RESUMO
Apicomplexan parasites are responsible for some of the most deadly parasitic diseases affecting humans and livestock. There is an urgent need for new medicines that will target apicomplexan-specific pathways. We characterized a Toxoplasma gondii C2H2 zinc finger protein, named TgZNF2, which is conserved among eukaryotes. We constructed an inducible KO strain (iKO-TgZNF2) for this gene where the tgznf2 gene expression is repressed in the presence of a tetracycline analog (ATc). We showed that the iKO-TgZNF2 parasites are unable to proliferate after depletion of the TgZNF2 protein. Complementation with a full length copy of the gene restores the phenotype Moreover, the homolog of this protein in the related apicomplexan Plasmodium falciparum was shown to efficiently rescue the phenotype, suggesting that this pathway is likely conserved among apicomplexan parasites. We demonstrated that the iKO-mutant lacking TgZNF2 are arrested during the cell cycle during the G1 phase. We identified potential protein partners of this protein among which are spliceosomal complex and mRNA nuclear export components. We confirmed that TgZNF2 is able to bind in vivo to transcripts but splicing is not perturbed in the ATc-treated parasites. Instead, we demonstrated that TgZNF2 depletion leads to the sequestration of polyA+ mRNAs in the nucleus while ribosomal RNAs are not affected. We discovered a conserved protein with specific apicomplexan functional properties that is essential for the survival of T. gondii. TgZNF2 may be crucial to ensure the correct polyA+ mRNA nuclear export, a function that is conserved in P. falciparum.
Assuntos
Transporte Ativo do Núcleo Celular , Dedos de Zinco CYS2-HIS2 , Fatores de Transcrição Kruppel-Like/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Toxoplasma/crescimento & desenvolvimento , Pontos de Checagem do Ciclo Celular , Técnicas de Silenciamento de Genes , Teste de Complementação Genética , Humanos , Fatores de Transcrição Kruppel-Like/deficiência , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Toxoplasma/genéticaRESUMO
Apicomplexan parasites have unique apical rhoptry and microneme secretory organelles that are crucial for host infection, although their role in protection against Toxoplasma gondii infection is not thoroughly understood. Here, we report a novel function of the endolysosomal T. gondii sortilin-like receptor (TgSORTLR), which mediates trafficking to functional apical organelles and their subsequent secretion of virulence factors that are critical to the induction of sterile immunity against parasite reinfection. We further demonstrate that the T. gondii armadillo repeats-only protein (TgARO) mutant, which is deficient only in apical secretion of rhoptries, is also critical in mounting protective immunity. The lack of TgSORTLR and TgARO proteins completely inhibited T-helper 1-dependent adaptive immunity and compromised the function of natural killer T-cell-mediated innate immunity. Our findings reveal an essential role for apical secretion in promoting sterile protection against T. gondii and provide strong evidence for rhoptry-regulated discharge of antigens as a key effector for inducing protective immunity.
Assuntos
Imunidade Adaptativa , Imunidade Inata , Organelas/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Antígenos de Protozoários/sangue , Linhagem Celular , Interações Hospedeiro-Parasita , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-1beta/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Transporte Proteico/imunologia , Toxoplasmose/imunologiaRESUMO
In addition to catalyzing a central step in glycolysis, enolase assumes a remarkably diverse set of secondary functions in different organisms, including transcription regulation as documented for the oncogene c-Myc promoter-binding protein 1. The apicomplexan parasite Toxoplasma gondii differentially expresses two nuclear-localized, plant-like enolases: enolase 1 (TgENO1) in the latent bradyzoite cyst stage and enolase 2 (TgENO2) in the rapidly replicative tachyzoite stage. A 2.75â Å resolution crystal structure of bradyzoite enolase 1, the second structure to be reported of a bradyzoite-specific protein in Toxoplasma, captures an open conformational state and reveals that distinctive plant-like insertions are located on surface loops. The enolase 1 structure reveals that a unique residue, Glu164, in catalytic loop 2 may account for the lower activity of this cyst-stage isozyme. Recombinant TgENO1 specifically binds to a TTTTCT DNA motif present in the cyst matrix antigen 1 (TgMAG1) gene promoter as demonstrated by gel retardation. Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays. Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development. Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.
