A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor
TL;DR: Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle, and suggests that this peripheral plastid protein, PPP 1, is likely localized to the periplastid compartment.
Abstract: Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.
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TL;DR: In this article, the authors adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts.
548 citations
01 Sep 2016
TL;DR: The first genome-wide genetic screen of an apicomplexan parasite is presented, revealing essential functions during infection of human cells and providing broad-based functional information on T. gondii genes that will facilitate future approaches to expand the horizon of antiparasitic interventions.
Abstract: National Institute of General Medical Sciences (U.S.) (Center for Integrative Synthetic Biology Grant P50GM098792)
456 citations
Cites methods from "A Systematic Screen to Discover and..."
..., 1996) flanked by homology regions to the C terminus of each gene (P115–P148) were co-transfected with 100 μg of pU6-Universal carrying the appropriate sgRNA into TATi/ΔKU80 parasites (Sheiner et al., 2011)....
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...30 μg of a repair oligonucleotide containing an in-frame Ty epitope (Bastin et al., 1996) flanked by homology regions to the C terminus of each gene (P115–P148) were co-transfected with 100 μg of pU6-Universal carrying the appropriate sgRNA into TATi/ΔKU80 parasites (Sheiner et al., 2011)....
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TL;DR: Parasites lacking the GABA shunt exhibit attenuated growth and are unable to sustain motility under nutrient-limited conditions, suggesting that GABA functions as a short-term energy reserve.
195 citations
Cites methods from "A Systematic Screen to Discover and..."
...The growth of parental and Dgad lines (Huynh and Carruthers, 2009), expressing a tandem red fluorescent protein (RFP-RFP) transgene, was measured using a real-time fluorescence assay (Sheiner et al., 2011)....
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TL;DR: This work determines the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.
158 citations
Cites background from "A Systematic Screen to Discover and..."
...Some, but Not All, Compartments Show Tight Transcriptional Regulatory Control In Toxoplasma, previous efforts to identify candidate proteins for select compartments have used the correlation of transcriptabundance profiles across the cell cycle assuming that colocated proteins are co-expressed (Bai et al., 2018; Lacombe et al., 2019; Long et al., 2017; Sheiner et al., 2011)....
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TL;DR: The novel term ‘T. gondii–rat manipulation–schizophrenia model’ is introduced and it is proposed how future behavioural research on this model should be performed from a biological, clinical and ethically appropriate perspective.
Abstract: Summary We examine the role of the protozoan Toxoplasma gondii as a manipulatory parasite and question what role study of infections in its natural intermediate rodent hosts and other secondary hosts, including humans, may elucidate in terms of the epidemiology, evolution and clinical applications of infection. In particular, we focus on the potential association between T. gondii and schizophrenia. We introduce the novel term ‘ T. gondii –rat manipulation–schizophrenia model’ and propose how future behavioural research on this model should be performed from a biological, clinical and ethically appropriate perspective.
149 citations
Cites background from "A Systematic Screen to Discover and..."
...Whilst no longer photosynthetic, the apicoplast, a red algal endosymbiont, is a centre of metabolic activity harbouring several major anabolic pathways, and these studies have revealed new apicoplast proteins, currently with no assigned function (Sheiner et al., 2011) and which, hypothetically at least, could perhaps be relevant to behavioural alterations....
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...…endosymbiont, is a centre of metabolic activity harbouring several major anabolic pathways, and these studies have revealed new apicoplast proteins, currently with no assigned function (Sheiner et al., 2011) and which, hypothetically at least, could perhaps be relevant to behavioural alterations....
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...Within Type I, for instance, new conditional mutant RH lines have recently been constructed via promoter replacement strategies that target genes encoding proteins that are conserved and unique to these apicomplexan parasites (Sheiner et al., 2011)....
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References
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TL;DR: Analysis of the complete asexual intraerythrocytic developmental cycle (IDC) transcriptome of the HB3 strain of P. falciparum demonstrates that this parasite has evolved an extremely specialized mode of transcriptional regulation that produces a continuous cascade of gene expression, beginning with genes corresponding to general cellular processes, such as protein synthesis, and ending with Plasmodium-specific functionalities.
Abstract: Plasmodium falciparum is the causative agent of the most burdensome form of human malaria, affecting 200–300 million individuals per year worldwide. The recently sequenced genome of P. falciparum revealed over 5,400 genes, of which 60% encode proteins of unknown function. Insights into the biochemical function and regulation of these genes will provide the foundation for future drug and vaccine development efforts toward eradication of this disease. By analyzing the complete asexual intraerythrocytic developmental cycle (IDC) transcriptome of the HB3 strain of P. falciparum, we demonstrate that at least 60% of the genome is transcriptionally active during this stage. Our data demonstrate that this parasite has evolved an extremely specialized mode of transcriptional regulation that produces a continuous cascade of gene expression, beginning with genes corresponding to general cellular processes, such as protein synthesis, and ending with Plasmodium-specific functionalities, such as genes involved in erythrocyte invasion. The data reveal that genes contiguous along the chromosomes are rarely coregulated, while transcription from the plastid genome is highly coregulated and likely polycistronic. Comparative genomic hybridization between HB3 and the reference genome strain (3D7) was used to distinguish between genes not expressed during the IDC and genes not detected because of possible sequence variations. Genomic differences between these strains were found almost exclusively in the highly antigenic subtelomeric regions of chromosomes. The simple cascade of gene regulation that directs the asexual development of P. falciparum is unprecedented in eukaryotic biology. The transcriptome of the IDC resembles a “just-in-time” manufacturing process whereby induction of any given gene occurs once per cycle and only at a time when it is required. These data provide to our knowledge the first comprehensive view of the timing of transcription throughout the intraerythrocytic development of P. falciparum and provide a resource for the identification of new chemotherapeutic and vaccine candidates.
