ABSTRACT
Twenty years ago, the first transcriptome of the intraerythrocytic developmental cycle of the malaria parasite Plasmodium falciparum was published in PLOS Biology. Since then, transcriptomics studies have transformed the study of parasite biology.
Subject(s)
Parasites , Plasmodium falciparum , Animals , Plasmodium falciparum/genetics , Transcriptome/genetics , Parasites/genetics , Gene Expression Profiling , BiologyABSTRACT
Babesia is a genus of apicomplexan parasites that infect red blood cells in vertebrate hosts. Pathology occurs during rapid replication cycles in the asexual blood stage of infection. Current knowledge of Babesia replication cycle progression and regulation is limited and relies mostly on comparative studies with related parasites. Due to limitations in synchronizing Babesia parasites, fine-scale time-course transcriptomic resources are not readily available. Single-cell transcriptomics provides a powerful unbiased alternative for profiling asynchronous cell populations. Here, we applied single-cell RNA sequencing to 3 Babesia species (B. divergens, B. bovis, and B. bigemina). We used analytical approaches and algorithms to map the replication cycle and construct pseudo-synchronized time-course gene expression profiles. We identify clusters of co-expressed genes showing "just-in-time" expression profiles, with gradually cascading peaks throughout asexual development. Moreover, clustering analysis of reconstructed gene curves reveals coordinated timing of peak expression in epigenetic markers and transcription factors. Using a regularized Gaussian graphical model, we reconstructed co-expression networks and identified conserved and species-specific nodes. Motif analysis of a co-expression interactome of AP2 transcription factors identified specific motifs previously reported to play a role in DNA replication in Plasmodium species. Finally, we present an interactive web application to visualize and interactively explore the datasets.
Subject(s)
Babesia , Babesia/genetics , Erythrocytes/parasitology , Transcription Factors/genetics , Transcriptome/geneticsABSTRACT
The role of specific host cell surface receptors during Toxoplasma gondii invasion of host cells is poorly defined. Here, we interrogated the role of the well-known malarial invasion receptor, basigin, in T. gondii infection of astrocytes. We found that primary astrocytes express two members of the BASIGIN (BSG) immunoglobulin family, basigin and embigin, but did not express neuroplastin. Antibody blockade of either basigin or embigin caused a significant reduction of parasite infectivity in astrocytes. The specific role of basigin during T. gondii invasion was further examined using a mouse astrocytic cell line (C8-D30), which exclusively expresses basigin. CRISPR-mediated deletion of basigin in C8-D30 cells resulted in decreased T. gondii infectivity. T. gondii replication and invasion efficiency were not altered by basigin deficiency, but parasite attachment to astrocytes was markedly reduced. We also conducted a proteomic screen to identify T. gondii proteins that interact with basigin. Toxoplasma-encoded cyclophilins, the protein 14-3-3, and protein disulfide isomerase (TgPDI) were among the putative basigin-ligands identified. Recombinant TgPDI produced in E. coli bound to basigin and pretreatment of tachyzoites with a PDI inhibitor decreased parasite attachment to host cells. Finally, mutagenesis of the active site cysteines of TgPDI abolished enzyme binding to basigin. Thus, basigin and its related immunoglobulin family members may represent host receptors that mediate attachment of T. gondii to diverse cell types.
Subject(s)
Toxoplasma , Toxoplasmosis , Basigin , Escherichia coli , Humans , ProteomicsABSTRACT
The apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2, and an apical annuli methyltransferase. Moreover, inner membrane complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy identified a concentric organisation comprising four rings with diameters ranging from 200 to 400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggests that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed that the AAPs are conserved narrowly in coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signalling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signalling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.