Assuntos
Núcleo Celular , Citoplasma , Proteínas Nucleares , Fosfopiruvato Hidratase , Proteínas de Protozoários , Toxoplasma , Núcleo Celular/enzimologia , Núcleo Celular/genética , Cristalografia por Raios X , Citoplasma/enzimologia , Citoplasma/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Toxoplasma/genéticaRESUMO
Toxoplasma (toxoplasmosis) and Plasmodium (malaria) use unique secretory organelles for migration, cell invasion, manipulation of host cell functions, and cell egress. In particular, the apical secretory micronemes and rhoptries of apicomplexan parasites are essential for successful host infection. New findings reveal that the contents of these organelles, which are transported through the endoplasmic reticulum (ER) and Golgi, also require the parasite endosome-like system to access their respective organelles. In this review, we discuss recent findings that demonstrate that these parasites reduced their endosomal system and modified classical regulators of this pathway for the biogenesis of apical organelles.
Assuntos
Retículo Endoplasmático/metabolismo , Malária/metabolismo , Plasmodium/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/metabolismo , Animais , Endossomos/metabolismo , Humanos , Malária/patologia , Transporte Proteico , Toxoplasmose/patologiaRESUMO
Gene regulation in apicomplexan parasites, a phylum containing important protozoan parasites such as Plasmodium and Toxoplasma, is poorly understood. The life cycle of Toxoplasma gondii is complex, with multiple proliferation and differentiation steps, of which tachyzoite proliferation is the most relevant to pathogenesis in humans and animals. Tachyzoites express invasion and virulence factors that are crucial for their survival and manipulation of host cell functions. The expression of those factors is tightly controlled during the tachyzoite cell cycle to permit their correct packaging in newly formed apical secretory organelles named micronemes and rhoptries in the daughter cells. However, little is known about the factors that control the expression of genes encoding the virulence factors present in these parasite-specific secretory organelles. We report that the plant-like nuclear factor TgAP2XI-5 targets more than 300 gene promoters and actively controls the transcription of these genes. Most of these target genes, including those that are essential for parasite virulence, showed a peak of expression in the S and M phases of the cell cycle. Furthermore, we identified the cis-regulatory element recognized by TgAP2XI-5 and demonstrated its ability to actively drive gene transcription. Our results demonstrated that TgAP2XI-5 is a novel DNA sequence-specific transcription factor associated with promoter activation. TgAP2XI-5 may regulate gene transcription of crucial virulence factors in T. gondii.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas de Protozoários/metabolismo , Elementos de Resposta , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Genes de Protozoários/fisiologia , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasmose/genética , Toxoplasmose/metabolismo , Fatores de Transcrição/genéticaRESUMO
Toxoplasma gondii undergoes many phenotypic changes during its life cycle. The recent identification of AP2 transcription factors in T. gondii has provided a platform for studying the mechanisms controlling gene expression. In the present study, we report that a recombinant protein encompassing the TgAP2XI-4 AP2 domain was able to specifically bind to a DNA motif using gel retardation assays. TgAP2XI-4 protein is localized in the parasite nucleus throughout the tachyzoite life cycle in vitro, with peak expression occurring after cytokinesis. We found that the TgAP2XI-4 transcript level was higher in bradyzoite cysts isolated from brains of chronically infected mice than in the rapidly replicating tachyzoites. A knockout of the TgAP2XI-4 gene in both T. gondii virulent type I and avirulent type II strains reveals its role in modulating expression and promoter activity of genes involved in stage conversion of the rapidly replicating tachyzoites to the dormant cyst forming bradyzoites. Furthermore, mice infected with the type II KO mutants show a drastically reduced brain cyst burden. Thus, our results validate TgAP2XI-4 as a novel nuclear factor that regulates bradyzoite gene expression during parasite differentiation and cyst formation.
