = 149.7 Å, α = β = 90, γ = 120°) was confirmed by molecular replacement. Refinement is in progress. PMID:25760711

  14. Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences

    Directory of Open Access Journals (Sweden)

    Michelle Klein Sercundes

    2016-03-01

    Full Text Available Abstract Phylogenies within Toxoplasmatinae have been widely investigated with different molecular markers. Here, we studied molecular phylogenies of the Toxoplasmatinae subfamily based on apicoplast and mitochondrial genes. Partial sequences of apicoplast genes coding for caseinolytic protease (clpC and beta subunit of RNA polymerase (rpoB, and mitochondrial gene coding for cytochrome B (cytB were analyzed. Laboratory-adapted strains of the closely related parasites Sarcocystis falcatula and Sarcocystis neurona were investigated, along with Neospora caninum, Neospora hughesi, Toxoplasma gondii (strains RH, CTG and PTG, Besnoitia akodoni, Hammondia hammondiand two genetically divergent lineages of Hammondia heydorni. The molecular analysis based on organellar genes did not clearly differentiate between N. caninum and N. hughesi, but the two lineages of H. heydorni were confirmed. Slight differences between the strains of S. falcatula and S. neurona were encountered in all markers. In conclusion, congruent phylogenies were inferred from the three different genes and they might be used for screening undescribed sarcocystid parasites in order to ascertain their phylogenetic relationships with organisms of the family Sarcocystidae. The evolutionary studies based on organelar genes confirm that the genusHammondia is paraphyletic. The primers used for amplification of clpC and rpoB were able to amplify genetic sequences of organisms of the genus Sarcocystisand organisms of the subfamily Toxoplasmatinae as well.

  15. Autophagy-related Atg8 localizes to the apicoplast of the human malaria parasite Plasmodium falciparum.

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    Kei Kitamura

    Full Text Available 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 Plasmodium falciparum (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 Plasmodium parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.

  16. Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences.

    Science.gov (United States)

    Sercundes, Michelle Klein; Valadas, Samantha Yuri Oshiro Branco; Keid, Lara Borges; Oliveira, Tricia Maria Ferreira Souza; Ferreira, Helena Lage; Vitor, Ricardo Wagner de Almeida; Gregori, Fábio; Soares, Rodrigo Martins

    2016-01-01

    Phylogenies within Toxoplasmatinae have been widely investigated with different molecular markers. Here, we studied molecular phylogenies of the Toxoplasmatinae subfamily based on apicoplast and mitochondrial genes. Partial sequences of apicoplast genes coding for caseinolytic protease (clpC) and beta subunit of RNA polymerase (rpoB), and mitochondrial gene coding for cytochrome B (cytB) were analyzed. Laboratory-adapted strains of the closely related parasites Sarcocystis falcatula and Sarcocystis neurona were investigated, along with Neospora caninum, Neospora hughesi, Toxoplasma gondii (strains RH, CTG and PTG), Besnoitia akodoni, Hammondia hammondiand two genetically divergent lineages of Hammondia heydorni. The molecular analysis based on organellar genes did not clearly differentiate between N. caninum and N. hughesi, but the two lineages of H. heydorni were confirmed. Slight differences between the strains of S. falcatula and S. neurona were encountered in all markers. In conclusion, congruent phylogenies were inferred from the three different genes and they might be used for screening undescribed sarcocystid parasites in order to ascertain their phylogenetic relationships with organisms of the family Sarcocystidae. The evolutionary studies based on organelar genes confirm that the genus Hammondia is paraphyletic. The primers used for amplification of clpC and rpoB were able to amplify genetic sequences of organisms of the genus Sarcocystisand organisms of the subfamily Toxoplasmatinae as well. PMID:27007245

  17. Plasmodium Apicoplast Gln-tRNA Gln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites

    KAUST Repository

    Mailu, Boniface M.

    2015-08-28

    © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl-tRNA synthetase to synthesize Glu-tRNAGln and a glutaminyl-tRNA amidotransferase to convert Glu-tRNAGln to Gln-tRNAGln. Here, we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyltRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNAGln to Gln-tRNAGln in vitro. We demonstrate that the apicoplast GatAB-catalyzed reaction is essential to the parasite blood stages because we could not delete the Plasmodium berghei gene encoding GatA in blood stage parasites in vivo. A phylogenetic analysis placed the split between Plasmodium GatB, archaeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Moreover, Plasmodium GatA also appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, although GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria.

