The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Ellen Yeh - One of the best experts on this subject based on the ideXlab platform.
-
caax like protease of cyanobacterial origin is required for complex plastid biogenesis in malaria parasites
Mbio, 2020Co-Authors: Thomas R Meister, Yong Tang, Michael J Pulkoskigross, Ellen YehAbstract:ABSTRACT Plasmodium parasites and related apicomplexans contain an essential “complex plastid” organelle of secondary endosymbiotic origin, the apicoplast. Biogenesis of this complex plastid poses a unique challenge requiring evolution of new cellular machinery. We previously conducted a mutagenesis screen for essential apicoplast biogenesis genes to discover organellar pathways with evolutionary and biomedical significance. Here we validate and characterize a gene candidate from our screen, Pf3D7_0913500. Using a conditional knockdown strain, we show that Pf3D7_0913500 depletion causes growth inhibition that is rescued by the sole essential product of the apicoplast, isopentenyl pyrophosphate (IPP), and results in apicoplast loss. Because Pf3D7_0913500 had no previous functional annotation, we name it apicoplast-minus IPP-rescued 4 (AMR4). AMR4 has an annotated CaaX protease and bacteriocin processing (CPBP) domain, which in eukaryotes typically indicates a role in CaaX postprenylation processing. Indeed, AMR4 is the only putative CaaX-like protease in Plasmodium parasites which are known to require protein prenylation, and we confirm that the conserved catalytic residue of AMR4 (E352) is required for its apicoplast function. However, we unexpectedly find that AMR4 does not act in a CaaX postprenylation processing pathway in Plasmodium falciparum. Instead, we find that AMR4 is imported into the apicoplast and is derived from a cyanobacterial CPBP gene which was retained through both primary and secondary endosymbiosis. Our findings suggest that AMR4 is not a true CaaX protease, but instead it performs a conserved, uncharacterized chloroplast function that has been retained for complex plastid biogenesis. IMPORTANCEPlasmodium parasites, which cause malaria, and related apicomplexans are important human and veterinary pathogens. These parasites represent a highly divergent and understudied branch of eukaryotes, and as such often defy the expectations set by model organisms. One striking example of unique apicomplexan biology is the apicoplast, an essential but nonphotosynthetic plastid derived from an unusual secondary (eukaryote-eukaryote) endosymbiosis. Endosymbioses are a major driver of cellular innovation, and apicoplast biogenesis pathways represent a hot spot for molecular evolution. We previously conducted an unbiased screen for apicoplast biogenesis genes in P. falciparum to uncover these essential and innovative pathways. Here, we validate a novel gene candidate from our screen and show that its role in apicoplast biogenesis does not match its functional annotation predicted by model eukaryotes. Our findings suggest that an uncharacterized chloroplast maintenance pathway has been reused for complex plastid biogenesis in this divergent branch of pathogens.
-
caax like protease of cyanobacterial origin is required for complex plastid biogenesis in malaria parasites
bioRxiv, 2020Co-Authors: Thomas R Meister, Yong Tang, Michael J Pulkoskigross, Ellen YehAbstract:Abstract Plasmodium parasites and related apicomplexans contain an essential “complex plastid” organelle of secondary endosymbiotic origin, the apicoplast. Biogenesis of this complex plastid poses a unique challenge requiring evolution of new cellular machinery. We previously conducted a mutagenesis screen for essential apicoplast biogenesis genes to discover organellar pathways with evolutionary and biomedical significance. Here we validate and characterize a gene candidate from our screen, Pf3D7_0913500. Using a conditional knockdown strain, we show that Pf3D7_0913500 depletion causes growth inhibition that is rescued by the sole essential product of the apicoplast, isopentenyl pyrophosphate (IPP), and results in apicoplast loss. Because Pf3D7_0913500 had no previous functional annotation, we name it apicoplast-minus IPP-rescued 4 (AMR4). AMR4 has an annotated CaaX Protease and Bacteriocin Processing (CPBP) domain, which in eukaryotes typically indicates a role in CaaX post-prenylation processing. Indeed, AMR4 is the only CaaX-like protease in Plasmodium parasites which are known to require protein prenylation, and we confirm that the conserved catalytic residue of AMR4 is required for its apicoplast function. However, we unexpectedly find that AMR4 does not act in a CaaX post-prenylation processing pathway in P. falciparum. Instead, we find that AMR4 is imported into the apicoplast and is derived from a cyanobacterial CPBP gene which was retained through both primary and secondary endosymbiosis. Our findings suggest that AMR4 is not a true CaaX protease, but instead acts in a conserved, uncharacterized chloroplast pathway that has been retained for complex plastid biogenesis. Importance Plasmodium parasites, which cause malaria, and related apicomplexans are important human and veterinary pathogens. These parasites represent a highly divergent and understudied branch of eukaryotes, and as such often defy the expectations set by model organisms. One striking example of unique apicomplexan biology is the apicoplast, an essential but non-photosynthetic plastid derived from an unusual secondary (eukaryote-eukaryote) endosymbiosis. Endosymbioses are a major driver of cellular innovation, and apicoplast biogenesis pathways represent a hotspot for molecular evolution. We previously conducted an unbiased screen for apicoplast biogenesis genes in P. falciparum to uncover these essential and innovative pathways. Here, we validate a novel gene candidate from our screen and show that its role in apicoplast biogenesis does not match its functional annotation predicted by model eukaryotes. Our findings suggest that an uncharacterized chloroplast maintenance pathway has been reused for complex plastid biogenesis in this divergent branch of pathogens.