Assuntos
Regulação da Expressão Gênica , Toxoplasma/citologia , Toxoplasma/genética , Fatores de Transcrição/metabolismo , Animais , Encéfalo/parasitologia , Encéfalo/patologia , DNA de Protozoário/metabolismo , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Técnicas de Inativação de Genes , Camundongos , Ligação Proteica , Esporos de Protozoários/citologia , Esporos de Protozoários/genética , Toxoplasmose Animal/parasitologia , Toxoplasmose Animal/patologia , Fatores de Transcrição/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Apicomplexan parasites possess specialized secretory organelles called rhoptries, micronemes, and dense granules that play a vital role in host infection. In this study, we demonstrate that TgREMIND, a protein found in Toxoplasma gondii, is necessary for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes-CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, which binds to membrane phospholipids, as well as a novel uncharacterized domain that we have named REMIND (regulator of membrane-interacting domain). Both the F-BAR domain and the REMIND are crucial for TgREMIND functions. When TgREMIND is depleted, there is a significant decrease in the abundance of dense granules and abnormal transparency of rhoptries, leading to a reduction in protein secretion from these organelles. The absence of TgREMIND inhibits host invasion and parasite dissemination, demonstrating that TgREMIND is essential for the proper function of critical secretory organelles required for successful infection by Toxoplasma.
Assuntos
Parasitos , Toxoplasma , Animais , Toxoplasma/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Organelas/metabolismo , Parasitos/metabolismo , Fosfatidilinositóis/metabolismoRESUMO
In Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii.
Assuntos
Nucléolo Celular/metabolismo , Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas de Protozoários/metabolismo , Toxoplasma/patogenicidade , Anticorpos Antiprotozoários , Nucléolo Celular/genética , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Espectrometria de Massas , Microscopia Eletrônica , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteômica , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequências Reguladoras de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/metabolismo , Análise de Sequência de Proteína , Coloração e Rotulagem , Proteínas de Ligação a Tacrolimo/química , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.
Assuntos
Antígenos de Protozoários/metabolismo , Apicomplexa/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Proteínas de Protozoários/metabolismo , Internalização do Vírus , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Apicomplexa/genética , Apicomplexa/metabolismo , Células Cultivadas , Chlorocebus aethiops , Conexinas/metabolismo , Sequência Conservada , Interações Hospedeiro-Parasita/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Modelos Biológicos , Modelos Moleculares , Parasitos/genética , Parasitos/metabolismo , Parasitos/fisiologia , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Plasmodium falciparum/fisiologia , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/metabolismo , Toxoplasma/fisiologia , Células VeroRESUMO
Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.
Assuntos
Movimento Celular , Retículo Endoplasmático/metabolismo , Glicoproteínas/química , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/parasitologia , Glicômica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Interações Hospedeiro-Parasita/genética , Humanos , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas Motores Moleculares/genética , Mutagênese Sítio-Dirigida , Plasmídeos , Ligação Proteica , Transporte Proteico/fisiologia , Proteômica , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxoplasma/genética , TransfecçãoRESUMO
Apicomplexa phylum includes numerous obligate intracellular protozoan parasites that are life threatening for humans and animals. In this context, Plasmodium falciparum and Toxoplasma gondii are of particular interest, as they are responsible for malaria and toxoplasmosis, respectively, for which efficient vaccines are presently lacking and therapies need to be improved. Apicomplexan parasites have a highly polarized morphology, with their apical end containing specific secretory organelles named rhoptries and micronemes, which depend on the unique receptor and transporter sortilin TgSORT for their biogenesis. In the present study, we took advantage of the subcellular polarity of the parasite to engineer a clonal transgenic Toxoplasma line that expresses simultaneously the green fluorescent protein TgSORT-GFP in the post-Golgi-endosome-like compartment and the red fluorescent protein rhoptry ROP1-mCherry near the apical end. We utilized this fluorescent transgenic T. gondii to develop a miniaturized image-based phenotype assay coupled to an automated image analysis. By applying this methodology to 1,120 compounds, we identified 12 that are capable of disrupting the T. gondii morphology and inhibiting intracellular replication. Analysis of the selected compounds confirmed that all 12 are kinase inhibitors and intramembrane pumps, with some exhibiting potent activity against Plasmodium falciparum. Our findings highlight the advantage of comparative and targeted phenotypic analysis involving two related parasite species as a means of identifying molecules with a conserved mode of action.
