Project description:Malaria and related apicomplexan parasites have two highly conserved organellar genomes: one is of plastid (pl) origin, and the other is mitochondrial (mt). The organization of both organellar DNA molecules from the human malaria parasite Plasmodium falciparum has been determined, and they have been shown to be tightly packed with genes. The 35-kb circular DNA is the smallest known vestigial plastid genome and is presumed to be functional. All but two of its recognized genes are involved with genetic expression: one of the two encodes a member of the clp family of molecular chaperones, and the other encodes a conserved protein of unknown function found both in algal plastids and in eubacterial genomes. The possible evolutionary source and intracellular location of the plDNA are discussed. The 6-kb tandemly repeated mt genome is the smallest known and codes for only three proteins (cytochrome b and two subunits of cytochrome oxidase) as well as two bizarrely fragmented rRNAs. The organization of the mt genome differs somewhat among genera. The mtDNA sequence provides information not otherwise available about the structure of apicomplexan cytochrome b as well as the unusually fragmented rRNAs. The malarial mtDNA has a phage-like replication mechanism and undergoes extensive recombination like the mtDNA of some other lower eukaryotes.
Project description:Messenger RNA polyadenylation is a universal aspect of gene expression in eukaryotes. In well-established model organisms, this process is mediated by a conserved complex of 15-20 subunits. To better understand this process in apicomplexans, a group of unicellular parasites that causes serious disease in humans and livestock, a computational and high throughput sequencing study of the polyadenylation complex and poly(A) sites in several species was conducted. BLAST-based searches for orthologs of the human polyadenylation complex yielded clear matches to only two-poly(A) polymerase and CPSF73-of the 19 proteins used as queries in this analysis. As the human subunits that recognize the AAUAAA polyadenylation signal (PAS) were not immediately obvious, a computational analysis of sequences adjacent to experimentally-determined apicomplexan poly(A) sites was conducted. The results of this study showed that there exists in apicomplexans an A-rich region that corresponds in position to the AAUAAA PAS. The set of experimentally-determined sites in one species, Sarcocystis neurona, was further analyzed to evaluate the extent and significance of alternative poly(A) site choice in this organism. The results showed that almost 80% of S. neurona genes possess more than one poly(A) site, and that more than 780 sites showed differential usage in the two developmental stages-extracellular merozoites and intracellular schizonts-studied. These sites affected more than 450 genes, and included a disproportionate number of genes that encode membrane transporters and ribosomal proteins. Taken together, these results reveal that apicomplexan species seem to possess a poly(A) signal analogous to AAUAAA even though genes that may encode obvious counterparts of the AAUAAA-recognizing proteins are absent in these organisms. They also indicate that, as is the case in other eukaryotes, alternative polyadenylation is a widespread phenomenon in S. neurona that has the potential to impact growth and development.
Project description:Parasites of the genus Eimeria infect a wide range of vertebrate hosts, including chickens. We have recently reported a comparative analysis of the transcriptomes of Eimeria acervulina, Eimeria maxima and Eimeria tenella, integrating ORESTES data produced by our group and publicly available Expressed Sequence Tags (ESTs). All cDNA reads have been assembled, and the reconstructed transcripts have been submitted to a comprehensive functional annotation pipeline. Additional studies included orthology assignment across apicomplexan parasites and clustering analyses of gene expression profiles among different developmental stages of the parasites. To make all this body of information publicly available, we constructed the Eimeria Transcript Database (EimeriaTDB), a web repository that provides access to sequence data, annotation and comparative analyses. Here, we describe the web interface, available sequence data sets and query tools implemented on the site. The main goal of this work is to offer a public repository of sequence and functional annotation data of reconstructed transcripts of parasites of the genus Eimeria. We believe that EimeriaTDB will represent a valuable and complementary resource for the Eimeria scientific community and for those researchers interested in comparative genomics of apicomplexan parasites. Database URL: http://www.coccidia.icb.usp.br/eimeriatdb/
Project description:BACKGROUND: The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. RESULTS: We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the ?C helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. CONCLUSIONS: We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these lineage-specific variations and propose specific hypotheses for experimental investigation. The apicomplexan-specific kinase features reported in this study can be used in the design of selective kinase inhibitors.
