RESUMO
OBJECTIVES: The olfactory decline that often accompanies aging is thought to contribute to undernutrition in older adults. It is believed to negatively affect eating pleasure, appetite, food intake and subsequently nutritional status. We have evaluated the associations of olfactory function with BMI, appetite and prospective weight change in a cohort of Dutch community-dwelling older adults. DESIGN: Cross-sectional cohort study. PARTICIPANTS: Dutch community-dwelling older adults from the ongoing Longitudinal Aging Study Amsterdam (LASA). Measurements and setting: In 2012-2013, the 40-item University of Pennsylvania Smell Identification Test (UPSIT) was administered to 824 LASA participants to evaluate their olfactory function. Body weight, height, appetite, comorbidity, cognitive status and socio-demographic factors were also assessed. Follow-up weight was measured after three years. RESULTS: 673 participants (aged 55-65 years) were included in the regression analyses. Median UPSIT-score was 33. When adjusted for potential confounders, lower UPSIT-score (indicative of poorer olfactory function) was not associated with poor appetite (OR = 1.062, p = 0.137) or prospective weight change (B = -0.027, p = 0.548). It was, however, associated with lower BMI in smokers (B = 0.178, p = 0.032), but not in non-smokers (B = -0.015, p = 0.732). CONCLUSION: Lower olfactory function scores were associated with lower BMI in community-dwelling older adults who smoke, but not with appetite or prospective weight change. Therefore, smoking older adults with olfactory impairments may pose as a vulnerable group with respect to developing undernutrition.
Assuntos
Apetite/fisiologia , Índice de Massa Corporal , Peso Corporal/fisiologia , Transtornos do Olfato/etiologia , Idoso , Envelhecimento , Estudos de Coortes , Estudos Transversais , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Países Baixos , Estudos ProspectivosRESUMO
Primary infection of IL-10 knockout (KO) mice with the protozoan parasite Toxoplasma gondii leads to a CD4(+)-T-cell dependent shock-like reaction with high systemic levels of IL-12 and IFN-gamma, severe liver pathology and death of mice. In the present study, this immune-mediated pathology was prevented by treatment of IL-10 KO mice with the anti-parasitic drug sulfadiazine, allowing these mice to progress to the chronic phase of infection. To address the role of endogenous IL-10 in the regulation of secondary immune responses to T. gondii, IL-10 KO mice were infected with the avirulent Me49 strain of this parasite, treated with sulfadiazine for 2 weeks starting at day 3 p.i., and were rechallenged 6 weeks p.i. with RH, a highly virulent strain of T. gondii. In these studies, chronically infected IL-10 KO mice survived secondary infection with RH and controlled parasite load. Although serum levels of IL-12 and IFN-gamma were higher in IL-10 KO mice than in wild type (WT) mice 8 days after RH rechallenge, these levels were well controlled in the absence of endogenous IL-10, suggesting that IL-10 is not required to down-regulate cytokine production during the memory response. Antigen-specific ex vivo recall responses further revealed that splenocytes from chronically infected WT and IL-10 KO mice responded to parasite antigen with similar production of IL-12 and IFN-gamma, and there was also no significant difference in ex vivo production of these cytokines by splenocytes in response to parasite antigen 7 days after secondary infection with T. gondii. Furthermore, IL-10 KO mice immunized with the Ts-4 vaccine-strain of T. gondii were protected when rechallenged with the virulent RH strain. Together, these studies demonstrate that the inhibitory effect of IL-10, which is required to prevent immune-mediated pathology during primary infection, is not required to prevent immune hyperactivity during a secondary response to T. gondii, and a highly effective memory response is generated in the absence of endogenous IL-10.