Assuntos
Parasitos , Toxoplasma , Animais , Humanos , Toxoplasma/genética , Toxoplasma/metabolismo , Parasitos/metabolismo , Plasmodium falciparum , Proteínas de Protozoários/metabolismo , Endossomos/metabolismo , Proteínas de Fluorescência Verde/genéticaRESUMO
Cerebral malaria is a neuroinflammatory disease induced by P. falciparum infection. In animal models, the neuro-pathophysiology of cerebral malaria results from the sequestration of infected red blood cells (iRBCs) in microvessels that promotes the activation of glial cells in the brain. This activation provokes an exacerbated inflammatory response characterized by the secretion of proinflammatory cytokines and chemokines, leading to brain infiltration by pathogenic CD8+ T lymphocytes. Astrocytes are a major subtype of brain glial cells that play an important role in maintaining the homeostasis of the central nervous system, the integrity of the brain-blood barrier and in mounting local innate immune responses. We have previously shown that parasitic microvesicles (PbA-MVs) are transferred from iRBCs to astrocytes. The present study shows that an unconventional LC3-mediated autophagy pathway independent of ULK1 is involved in the transfer and degradation of PbA-MVs inside the astrocytes. We further demonstrate that inhibition of the autophagy process by treatment with 3-methyladenine blocks the transfer of PbA-MVs, which remain localized in the astrocytic cell membrane and are not internalized. Moreover, bafilomycin A1, another drug against autophagy promotes the accumulation of PbA-MVs inside the astrocytes by inhibiting the fusion with lysosomes, and prevents ECM in mice infected with PbA. Finally, we establish that RUBCN/rubicon or ATG5 silencing impede astrocyte production in CCL2 and CXCL10 chemokines induced by PbA stimulation. Altogether, our data suggest that a non-canonical autophagy-lysosomal pathway may play a key role in cerebral malaria through regulation of brain neuro-inflammation by astrocytes.
Assuntos
Malária Cerebral , Plasmodium , Animais , Astrócitos/metabolismo , Autofagia , Malária Cerebral/parasitologia , Malária Cerebral/patologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium bergheiRESUMO
The purpose of this study was to assess the direct effect of CCL18, a chemokine elevated in allergic diseases and induced by Th2 cytokines, on the polarization of human CD4(+) T cells. Purified human T cells from healthy subjects were pretreated or not with CCL18, and evaluated for cytokine production. CCL18-pretreated memory but not naive CD4(+) T cells exhibited an increased production of IL-10 (12.3 ± 2.6 vs. 5.6 ± 0.9 ng/ml for medium) and TGF-ß1 but not IL-4, IFN-γ, and IL-17 compared with control cells. Pretreatment of highly purified CD4(+)CD25(-) memory T cells with CCL18 led to their conversion to CD4(+)CD25(+)Foxp3(+) regulatory T cells able to inhibit the proliferation of CD4(+)CD25(-) effector T cells by both cytokine and cell contact-dependent mechanisms. However, this regulatory effect of CCL18 was lost when T cells originated from allergic subjects in relation with a decreased binding of CCL18 to these cells [0.7 ± 0.3 mean fluorescence intensity (MFI)] as compared to those from healthy subjects (6.0 ± 1.7 MFI). This study is the first to define a chemokine that generates adaptive regulatory T cells from CD4(+)CD25(-) memory T cells. This mechanism appears defective in allergic patients and may underlie the decreased tolerance observed in allergic diseases.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Quimiocinas CC/farmacologia , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Humanos , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.