Project description:Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs), factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for Guillardiatheta and Bigelowiellanatans. The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate Perkinsus marinus. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during, and possibly pre-dating, the move from free-living marine algae to deadly human parasites.
Project description:Emerging lipidomic technologies have enabled researchers to dissect the complex roles of phospholipases in lipid metabolism, cellular signaling and immune regulation. Host phospholipase products are involved in stimulating and resolving the inflammatory response to pathogens. While many pathogen-derived phospholipases also manipulate the immune response, they have recently been shown to be involved in lipid remodeling and scavenging during replication. Animal and plant hosts as well as many pathogens contain a family of patatin-like phospholipases, which have been shown to have phospholipase A2 activity. Proteins containing patatin-like phospholipase domains have been identified in protozoan parasites within the Apicomplexa phylum. These parasites are the causative agents of some of the most widespread human diseases. Malaria, caused by Plasmodium spp., kills nearly half a million people worldwide each year. Toxoplasma and Cryptosporidium infect millions of people each year with lethal consequences in immunocompromised populations. Parasite-derived patatin-like phospholipases are likely effective drug targets and progress in the tools available to the Apicomplexan field will allow for a closer look at the interplay of lipid metabolism and immune regulation during host infection.
Project description:The peroxisome was the last organelle to be discovered and five decades later it is still the Cinderella of eukaryotic compartments. Peroxisomes have a crucial role in the detoxification of reactive oxygen species, the beta-oxidation of fatty acids, and the biosynthesis of etherphospholipids, and they are assumed to be present in virtually all aerobic eukaryotes. Apicomplexan parasites including the malaria and toxoplasmosis agents were described as the first group of mitochondriate protists devoid of peroxisomes. This study was initiated to reassess the distribution and evolution of peroxisomes in the superensemble Alveolata (apicomplexans, dinoflagellates, ciliates). We established transcriptome data from two chromerid algae (Chromera velia, Vitrella brassicaformis), and two dinoflagellates (Prorocentrum minimum, Perkinsus olseni) and identified the complete set of essential peroxins in all four reference species. Our comparative genome analysis provides unequivocal evidence for the presence of peroxisomes in Toxoplasma gondii and related genera. Our working hypothesis of a common peroxisomal origin of all alveolates is supported by phylogenetic analyses of essential markers such as the import receptor Pex5. Vitrella harbors the most comprehensive set of peroxisomal proteins including the catalase and the glyoxylate cycle and it is thus a promising model organism to investigate the functional role of this organelle in Apicomplexa.
Project description:BACKGROUND:Chicken coccidiosis, caused by the infection of Eimeria species, leads to important economic losses to the poultry industry. Vaccination with attenuated live parasites seems to be the best way to control this disease. Attenuated eimerian parasites with shortened prepatent times show great changes in intracellular development compared to their parent strains but the mechanisms involved in these biological differences are still unclear. RESULTS:In this study, we obtained a precocious line of E. maxima by sequential selection of 22 generations of early shed oocysts in chickens and performed a comparative transcriptome analysis of three different developmental stages of the precocious line and its parent strain using Illumina high-throughput sequencing. Our E. maxima precocious line showed decreased pathogenicity, reduced fecundity and a greatly shorted prepatent time of only 98 h. We found that typical gene changes in the stage development from unsporulated to sporulated oocyst and from sporulated oocyst to merozoite were marked by upregulated organelle genes and protein translation related genes, respectively. Additionally, major differences between the precocious line and its parent strain were detected in the merozoite stage, characterized by downregulated genes involved in protein cleavage and DNA replication activities. CONCLUSIONS:Our study generated and characterized an E. maxima precocious line, illustrating gene expression landscapes during parasite development by transcriptome analysis. We also show that the suppressed DNA replication progress in the merozoite stage in the precocious line may result in its reduced fecundity. These results provide the basis for a better understanding of the mechanism of precocity in Eimeria species, which can be useful in studies in early gametocytogenesis in apicomplexan parasites.