Subject(s)
Cytoskeleton/chemistry , Phylogeny , Protozoan Proteins/chemistry , Signal Transduction , Toxoplasma/chemistry , Toxoplasma/cytology , Animals , Methyltransferases/chemistry , Methyltransferases/genetics , Mice, Inbred C57BL , Microscopy , Protein Domains , Protein Interaction Maps , Protozoan Proteins/geneticsABSTRACT
Toxoplasmosis is the clinical and pathological consequence of acute infection with the obligate intracellular apicomplexan parasite Toxoplasma gondii. Symptoms result from tissue destruction that accompanies lytic parasite growth. This review updates current understanding of the host cell invasion, parasite replication, and eventual egress that constitute the lytic cycle, as well as the ways T. gondii manipulates host cells to ensure its survival. Since the publication of a previous iteration of this review 15 years ago, important advances have been made in our molecular understanding of parasite growth and mechanisms of host cell egress, and knowledge of the parasite's manipulation of the host has rapidly progressed. Here we cover molecular advances and current conceptual frameworks that include each of these topics, with an eye to what may be known 15 years from now.
Subject(s)
Toxoplasma/growth & development , Toxoplasmosis/parasitology , Animals , Host-Parasite Interactions , Humans , Protozoan Proteins/metabolism , Toxoplasma/cytology , Toxoplasmosis/immunology , Toxoplasmosis/pathology , Vacuoles/parasitologyABSTRACT
The phylum Apicomplexa encompasses deadly pathogens such as malaria and Cryptosporidium. Apicomplexa cell division is mechanistically divergent from that of their mammalian host, potentially representing an attractive source of drug targets. Depending on the species, apicomplexan parasites can modulate the output of cell division, producing two to thousands of daughter cells at once. The inherent flexibility of their cell division mechanisms allows these parasites to adapt to different niches, facilitating their dissemination. Toxoplasma gondii tachyzoites divide using a unique form of cell division called endodyogeny. This process involves a single round of DNA replication, closed nuclear mitosis, and assembly of two daughter cells within a mother. In higher Eukaryotes, the four-subunit chromosomal passenger complex (CPC) (Aurora kinase B (ARKB)/INCENP/Borealin/Survivin) promotes chromosome bi-orientation by detaching incorrect kinetochore-microtubule attachments, playing an essential role in controlling cell division fidelity. Herein, we report the characterization of the Toxoplasma CPC (Aurora kinase 1 (Ark1)/INCENP1/INCENP2). We show that the CPC exhibits dynamic localization in a cell cycle-dependent manner. TgArk1 interacts with both TgINCENPs, with TgINCENP2 being essential for its translocation to the nucleus. While TgINCENP1 appears to be dispensable, interfering with TgArk1 or TgINCENP2 results in pronounced division and growth defects. Significant anti-cancer drug development efforts have focused on targeting human ARKB. Parasite treatment with low doses of hesperadin, a known inhibitor of human ARKB at higher concentrations, phenocopies the TgArk1 and TgINCENP2 mutants. Overall, our study provides new insights into the mechanisms underpinning cell cycle control in Apicomplexa, and highlights TgArk1 as potential drug target.
Subject(s)
Chromosome Segregation , Chromosomes/genetics , Spindle Apparatus/metabolism , Toxoplasma/genetics , Animals , Aurora Kinase A/genetics , Aurora Kinase A/metabolism , Cell Cycle Checkpoints/genetics , Chromosomes/metabolism , DNA Replication/genetics , Gene Expression , Host-Parasite Interactions , Humans , Microscopy, Electron, Transmission , Mitosis/genetics , Toxoplasma/physiology , Toxoplasma/ultrastructure , Toxoplasmosis/parasitologyABSTRACT
When Toxoplasma gondii egresses from the host cell, glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1), which is primary a glycolysis enzyme but actually a quintessential multifunctional protein, translocates to the unique cortical membrane skeleton. Here, we report the 2.25 Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex providing the basis for the molecular dissection of GAPDH1 structure-function relationships Knockdown of GAPDH1 expression and catalytic site disruption validate the essentiality of GAPDH1 in intracellular replication but we confirmed that glycolysis is not strictly essential. We identify, for the first time, S-loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S-loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by the N-terminus, likely palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post-translational modifications.