Assuntos
Glicômica , Glicoproteínas/metabolismo , Interações Hospedeiro-Parasita , Proteômica , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Animais , Sequência de Carboidratos , Células Cultivadas , Glicoproteínas/análise , Glicosilação , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Oligossacarídeos/análise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Lectinas de Plantas/química , Polissacarídeos/química , Proteínas de Protozoários/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Toxoplasma/química , Toxoplasma/metabolismoRESUMO
The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model heterotrophic dinoflagellate Crypthecodinium cohnii. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of green algae and land plant starch. Preliminary characterization of the starch pathway demonstrated that C. cohnii contains multiple forms of soluble starch synthases and one major 110-kDa granule-bound starch synthase. All purified enzymes displayed a marked substrate preference for UDP-glucose. At variance with most other microorganisms, the accumulation of starch in the dinoflagellate occurs during early and mid-log phase, with little or no synthesis witnessed when approaching stationary phase. In order to establish a genetic system allowing the study of cytoplasmic starch metabolism in eukaryotes, we describe the isolation of marker mutations and the successful selection of random recombinant populations after homothallic crosses.
Assuntos
Citoplasma/metabolismo , Dinoflagellida/genética , Dinoflagellida/metabolismo , Modelos Genéticos , Amido/metabolismo , Proteínas de Algas/análise , Proteínas de Algas/metabolismo , Animais , Cruzamentos Genéticos , Dinoflagellida/enzimologia , Dinoflagellida/crescimento & desenvolvimento , Processos Heterotróficos , Mutagênese , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , Recombinação Genética , Amido/isolamento & purificação , Amido/ultraestrutura , Amido Fosforilase/análise , Amido Fosforilase/metabolismo , Sintase do Amido/análise , Sintase do Amido/metabolismo , Uridina Difosfato Glucose/metabolismoRESUMO
After entry into the host cell, the intracellular parasite Toxoplasma gondii resides within a membrane-bound compartment, the parasitophorous vacuole (PV). The PV defines an intracellular, parasite-specific niche surrounded by host organelles, including the Golgi apparatus. The mechanism by which T. gondii hijacks the host Golgi and subverts its functions remains unknown. Here, we present evidence that the dense granule protein TgGRA3 interacts with host Golgi, leading to the formation of tubules and the entry of host Golgi material into the PV. Targeted disruption of the TgGRA3 gene delays this engulfment of host Golgi. We also demonstrate that TgGRA3 oligomerizes and binds directly to host Golgi membranes. In addition, we show that TgGRA3 dysregulates anterograde transport in the host cell, thereby revealing one of the mechanisms employed by T. gondii to recruit host organelles and divert their functions. This article has an associated First Person interview with the first author of the paper.
RESUMO
The protozoan parasite Toxoplasma gondii differentially expresses two distinct enolase isoenzymes known as ENO1 and ENO2, respectively. To understand differential gene expression during tachyzoite to bradyzoite conversion, we have characterized the two T.gondii enolase promoters. No homology could be found between these sequences and no TATA or CCAAT boxes were evident. The differential activation of the ENO1 and ENO2 promoters during tachyzoite to bradyzoite differentiation was investigated by deletion analysis of 5'-flanking regions fused to the chloramphenicol acetyltransferase reporter followed by transient transfection. Our data indicate that in proliferating tachyzoites, the repression of ENO1 involves a negative distal regulatory region (nucleotides -1245 to -625) in the promoter whereas a proximal regulatory region in the ENO2 promoter directs expression at a low level. In contrast, the promoter activity of ENO1 is highly induced following the conversion of tachyzoites into resting bradyzoites. The ENO2 promoter analysis in bradyzoites showed that there are two upstream repression sites (nucleotides -1929 to -1067 and -456 to -222). Furthermore, electrophoresis mobility shift assays demonstrated the presence of DNA-binding proteins in tachyzoite and bradyzoite nuclear lysates that bound to stress response elements (STRE), heat shock-like elements (HSE) and other cis-regulatory elements in the upstream regulatory regions of ENO1 and ENO2. Mutation of the consensus AGGGG sequence, completely abolished protein binding to an oligonucleotide containing this element. This study defines the first characterization of cis-regulatory elements and putative transcription factors involved in gene regulation of the important pathogen T.gondii.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fosfopiruvato Hidratase/genética , Regiões Promotoras Genéticas , Toxoplasma/genética , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Genes de Protozoários , Humanos , Dados de Sequência Molecular , Mutação , Elementos de Resposta , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Ativação TranscricionalRESUMO
