RESUMO
Toxoplasma gondii is an obligate intracellular protozoan parasite. This apicomplexan is the causative agent of toxoplasmosis, a leading cause of central nervous system disease in AIDS. It has long been known that T. gondii interferes with major histocompatibility complex class II (MHC-II) antigen presentation to attenuate CD4(+) T cell responses and establish persisting infections. Transcriptional downregulation of MHC-II genes by T. gondii was previously established, but the precise mechanisms inhibiting MHC-II function are currently unknown. Here, we show that, in addition to transcriptional regulation of MHC-II, the parasite modulates the expression of key components of the MHC-II antigen presentation pathway, namely, the MHC-II-associated invariant chain (Ii or CD74) and the peptide editor H2-DM, in professional antigen-presenting cells (pAPCs). Genetic deletion of CD74 restored the ability of infected dendritic cells to present a parasite antigen in the context of MHC-II in vitro. CD74 mRNA and protein levels were, surprisingly, elevated in infected cells, whereas MHC-II and H2-DM expression was inhibited. CD74 accumulated mainly in the endoplasmic reticulum (ER), and this phenotype required live parasites, but not active replication. Finally, we compared the impacts of genetic deletion of CD74 and H2-DM genes on parasite dissemination toward lymphoid organs in mice, as well as activation of CD4(+) T cells and interferon gamma (IFN-γ) levels during acute infection. Cyst burdens and survival during the chronic phase of infection were also evaluated in wild-type and knockout mice. These results highlight the fact that the infection is influenced by multiple levels of parasite manipulation of the MHC-II antigen presentation pathway.
Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos T CD4-Positivos , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Interferon gama/genética , Interferon gama/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Toxoplasma/genética , Toxoplasma/fisiologia , Toxoplasmose/genética , Toxoplasmose/parasitologiaRESUMO
Protozoa in the phylum Apicomplexa are a large group of obligate intracellular parasites. Toxoplasma gondii and other apicomplexan parasites, such as Plasmodium falciparum, cause diseases by reiterating their lytic cycle, comprising host cell invasion, parasite replication, and parasite egress. The successful completion of the lytic cycle requires that the parasite senses changes in its environment and switches between the non-motile (for intracellular replication) and motile (for invasion and egress) states appropriately. Although the signaling pathway that regulates the motile state switch is critical to the pathogenesis of the diseases caused by these parasites, it is not well understood. Here we report a previously unknown mechanism of regulating the motility activation in Toxoplasma, mediated by a protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). AKMT depletion greatly inhibits activation of motility, compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Interestingly, AKMT redistributes from the apical complex to the parasite body rapidly in the presence of egress-stimulating signals that increase [Ca²âº] in the parasite cytoplasm, suggesting that AKMT regulation of parasite motility might be accomplished by the precise temporal control of its localization in response to environmental changes.
Assuntos
Movimento Celular/fisiologia , Interações Hospedeiro-Parasita , Metiltransferases/fisiologia , Toxoplasma/fisiologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Cultivadas , Humanos , Masculino , Metiltransferases/deficiênciaRESUMO
Comparing the steady-state expression levels of recombinant proteins in Toxoplasma gondii parasites indicates considerable variability, and this has sometimes caused difficulties in the engineering of transgenic parasites. Anecdotal observations suggested that alteration of the N-terminus, e.g. by engineering as a fusion protein, permits stable expression of various transgenes that were previously difficult to express in their native form. We have exploited the sensitivity and quantitative nature of fire-fly luciferase (LUC) to examine expression levels in further detail. Fusing the 26 N-terminal residues derived from chloramphenicol acetyl transferase (DeltaCAT) to LUC permits efficient transient or stable luciferase expression in transgenic parasite tachyzoites, providing a useful reporter for studies in T. gondii. Site-directed mutagenesis was used to alter the second codon of DeltaCAT-LUC to encode all 20 possible amino acids, and these constructs showed that changes in the second amino acid can have dramatic effects on luciferase activity, with Ala, Glu, and Asp codons yielding the highest expression levels. Similar results were observed for the expression of both GFP and the T. gondii HXGPRT gene, demonstrating the generality of this effect.