Subject(s)
Apoptosis/physiology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Glycolysis/physiology , Protein Processing, Post-Translational , Protozoan Proteins/metabolism , Toxoplasma/enzymology , Toxoplasma/metabolism , Allosteric Regulation , Amino Acid Sequence , Crystallography, X-Ray , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Lipoylation , Phosphorylation , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Transport/physiology , Protozoan Proteins/genetics , Structure-Activity Relationship , rab2 GTP-Binding Protein/metabolismABSTRACT
The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon infection, parasitized dendritic cells (DCs) and microglia exhibit a hypermigratory phenotype in vitro that has been associated with enhancing parasite dissemination in vivo in mice. One unresolved question is how parasites commandeer parasitized cells to achieve systemic dissemination by a 'Trojan-horse' mechanism. By chromatography and mass spectrometry analyses, we identified an orthologue of the 14-3-3 protein family, T. gondii 14-3-3 (Tg14-3-3), as mediator of DC hypermotility. We demonstrate that parasite-derived polypeptide fractions enriched for Tg14-3-3 or recombinant Tg14-3-3 are sufficient to induce the hypermotile phenotype when introduced by protein transfection into murine DCs, human DCs or microglia. Further, gene transfer of Tg14-3-3 by lentiviral transduction induced hypermotility in primary human DCs. In parasites expressing Tg14-3-3 in a ligand-regulatable fashion, overexpression of Tg14-3-3 was correlated with induction of hypermotility in parasitized DCs. Localization studies in infected DCs identified Tg14-3-3 within the parasitophorous vacuolar space and a rapid recruitment of host cell 14-3-3 to the parasitophorous vacuole membrane. The present work identifies a determinant role for Tg14-3-3 in the induction of the migratory activation of immune cells by T. gondii. Collectively, the findings reveal Tg14-3-3 as a novel target for an intracellular pathogen that acts by hijacking the host cell's migratory properties to disseminate.
Subject(s)
14-3-3 Proteins/physiology , Dendritic Cells/physiology , Protozoan Proteins/physiology , Toxoplasma/physiology , Animals , Cell Movement , Cells, Cultured , Dendritic Cells/parasitology , Host-Parasite Interactions , Humans , Mice, Inbred C57BL , Vacuoles/metabolism , Vacuoles/parasitologyABSTRACT
Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target.
Subject(s)
Aurora Kinases/physiology , Protozoan Proteins/physiology , Toxoplasma/enzymology , Toxoplasmosis/parasitology , Animals , Female , Host-Parasite Interactions , Mice , Protein Transport , Toxoplasma/physiology , Toxoplasma/ultrastructure , VirulenceABSTRACT
Apicomplexan parasites replicate by several budding mechanisms with two well-characterized examples being Toxoplasma endodyogeny and Plasmodium schizogony. Completion of budding requires the tapering of the nascent daughter buds toward the basal end, driven by contraction of the basal complex. This contraction is not executed by any of the known cell division associated contractile mechanisms and in order to reveal new components of the unusual basal complex we performed a yeast two-hybrid screen with its major scaffolding protein, TgMORN1. Here we report on a conserved protein with a haloacid dehalogenase (HAD) phosphatase domain, hereafter named HAD2a, identified by yeast two-hybrid. HAD2a has demonstrated enzyme-activity in vitro, localizes to the nascent daughter buds, and co-localizes with MORN1 to the basal complex during its contraction. Conditional knockout of HAD2a in Toxoplasma interferes with basal complex assembly, which leads to incomplete cytokinesis and conjoined daughters that ultimately results in disrupted proliferation. In Plasmodium, we further confirmed localization of the HAD2a ortholog to the basal complex toward the end of schizogony. In conclusion, our work highlights an essential role for this HAD phosphatase across apicomplexan budding and suggests a regulatory mechanism of differential phosphorylation on the structure and/or contractile function of the basal complex.
