RESUMO
Apoptosis has been found to help in the defence against pathogens. Infection with the obligate intracellular parasite Toxoplasma gondii is known to trigger host-cell apoptosis. When using a T. gondii-infected macrophage cell line, J774A.1, treatment with IFN-gamma significantly enhanced apoptosis in noninfected bystander cells while parasitized cells became relatively resistant. Infection and IFN-gamma treatment activated the expression of inducible nitric oxide synthase (iNOS), and the production of nitric oxide (NO) and treatment of cells with an iNOS inhibitor, N(G)-monomethlyl-L-arginine acetate (L-NMMA) reduced the apoptosis frequency. However, the reversal was only partial suggesting that not only NO, but also other, as of yet, unknown factors are induced. Finally, we studied the effect in vivo by infecting mice with either a virulent or an avirulent strain. Challenge with the virulent strain lead to a higher parasite burden, induced host-cell apoptosis in peritoneal cells, and produced higher levels of IFN-gamma and NO. Moreover, treatment of mice with a NO synthase inhibitor, aminoguanidine, partially inhibited the host-cell apoptosis induced by the parasite infection. Altogether, our findings indicate that apoptosis in bystander host cells is due to the secretion of NO and other soluble factors released by parasite-infected cells.
Assuntos
Apoptose , Interferon gama/biossíntese , Macrófagos/fisiologia , Óxido Nítrico/biossíntese , Toxoplasma/patogenicidade , Animais , Linhagem Celular , Feminino , Interações Hospedeiro-Parasita , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/imunologia , Toxoplasmose/parasitologiaRESUMO
A fundamental property of any eukaryotic cell is endocytosis, that is the ability to take up external fluid, solutes and particulate matter into membrane-bound intracellular vesicles by various mechanisms. Toxoplasma gondii is an intracellular protozoan parasite of the phylum Apicomplexa with a wide geographical and host range distribution. Significant progress in studying the cell biology of this parasite has been accomplished over the last few years. Only recently endocytic compartments and endocytic trafficking have come to a closer dissection in T. gondii. In this review, we discuss the evidence for an endocytic compartment and present a model for an endocytic pathway in Toxoplasma against a background of endocytosis in kinetoplastida and the extensive insights gained from mammalian and yeast cells.
Assuntos
Endocitose/fisiologia , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Animais , Apicomplexa/citologia , Apicomplexa/metabolismo , Apicomplexa/fisiologia , Endossomos , Humanos , Kinetoplastida/citologia , Kinetoplastida/metabolismo , Kinetoplastida/fisiologia , Toxoplasma/citologia , Toxoplasma/metabolismo , Toxoplasmose/parasitologiaRESUMO
Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/fisiologia , Toxoplasma/química , Vacúolos/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetinae , Citoplasma/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Matriz Nuclear/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Frações Subcelulares , Transfecção , Tripsina/farmacologiaRESUMO
Toxoplasma gondii dense granules are morphologically similar to dense matrix granules in specialized secretory cells, yet are secreted in a constitutive, calcium-independent fashion. We previously demonstrated that secretion of dense granule proteins in permeabilized parasites was augmented by the non-hydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (Chaturvedi, S., Qi, H., Coleman, D. L., Hanson, P., Rodriguez, A., and Joiner, K. A. (1998) J. Biol. Chem. 274, 2424-2431). As now demonstrated by pharmacological and electron microscopic approaches, GTPgammaS enhanced release of dense granule proteins in the permeabilized cell system. To investigate the role of ADP-ribosylation factor 1 (ARF1) in this process, a cDNA encoding T. gondii ARF1 (TgARF1) was isolated. Endogenous and transgenic TgARF1 localized to the Golgi of T. gondii, but not to dense granules. An epitope-tagged mutant of TgARF1 predicted to be impaired in GTP hydrolysis (Q71L) partially dispersed the Golgi signal, with localization to scattered vesicles, whereas a mutant impaired in nucleotide binding (T31N) was cytosolic in location. Both mutants caused partial dispersion of a Golgi/trans-Golgi network marker. TgARF1 mutants inhibited delivery of the secretory reporter, Escherichia coli alkaline phosphatase, to dense granules, precluding an in vivo assessment of the role of TgARF1 in release of intact dense granules. To circumvent this limitation, recombinant TgARF1 was purified using two separate approaches, and used in the permeabilized cell assay. TgARF1 protein purified on a Cibacron G3 column and able to bind GTP stimulated dense granule secretion in the permeabilized cell secretion assay. These results are the first to show that ARF1 can augment release of constitutively secreted vesicles at the target membrane.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Fator 1 de Ribosilação do ADP/genética , Sequência de Aminoácidos , Animais , Complexo de Golgi/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vesículas Secretórias/metabolismo , Toxoplasma/genética , Toxoplasma/ultraestruturaRESUMO
The adequacy of fellowship training in the field of infectious diseases was assessed by means of a survey of recently graduated fellows. Surveys were mailed to all individuals who had passed the American Board of Internal Medicine's board certification examination in infectious diseases since 1992. A total of 666 completed surveys were returned by the deadline (response rate, 36%). Although most recent graduates thought that training in the standard components of clinical infectious diseases was adequate, only 50% thought that training in infection control was adequate. Fewer than 1 in 3 believed that they had received adequate training in the business aspects of infectious diseases practice. The adequacy and duration of research training were linked to ultimate career choice. These results form the basis for the Infectious Diseases Society of America's new initiatives to assist with more-diversified and relevant fellowship training.