  18. A Toxoplasma MORN1 null mutant undergoes repeated divisions but is defective in basal assembly, apicoplast division and cytokinesis.

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    Alexander Lorestani

    Full Text Available The membrane occupation and recognition nexus protein 1 (MORN1 is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother's cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation.

  19. Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii.

    Science.gov (United States)

    Martins-Duarte, Érica S; Carias, Maira; Vommaro, Rossiane; Surolia, Namita; de Souza, Wanderley

    2016-09-01

    The apicomplexan protozoan Toxoplasma gondii, the causative agent of toxoplasmosis, harbors an apicoplast, a plastid-like organelle with essential metabolic functions. Although the FASII fatty acid biosynthesis pathway located in the apicoplast is essential for parasite survival, the cellular effects of FASII disruption in T. gondii had not been examined in detail. Here, we combined light and electron microscopy techniques - including focused ion beam scanning electron microscopy (FIB-SEM) - to characterize the effect of FASII disruption in T. gondii, by treatment with the FASII inhibitor triclosan or by inducible knockdown of the FASII component acyl carrier protein. Morphological analyses showed that FASII disruption prevented cytokinesis completion in T. gondii tachyzoites, leading to the formation of large masses of 'tethered' daughter cells. FIB-SEM showed that tethered daughters had a mature basal complex, but a defect in new membrane addition between daughters resulted in incomplete pellicle formation. Addition of exogenous fatty acids to medium suppressed the formation of tethered daughter cells and supports the notion that FASII is essential to generate lipid substrates required for the final step of parasite division. PMID:27457282

  20. Apicoplast-Localized Lysophosphatidic Acid Precursor Assembly Is Required for Bulk Phospholipid Synthesis in Toxoplasma gondii and Relies on an Algal/Plant-Like Glycerol 3-Phosphate Acyltransferase.

    Science.gov (United States)

    Amiar, Souad; MacRae, James I; Callahan, Damien L; Dubois, David; van Dooren, Giel G; Shears, Melanie J; Cesbron-Delauw, Marie-France; Maréchal, Eric; McConville, Malcolm J; McFadden, Geoffrey I; Yamaryo-Botté, Yoshiki; Botté, Cyrille Y

    2016-08-01

    Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii. PMID:27490259

  1. 系统发育分析指示细菌向Apicoplast的水平基因转移%Phylogenetic Analysis Indicates Bacteria-to-Apicoplast Lateral Gene Transfer

    Institute of Scientific and Technical Information of China (English)

    朱新宇

    2004-01-01

    Apicomplexan protozoa contains a highly reduced plastid-like organelle termed apicoplast.Data from clpC gene in apicoplast and their homologs in other plastids and bacteria were used to reconstruct phylogeny of apicoplast.Trees were reconstructed using neighbor-joining,minimum evolution,maximum parsimony and maximum likelihood.The reconstructions robustly support the monophyly of apicoplast and B.burgdorferi.This result underpins the mixture-genome hypotheses of apicoplast,furthermore,provides a new insight into the origin of this mixture genome.%顶复合器门的原生动物(Apicomplexan protozoa)含有一个高度退化的质体样(plastid-like)细胞器,定名为apicoplast.来自apicoplast的clpC基因和它在其他质体和细菌中的同源物用来重建apicoplast的系统发育史.使用邻接法(Neighbor-Joining)、最小进化法(Minimum Evolution)、最大简约法(Maximum Parsimony)和最大似然法(Maximum Likelihood)建立进化树.此外为了避免由于序列之间相似的碱基组成而引起的虚假聚类,建立了基于LogDet距离的核苷酸NJ树;以及为了避免由于在核苷酸和氨基酸水平上的突变饱和而引起的长分枝吸引(Long Branch Attraction,LBA),建立了基于非饱和位点的核苷酸和氨基酸序列的系统发育树.重建结果强有力地支持apicoplast和细菌B.buigdorferi之间的单系(monophyly)起源关系,也强化了apicoplasst属于混合基因组的假设,并且提供了对这个混合基因组起源的新的认识.