Assuntos
Memória Imunológica/imunologia , Interleucina-10/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Antiprotozoários/uso terapêutico , Encéfalo/parasitologia , Feminino , Histocitoquímica , Interferon gama/imunologia , Interleucina-10/deficiência , Interleucina-12/imunologia , Subunidade p40 da Interleucina-12 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Cavidade Peritoneal/parasitologia , Subunidades Proteicas/imunologia , Estatísticas não Paramétricas , Sulfadiazina/uso terapêutico , Toxoplasmose/tratamento farmacológico , Toxoplasmose/parasitologia , Toxoplasmose/patologiaRESUMO
Tachyzoite endodyogeny is characterized by a three phase cell cycle comprised of major G1 and S phases with mitosis following immediately upon the conclusion of DNA replication. Cytokinesis, which begins with the formation of daughter apical complexes, initiates in late S phase and overlaps mitosis. There is no evidence to support an extended G2 period in these parasites. In all strains, parasites with a 2 N DNA content are a relatively small subpopulation and when tachyzoites expressing a fluorescent nuclear marker (green-fluorescent-protein fused to proliferating-cell-nuclear-antigen) were observed by time-lapse microscopy, there appeared to be little delay between S phase and mitosis. Measurements of the DNA content of RH parasites by flow cytometry demonstrated that the G1 and S periods were approximately 60 and approximately 30% of a single division cycle, although these phases were longer in strains that display a slower growth rate. The overall length of S phase was determined by [3H]-thymidine autoradiography using transgenic parasites expressing herpes simplex thymidine kinase and validated by Northern analysis of S phase specific genes during synchronous growth. The fraction of S phase parasites by flow cytometry paralleled autoradiography, however, within S phase, the distribution of parasites was bimodal in all strains examined. Parasites containing a 1-1.7 N DNA complement were a small fraction when compared to the major S phase population which contained a near-diploid ( approximately 1.8 N) complement, suggesting parasites in late S phase have a slower rate of DNA replication. In lieu of a short or missing G2, where checkpoints are thought to operate in other eukaryotes, the bimodal replication of tachyzoite chromosomes may represent a distinct premitotic checkpoint associated with endodyogeny.
Assuntos
Ciclo Celular/fisiologia , Toxoplasma/crescimento & desenvolvimento , Animais , Divisão Celular , DNA de Protozoário/análise , Citometria de Fluxo , Imunofluorescência , Fase G1 , Fase G2 , Expressão Gênica , Mitose , Antígeno Nuclear de Célula em Proliferação/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fase S , Toxoplasma/citologia , Toxoplasma/genéticaRESUMO
Interleukin (IL)-10 is an inhibitor of cell mediated immunity and an antagonist of the development of protective immune responses associated with resistance to T. gondii. These observations led to the hypothesis that the production of IL-10 could contribute to the ability of T. gondii to replicate and survive in an immune competent host. To determine whether the production of IL-10 affects the ability of the RH strain of T. gondii to cause a lethal infection in mice, we compared the immune response to RH in IL-10+/+ and IL-10-/- BALB/c mice. Both groups of mice produced comparable amounts of IL-12 and interferon (IFN)-gamma and had similar mortality curves and parasite burdens. The use of green fluorescent protein-labelled parasites allowed us to infect IL-10+/+ and IL-10-/- mice and use a fluorescence-activated cell sorter to distinguish infected and uninfected populations of macrophages and compare their expression of CD80, CD86 and major histocompatibility complex (MHC) class II. Although infected cells expressed higher overall levels of these molecules than uninfected cells, there were no differences between cells isolated from IL-10+/+ and IL-10-/- mice. Taken together, these results indicate that IL-10 is not required for the virulence of the RH strain of T. gondii, nor is it involved in the regulation of the CD80, CD86 and MHC class II molecules during RH-infection.