Subject(s)
Hydrolases/chemistry , Phosphoric Monoester Hydrolases/chemistry , Protozoan Proteins/chemistry , Toxoplasma/enzymology , Amino Acid Sequence , Cytokinesis , Cytoskeleton/enzymology , Genes, Essential , Hydrolases/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Transport , Protozoan Proteins/metabolism , Two-Hybrid System TechniquesABSTRACT
The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite Toxoplasma gondii at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the Toxoplasma MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following Toxoplasma infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.
Subject(s)
Activin Receptors, Type I/antagonists & inhibitors , Hypoxia-Inducible Factor 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Toxoplasma/growth & development , Animals , Base Sequence , Benzodioxoles/pharmacology , Catalytic Domain/drug effects , Catalytic Domain/genetics , DNA, Protozoan/genetics , Drug Resistance/genetics , Genome, Protozoan/genetics , Host-Parasite Interactions/genetics , Hypoxia-Inducible Factor 1/genetics , Imidazoles/pharmacology , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 1/genetics , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Pyridines/pharmacology , Sequence Analysis, DNA , Signal Transduction/drug effects , Signal Transduction/genetics , Toxoplasma/geneticsABSTRACT
The pathology and severity of toxoplasmosis results from the rapid replication cycle of the apicomplexan parasite Toxoplasma gondii. The tachyzoites divide asexually through endodyogeny, wherein two daughter cells bud inside the mother cell. Before mitosis is completed, the daughter buds form around the duplicated centrosomes and subsequently elongate to serve as the scaffold for organellogenesis and organelle partitioning. The molecular control mechanism of this process is poorly understood. Here, we characterized a T. gondii NIMA-related kinase (Nek) ortholog that was identified in a chemical mutagenesis screen. A temperature-sensitive mutant, V-A15, possesses a Cys316Arg mutation in TgNek1 (a novel mutant allele in Neks), which is responsible for growth defects at the restrictive temperature. Phenotypic analysis of V-A15 indicated that TgNek1 is essential for centrosome splitting, proper formation of daughter cells and faithful segregation of genetic material. In vitro kinase assays showed that the mutation abolishes the kinase activity of TgNek1. TgNek1 is recruited to the centrosome prior to its duplication and localizes on the duplicated centrosomes facing the spindle poles in a cell-cycle-dependent manner. Mutational analysis of the activation loop suggests that localization and activity are spatio-temporally regulated by differential phosphorylation. Collectively, our results identified a novel temperature-sensitive allele for a Nek kinase and highlight its essential function in centrosome splitting in Toxoplasma. Moreover, these results conclusively show for the first time that Toxoplasma bud assembly is facilitated by the centrosome because defective centrosome splitting results in single daughter cell budding.
Subject(s)
Cell Cycle Proteins/genetics , Centrosome/physiology , Protein Serine-Threonine Kinases/genetics , Toxoplasma/ultrastructure , Cell Cycle Proteins/metabolism , DNA Replication , NIMA-Related Kinase 1 , Protein Serine-Threonine Kinases/metabolism , Toxoplasma/genetics , Toxoplasma/growth & development , Toxoplasma/metabolism , Toxoplasmosis/parasitologyABSTRACT
The kinetochore is a multi-protein structure assembled on eukaryotic centromeres mediating chromosome attachment to spindle microtubules. Here we identified the kinetochore proteins Nuf2 and Ndc80 in the apicomplexan parasite Toxoplasma gondii. Localization revealed that kinetochores remain clustered throughout the cell cycle and colocalize with clustered centromeres at the centrocone, a structure containing the spindle pole embedded in the nuclear envelope. Pharmacological disruption of microtubules resulted in partial loss of some kinetochore and centromere clustering, indicating microtubules are necessary but not strictly required for kinetochore clustering. Generation of a TgNuf2 conditional knock-down strain revealed it is essential for chromosome segregation, but dispensable for centromere clustering. The centromeres actually remained associated with the centrocone suggesting microtubule binding is not required for their interaction with the spindle pole. The most striking observation upon TgNuf2 depletion was that the centrosome behaved normally, but that it lost its association with the centrocone. This suggests that microtubules are essential to maintain contact between the centrosome and chromosomes, and this interaction is critical for the partitioning of the nuclei into the two daughter parasites. Finally, genetic complementation experiments with mutated TgNuf2 constructs highlighted an apicomplexan-specific motif with a putative role in nuclear localization.