  2. Identification of vital and dispensable sulfur utilization factors in the Plasmodium apicoplast

    NARCIS (Netherlands)

    Haussig, J.M.; Matuschewski, K.; Kooij, T.W.A.

    2014-01-01

    Iron-sulfur [Fe-S] clusters are ubiquitous and critical cofactors in diverse biochemical processes. They are assembled by distinct [Fe-S] cluster biosynthesis pathways, typically in organelles of endosymbiotic origin. Apicomplexan parasites, including Plasmodium, the causative agent of malaria, harb

  3. 质体样细胞器APICOPLAST起源的分子证据评述%Molecular Evidence for Origin of Plastid-like Organelle Apicoplast: a Review

    Institute of Scientific and Technical Information of China (English)

    朱新宇; 张瑶; 周鸣鸣

    2005-01-01

    顶复合器门的原生动物(Apicomplexan protozoa)含有一个高度退化的质体样(pIastid-like)细胞器,定名为apicoplast.Apicoplast的进化起源是一个长期激烈争论的问题,尽管使用了多种分子技术,但尚未取得一致的结论,以致成为质体起源研究的典型案例.文章评述了apicoplast起源研究的分子证据,分析了新的分子证据的可能来源,为进一步研究提供线索.

  4. Telithromycin and Quinupristin-Dalfopristin Induce Delayed Death in Plasmodium falciparum▿

    OpenAIRE

    Barthel, Diana; Schlitzer, Martin; Pradel, Gabriele

    2007-01-01

    Antibacterial agents are used in malaria therapy due to their effect on two prokaryote organelles, the mitochondrion and the apicoplast. We demonstrate here that the ribosome-blocking antibiotics telithromycin and quinupristin-dalfopristin, but not linezolid, inhibit the growth of Plasmodium falciparum. Both drugs induce delayed death in the parasite, suggesting that their effect involves the impairment of apicoplast translation processes.

  5. Repurposing of conserved autophagy-related protein ATG8 in a divergent eukaryote.

    Science.gov (United States)

    Lévêque, Maude F; Nguyen, Hoa Mai; Besteiro, Sébastien

    2016-01-01

    Toxoplasma gondii and other apicomplexan parasites contain a peculiar non-photosynthetic plastid called the apicoplast, which is essential for their survival. The localization of autophagy-related protein ATG8 to the apicoplast in several apicomplexan species and life stages has recently been described, and we have shown this protein is essential for proper inheritance of this complex plastid into daughter cells during cell division. Although the mechanism behind ATG8 association to the apicoplast in T. gondii is related to the canonical conjugation system leading to autophagosome formation, its singular role seems independent from the initial catabolic purpose of autophagy. Here we also discuss further the functional evolution and innovative adaptations of the autophagy machinery to maintain this organelle during parasite division. PMID:27574540

  6. Role of Different Pfcrt and Pfmdr-1 Mutations in Conferring Resistance to Antimalaria Drugs in Plasmodium falciparum

    OpenAIRE

    Zaid O. Ibraheem; Abd Majid, R.; S. Mohd Noor; H. Mohd. Sedik; Basir, R.

    2014-01-01

    Emergence of drugs resistant strains of Plasmodium falciparum has augmented the scourge of malaria in endemic areas. Antimalaria drugs act on different intracellular targets. The majority of them interfere with digestive vacuoles (DVs) while others affect other organelles, namely, apicoplast and mitochondria. Prevention of drug accumulation or access into the target site is one of the mechanisms that plasmodium adopts to develop resistance. Plasmodia are endowed with series of transporters th...

  7. Ciprofloxacin Derivatives Affect Parasite Cell Division and Increase the Survival of Mice Infected with Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Erica S Martins-Duarte

    Full Text Available Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against T. gondii in vitro, compared with the original compound. Cipro derivatives are expected to target the parasite's DNA gyrase complex in the apicoplast. The activity of these compounds in vivo, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives in vivo, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects T. gondii tachyzoites in vitro, by immunofluorescence and transmission electron microscopy (TEM. When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13-25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment. Light microscopy examination early (6 and 24h post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition--with the appearance of 'tethered' parasites--malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against C. parvum, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in T. gondii tachyzoites, supporting that these compounds targets the apicoplast. Our results

  8. Ciprofloxacin Derivatives Affect Parasite Cell Division and Increase the Survival of Mice Infected with Toxoplasma gondii.