Assuntos
Educação de Pós-Graduação em Medicina , Bolsas de Estudo , Avaliação de Programas e Projetos de Saúde , Coleta de Dados , Feminino , Apoio Financeiro , Humanos , Controle de Infecções/tendências , Masculino , Microbiologia/educação , Estados UnidosRESUMO
Apicomplexan parasites possess a highly specialized secretory apparatus. The timed secretion of proteins from three different organelles--micronemes, rhoptries and dense granules--serves to establish and maintain a parasitophorous vacuole inside the host cell in which the parasites can divide. Recent efforts have identified components that sort apicomplexan proteins to these unusual secretory organelles and have shown that this machinery is evolutionarily conserved across species. Concise amino acid sequences (e.g. tyrosine-based motifs) within the targeted protein determine their destination in Apicomplexa in a way similar to mammalian cells. Additionally, the parasite exploits new or unusual mechanisms of protein targeting (e.g. post-secretory membrane insertion).
Assuntos
Organelas/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Animais , Camundongos , Transporte Proteico , Toxoplasma/patogenicidade , Toxoplasmose/parasitologiaRESUMO
We have previously shown that an antisense RNA strategy can be used to inhibit the expression of hypoxanthine-xanthine-guanine-phosphoribosyltransferase (HXGPRT) in Toxoplasma gondii [Nakaar et al., J. Biol. Chem. 1999;274:5083-5087]. Here, we report that parasites rendered deficient in HXGPRT by antisense RNA are resistant to high doses of 6-thioxanthine (6-TX). We have exploited this finding to develop a selection procedure. In this scheme, parasites transfected with a chimeric construct harboring the bacterial chloramphenicol acetyl transferase (CAT) reporter gene linked to antisense HXGPRT gene were selected in 6-TX to inhibit the growth of tachyzoites expressing endogenous HXGPRT. Concomitant with a reduction in HXGPRT levels by antisense RNA, 6-TX(R) parasites displayed reporter CAT activity. These data indicate that transfection of antisense HXGPRT gene provides a means to select for parasites expressing foreign or altered genes in T. gondii. These findings also suggest, in principle, that antisense RNA can be used as a strategy to generate selectable markers employing genes that encode enzymes with known subversive substrates.
Assuntos
Pentosiltransferases/genética , Pentosiltransferases/metabolismo , RNA Antissenso/metabolismo , Seleção Genética , Toxoplasma/genética , Animais , Antimetabólitos/farmacologia , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Resistência a Medicamentos , Genes Reporter , Humanos , Immunoblotting , Plasmídeos , RNA Antissenso/genética , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , Toxoplasma/crescimento & desenvolvimento , Transfecção , Xantinas/farmacologiaRESUMO
Protein targeting in malaria parasites is a complex process, involving several cellular compartments that distinguish these cells from more familiar systems, such as yeast or mammals. At least a dozen distinct protein destinations are known. The best studied of these is the vestigial chloroplast (the apicoplast), but new tools promise rapid progress in understanding how Plasmodium falciparum and related apicomplexan parasites traffic proteins to their invasion-related organelles, and how they modify the host by trafficking proteins into its cytoplasm and plasma membrane. Here, Giel van Dooren and colleagues discuss recent insights into protein targeting via the secretory pathway in this fascinating and important system. This topic emerged as a major theme at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000.