-
A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites.
Public Library of Science (PLoS), 2019Co-Authors: Yong Tang, Katherine Amberg-johnson, Sanjay B. Hari, Robert T Sauer, Thomas R Meister, Marta Walczak, Michael J Pulkoski-gross, Ellen YehAbstract:Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid-the apicoplast-which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(-) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases
-
Disruption of Apicoplast Biogenesis by Chemical Stabilization of an Imported Protein Evades the Delayed-Death Phenotype in Malaria Parasites
American Society for Microbiology, 2019Co-Authors: Michael J. Boucher, Ellen YehAbstract:Malaria is a major cause of global childhood mortality. To sustain progress in disease control made in the last decade, new antimalarial therapies are needed to combat emerging drug resistance. Malaria parasites contain a relict chloroplast called the apicoplast, which harbors new targets for drug discovery. Unfortunately, some drugs targeting apicoplast pathways exhibit a delayed-death phenotype, which results in a slow onset-of-action that precludes their use as fast-acting, frontline therapies. Identification of druggable apicoplast biogenesis factors that will avoid the delayed-death phenotype is an important priority. Here, we find that chemical stabilization of an apicoplast-targeted mDHFR domain disrupts apicoplast biogenesis and inhibits parasite growth after a single lytic cycle, suggesting a non-delayed-death target. Our finding indicates that further interrogation of the mechanism-of-action of this exogenous fusion protein may reveal novel therapeutic avenues.Malaria parasites (Plasmodium spp.) contain a nonphotosynthetic plastid organelle called the apicoplast, which houses essential metabolic pathways and is required throughout the parasite life cycle. The biogenesis pathways responsible for apicoplast growth, division, and inheritance are of key interest as potential drug targets. Unfortunately, several known apicoplast biogenesis inhibitors are of limited clinical utility because they cause a peculiar “delayed-death” phenotype in which parasites do not stop replicating until the second lytic cycle posttreatment. Identifying apicoplast biogenesis pathways that avoid the delayed-death phenomenon is a priority. Here, we generated parasites targeting a murine dihydrofolate reductase (mDHFR) domain, which can be conditionally stabilized with the compound WR99210, to the apicoplast. Surprisingly, chemical stabilization of this exogenous fusion protein disrupted parasite growth in an apicoplast-specific manner after a single lytic cycle. WR99210-treated parasites exhibited an apicoplast biogenesis defect beginning within the same lytic cycle as drug treatment, indicating that stabilized mDHFR perturbs a non-delayed-death biogenesis pathway. While the precise mechanism-of-action of the stabilized fusion is still unclear, we hypothesize that it inhibits apicoplast protein import by stalling within and blocking translocons in the apicoplast membranes
-
host cell metabolism contributes to delayed death kinetics of apicoplast inhibitors in toxoplasma gondii
Antimicrobial Agents and Chemotherapy, 2018Co-Authors: Katherine Ambergjohnson, Ellen YehAbstract:Toxoplasma gondii and related human parasites contain an essential plastid organelle called the apicoplast. Clinically used antibiotics and other inhibitors that disrupt apicoplast biogenesis cause a mysterious "delayed-death" phenotype in which parasite growth is unaffected during the first lytic cycle of inhibitor treatment but is severely inhibited in the second lytic cycle even after drug removal. Critical to understanding the complex downstream cellular effects of these drug classes are the timing of apicoplast loss during inhibitor treatment and how it relates to this peculiar growth phenotype. Here we show that, upon treatment with diverse classes of apicoplast inhibitors, newly replicated T. gondii parasites in the first lytic cycle initially form Apicoplasts with defects in protein import or genome replication and eventually fail to inherit the apicoplast altogether. Despite the accumulation of parasites with defective or missing Apicoplasts, growth is unaffected during the first lytic cycle, as previously observed. Strikingly, concomitant inhibition of host cell isoprenoid biosynthesis results in growth inhibition in the first lytic cycle and unmasks the apicoplast defects. These results suggest that defects in and even the complete loss of the apicoplast in T. gondii are partially rescued by scavenging of host cell metabolites, leading to death that is delayed. Our findings uncover host cell interactions that can alleviate apicoplast inhibition and highlight key differences in delayed-death inhibitors between T. gondii and Plasmodium falciparum.