    Science.gov (United States)

    Martins-Duarte, Erica S; Dubar, Faustine; Lawton, Philippe; da Silva, Cristiane França; Soeiro, Maria de Nazaré C; de Souza, Wanderley; Biot, Christophe; Vommaro, Rossiane C

    2015-01-01

    Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against T. gondii in vitro, compared with the original compound. Cipro derivatives are expected to target the parasite's DNA gyrase complex in the apicoplast. The activity of these compounds in vivo, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives in vivo, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects T. gondii tachyzoites in vitro, by immunofluorescence and transmission electron microscopy (TEM). When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13-25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment). Light microscopy examination early (6 and 24h) post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition--with the appearance of 'tethered' parasites--malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against C. parvum, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in T. gondii tachyzoites, supporting that these compounds targets the apicoplast. Our results show that Cipro

  9. Characterization of the autophagy marker protein Atg8 reveals atypical features of autophagy in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Rahul Navale

    Full Text Available 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

  10. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    M El Bakkouri; A Pow; A Mulichak; K Cheung; J Artz; M Amani; S Fell; T de Koning-Ward; C Goodman; et al.

    2011-12-31

    The Clpchaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clpchaperones and proteases in the humanmalariaparasitePlasmodiumfalciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clpchaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  11. Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate

    KAUST Repository

    Gornik, Sebastian G.

    2015-04-20

    Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes - notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium - highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite\\'s host. Hematodinium sp. thus represents a further dimension of endosymbiosis-life after the organelle. © 2015, National Academy of Sciences. All rights reserved.

  12. Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.

    Science.gov (United States)

    Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W; Ben Mamoun, Choukri; Altman, Sidney

    2015-09-22

    Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome.

  13. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A. (McMaster U.); (Melbourne); (Toronto); (Deakin); (HWMRI)

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  14. Genome sequence of Babesia bovis and comparative analysis of apicomplexan hemoprotozoa.

    Directory of Open Access Journals (Sweden)

    Kelly A Brayton

    2007-10-01

    Full Text Available Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The approximately 150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast

  15. A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains

    Science.gov (United States)

    Preston, Mark D.; Campino, Susana; Assefa, Samuel A.; Echeverry, Diego F.; Ocholla, Harold; Amambua-Ngwa, Alfred; Stewart, Lindsay B.; Conway, David J.; Borrmann, Steffen; Michon, Pascal; Zongo, Issaka; Ouédraogo, Jean-Bosco; Djimde, Abdoulaye A.; Doumbo, Ogobara K.; Nosten, Francois; Pain, Arnab; Bousema, Teun; Drakeley, Chris J.; Fairhurst, Rick M.; Sutherland, Colin J.; Roper, Cally; Clark, Taane G.

    2014-01-01

    Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A simple genetic marker that quickly and accurately identifies the geographic origin of infections would be a valuable public health tool for locating the source of imported outbreaks. Here we analyse the mitochondrion and apicoplast genomes of 711 Plasmodium falciparum isolates from 14 countries, and find evidence that they are non-recombining and co-inherited. The high degree of linkage produces a panel of relatively few single-nucleotide polymorphisms (SNPs) that is geographically informative. We design a 23-SNP barcode that is highly predictive (~92%) and easily adapted to aid case management in the field and survey parasite migration worldwide. PMID:24923250

  16. A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains

    KAUST Repository

    Preston, Mark D.

    2014-06-13

    Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A simple genetic marker that quickly and accurately identifies the geographic origin of infections would be a valuable public health tool for locating the source of imported outbreaks. Here we analyse the mitochondrion and apicoplast genomes of 711 Plasmodium falciparum isolates from 14 countries, and find evidence that they are non-recombining and co-inherited. The high degree of linkage produces a panel of relatively few single-nucleotide polymorphisms (SNPs) that is geographically informative. We design a 23-SNP barcode that is highly predictive (?92%) and easily adapted to aid case management in the field and survey parasite migration worldwide. 2014 Macmillan Publishers Limited. All rights reserved.