Assuntos
Malária Falciparum/parasitologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , Animais , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Organelas/metabolismo , Plasmodium falciparum/metabolismo , Transporte Proteico , VirulênciaRESUMO
Intracellular pathogens have evolved distinct strategies to subvert host cell defenses. At diametrically opposed ends of the spectrum with regard to the host endosomal/lysosomal defenses are the obligate intracellular protozoan Toxoplasma gondii and the bacterium Coxiella burnetti. While the intracellular replication of T. gondii requires complete avoidance of the host endocytic cascade, C. burnetti actively subverts it. This results in these organisms establishing and growing in very different vacuolar compartments. In this study we examined the potential interaction between these distinct compartments following coinfection of mammalian fibroblasts. When present within the same cell, these organisms exhibit minimal interaction with each other. Colocalization of T. gondii and C. burnetti within the same vacuole occurs at a low frequency in doubly infected cells. In such instances only one of the organisms appears to be replication competent, emphasizing the different requirements for survival and/or intracellular growth. The potential basis for both the lack of interaction between these distinct pathogen-containing compartments, and the mechanisms to address their low frequency of colocalization are discussed in the context of our understanding of the biology of the organisms and membrane traffic in eukaryotic cells.
Assuntos
Coxiella/patogenicidade , Fibroblastos/microbiologia , Fibroblastos/parasitologia , Toxoplasma/patogenicidade , Animais , Compartimento Celular , Linhagem Celular , Chlorocebus aethiops , Coxiella/crescimento & desenvolvimento , Humanos , Lisossomos/ultraestrutura , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Fagossomos/ultraestrutura , Superinfecção , Toxoplasma/crescimento & desenvolvimento , Vacúolos/ultraestrutura , Células VeroAssuntos
Nucleotídeos de Adenina/metabolismo , Adenosina/metabolismo , Purinas/metabolismo , Toxoplasma/metabolismo , 5'-Nucleotidase/química , 5'-Nucleotidase/genética , Animais , Sequência Conservada , Primers do DNA/química , Histocitoquímica , Reação em Cadeia da Polimerase , Alinhamento de SequênciaRESUMO
Intracellular parasites of the phylum Apicomplexa contain specialized rhoptry secretory organelles that have a crucial function in host-cell invasion and establishment of the parasitophorous vacuole. Here we show that localization of the Toxoplasma gondii rhoptry protein ROP2 is dependent on a YEQL sequence in the cytoplasmic tail that binds to micro-chain subunits of T. gondii and mammalian adaptors, and conforms to the YXXstraight phi mammalian sorting motif. Chimaeric reporters, containing the transmembrane domains and cytoplasmic tails of the low-density lipoprotein receptor and of Lamp-1, are sorted to the Golgi or the trans-Golgi network (TGN), and partially to apical microneme organelles of the parasite, respectively. Targeting of these reporters is mediated by YXXstraight phi- and NPXY-type signals. This is the first demonstration of tyrosine-dependent sorting in protozoan parasites, indicating that T. gondii proteins may be targeted to, and involved in biogenesis of, morphologically unique organelles through the use of evolutionarily conserved signals and machinery.
Assuntos
Sequência Conservada/fisiologia , Evolução Molecular , Organelas/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Toxoplasma/citologia , Toxoplasma/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Transporte Biológico , Clatrina/metabolismo , Sequência Conservada/genética , Imunofluorescência , Complexo de Golgi/metabolismo , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Organelas/ultraestrutura , Ligação Proteica , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Receptores de LDL/química , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/genética , Toxoplasma/ultraestrutura , Técnicas do Sistema de Duplo-Híbrido , Tirosina/genética , Tirosina/metabolismoRESUMO
The protozoan parasite Toxoplasma gondii has a very specialized secretory apparatus. In this review we will discuss how different methodological approaches, including morphological studies, biochemistry and genetics, are revealing a novel secretory organization, whose function is performed, mainly, by a highly conserved protein machinery found in mammalian cells.
Assuntos
Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Animais , Células Eucarióticas/metabolismo , Humanos , Organelas/metabolismoRESUMO
The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.