Christopher D. Goodman - One of the best experts on this subject based on the ideXlab platform.
-
a genetic screen in rodent malaria parasites identifies five new apicoplast putative membrane transporters one of which is essential in human malaria parasites
Cellular Microbiology, 2018Co-Authors: Claire P. Sayers, Hayley D. Buchanan, Vanessa Mollard, Christopher D. GoodmanAbstract:The malaria-causing parasite, Plasmodium, contains a unique non-photosynthetic plastid known as the apicoplast. The apicoplast is an essential organelle bound by four membranes. Although membrane transporters are attractive drug targets, only two transporters have been characterised in the malaria parasite apicoplast membranes. We selected 27 candidate apicoplast membrane proteins, 20 of which are annotated as putative membrane transporters, and performed a genetic screen in Plasmodium berghei to determine blood stage essentiality and subcellular localisation. Eight apparently essential blood stage genes were identified, three of which were apicoplast-localised: PbANKA_0614600 (DMT2), PbANKA_0401200 (ABCB4), and PbANKA_0505500. Nineteen candidates could be deleted at the blood stage, four of which were apicoplast-localised. Interestingly, three apicoplast-localised candidates lack a canonical apicoplast targeting signal but do contain conserved N-terminal tyrosines with likely roles in targeting. An inducible knockdown of an essential apicoplast putative membrane transporter, PfDMT2, was only viable when supplemented with isopentenyl diphosphate. Knockdown of PfDMT2 resulted in loss of the apicoplast, identifying PfDMT2 as a crucial apicoplast putative membrane transporter and a candidate for therapeutic intervention.
-
Validation of Putative Apicoplast-Targeting Drugs Using a Chemical Supplementation Assay in Cultured Human Malaria Parasites.
Antimicrobial agents and chemotherapy, 2017Co-Authors: Taher Uddin, Christopher D. GoodmanAbstract:Malaria parasites contain a relict plastid, the apicoplast, which is considered an excellent drug target due to its bacterial-like ancestry. Numerous parasiticidals have been proposed to target the apicoplast, but few have had their actual targets substantiated. Isopentenyl pyrophosphate (IPP) production is the sole required function of the apicoplast in the blood stage of the parasite life cycle, and IPP supplementation rescues parasites from apicoplast-perturbing drugs. Hence, any drug that kills parasites when IPP is supplied in culture must have a nonapicoplast target. Here, we use IPP supplementation to discriminate whether 23 purported apicoplast-targeting drugs are on- or off-target. We demonstrate that a prokaryotic DNA replication inhibitor (ciprofloxacin), several prokaryotic translation inhibitors (chloramphenicol, doxycycline, tetracycline, clindamycin, azithromycin, erythromycin, and clarithromycin), a tRNA synthase inhibitor (mupirocin), and two IPP synthesis pathway inhibitors (fosmidomycin and FR900098) have apicoplast targets. Intriguingly, fosmidomycin and FR900098 leave the apicoplast intact, whereas the others eventually result in apicoplast loss. Actinonin, an inhibitor of bacterial posttranslational modification, does not produce a typical delayed-death response but is rescued with IPP, thereby confirming its apicoplast target. Parasites treated with putative apicoplast fatty acid pathway inhibitors could not be rescued, demonstrating that these drugs have their primary targets outside the apicoplast, which agrees with the dispensability of the apicoplast fatty acid synthesis pathways in the blood stage of malaria parasites. IPP supplementation provides a simple test of whether a compound has a target in the apicoplast and can be used to screen novel compounds for mode of action.