1,598 citations
"A Systematic Screen to Discover and..." refers background in this paper
...Similar time-ordered relationships leading to shared transcription patterns had previously been observed for organellar biogenesis during replication of Plasmodium merozoites in the red blood cell [34]....
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TL;DR: A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria and the presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprene biosynthesis in P. falcIParum depends on the DOXP pathway.
Abstract: A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.
1,136 citations
TL;DR: In this paper, 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) were identified.
Abstract: 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.
699 citations
01 Jan 1998
TL;DR: This work has identified nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), beta-ketoacyl-ACP synthase III (FabH), and beta-hydroxyacyL-ACP dehydratase (FabZ) that are potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.
Abstract: 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 Plasmo- dium 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), b-ketoacyl-ACP synthase III (FabH), and b-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 pre- sequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green f luorescent protein reporter to the apicoplast in trans- genic 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 plastidybacterial type, and distinct from those of the equiv- alent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases. The phylum Apicomplexa is a group of obligate endoparasites that includes Plasmodium spp. (the causative agents of malar- ia), Toxoplasma gondii (an important opportunistic pathogen associated with AIDS and congenital birth defects), and several other parasites of medical and economic significance (Cryptosporidium, Eimeria, Babesia, and Theileria). It has been
672 citations
"A Systematic Screen to Discover and..." refers background in this paper
...For most stromal apicoplast proteins, trafficking depends on a bipartite leader at their N terminus [21,22]....
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...Like many apicoplast proteins, ACP is synthesized with a bipartite Nterminal signal, which is cleaved upon reaching the organelle lumen, and this modification depends on import of the protein into the lumen of the organelle [13,21,32]....
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TL;DR: It is argued that dinoflagellate and sporozoan plastids are directly related and that the common ancestor of dinof lagellates and Sporozoa was photosynthetic, and a novel theory for the function of periplastid vesicles in membrane biogenesis of chlorarachneans and chromists is proposed.
Abstract: The biggest unsolved problems in chloroplast evolution are the origins of dinoflagellate and euglenoid chloroplasts,which have envelopes of three membranes not two like plants and chromists, and of the sporozoan plastid, bounded by four smooth membranes. I review evidence that all three of these protozoan plastid types originated by secondary symbiogenesis from eukaryotic symbionts. Instead of separate symbiogenetic events, I argue that dinoflagellate and sporozoan plastids are directly related and that the common ancestor of dinoflagellates and Sporozoa was photosynthetic. I suggest that the last common ancestor of all Alveolata was photosynthetic and acquired its chlorophyll c-containing plastids in the same endosymbiogenetic event as those of Chromista. Chromistaand Alveolata are postulated to be a clade designated chrornalveolates. I propose that euglenoids obtained their plastids from the same(possibly ulvophycean) green alga as chlorarachneans and that Discicristata (Euglenozoa plus Percolozoa) and Cercozoa (the group including chlorarachneans) form a clade designated cabozoa (protozoa with chlorophyll a + b). If both theories are correct, there were only two secondary symbiogenetic events (witnessed by the chlorarachnean and cryptomonad nucleormorphs) in the history of life, not seven as commonly assumed. This greatly reduces the postulated number of independent origins of chloroplast protein-targeting machinery and of gene transfers from endosymbiont to host nuclei. I discuss the membrane and plastid losses and innovations in protein targeting implied by these theories, the comparative evidence for them, and their implications for eukaryote megaphylogeny. The principle of evolutionary conservatism leads to a novel theory for the function of periplastid vesicles in membrane biogenesis ofchlorarachneans and chromists and of the key steps in secondary symbiogenesis. Protozoan classification is also slightly revised by abandoning the probably polyphyletic infrakingdom Actinopoda, grouping Foraminifera and Radiolaria as a new infrakingdom Retaria,placing Heliozoa within a revised infrakingdom Sarcomastigota, establishing a new flagellate phylum Loukozoa for Jakobea plus Anaeromonadea within an emended subkingdom Eozoa, and ranking Archezoa as an infrakingdom within Eozoa.
599 citations
"A Systematic Screen to Discover and..." refers background in this paper
...The chromealveolates span all the way from singlecelled predators and parasites to complex multicellular organisms like the large kelps [11]....
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