In malaria, CD4 Th1 and T follicular helper (TFH) cells are important for controlling parasite growth, but Th1 cells also contribute to immunopathology. Moreover, various regulatory CD4 T-cell subsets are critical to hamper pathology. Yet the antigen-presenting cells controlling Th functionality, as well as the antigens recognized by CD4 T cells, are largely unknown. Here, we characterize the MHC II immunopeptidome presented by DC during blood-stage malaria in mice. We establish the immunodominance hierarchy of 14 MHC II ligands derived from conserved parasite proteins. Immunodominance is shaped differently whether blood stage is preceded or not by liver stage, but the same ETRAMP-specific dominant response develops in both contexts. In naïve mice and at the onset of cerebral malaria, CD8α+ dendritic cells (cDC1) are superior to other DC subsets for MHC II presentation of the ETRAMP epitope. Using in vivo depletion of cDC1, we show that cDC1 promote parasite-specific Th1 cells and inhibit the development of IL-10+ CD4 T cells. This work profiles the P. berghei blood-stage MHC II immunopeptidome, highlights the potency of cDC1 to present malaria antigens on MHC II, and reveals a major role for cDC1 in regulating malaria-specific CD4 T-cell responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Malária Cerebral/imunologia , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Cromatografia Líquida de Alta Pressão , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Antígenos de Histocompatibilidade Classe II/química , Imunoprecipitação , Interferon gama/metabolismo , Interleucina-10/metabolismo , Malária Cerebral/patologia , Malária Cerebral/veterinária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/análise , Peptídeos/imunologia , Plasmodium berghei/imunologia , Células Th1/citologia , Células Th1/metabolismo , Células Th1/parasitologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pathogens have evolved a range of mechanisms to counteract host defenses, notably to survive harsh acidic conditions in phagosomes. In the case of Mycobacterium tuberculosis, it has been shown that regulation of phagosome acidification could be achieved by interfering with the retention of the V-ATPase complexes at the vacuole. Here, we present evidence that M. tuberculosis resorts to yet another strategy to control phagosomal acidification, interfering with host suppressor of cytokine signaling (SOCS) protein functions. More precisely, we show that infection of macrophages with M. tuberculosis leads to granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion, inducing STAT5-mediated expression of cytokine-inducible SH2-containing protein (CISH), which selectively targets the V-ATPase catalytic subunit A for ubiquitination and degradation by the proteasome. Consistently, we show that inhibition of CISH expression leads to reduced replication of M. tuberculosis in macrophages. Our findings further broaden the molecular understanding of mechanisms deployed by bacteria to survive.
Assuntos
Mycobacterium tuberculosis/patogenicidade , Fagossomos/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Camundongos , Mycobacterium tuberculosis/metabolismo , Transdução de SinaisRESUMO
Mycobacterium tuberculosis (Mtb) encodes several bacterial effectors impacting the colonization of phagocytes. LppM (Rv2171) is both implicated in phagocytosis and able to efficiently block phagosomal acidification in the macrophage, two key processes contributing to Mtb persistence. We show that LppM is anchored to the mycobacterial cell wall by a C-terminal membrane domain. However, the protein also exists as a truncated protein secreted into the culture medium. The LppM solution structure we solve here displays no similarity with other Mtb lipoproteins also involved in phagosomal maturation (i.e., LprG). In addition, we demonstrate that the protein may be able to bind rare molecular species of phosphatidylinositol mannosides, bacterial compounds known to affect the host immune response. Thus, our data demonstrate a dual localization of LppM and provide a unique perspective on the regulation of protein secretion and localization in Mtb.