-
Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum.
PloS one, 2016Co-Authors: Liting Lim, Claire P. Sayers, Christopher D. GoodmanAbstract: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.
-
Ycf93 (Orf105), a Small Apicoplast-Encoded Membrane Protein in the Relict Plastid of the Malaria Parasite Plasmodium falciparum That Is Conserved in Apicomplexa
PLOS ONE, 2014Co-Authors: Christopher D. GoodmanAbstract:Malaria parasites retain a relict plastid (apicoplast) from a photosynthetic ancestor shared with dinoflagellate algae. The apicoplast is a useful drug target; blocking housekeeping pathways such as genome replication and translation in the organelle kills parasites and protects against malaria. The apicoplast of Plasmodium falciparum encodes 30 proteins and a suite of rRNAs and tRNAs that facilitate their expression. orf105 is a hypothetical apicoplast gene that would encode a small protein (PfOrf105) with a predicted C-terminal transmembrane domain. We produced antisera to a predicted peptide within PfOrf105. Western blot analysis confirmed expression of orf105 and immunofluorescence localised the gene product to the apicoplast. Pforf105 encodes a membrane protein that has an apparent mass of 17.5 kDa and undergoes substantial turnover during the 48-hour asexual life cycle of the parasite in blood stages. The effect of actinonin, an antimalarial with a putative impact on post-translational modification of apicoplast proteins like PfOrf105, was examined. Unlike other drugs perturbing apicoplast housekeeping that induce delayed death, actinonin kills parasites immediately and has an identical drug exposure phenotype to the isopentenyl diphosphate synthesis blocker fosmidomycin. Open reading frames of similar size to PfOrf105, which also have predicted C-terminal trans membrane domains, occur in syntenic positions in all sequenced apicoplast genomes from Phylum Apicomplexa. We therefore propose to name these genes ycf93 (hypothetical chloroplast reading frame 93) according to plastid gene nomenclature convention for conserved proteins of unknown function.
-
apicoplast acetyl co a carboxylase of the human malaria parasite is not targeted by cyclohexanedione herbicides
International Journal for Parasitology, 2014Co-Authors: Christopher D. Goodman, Vanessa Mollard, Theola Louie, Georgina A Holloway, Keith G Watson, Geoffrey I. McfaddenAbstract:Malaria parasites retain a relict plastid (apicoplast) from a photosynthetic ancestor. The apicoplast is a useful drug target but the specificity of compounds believed to target apicoplast fatty acid biosynthesis has become uncertain, as this pathway is not essential in blood stages of the parasite. Herbicides that inhibit the plastid acetyl Coenzyme A (Co-A) carboxylase of plants also kill Plasmodium falciparum in vitro, but their mode of action remains undefined. We characterised the gene for acetyl Co-A carboxylase in P. falciparum. The P. falciparum acetyl-CoA carboxylase gene product is expressed in blood stage parasites and accumulates in the apicoplast. Ablation of the gene did not render parasites insensitive to herbicides, suggesting that these compounds are acting off-target in blood stages of P. falciparum.
David S Roos - One of the best experts on this subject based on the ideXlab platform.
-
n terminal positively charged amino acids but not their exact position are important for apicoplast transit peptide fidelity in toxoplasma gondii
Molecular and Biochemical Parasitology, 2006Co-Authors: Christopher J. Tonkin, David S Roos, Geoffrey I. McfaddenAbstract:The non-photosynthetic plastid - or apicoplast - of Toxoplasma gondii and other apicomplexan parasites is an essential organelle and promising drug target. Most apicoplast proteins are encoded in the nucleus and targeted into the organelle through the apicoplast's four membranes courtesy of a bipartite N-terminal leader sequence comprising of an endomembrane signal peptide followed by a plastid transit peptide. Apicoplast transit peptides, like plant plastid transit peptides, have no primary consensus, are variable in length and may be distinguishable only by a relative depletion of negative charged residues and consequent enrichment in basic residues. In this study we examine the role of charged residues within an apicoplast transit peptide in T. gondii by point mutagenesis. We demonstrate that positive charged residues, combined with the absence of negatively charged amino acids, are essential for apicoplast transit peptide fidelity, as also observed in P. falciparum. Furthermore, we show that positive charge is more important at the transit peptide's N-terminus than its C-terminus, and that the nature of the positive residue and the exact position of the N-terminal positive charge are not important. These results suggest that a simple, rule-based prediction for T. gondii transit peptides, similar to that successfully implemented for P. falciparum should help to identify apicoplast proteins and facilitate the identification of drug targets in this important human pathogen.