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Division , Centrosome/metabolism , Kinetochores/metabolism , Nuclear Proteins/metabolism , Spindle Poles/metabolism , Toxoplasma/physiology , Cell Cycle Proteins/genetics , Chromosome Segregation , Gene Knockdown Techniques , Genetic Complementation Test , Nuclear Proteins/geneticsABSTRACT
Apicomplexan parasites express various calcium-dependent protein kinases (CDPKs), and some of them play essential roles in invasion and egress. Five of the six CDPKs conserved in most Apicomplexa have been studied at the molecular and cellular levels in Plasmodium species and/or in Toxoplasma gondii parasites, but the function of CDPK7 was so far uncharacterized. In T. gondii, during intracellular replication, two parasites are formed within a mother cell through a unique process called endodyogeny. Here we demonstrate that the knock-down of CDPK7 protein in T. gondii results in pronounced defects in parasite division and a major growth deficiency, while it is dispensable for motility, egress and microneme exocytosis. In cdpk7-depleted parasites, the overall DNA content was not impaired, but the polarity of daughter cells budding and the fate of several subcellular structures or proteins involved in cell division were affected, such as the centrosomes and the kinetochore. Overall, our data suggest that CDPK7 is crucial for proper maintenance of centrosome integrity required for the initiation of endodyogeny. Our findings provide a first insight into the probable role of calcium-dependent signalling in parasite multiplication, in addition to its more widely explored role in invasion and egress.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Division , Protein Kinases/metabolism , Toxoplasma/enzymology , Toxoplasma/physiology , Cell Cycle Proteins/genetics , Cell Survival , Centrosome/metabolism , Gene Knockdown Techniques , Protein Kinases/genetics , Toxoplasma/growth & developmentABSTRACT
BACKGROUND: Next generation sequencing is helping to overcome limitations in organisms less accessible to classical or reverse genetic methods by facilitating whole genome mutational analysis studies. One traditionally intractable group, the Apicomplexa, contains several important pathogenic protozoan parasites, including the Plasmodium species that cause malaria.Here we apply whole genome analysis methods to the relatively accessible model apicomplexan, Toxoplasma gondii, to optimize forward genetic methods for chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) and ethylmethane sulfonate (EMS) at varying dosages. RESULTS: By comparing three different lab-strains we show that spontaneously generated mutations reflect genome composition, without nucleotide bias. However, the single nucleotide variations (SNVs) are not distributed randomly over the genome; most of these mutations reside either in non-coding sequence or are silent with respect to protein coding. This is in contrast to the random genomic distribution of mutations induced by chemical mutagenesis. Additionally, we report a genome wide transition vs transversion ratio (ti/tv) of 0.91 for spontaneous mutations in Toxoplasma, with a slightly higher rate of 1.20 and 1.06 for variants induced by ENU and EMS respectively. We also show that in the Toxoplasma system, surprisingly, both ENU and EMS have a proclivity for inducing mutations at A/T base pairs (78.6% and 69.6%, respectively). CONCLUSIONS: The number of SNVs between related laboratory strains is relatively low and managed by purifying selection away from changes to amino acid sequence. From an experimental mutagenesis point of view, both ENU (24.7%) and EMS (29.1%) are more likely to generate variation within exons than would naturally accumulate over time in culture (19.1%), demonstrating the utility of these approaches for yielding proportionally greater changes to the amino acid sequence. These results will not only direct the methods of future chemical mutagenesis in Toxoplasma, but also aid in designing forward genetic approaches in less accessible pathogenic protozoa as well.