  17. P. falciparum cpn20 is a bona fide co-chaperonin that can replace GroES in E. coli.

    Directory of Open Access Journals (Sweden)

    Anna Vitlin Gruber

    Full Text Available Human malaria is among the most ubiquitous and destructive tropical, parasitic diseases in the world today. The causative agent, Plasmodium falciparum, contains an unusual, essential organelle known as the apicoplast. Inhibition of this degenerate chloroplast results in second generation death of the parasite and is the mechanism by which antibiotics function in treating malaria. In order to better understand the biochemistry of this organelle, we have cloned a putative, 20 kDa, co-chaperonin protein, Pf-cpn20, which localizes to the apicoplast. Although this protein is homologous to the cpn20 that is found in plant chloroplasts, its ability to function as a co-chaperonin was questioned in the past. In the present study, we carried out a structural analysis of Pf-cpn20 using circular dichroism and analytical ultracentrifugation and then used two different approaches to investigate the ability of this protein to function as a co-chaperonin. In the first approach, we purified recombinant Pf-cpn20 and tested its ability to act as a co-chaperonin for GroEL in vitro, while in the second, we examined the ability of Pf-cpn20 to complement an E. coli depletion of the essential bacterial co-chaperonin GroES. Our results demonstrate that Pf-cpn20 is fully functional as a co-chaperonin in vitro. Moreover, the parasitic co-chaperonin is able to replace GroES in E. coli at both normal and heat-shock temperatures. Thus, Pf-cpn20 functions as a co-chaperonin in chaperonin-mediated protein folding. The ability of the malarial protein to function in E. coli suggests that this simple system can be used as a tool for further analyses of Pf-cpn20 and perhaps other chaperone proteins from P. falciparum.

  18. The Complete Mitochondrial Genome of the Foodborne Parasitic Pathogen Cyclospora cayetanensis.

    Directory of Open Access Journals (Sweden)

    Hediye Nese Cinar

    Full Text Available Cyclospora cayetanensis is a human-specific coccidian parasite responsible for several food and water-related outbreaks around the world, including the most recent ones involving over 900 persons in 2013 and 2014 outbreaks in the USA. Multicopy organellar DNA such as mitochondrion genomes have been particularly informative for detection and genetic traceback analysis in other parasites. We sequenced the C. cayetanensis genomic DNA obtained from stool samples from patients infected with Cyclospora in Nepal using the Illumina MiSeq platform. By bioinformatically filtering out the metagenomic reads of non-coccidian origin sequences and concentrating the reads by targeted alignment, we were able to obtain contigs containing Eimeria-like mitochondrial, apicoplastic and some chromosomal genomic fragments. A mitochondrial genomic sequence was assembled and confirmed by cloning and sequencing targeted PCR products amplified from Cyclospora DNA using primers based on our draft assembly sequence. The results show that the C. cayetanensis mitochondrion genome is 6274 bp in length, with 33% GC content, and likely exists in concatemeric arrays as in Eimeria mitochondrial genomes. Phylogenetic analysis of the C. cayetanensis mitochondrial genome places this organism in a tight cluster with Eimeria species. The mitochondrial genome of C. cayetanensis contains three protein coding genes, cytochrome (cytb, cytochrome C oxidase subunit 1 (cox1, and cytochrome C oxidase subunit 3 (cox3, in addition to 14 large subunit (LSU and nine small subunit (SSU fragmented rRNA genes.