-
multiple functionally redundant signals mediate targeting to the apicoplast in the apicomplexan parasite toxoplasma gondii
Eukaryotic Cell, 2004Co-Authors: Omar S Harb, Martin Fraunholz, Bithi Chatterjee, Michael Crawford, Manami Nishi, David S RoosAbstract:Most species of the protozoan phylum Apicomplexa harbor an endosymbiotic organelle—the apicoplast—acquired when an ancestral parasite engulfed a eukaryotic plastid-containing alga. Several hundred proteins are encoded in the parasite nucleus and are posttranslationally targeted to the apicoplast by a distinctive bipartite signal. The N-terminal 20 to 30 amino acids of nucleus-encoded apicoplast targeted proteins function as a classical signal sequence, mediating entry into the secretory pathway. Cleavage of the signal sequence exposes a transit peptide of variable length (50 to 200 amino acids) that is required for directing proteins to the apicoplast. Although these peptides are enriched in basic amino acids, their structural and functional characteristics are not well understood, which hampers the identification of apicoplast proteins that may constitute novel chemotherapeutic targets. To identify functional domains for a model apicoplast transit peptide, we generated more than 80 deletions and mutations throughout the transit peptide of Toxoplasma gondii ferredoxin NADP+ reductase (TgFNR) and examined the ability of these altered transit peptides to mediate proper targeting and processing of a fluorescent protein reporter. These studies revealed the presence of numerous functional domains. Processing can take place at multiple sites in the protein sequence and may occur outside of the apicoplast lumen. The TgFNR transit peptide contains at least two independent and functionally redundant targeting signals, each of which contains a subdomain that is required for release from or proper sorting within the endoplasmic reticulum. Certain deletion constructs traffic to multiple locations, including the apicoplast periphery, the rhoptries, and the parasitophorous vacuole, suggesting a common thread for targeting to these specialized compartments.
-
tropical infectious diseases metabolic maps and functions of the plasmodium falciparum apicoplast
Nature Reviews Microbiology, 2004Co-Authors: Stuart A Ralph, Ross F. Waller, Christopher J. Tonkin, Bernardo J. Foth, Giel G Van Dooren, Michael J Crawford, Martin Fraunholz, David S RoosAbstract:Discovery of a relict chloroplast (the apicoplast) in malarial parasites presented new opportunities for drug development. The apicoplast – although no longer photosynthetic – is essential to parasites. Combining bioinformatics approaches with experimental validation in the laboratory, we have identified more than 500 proteins predicted to function in the apicoplast. By comparison with plant chloroplasts, we have reconstructed several anabolic pathways for the parasite plastid that are fundamentally different to the analogous pathways in the human host and are potentially good targets for drug development. Products of these pathways seem to be exported from the apicoplast and might be involved in host-cell invasion.
-
a plastid segregation defect in the protozoan parasite toxoplasma gondii
The EMBO Journal, 2001Co-Authors: Michael K Shaw, Charles H Pletcher, Lewis G Tilney, David S RoosAbstract:Apicomplexan parasites—including the causative agents of malaria (Plasmodium sp.) and toxoplasmosis (Toxoplasma gondii)—harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear-encoded apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast-deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence-activated cell sorting, but die in the subsequent host cell, confirming the ‘delayed death’ phenotype previously described pharmacologically, and validating the apicoplast as essential for parasite viability.
-
nuclear encoded proteins target to the plastid in toxoplasma gondii and plasmodium falciparum
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Ross F. Waller, Alan F Cowman, Patrick J Keeling, Robert G K Donald, Emanuela Handman, Naomi Langunnasch, Gurdyal S Besra, David S RoosAbstract:A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or “apicoplast,” is indispensable, but the complete sequence of both the Plasmodium falciparum and Toxoplasma gondii apicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the apicoplast in Plasmodium and Toxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), β-ketoacyl-ACP synthase III (FabH), and β-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green fluorescent protein reporter to the apicoplast in transgenic T. gondii. Localization of ACP, and very probably FabH and FabZ, in the apicoplast implicates fatty acid biosynthesis as a likely function of the apicoplast. Moreover, inhibition of P. falciparum growth by thiolactomycin, an inhibitor of FabH, indicates a vital role for apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid/bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.