Assuntos
Regulação da Expressão Gênica , Toxoplasma/genética , Toxoplasma/metabolismo , Animais , Animais Geneticamente Modificados , Composição de Bases , Western Blotting , Códon/genética , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutagênese Sítio-Dirigida , Plasmodium falciparum/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A forkhead transcription factor gene, fktf-1, which we propose to be orthologous to the Caenorhabditis elegans dauer-regulatory gene daf-16 has been discovered in the parasitic nematode Strongyloides stercoralis. Genomic and cDNA sequences from both species predict alternately spliced a and b message isoforms. In contrast to C. elegans, where two a isoforms, daf-16a1 and daf-16a2, are found, a single fktf-1a isoform is found in S. stercoralis. Five of the 10 introns found in the C. elegans gene are found in the proposed S. stercoralis ortholog. Functional motifs common to DAF-16 and several mammalian forkhead transcription factors are conserved in FKTF-1. These include the forkhead DNA binding domain, four Akt/protein kinase B phosphorylation sites and a C-terminal domain that may associate with factors such as the steroid receptor coactivator and other factors necessary for transcriptional regulation. An N-terminal serine-rich domain found in DAF-16A is greatly expanded in FKTF-1A. This domain is missing in DAF-16B, FKTF-1B and all mammalian orthologs. FKTF-1 shows the closest phylogenetic relationship to DAF-16 among all known mammalian and nematode forkhead transcription factors. Like its proposed Caenorhabditis ortholog, the fktf-1 message is expressed at all stages of the life cycle examined thus far. Discovery of fktf-1 indicates the presence of an insulin-like signalling pathway in S. stercoralis similar to that known to regulate dauer development in C. elegans. This pathway is a likely candidate to control infective larval arrest and reactivation as well as regulation of the switch between parasitic and free-living development in the parasite.
Assuntos
Proteínas de Caenorhabditis elegans , Genes de Helmintos , Strongyloides stercoralis/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Fatores de Transcrição Forkhead , Expressão Gênica , Humanos , Larva , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , Alinhamento de Sequência , Homologia de Sequência , Strongyloides stercoralis/fisiologiaRESUMO
The protozoan phylum Apicomplexa encompasses approximately 5000 species of obligate intracellular parasites, including those responsible for malaria and toxoplasmosis. Rather than dividing by binary fission, apicomplexans use a remarkable mechanism for replication, assembling daughters de novo within the cytoplasm. Here, we exploit time-lapse microscopy of fluorescent markers targeted to various subcellular structures in Toxoplasma gondii tachyzoites to determine how these unicellular eukaryotes efficiently package a complete set of organelles, maintaining the highly polarized organization necessary for host cell invasion and pathogenesis. Golgi division and elongation of the apicoplast are among the first morphologically observable events, associated with an unusual pattern of centriolar migration. Daughter parasites are assembled on cytoskeletal scaffolding, whose growth proceeds from the apical end, first encapsulating the divided Golgi. Further extension of the cytoskeletal scaffold results in partitioning of the apicoplast, nucleus, endoplasmic reticulum, and finally the mitochondrion, which enters the developing daughters rapidly, but only very late during the division cycle. The specialized secretory organelles (micronemes and rhoptries) form de novo. This distinctive pattern of replication -- in which organellar segregation spans approximately 75% of the cell cycle, completely encompassing S phase -- suggests an unusual mechanism of cell cycle regulation.
Assuntos
Ciclo Celular , Organelas/metabolismo , Toxoplasma/citologia , Animais , Ciclo Celular/efeitos dos fármacos , Dinitrobenzenos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Organelas/efeitos dos fármacos , Organelas/ultraestrutura , Sulfanilamidas/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/ultraestruturaRESUMO
We previously reported a cytosolic pyruvate kinase (EC 2.7.1.40) from Toxoplasma gondii (TgPyKI) that differs from most eukaryotic pyruvate kinases in being regulated by glucose 6-phosphate rather than fructose 1,6-diphosphate. Another putative pyruvate kinase (TgPyKII) was identified from parasite genome, which exhibits 32% amino acid sequence identity to TgPyKI and retains pyruvate kinase signature motifs and amino acids essential for substrate binding and catalysis. Whereas TgPyKI is most closely related to plant/algal enzymes, phylogenetic analysis suggests a proteobacterial origin for TgPyKII. Enzymatic characterization of recombinant TgPyKII shows a high pH optimum at 8.5, and a preference for GDP as a phosphate recipient. Catalytic activity is independent of K+, and no allosteric or regulatory effects were observed in the presence of fructose 1,6-diphosphate, fructose 2,6-diphosphate, glucose 6-phosphate, ribose 5-phosphate, AMP, or ATP. Unlike TgPyKI, native TgPyKII activity was exclusively associated with the membranous fraction of a T. gondii tachyzoite lysate. TgPyKII possesses a long N-terminal extension containing five putative start codons before the conserved region and localizes to both apicoplast and mitochondrion by immunofluorescence assay using native antibody and fluorescent protein fusion to the N-terminal extension. Further deletional and site-directed mutagenesis suggests that a translation product from 1st Met is responsible for the localization to the apicoplast, whereas one from 3rd Met is for the mitochondrion. This is the first study of a potential mitochondrial pyruvate kinase in any system.