Assuntos
Interleucina-10/fisiologia , Toxoplasma/patogenicidade , Toxoplasmose/imunologia , Animais , Antígenos CD/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-2 , Antígenos de Histocompatibilidade Classe II/biossíntese , Interferon gama/biossíntese , Interleucina-10/deficiência , Interleucina-12/biossíntese , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos KnockoutRESUMO
In the protozoan parasite Toxoplasma gondii, cell division occurs by an unusual internal budding process whereby two daughter cells develop within and eventually subsume the mother cell. We have examined this process using inhibitors targeted at specific events in the cell cycle. By adding inhibitors to newly established parasites we were able to examine the effects of the inhibitors on parasites treated at the start of intracellular development and many hours prior to the onset of daughter cell budding. As with other eukaryotes, inhibitors of nuclear DNA synthesis blocked parasite DNA synthesis and prevented cell division. Examination of parasites treated with the nuclear DNA synthesis inhibitor aphidicolin showed that the formation of daughter apical complexes and the initiation of budding occurred as normal and only the inability of the nucleus to become incorporated into the daughter cells prevented successful cell division. Moreover, these inhibitory effects of aphidicolin were not reversible. The initiation of nuclear DNA synthesis and cell division in newly invaded Toxoplasma required both gene transcription and protein synthesis, although inhibitors of mitochondrial DNA synthesis, transcription and protein synthesis did not block parasite division. Thus, unlike most eukaryotes, Toxoplasma tachyzoites have separated nuclear DNA replication and mitosis from the events associated with cell division (daughter cell budding). This implies that Toxoplasma tachyzoites may have dispensed with specific cell cycle checkpoints present in other eukaryotes with, in particular, a DNA-replication checkpoint control either missing, or downregulated in this stage of the parasite life cycle.
Assuntos
Replicação do DNA , DNA de Protozoário/biossíntese , Toxoplasma/citologia , Toxoplasma/crescimento & desenvolvimento , Animais , Afidicolina/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Inibidores do Crescimento/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Organelas/efeitos dos fármacos , Organelas/fisiologia , Organelas/ultraestrutura , Inibidores da Topoisomerase II , Toxoplasma/efeitos dos fármacos , Toxoplasma/ultraestrutura , Transcrição Gênica , Vacúolos/efeitos dos fármacosRESUMO
The apicoplast is a distinctive organelle associated with apicomplexan parasites, including Plasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant "poison" that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity.
Assuntos
Núcleo Celular/metabolismo , Citometria de Fluxo/métodos , Plastídeos/genética , Toxoplasma/genética , Animais , Membrana Celular/metabolismo , Separação Celular , Cloranfenicol O-Acetiltransferase/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Plasmídeos/metabolismo , Plastídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares , Proteína Vermelha FluorescenteRESUMO
Pyrimethamine is a potent inhibitor of dihydrofolate reductase and is widely used in the treatment of opportunistic infections caused by the protozoan parasite Toxoplasma gondii. In order to assess the potential role of dhfr sequence polymorphisms in drug treatment failures, we examined the dhfr-ts genes of representative isolates for T. gondii virulence types I, II, and III. These strains exhibit differences in their sensitivities to pyrimethamine but no differences in predicted dhfr-ts protein sequences. To assess the potential for pyrimethamine-resistant dhfr mutants to emerge, three drug-sensitive variants of the T. gondii dhfr-ts gene (the wild-type T. gondii sequence and two mutants engineered to reflect polymorphisms observed in drug-sensitive Plasmodium falciparum) were subjected to random mutagenesis and transfected into either wild-type T. gondii parasites or dhfr-deficient Saccharomyces cerevisiae under pyrimethamine selection. Three resistance mutations were identified, at amino acid residues 25 (Trp-->Arg), 98 (Leu-->Ser), and 134 (Leu-->His).
Assuntos
Antiprotozoários/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Pirimetamina/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , Alelos , Animais , Animais Geneticamente Modificados , Linhagem Celular , Resistência a Medicamentos , Genes de Protozoários , Mutagênese , Testes de Sensibilidade Parasitária , Mutação Puntual , Saccharomyces cerevisiae/genética , Toxoplasma/genética , TransfecçãoRESUMO
Rhoptry and microneme organelles of the protozoan parasite Toxoplasma gondii are closely associated with host cell adhesion/invasion and establishment of the intracellular parasitophorous vacuole. In order to study the targeting of proteins to these specialized secretory organelles, we have engineered green fluorescent protein (GFP) fusions to the rhoptry protein ROP1 and the microneme protein MIC3. Both chimeras are correctly targeted to the appropriate organelles, permitting deletion analysis to map protein subdomains critical for targeting. The propeptide and a central 146 amino acid region of ROP1 are sufficient to target GFP to the rhoptries. More extensive deletions result in a loss of rhoptry targeting; the GFP reporter is diverted into the parasitophorous vacuole via dense granules. Certain MIC3 deletion mutants were also secreted into the parasitophorous vacuole via dense granules, supporting the view that this route constitutes the default pathway in T. gondii, and that specific signals are required for sorting to rhoptries and micronemes. Deletions within the cysteine-rich central region of MIC3 cause this protein to be arrested at various locations within the secretory pathway, presumably due to improper folding. Although correctly targeted to the appropriate organelles in living parasites, ROP1-GFP and MIC3-GFP fusion proteins were not secreted during invasion. GFP fusion proteins were readily secreted from dense granules, however, suggesting that protein secretion from rhoptries and micronemes might involve more than a simple release of organellar contents.