Giel G. Van Dooren - One of the best experts on this subject based on the ideXlab platform.
-
TgATS1 is critical for normal intracellular development and division.
2016Co-Authors: Souad Amiar, Giel G. Van Dooren, James I Macrae, Melanie J Shears, Malcolm J Mcconville, Damien L. Callahan, David Dubois, Marie-france Cesbron-delauw, Eric Maréchal, Geoffrey I. McfaddenAbstract:(A) Inducible knockdown of TgATS1 in the TgATS1-HA-iKO line. TgATS1 was detected by Western blot analysis using anti-HA antibody as two bands: the pre-mature form (pATS1, ~75KDa) and the mature form (mATS1, ~55KDa). Protein expression was down-regulated to undetectable levels by 3 or 4 days of ATc treatment (1 mg/mL ATc, numbers indicate days of culture). GRA1 (lower panel) served as a loading control. (B) Plaque assays performed in the absence (-) or presence (+) of ATc and fixed after 10 days show an impaired lytic cycle of TgATS1-HA-iKO parasites in the presence of ATc. (C) IFA of TgATS1-HA-iKO parasites using anti-HA antibody and apicoplast stromal markers FNR-RFP (two upper panels) and CPN60 antibody (lower panel) indicates loss of HA and apicoplast signals in the presence of ATc for 4 days (white arrows indicate normal apicoplast while red arrows indicate loss of apicoplast signal), as well as the cytosolic mis-localisation of apicoplast CPN60 (lower panel, zoomed area). (D) IFA of TgATS1-HA-iKO parasites using antibodies against the apicoplast stromal marker CPN60 and the apicoplast outer membrane marker ATRx1 confirms mis-localisation of CPN60 and ATRx1, indicating loss of apicoplast structure (lower panels, zoomed areas). White arrows indicate normal apicoplast and red arrows indicate a normal apicoplast. (E) Quantification of the number of intact Apicoplasts relative to parasites and vacuoles in TgATS1-HA-iKO parasites following ATc treatment. A significant loss of Apicoplasts was observed in the presence of ATc (upper graph) of up to 60% at days 5 and 6, compared to the wild type (parental) strain that contained 100% Apicoplasts in all vacuoles regardless of the number of parasites per vacuole (lower panel). (n = 100 vacuoles). (F) IFA of TgATS1-HA-iKO parasites using anti-IMC1 antibody grown in the presence and absence of ATc indicates IMC structure defect (zoomed areas). Scale bars: 2.5 μm.
-
13C-carbon source labelling strategies to assess the role of FASII and TgATS1 in T. gondii membrane biogenesis.
2016Co-Authors: Souad Amiar, Giel G. Van Dooren, James I Macrae, Melanie J Shears, Malcolm J Mcconville, Damien L. Callahan, David Dubois, Marie-france Cesbron-delauw, Eric Maréchal, Geoffrey I. McfaddenAbstract:TgATS1-iKO parasites were grown in the presence of 12C-glucose or U-13C-glucose in the presence or absence of ATc. Glucose is metabolized via the parasite glycolytic pathway into dihydroxyacetone-phosphate (DHAP) and phosphoenolpyruvate (PEP). These precursors are imported into the apicoplast via the apicoplast phosphate transporter (APT). PEP can then be transformed into acetyl-CoA via the action of the apicoplast pyruvate kinase (PK) and the pyruvate dehydrogenase complex (PDH). Acetyl-CoA serves as a substrate for the FASII pathway to generate growing acyl chains on an acyl carrier protein (ACP) scaffold. Glycerol 3-phosphate (G3P) can be generated from imported DHAP by the apicoplast glycerol 3-phosphate dehydrogenase (Gpda), or potentially by an as yet unidentified cytosolic G3P dehydrogenase (G3PDH). Both labelled and/or unlabelled G3P and FA can be used as a substrate by the apicoplast TgATS1 (glycerol 3-phosphate acyltransferase) to form lysophosphatidic acid (LPA). These LPA species can potentially be exported from the apicoplast towards the ER to be assembled into phospholipids (PL) and/or have its FA elongated via the three ER elongases (ELO). In order to assess ER elongation, TgATS1-iKO parasites were grown in the presence or absence of ATc together with 12C-acetate or U-13C-acetate, which is metabolized to cytosolic acetyl-CoA by acetyl-CoA synthetase (ACS). Collectively, this strategy enabled the determination of (i) apicoplast FASII-generated FA products, (ii) products assembled via TgATS1, and (iii) PL species generated via the apicoplast. Total lipid extracts from TgATS1-iKO parasites grown in the described conditions were analyzed by two mass spectrometry techniques. GC-MS was used for the measurement of total FA profiles and LC-MS/MS was used to determine the relative amounts of 13C incorporation into individual PL molecular species. In the cartoon, unlabelled and labelled moieties are shown in black and red, respectively.
