Highlights d K13 artemisinin resistance-conferring mutations lead to reduced K13 abundance d K13 is localized to cytostome-like structures at the parasite periphery d Reduced K13 abundance impairs hemoglobin catabolism and lessens artemisinin activation d Reduced artemisinin activation limits cell damage, permitting parasite survival
Outside of well characterized model eukaryotes, relatively little is known about the translocons that transport proteins across the two membranes that surround the mitochondrion. Apicomplexans are a phylum of intracellular parasites that cause major diseases in humans and animals and are evolutionarily distant from model eukaryotes such as yeast. Apicomplexans harbor a mitochondrion that is essential for parasite survival and is a validated drug target. Here, we demonstrate that the apicomplexan Toxoplasma gondii harbors homologues of proteins from all the major mitochondrial protein translocons present in yeast, suggesting these arose early in eukaryotic evolution. We demonstrate that a T. gondii homologue of Tom22 (TgTom22), a central component of the translocon of the outer mitochondrial membrane (TOM) complex, is essential for parasite survival, mitochondrial protein import, and assembly of the TOM complex. We also identify and characterize a T. gondii homologue of Tom7 (TgTom7) that is important for parasite survival and mitochondrial protein import. Contrary to the role of Tom7 in yeast, TgTom7 is important for TOM complex stability, suggesting the role of this protein has diverged during eukaryotic evolution. Together, our study identifies conserved and modified features of mitochondrial protein import in apicomplexan parasites.
BackgroundThe clinical symptoms of malaria are caused by the asexual replication of Plasmodium parasites in the blood of the vertebrate host. To spread to new hosts, however, the malaria parasite must differentiate into sexual forms, termed gametocytes, which are ingested by a mosquito vector. Sexual differentiation produces either female or male gametocytes, and involves significant morphological and biochemical changes. These transformations prepare gametocytes for the rapid progression to gamete formation and fertilisation, which occur within 20 min of ingestion. Here we present the transcriptomes of asexual, female, and male gametocytes in P. berghei, and a comprehensive statistically-based differential-expression analysis of the transcriptional changes that underpin this sexual differentiation.ResultsRNA-seq analysis revealed numerous differences in the transcriptomes of female and male gametocytes compared to asexual stages. Overall, there is net downregulation of transcripts in gametocytes compared to asexual stages, with this trend more marked in female gametocytes. Our analysis identified transcriptional changes in previously-characterised gametocyte-specific pathways, which validated our approach. We also detected many previously-unreported female- and male-specific pathways and genes. Transcriptional biases in stage and gender were then used to investigate sex-specificity and sexual dimorphism of Plasmodium in an evolutionary context. Sex-related gene expression is well conserved between Plasmodium species, but relatively poorly conserved in related organisms outside this genus. This pattern of conservation is most evident in genes necessary for both male and female gametocyte formation. However, this trend is less pronounced for male-specific genes, which are more highly conserved outside the genus than genes specific to female development.ConclusionsWe characterised the transcriptional changes that are integral to the development of the female and male sexual forms of Plasmodium. These differential-expression patterns provide a vital insight into understanding the gender-specific characteristics of this essential stage that is the primary target for treatments that block parasite transmission. Our results also offer insight into the evolution of sex genes through Alveolata, and suggest that many Plasmodium sex genes evolved within the genus. We further hypothesise that male gametocytes co-opted pre-existing cellular machinery in their evolutionary history, whereas female gametocytes evolved more through the development of novel, parasite-specific pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-4100-0) contains supplementary material, which is available to authorized users.
Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes.
Plasmodium parasites possess two endosymbiotic organelles: a mitochondrion and a relict plastid called the apicoplast. To accommodate the translational requirements of these organelles in addition to its cytosolic translation apparatus, the parasite must maintain a supply of charged tRNA molecules in each of these compartments. In the present study we investigate how the parasite manages these translational requirements for charged tRNACys with only a single gene for CysRS (cysteinyl-tRNA synthetase). We demonstrate that the single PfCysRS (Plasmodium falciparum CysRS) transcript is alternatively spliced, and, using a combination of endogenous and heterologous tagging experiments in both P. falciparum and Toxoplasma gondii, we show that CysRS isoforms traffic to the cytosol and apicoplast. PfCysRS can recognize and charge the eukaryotic tRNACys encoded by the Plasmodium nucleus as well as the bacterial-type tRNA encoded by the apicoplast genome, albeit with a preference for the eukaryotic type cytosolic tRNA. The results of the present study indicate that apicomplexan parasites have lost their original plastidic cysteinyl-tRNA synthetase, and have replaced it with a dual-targeted eukaryotic type CysRS that recognizes plastid and nuclear tRNACys. Inhibitors of the Plasmodium dual-targeted CysRS would potentially offer a therapy capable of the desirable immediate effects on parasite growth as well as the irreversibility of inhibitors that disrupt apicoplast inheritance.
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