  19. An FtsH protease is recruited to the mitochondrion of Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Aiman Tanveer

    Full Text Available The two organelles, apicoplast and mitochondrion, of the malaria parasite Plasmodium falciparum have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. Little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. We report the identification of an AAA+/FtsH protease homolog (PfFtsH1 that exhibits ATP- and Zn(2+-dependent protease activity. PfFtsH1 undergoes processing, forms oligomeric assemblies, and is associated with the membrane fraction of the parasite cell. Generation of a transfectant parasite line with hemagglutinin-tagged PfFtsH1, and immunofluorescence assay with anti-PfFtsH1 Ab demonstrated that the protein localises to P. falciparum mitochondria. Phylogenetic analysis and the single transmembrane region identifiable in PfFtsH1 suggest that it is an i-AAA like inner mitochondrial membrane protein. Expression of PfFtsH1 in Escherichia coli converted a fraction of bacterial cells into division-defective filamentous forms implying a sequestering effect of the Plasmodium factor on the bacterial homolog, indicative of functional conservation with EcFtsH. These results identify a membrane-associated mitochondrial AAA+/FtsH protease as a candidate regulatory protein for organelle biogenesis in P. falciparum.

  20. Role of Different Pfcrt and Pfmdr-1 Mutations in Conferring Resistance to Antimalaria Drugs in Plasmodium falciparum.

    Science.gov (United States)

    Ibraheem, Zaid O; Abd Majid, R; Noor, S Mohd; Sedik, H Mohd; Basir, R

    2014-01-01

    Emergence of drugs resistant strains of Plasmodium falciparum has augmented the scourge of malaria in endemic areas. Antimalaria drugs act on different intracellular targets. The majority of them interfere with digestive vacuoles (DVs) while others affect other organelles, namely, apicoplast and mitochondria. Prevention of drug accumulation or access into the target site is one of the mechanisms that plasmodium adopts to develop resistance. Plasmodia are endowed with series of transporters that shuffle drugs away from the target site, namely, pfmdr (Plasmodium falciparum multidrug resistance transporter) and pfcrt (Plasmodium falciparum chloroquine resistance transporter) which exist in DV membrane and are considered as putative markers of CQ resistance. They are homologues to human P-glycoproteins (P-gh or multidrug resistance system) and members of drug metabolite transporter (DMT) family, respectively. The former mediates drifting of xenobiotics towards the DV while the latter chucks them outside. Resistance to drugs whose target site of action is intravacuolar develops when the transporters expel them outside the DVs and vice versa for those whose target is extravacuolar. In this review, we are going to summarize the possible pfcrt and pfmdr mutation and their role in changing plasmodium sensitivity to different anti-Plasmodium drugs. PMID:25506039

  1. Multigenomic Delineation of Plasmodium Species of the Laverania Subgenus Infecting Wild-Living Chimpanzees and Gorillas

    Science.gov (United States)

    Liu, Weimin; Sundararaman, Sesh A.; Loy, Dorothy E.; Learn, Gerald H.; Li, Yingying; Plenderleith, Lindsey J.; Ndjango, Jean-Bosco N.; Speede, Sheri; Atencia, Rebeca; Cox, Debby; Shaw, George M.; Ayouba, Ahidjo; Peeters, Martine; Rayner, Julian C.; Hahn, Beatrice H.; Sharp, Paul M.

    2016-01-01

    Plasmodium falciparum, the major cause of malaria morbidity and mortality worldwide, is only distantly related to other human malaria parasites and has thus been placed in a separate subgenus, termed Laverania. Parasites morphologically similar to P. falciparum have been identified in African apes, but only one other Laverania species, Plasmodium reichenowi from chimpanzees, has been formally described. Although recent studies have pointed to the existence of additional Laverania species, their precise number and host associations remain uncertain, primarily because of limited sampling and a paucity of parasite sequences other than from mitochondrial DNA. To address this, we used limiting dilution polymerase chain reaction to amplify additional parasite sequences from a large number of chimpanzee and gorilla blood and fecal samples collected at two sanctuaries and 30 field sites across equatorial Africa. Phylogenetic analyses of more than 2,000 new sequences derived from the mitochondrial, nuclear, and apicoplast genomes revealed six divergent and well-supported clades within the Laverania parasite group. Although two of these clades exhibited deep subdivisions in phylogenies estimated from organelle gene sequences, these sublineages were geographically defined and not present in trees from four unlinked nuclear loci. This greatly expanded sequence data set thus confirms six, and not seven or more, ape Laverania species, of which P. reichenowi, Plasmodium gaboni, and Plasmodium billcollinsi only infect chimpanzees, whereas Plasmodium praefalciparum, Plasmodium adleri, and Pladmodium blacklocki only infect gorillas. The new sequence data also confirm the P. praefalciparum origin of human P. falciparum. PMID:27289102

  2. Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii.