-
An apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins
PLoS Pathogens, 2013Co-Authors: Swati Agrawal, Giel G. Van Dooren, Duk-won D Chung, Nadia Ponts, Jacques Prudhomme, Carrie F. Brooks, Elisadra M. Rodrigues, John C. Tan, Michael T. FerdigAbstract:Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.
-
Genetic ablation of E2Ap in T. gondii results in a block of apicoplast protein import and parasite growth.
2013Co-Authors: Swati Agrawal, Giel G. Van Dooren, Duk-won D Chung, Nadia Ponts, Jacques Prudhomme, Carrie F. Brooks, Elisadra M. Rodrigues, John C. Tan, Michael T. Ferdig, Boris StriepenAbstract:(A) The TgE2Ap locus was modified homologous recombination and insertion of a DHFR selectable marker and a regulatable promoter (arrow, black circles indicate tet operator elements). Positions of diagnostic primers (half arrows) and probe (black line) are indicated. (B) PCR test for insertion, note that only the recombined locus is suitable to template the 2.8 kbp product. (C) Southern analysis hybridizing genomic DNA form parental (ΔKu80/TATI) or mutant ((i)ΔTgE2Ap) parasites with a probe derived from the first exon of TgE2Ap. (D) Real time PCR analysis of TgE2Ap expression in (i)ΔTgE2Ap parasites upon ATc treatment (normalized to the mRNA of the major surface protein SAG1, untreated control set to 100%, error bars show SD, n = 3) detects the expected DNA fragments. PCR analysis of clones shows integration of the promoter. (E) Growth of (i)ΔTgE2Ap was measured by fluorescence (circles no drug, squares ATc, triangles 3 days of pretreatment with ATc prior to plate inoculation) and (F) plaque assay. (G) Apicoplast protein import in (i)ΔTgE2Ap was measured by following PDH(E2) lipoylation ( [6] highlighted by box). Note loss of band upon ATc treatment and persistence of mitochondrial lipoylation (Mito (E2)-LA); Please note that there are two lipoylated proteins in the T. gondii mitochondrion [42], [43], * human PDH-E2. (H) Quantification of protein import (as measured in G, squares, data shown is representative of four experiments) and number of Apicoplasts per parasite (circles, n = 3, SD shown) in (i)ΔTgE2Ap over the course of ATc treatment. (I) Western blots probing FNR-RFP in (i)ΔTgE2Ap upon ATc treatment (p, precursor; m, mature protein). Note reduction in mature band upon treatment. Immunofluorescence assays showing (i)ΔTgE2Ap in the absence (J) or presence (K) of ATc. 38% of parasite vacuoles showed labeling outside the apicoplast, likely due to back up of cargo into the ER at 48 h of treatment (
-
Tic22 Is an Essential Chaperone Required for Protein Import into the Apicoplast
The Journal of biological chemistry, 2012Co-Authors: Stephanie Glaser, Giel G. Van Dooren, Swati Agrawal, Carrie F. Brooks, Matthew K. HigginsAbstract:Most plastids proteins are post-translationally imported into organelles through multisubunit translocons. The TIC and TOC complexes perform this role in the two membranes of the plant chloroplast and in the inner two membranes of the Apicoplasts of the apicomplexan parasites, Toxoplasma gondii and Plasmodium falciparum. Tic22 is a ubiquitous intermembrane translocon component that interacts with translocating proteins. Here, we demonstrate that T. gondii Tic22 is an apicoplast-localized protein, essential for parasite survival and protein import into the apicoplast stroma. The structure of Tic22 from P. falciparum reveals a fold conserved from cyanobacteria to plants, which displays a non-polar groove on each side of the molecule. We show that these grooves allow Tic22 to act as a chaperone. General chaperones are common components of protein translocation systems where they maintain cargo proteins in an unfolded conformation during transit. Such a chaperone had not been identified in the intermembrane space of plastids and we propose that Tic22 fulfills this role.