Subject(s)
Genome , Toxoplasma/genetics , Adenosine/genetics , Adenosine/metabolism , Amino Acid Sequence , Base Pairing , Cell Line , Ethyl Methanesulfonate/toxicity , Ethylnitrosourea/toxicity , High-Throughput Nucleotide Sequencing , Humans , Molecular Sequence Data , Pentosyltransferases/genetics , Pentosyltransferases/metabolism , Phenotype , Point Mutation , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Thymidine/genetics , Thymidine/metabolism , Toxoplasma/drug effectsABSTRACT
One of the defining features of apicomplexan parasites is their cytoskeleton composed of alveolar vesicles, known as the inner membrane complex (IMC) undergirded by intermediate-like filament network and an array of subpellicular microtubules (SPMTs). In Toxoplasma gondii, this specialized cytoskeleton is involved in all aspects of the disease-causing lytic cycle, and notably acting as a scaffold for parasite offspring in the internal budding process. Despite advances in our understanding of the architecture and molecular composition, insights pertaining to the coordinated assembly of the scaffold are still largely elusive. Here, T. gondii tachyzoites were dissected by advanced, iterative expansion microscopy (pan-ExM) revealing new insights into the very early sequential formation steps of the tubulin scaffold. A comparative study of the related parasite Sarcocystis neurona revealed that different MT bundling organizations of the nascent SPMTs correlate with the number of central and basal alveolar vesicles. In absence of a so far identified MT nucleation mechanism, we genetically dissected T. gondii γ-tubulin and γ-tubulin complex protein 4 (GCP4). While γ-tubulin depletion abolished the formation of the tubulin scaffold, a set of MTs still formed that suggests SPMTs are nucleated at the outer core of the centrosome. Depletion of GCP4 interfered with the correct assembly of SPMTs into the forming daughter buds, further indicating that the parasite utilizes the γ-tubulin complex in tubulin scaffold formation .
ABSTRACT
Sequential lytic cycles driven by cascading transcriptional waves underlie pathogenesis in the apicomplexan parasite Toxoplasma gondii. This parasite's unique division by internal budding, short cell cycle, and jumbled up classically defined cell cycle stages have restrained in-depth transcriptional program analysis. Here, unbiased transcriptome and chromatin accessibility maps throughout the lytic cell cycle are established at the single-cell level. Correlated pseudo-timeline assemblies of expression and chromatin profiles maps transcriptional versus chromatin level transition points promoting the cell division cycle. Sequential clustering analysis identifies functionally related gene groups promoting cell cycle progression. Promoter DNA motif mapping reveals patterns of combinatorial regulation. Pseudo-time trajectory analysis reveals transcriptional bursts at different cell cycle points. The dominant burst in G1 is driven largely by transcription factor AP2XII-8, which engages a conserved DNA motif, and promotes the expression of 44 ribosomal proteins encoding regulon. Overall, the study provides integrated, multi-level insights into apicomplexan transcriptional regulation.