Assuntos
Colesterol/metabolismo , Endocitose , Receptores de LDL/metabolismo , Toxoplasma/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Colesterol/biossíntese , Colesterol/farmacologia , Cricetinae , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Endocitose/efeitos dos fármacos , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/fisiologia , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Ácido Mevalônico/metabolismo , Modelos Biológicos , Mutação/genética , Receptores de LDL/antagonistas & inibidores , Reprodução/efeitos dos fármacos , Toxoplasma/citologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/crescimento & desenvolvimento , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Toxoplasma gondii uses a highly coordinated arsenal of three structurally and biochemically distinct secretory granules to invade and develop in a wide range of host cells. Proteins of these secretory granules are sorted to strategic subcellular locations using distinctive sorting signals and are then triggered differentially for exocytosis. These secreted proteins are subsequently targeted and inserted into membrane domains.
Assuntos
Interações Hospedeiro-Parasita , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Animais , Apicomplexa , Grânulos Citoplasmáticos , Humanos , Sinais Direcionadores de Proteínas , Toxoplasma/metabolismo , Toxoplasmose/metabolismoRESUMO
We sought to identify and characterize peroxisomes in the apicomplexan parasite Toxoplasma gondii. To initiate this process, we first cloned and sequenced the gene for T. gondii catalase (EC 1. 11.1.6), a marker enzyme for peroxisomes in eukaryotic cells. The gene predicts a protein of 57.2 kDa and 502 amino acids and has a strong homology to other eukaryotic catalases. A polyclonal antiserum raised against a glutathione S-transferase fusion protein recognized a single band with a molecular mass of 63 kDa by immunoblot. By immunofluorescence T. gondii catalase is present primarily in a punctate staining pattern anterior to the parasite nucleus. This compartment is distinguishable from other parasite organelles, namely micronemes, rhoptries, dense granules, and the apicoplast. Cytochemical visualization of catalase using diaminobenzidine precipitation gives a vesicular staining pattern anterior to the nucleus at the light level and round, vesicular structures with an estimated diameter of 100-300 nm by electron microscopy. T. gondii catalase has a putative C-terminal peroxisomal targeting signal in the last 3 amino acids (-AKM). Expression of T. gondii catalase in mammalian cells results in peroxisomal localization, whereas a construct lacking the targeting signal remains in the cytosol. Furthermore, addition of -AKM to the C terminus of chloramphenicol acetyltransferase is sufficient to target this protein to peroxisomes. These results provide the first evidence for peroxisomes in Apicomplexan parasites.
Assuntos
Catalase/genética , Catalase/metabolismo , Peroxissomos/enzimologia , Toxoplasma/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/enzimologia , Catalase/química , Primers do DNA , Humanos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Organelas/enzimologia , Organelas/ultraestrutura , Peroxissomos/ultraestrutura , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Frações Subcelulares/enzimologia , Toxoplasma/genética , Toxoplasma/ultraestruturaRESUMO
In mammalian cells and yeasts, amino acid motifs in the cytoplasmic tails of transmembrane proteins play a prominent role in protein targeting in the early secretory pathway by mediating localization to or rapid export from the endoplasmic reticulum (ER). However, early sorting events are poorly characterized in protozoan parasites. Here, we show that a C-terminal QKTT sequence mediates the ER localization of chimeric reporter constructs consisting of bacterial alkaline phosphatase (BAP) fused to the transmembrane domain (TMD) and truncated cytoplasmic tail of the human low-density lipoprotein receptor (LDL) receptor or of murine lysosome-associated membrane protein (lamp-1) in Toxoplasma gondii. The cytoplasmic tail of human TGN46 also determines ER localization of BAP chimeras in the parasite, but this can be overcome by the addition at the C-terminus of the tail of an acidic patch, which functions as an ER export signal in conjunction with an upstream tyrosine motif. These results suggest that COPI-dependent ER retrieval and COPII-dependent export mechanisms mediated by KKXX and DXE motifs of mammalian cells are generally conserved in T. gondii. In contrast, the failure of the QKTT motif and TGN46 cytoplasmic tail to induce steady-state ER localization of vesicular stomatitis virus glycoprotein (VSVG) chimeras in HeLa and NRK cells indicates that significant differences in early secretory trafficking also exist.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Toxoplasma/patogenicidade , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Bactérias/enzimologia , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Proteínas Ligadas por GPI , Genes Reporter , Humanos , Proteínas de Membrana/genética , Ratos , Receptores de LDL/química , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Transfecção , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The expression and distribution of dense granule proteins in the enteric (coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Experimental Parasitology 91, 203-211. The expression and location of the dense granule proteins (GRA1-6 and NTPase) in the merozoite and during asexual and sexual development of Toxoplasma gondii in the small intestine of the cat (definitive host) was examined by immuno-light and electron microscopy. This was compared with that of tachyzoites and bradyzoites present in the intermediate host. It was found that the merozoite contained the characteristic apical organelles plus a few large dense granules. By immunocytochemistry, dense granules in merozoites were negative for GRA proteins 1 to 6 in contrast to both tachyzoites and bradyzoites in which dense granules were positive for all six proteins. The GRA proteins were associated with the parasitophorous vacuole (PV) during tachyzoite and bradyzoite development but were absent from the PV of the enteric stages. However, the merozoite dense granules were positive for NTPase, which was similar to the tachyzoite while this antigen was down regulated in the bradyzoite. The apparent release of the NTPases into the PV formed by merozoites was also similar to that described for the tachyzoite, possibly reflecting the relative metabolic activity of the various stages. This study shows that the majority of GRA proteins have a similar stage-specific expression, which is independent of NTPases expression. These observations are consistent with T. gondii having a different host parasite relationship in the enteric forms, which does not involve the GRA proteins 1-6.