Ross F. Waller - One of the best experts on this subject based on the ideXlab platform.
-
The apicoplast: A review of the derived plastid of apicomplexan parasites
Current Issues in Molecular Biology, 2005Co-Authors: Ross F. WallerAbstract:The apicoplast is a plastid organelle, homologous to chloroplasts of plants, that is found in apicomplexan parasites such as the causative agents of Malaria Plasmodium spp. It occurs throughout the Apicomplexa and is an ancient feature of this group acquired by the process of endosymbiosis. Like plant chloroplasts, Apicoplasts are semi-autonomous with their own genome and expression machinery. In addition, Apicoplasts import numerous proteins encoded by nuclear genes. These nuclear genes largely derive from the endosymbiont through a process of intracellular gene relocation. The exact role of a plastid in parasites is uncertain but early clues indicate synthesis of lipids, heme and isoprenoids as possibilities. The various metabolic processes of the apicoplast are potentially excellent targets for drug therapy.
-
tropical infectious diseases metabolic maps and functions of the plasmodium falciparum apicoplast
Nature Reviews Microbiology, 2004Co-Authors: Stuart A Ralph, Ross F. Waller, Christopher J. Tonkin, Bernardo J. Foth, Giel G Van Dooren, Michael J Crawford, Martin Fraunholz, David S RoosAbstract:Discovery of a relict chloroplast (the apicoplast) in malarial parasites presented new opportunities for drug development. The apicoplast – although no longer photosynthetic – is essential to parasites. Combining bioinformatics approaches with experimental validation in the laboratory, we have identified more than 500 proteins predicted to function in the apicoplast. By comparison with plant chloroplasts, we have reconstructed several anabolic pathways for the parasite plastid that are fundamentally different to the analogous pathways in the human host and are potentially good targets for drug development. Products of these pathways seem to be exported from the apicoplast and might be involved in host-cell invasion.
-
protein trafficking to the plastid of plasmodium falciparum is via the secretory pathway
The EMBO Journal, 2000Co-Authors: Ross F. Waller, Michael B Reed, Alan F CowmanAbstract:The plastid of Plasmodium falciparum (or ‘apicoplast’) is the evolutionary homolog of the plant chloroplast and represents a vestige of a photosynthetic past. Apicoplast indispensability indicates that it still provides essential functions to parasites. Similar to plant chloroplasts, the apicoplast is dependent on many nucleus-encoded genes to provide these functions. The apicoplast is surrounded by four membranes, two more than plant chloroplasts. Thus, protein targeting to the apicoplast must overcome additional membrane barriers. In P.falciparum we have analyzed apicoplast targeting using green fluorescent protein (GFP). We demonstrate that protein targeting is at least a two-step process mediated by bipartite N-terminal pre-sequences that consist of a signal peptide for entry into the secretory pathway and a plant-like transit peptide for subsequent import into the apicoplast. The P.falciparum transit peptide is exceptional compared with other known plastid transit peptides in not requiring serine or threonine residues. The pre-sequence components are removed stepwise during apicoplast targeting. Targeting GFP to the apicoplast has also provided the first opportunity to examine apicoplast morphology in live P.falciparum.
-
nuclear encoded proteins target to the plastid in toxoplasma gondii and plasmodium falciparum
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Ross F. Waller, Alan F Cowman, Patrick J Keeling, Robert G K Donald, Emanuela Handman, Naomi Langunnasch, Gurdyal S Besra, David S RoosAbstract:A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or “apicoplast,” is indispensable, but the complete sequence of both the Plasmodium falciparum and Toxoplasma gondii apicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the apicoplast in Plasmodium and Toxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), β-ketoacyl-ACP synthase III (FabH), and β-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green fluorescent protein reporter to the apicoplast in transgenic T. gondii. Localization of ACP, and very probably FabH and FabZ, in the apicoplast implicates fatty acid biosynthesis as a likely function of the apicoplast. Moreover, inhibition of P. falciparum growth by thiolactomycin, an inhibitor of FabH, indicates a vital role for apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid/bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.