    Science.gov (United States)

    Stewart, Rebecca J; Ferguson, David J P; Whitehead, Lachlan; Bradin, Clare H; Wu, Hong J; Tonkin, Christopher J

    2016-02-01

    Upon infection, apicomplexan parasites quickly invade host cells and begin a replicative cycle rapidly increasing in number over a short period of time, leading to tissue lysis and disease. The secretory pathway of these highly polarized protozoan parasites tightly controls, in time and space, the biogenesis of specialized structures and organelles required for invasion and intracellular survival. In other systems, regulation of protein trafficking can occur by phosphorylation of vesicle fusion machinery. Previously, we have shown that Toxoplasma gondii αSNAP - a protein that controls the disassembly of cis-SNARE complexes--is phosphorylated. Here, we show that this post-translational modification is required for the correct function of αSNAP in controlling secretory traffic. We demonstrate that during intracellular development conditional expression of a non-phosphorylatable form of αSNAP results in Golgi fragmentation and vesiculation of all downstream secretory organelles. In addition, we show that the vestigial plastid (termed apicoplast), although reported not to be reliant on Golgi trafficking for biogenesis, is also affected upon overexpression of αSNAP and is much more sensitive to the levels of this protein than targeting to other organelles. This work highlights the importance of αSNAP and its phosphorylation in Toxoplasma organelle biogenesis and exposes a hereto fore-unexplored mechanism of regulation of vesicle fusion during secretory pathway trafficking in apicomplexan parasites.

  3. Multigenomic Delineation of Plasmodium Species of the Laverania Subgenus Infecting Wild-Living Chimpanzees and Gorillas.

    Science.gov (United States)

    Liu, Weimin; Sundararaman, Sesh A; Loy, Dorothy E; Learn, Gerald H; Li, Yingying; Plenderleith, Lindsey J; Ndjango, Jean-Bosco N; Speede, Sheri; Atencia, Rebeca; Cox, Debby; Shaw, George M; Ayouba, Ahidjo; Peeters, Martine; Rayner, Julian C; Hahn, Beatrice H; Sharp, Paul M

    2016-01-01

    Plasmodium falciparum, the major cause of malaria morbidity and mortality worldwide, is only distantly related to other human malaria parasites and has thus been placed in a separate subgenus, termed Laverania Parasites morphologically similar to P. falciparum have been identified in African apes, but only one other Laverania species, Plasmodium reichenowi from chimpanzees, has been formally described. Although recent studies have pointed to the existence of additional Laverania species, their precise number and host associations remain uncertain, primarily because of limited sampling and a paucity of parasite sequences other than from mitochondrial DNA. To address this, we used limiting dilution polymerase chain reaction to amplify additional parasite sequences from a large number of chimpanzee and gorilla blood and fecal samples collected at two sanctuaries and 30 field sites across equatorial Africa. Phylogenetic analyses of more than 2,000 new sequences derived from the mitochondrial, nuclear, and apicoplast genomes revealed six divergent and well-supported clades within the Laverania parasite group. Although two of these clades exhibited deep subdivisions in phylogenies estimated from organelle gene sequences, these sublineages were geographically defined and not present in trees from four unlinked nuclear loci. This greatly expanded sequence data set thus confirms six, and not seven or more, ape Laverania species, of which P. reichenowi, Plasmodium gaboni, and Plasmodium billcollinsi only infect chimpanzees, whereas Plasmodium praefalciparum, Plasmodium adleri, and Pladmodium blacklocki only infect gorillas. The new sequence data also confirm the P. praefalciparum origin of human P. falciparum. PMID:27289102

  4. Structural mapping of the ClpB ATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design.

    Science.gov (United States)

    AhYoung, Andrew P; Koehl, Antoine; Cascio, Duilio; Egea, Pascal F

    2015-09-01

    Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains-a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs. PMID:26130467

  5. Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections.

    Science.gov (United States)

    Bernotienė, Rasa; Palinauskas, Vaidas; Iezhova, Tatjana; Murauskaitė, Dovilė; Valkiūnas, Gediminas

    2016-04-01

    Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections.