Assuntos
Doenças do Gato/parasitologia , Intestino Delgado/parasitologia , Proteínas de Protozoários/biossíntese , Toxoplasma/metabolismo , Toxoplasmose Animal/parasitologia , Hidrolases Anidrido Ácido/análise , Hidrolases Anidrido Ácido/biossíntese , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/biossíntese , Gatos , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Imunoeletrônica/veterinária , Nucleosídeo-Trifosfatase , Proteínas de Protozoários/análise , Toxoplasma/enzimologia , Toxoplasma/imunologia , Toxoplasma/ultraestrutura , Vacúolos/química , Vacúolos/metabolismoRESUMO
Nucleoside triphosphate hydrolase (NTPase) is a very abundant protein secreted by the obligate intracellular parasite Toxoplasma gondii shortly after invasion of the host cell. When activated by dithiols, NTPase is one of the most potent apyrases known to date, but its physiological function remains unknown. The genes encoding NTPase have been cloned (Bermudes, D., Peck, K. R., Afifi-Afifi, M., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260). We have recently shown that the enzyme is tightly controlled within the vacuolar space and may influence parasite exit from the host cell (Silverman, J. A., Qi, H., Riehl, A., Beckers, C., Nakaar, V., and Joiner, K. A (1998) J. Biol. Chem. 273, 12352-12359). In the present study, we have generated an antisense NTP RNA construct in which the 3'-untranslated region is replaced by a hammerhead ribozyme. The constitutive synthesis of the chimeric antisense RNA-ribozyme construct in parasites that were stably transfected with this construct resulted in a dramatic reduction in the steady-state levels of NTPase. This inhibition was accompanied by a decrease in the capacity of the parasites to replicate. The reduction in parasite proliferation was due to a specific effect of antisense NTP RNA, since a drastic inhibition of hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) expression by a chimeric antisense HXGPRT RNA-ribozyme construct did not alter NTPase expression nor compromise parasite replication. These data implicate NTPase in an essential parasite function and suggest that NTPase may have more than one function in vivo. These results also establish that it is possible to study gene function in apicomplexan parasites using antisense RNA coupled to ribozymes.
Assuntos
Hidrolases Anidrido Ácido/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , RNA Antissenso/farmacologia , Toxoplasma/fisiologia , Hidrolases Anidrido Ácido/genética , Animais , RNA Antissenso/genética , Toxoplasma/enzimologia , Toxoplasma/genéticaRESUMO
The signals and the molecular machinery mediating release of dense matrix granules from pathogenic protozoan parasites are unknown. We compared the secretion of the endogenous dense granule marker GRA3 in Toxoplasma gondii with the release of a stably transfected foreign reporter, beta-lactamase, that localizes to parasite dense granules. Both proteins were released constitutively in a calcium-independent fashion, as shown using both intact and streptolysin O-permeabilized parasites. N-Ethylmaleimide and recombinant bovine Rab-guanine dissociation inhibitor inhibited beta-lactamase secretion in permeabilized parasites, whereas recombinant hamster N-ethylmaleimide-sensitive fusion protein and bovine alpha-SNAP augmented release. Guanosine 5'-3-O-(thio)triphosphate, but not cAMP, augmented secretion in the presence but not in the absence of ATP. The T. gondii NSF/SNAP/SNARE/Rab machinery participates in dense granule release using parasite protein components that can interact functionally with their mammalian homologues.