Assuntos
Guanosina Difosfato/química , Mitocôndrias/metabolismo , Piruvato Quinase/química , Toxoplasma/metabolismo , Sequência de Aminoácidos , Animais , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Ligação Proteica , Isoformas de Proteínas , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
A unique feature of the Toxoplasma gondii purine salvage pathway is the expression of two isoforms of the hypoxanthine-xanthine-guanine phosophoribosyltransferase (HXGPRT) of the parasite encoded by a single genetic locus. These isoforms differ in the presence or absence of a 49-amino acid insertion (which is specified by a single differentially spliced exon) but exhibit similar substrate specificity, kinetic characteristics, and temporal expression patterns. To examine possible functional differences between the two HXGPRT isoforms, fluorescent protein fusions were expressed in parasites lacking the endogenous hxgprt gene. Immunoblot analysis of fractionated cell extracts and fluorescence microscopy indicated that HXGPRT-I (which lacks the 49-amino acid insertion) is found in the cytosol, whereas HXGPRT-II (which contains the insertion) localizes to the inner membrane complex (IMC) of the parasite. Simultaneous expression of both isoforms resulted in the formation of hetero-oligomers, which distributed between the cytosol and IMC. Chimeric constructs expressing N-terminal peptides from either isoform I (11 amino acids) or isoform II (60 amino acids) fused to a chloramphenicol acetyl transferase (CAT) reporter demonstrated that the N-terminal domain of isoform II is both necessary and sufficient for membrane association. Metabolic labeling experiments with transgenic parasites showed that isoform II or an isoform II-CAT fusion protein (but not isoform I or isoform I-CAT) incorporate [(3)H]palmitate. Mutation of three adjacent cysteine residues within the isoform II-targeting domain to serines blocked both palmitate incorporation and IMC attachment without affecting enzyme activity, demonstrating that acylation of N-terminal isoform II cysteine residues is responsible for the association of HXGPRT-II with the IMC.
Assuntos
Pentosiltransferases/química , Toxoplasma/enzimologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Membrana Celular/metabolismo , Cisteína/química , Citosol/metabolismo , DNA Complementar/metabolismo , Éxons , Biblioteca Gênica , Immunoblotting , Imunoprecipitação , Cinética , Microscopia de Fluorescência , Dados de Sequência Molecular , Ácidos Palmíticos/metabolismo , Peptídeos/química , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos , Frações Subcelulares , Especificidade por Substrato , Fatores de Tempo , TransgenesRESUMO
Parasite differentiation is commonly associated with transitions between complex life cycle stages and with long-term persistence in the host, and it is therefore critical for pathogenesis. In the protozoan parasite Toxoplasma gondii, interconversion between rapidly growing tachyzoites and latent encysted bradyzoites is accompanied by numerous morphological and metabolic adaptations. In order to explore early cell biological events associated with this differentiation process, we have exploited fluorescent reporter proteins targeted to various subcellular locations. Combining these markers with efficient in vitro differentiation and time-lapse video microscopy provides a dynamic view of bradyzoite development in living cultures, demonstrating subcellular reorganization, maintenance of the mitochondrion, and missegregation of the apicoplast. Bradyzoites divide asynchronously, using both endodyogeny and endopolygeny, and are highly motile both within and between host cells. Cysts are able to proliferate without passing through an intermediate tachyzoite stage, via both the migration of free bradyzoites and the fission of bradyzoite cysts, suggesting a mechanism for dissemination during chronic infection.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/citologia , Toxoplasma/fisiologia , Animais , Forma Celular , Fibroblastos/citologia , Fibroblastos/parasitologia , Fibroblastos/fisiologia , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mitocôndrias/metabolismo , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Toxoplasma/genética , Vacúolos/parasitologiaRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Primers do DNA , Fibroblastos/citologia , Fibroblastos/parasitologia , Genes Reporter/genética , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Toxoplasma/citologiaRESUMO
Here we report the isolation and characterization of a type I vacuolar-type H(+)-pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation. The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category. Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T. gondii. Providing that the coding sequence for the entire N-terminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmid-borne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K(+)-activated pyrophosphate (PP(i)) hydrolysis and PP(i)-energized H(+) translocation. Immunofluorescence microscopy of both free and intracellular T. gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme. Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow. Although treatment of T. gondii with AMDP concentrations as high as 100 microm had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I(50) for the inhibition of TgVP1 activity in vitro (0.9 microm) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisome-like vesicular structures. A dynamic association of TgVP1 with the host cell invasion apparatus is invoked, one in which the effects of inhibitory V-PPase substrate analogs are exerted after rather than during host cell invasion.