Subject(s)
Chromatin , Protozoan Proteins , Regulon , Ribosomes , Single-Cell Analysis , Toxoplasma , Toxoplasma/genetics , Toxoplasma/metabolism , Chromatin/metabolism , Chromatin/genetics , Regulon/genetics , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Ribosomes/metabolism , Ribosomes/genetics , Gene Expression Regulation , Promoter Regions, Genetic/genetics , Cell Cycle/genetics , Humans , Nucleotide Motifs/genetics , Transcriptome , Ribosomal Proteins/metabolism , Ribosomal Proteins/geneticsABSTRACT
BACKGROUND: Toxoplasma gondii has a largely clonal population in North America and Europe, with types I, II and III clonal lineages accounting for the majority of strains isolated from patients. RH, a particular type I strain, is most frequently used to characterize Toxoplasma biology. However, compared to other type I strains, RH has unique characteristics such as faster growth, increased extracellular survival rate and inability to form orally infectious cysts. Thus, to identify candidate genes that could account for these parasite phenotypic differences, we determined genetic differences and differential parasite gene expression between RH and another type I strain, GT1. Moreover, as differences in host cell modulation could affect Toxoplasma replication in the host, we determined differentially modulated host processes among the type I strains through host transcriptional profiling. RESULTS: Through whole genome sequencing, we identified 1,394 single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) between RH and GT1. These SNPs/indels together with parasite gene expression differences between RH and GT1 were used to identify candidate genes that could account for type I phenotypic differences. A polymorphism in dense granule protein, GRA2, determined RH and GT1 differences in the evasion of the interferon gamma response. In addition, host transcriptional profiling identified that genes regulated by NF-ĸB, such as interleukin (IL)-12p40, were differentially modulated by the different type I strains. We subsequently showed that this difference in NF-ĸB activation was due to polymorphisms in GRA15. Furthermore, we observed that RH, but not other type I strains, recruited phosphorylated IĸBα (a component of the NF-ĸB complex) to the parasitophorous vacuole membrane and this recruitment of p- IĸBα was partially dependent on GRA2. CONCLUSIONS: We identified candidate parasite genes that could be responsible for phenotypic variation among the type I strains through comparative genomics and transcriptomics. We also identified differentially modulated host pathways among the type I strains, and these can serve as a guideline for future studies in examining the phenotypic differences among type I strains.
Subject(s)
Phenotype , Toxoplasma/genetics , Toxoplasma/physiology , Animals , Fibroblasts/parasitology , Gene Expression Regulation , Genes, Protozoan/genetics , HEK293 Cells , Humans , Interleukin-12 Subunit p40/metabolism , Intracellular Membranes/metabolism , Intracellular Membranes/parasitology , Macrophages/metabolism , Macrophages/parasitology , Mice , NF-kappa B/metabolism , Polymorphism, Single Nucleotide , Protein Transport , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Species Specificity , Toxoplasma/metabolism , Vacuoles/metabolismABSTRACT
Toxoplasma gondii is an obligate intracellular protozoan parasite whose rapid lytic replication cycles define its pathogenicity. We identified a temperature-sensitive growth mutant, FV-P6, which irreversibly arrests before the middle of the G1 stage of the tachyzoite cell cycle. This arrest is caused by a point mutation in a gene conserved across eukaryotes, Cactin, whose product localizes to the nucleus. To elucidate the role of TgCactin we performed genome-wide expression profiling. Besides the expected G1 expression profile, many genes associated with the extracellular state as well as with the bradyzoite cyst stage were identified. Consistent with these profiles were the expression of AP2 transcription factors typically associated with extracellular and bradyzoite stage parasites. This suggests a role for TgCactin in control of gene expression. As TgCactin does not contain any functionally defined domains we reasoned TgCactin exerts its function through interactions with other proteins. In support of this model we demonstrated that TgCactin is present in a protein complex and can oligomerize. Taken together, these results suggest that TgCactin acts as a pivotal protein potentially regulating gene expression at several transition points in parasite development.
Subject(s)
Carrier Proteins/metabolism , Drosophila Proteins/metabolism , G1 Phase , Protozoan Proteins/metabolism , Toxoplasma/cytology , Toxoplasma/metabolism , Amino Acid Sequence , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Cycle , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Humans , Molecular Sequence Data , Point Mutation , Protein Transport , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Sequence Alignment , Toxoplasma/chemistry , Toxoplasma/genetics , Toxoplasmosis/parasitologyABSTRACT
The cell division cycle of T. gondii is driven by cyclically expressed ApiAP2 transcription factors (AP2s) that promote gene sets (regulons) associated with specific biological functions. AP2s drive other AP2s, thereby propelling the progressive gene expression waves defining the lytic cycle. AP2s can act as dimers by themselves, in combination with other AP2s (constitutive or cyclical) or in complexes with epigenetic factors. Exit from the cell cycle into either the extracellular state or differentiation into bradyzoites results in major changes in gene expression. Surprisingly, both transitions lead to expression of a shared set of unique AP2s that suggest a shared stress response that, governed by the specific conditions, leads to different outcomes.