  6. The evolutionary host switches of Polychromophilus: a multi-gene phylogeny of the bat malaria genus suggests a second invasion of mammals by a haemosporidian parasite

    Directory of Open Access Journals (Sweden)

    Witsenburg Fardo

    2012-02-01

    Full Text Available Abstract Background The majority of Haemosporida species infect birds or reptiles, but many important genera, including Plasmodium, infect mammals. Dipteran vectors shared by avian, reptilian and mammalian Haemosporida, suggest multiple invasions of Mammalia during haemosporidian evolution; yet, phylogenetic analyses have detected only a single invasion event. Until now, several important mammal-infecting genera have been absent in these analyses. This study focuses on the evolutionary origin of Polychromophilus, a unique malaria genus that only infects bats (Microchiroptera and is transmitted by bat flies (Nycteribiidae. Methods Two species of Polychromophilus were obtained from wild bats caught in Switzerland. These were molecularly characterized using four genes (asl, clpc, coI, cytb from the three different genomes (nucleus, apicoplast, mitochondrion. These data were then combined with data of 60 taxa of Haemosporida available in GenBank. Bayesian inference, maximum likelihood and a range of rooting methods were used to test specific hypotheses concerning the phylogenetic relationships between Polychromophilus and the other haemosporidian genera. Results The Polychromophilus melanipherus and Polychromophilus murinus samples show genetically distinct patterns and group according to species. The Bayesian tree topology suggests that the monophyletic clade of Polychromophilus falls within the avian/saurian clade of Plasmodium and directed hypothesis testing confirms the Plasmodium origin. Conclusion Polychromophilus' ancestor was most likely a bird- or reptile-infecting Plasmodium before it switched to bats. The invasion of mammals as hosts has, therefore, not been a unique event in the evolutionary history of Haemosporida, despite the suspected costs of adapting to a new host. This was, moreover, accompanied by a switch in dipteran host.

  7. Plasmodium delichoni n. sp.: description, molecular characterisation and remarks on the exoerythrocytic merogony, persistence, vectors and transmission.

    Science.gov (United States)

    Valkiūnas, Gediminas; Ilgūnas, Mikas; Bukauskaitė, Dovilė; Žiegytė, Rita; Bernotienė, Rasa; Jusys, Vytautas; Eigirdas, Vytautas; Fragner, Karin; Weissenböck, Herbert; Iezhova, Tatjana A

    2016-07-01

    Malaria parasite Plasmodium (Novyella) delichoni n. sp. (Haemosporida, Plasmodiidae) was found in a widespread Eurasian songbird, the common house martin Delichon urbicum (Hirundinidae). It is described based on the morphology of its blood stages and segments of the mitochondrial cytochrome b and apicoplast genes, which can be used for molecular identification of this species. Erythrocytic meronts and gametocytes are strictly nucleophilic, and mature gametocytes possess pigment granules of markedly variable size, including large ones (1 μm in length). Due to these features, P. delichoni can be readily distinguished from all described species of avian malaria parasites belonging to subgenus Novyella. Additionally, mature erythrocytic merozoites contain a dense clump of chromatin, a rare character in avian malaria parasites. Erythrocytic merogony is asynchronous. Illustrations of blood stages of the new species are given, and phylogenetic analysis identifies DNA lineages closely related to this parasite. Domestic canary Serinus canaria and Eurasian siskin Carduelis spinus were infected after subinoculation of infected blood obtained from the house martin. Parasitemia was long lasting in both these hosts, but it was high (up to 70 %) in Eurasian siskins and low (up to 1 %) in canaries. Mortality was not observed, and histological examination and chromogenic in situ hybridisation did not reveal secondary exoerythrocytic meronts (phanerozoites) in the exposed birds. It is likely that persistence of this infection occurs due to long-lasting parasitemia in avian hosts. Sporogony was abortive in mosquitoes Culex pipiens pipiens form molestus, Culex quinquefasciatus and Aedes aegypti at gametogenesis or ookinete stages. The new species is absent from juvenile birds at breeding sites in Europe, indicating that transmission occurs at African wintering grounds. PMID:27000087