Assuntos
Adesinas Bacterianas , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Adesão Celular , Células Cultivadas , Deleção de Genes , Proteínas de Fluorescência Verde , Humanos , Immunoblotting , Indicadores e Reagentes , Proteínas Luminescentes , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação , Organelas/metabolismo , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Toxoplasma/genética , Transfecção , Proteína Vermelha FluorescenteRESUMO
The phylum Apicomplexa encompasses a large number of intracellular protozoan parasites, including the causative agents of malaria (Plasmodium), toxoplasmosis (Toxoplasma), and many other human and animal diseases. Apicomplexa have recently been found to contain a relic, nonphotosynthetic plastid that has attracted considerable interest as a possible target for therapeutics. This plastid is known to have been acquired by secondary endosymbiosis, but when this occurred and from which type of alga it was acquired remain uncertain. Based on the molecular phylogeny of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, we provide evidence that the apicomplexan plastid is homologous to plastids found in dinoflagellates-close relatives of apicomplexa that contain secondary plastids of red algal origin. Surprisingly, apicomplexan and dinoflagellate plastid-targeted GAPDH sequences were also found to be closely related to the plastid-targeted GAPDH genes of heterokonts and cryptomonads, two other groups that contain secondary plastids of red algal origin. These results address several outstanding issues: (1) apicomplexan and dinoflagellate plastids appear to be the result of a single endosymbiotic event which occurred relatively early in eukaryotic evolution, also giving rise to the plastids of heterokonts and perhaps cryptomonads; (2) apicomplexan plastids are derived from a red algal ancestor; and (3) the ancestral state of apicomplexan parasites was photosynthetic.
Assuntos
Dinoflagellida/genética , Eucariotos/genética , Marcação de Genes , Plastídeos , Animais , Sequência de Bases , Primers do DNA , Gliceraldeído-3-Fosfato Desidrogenases/genética , Dados de Sequência Molecular , FilogeniaRESUMO
Apicomplexan parasites such as Toxoplasma gondii contain a primitive plastid, the apicoplast, whose genome consists of a 35-kb circular DNA related to the plastid DNA of plants. Plants synthesize fatty acids in their plastids. The first committed step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC). This enzyme is encoded in the nucleus, synthesized in the cytosol, and transported into the plastid. In the present work, two genes encoding ACC from T. gondii were cloned and the gene structure was determined. Both ORFs encode multidomain proteins, each with an N-terminal extension, compared with the cytosolic ACCs from plants. The N-terminal extension of one isozyme, ACC1, was shown to target green fluorescent protein to the apicoplast of T. gondii. In addition, the apicoplast contains a biotinylated protein, consistent with the assertion that ACC1 is localized there. The second ACC in T. gondii appears to be cytosolic. T. gondii mitochondria also contain a biotinylated protein, probably pyruvate carboxylase. These results confirm the essential nature of the apicoplast and explain the inhibition of parasite growth in cultured cells by herbicides targeting ACC.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Frações Subcelulares/enzimologia , Toxoplasma/enzimologia , Acetil-CoA Carboxilase/química , Acetil-CoA Carboxilase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , DNA Complementar , Ácidos Graxos/biossíntese , Genoma de Protozoário , Microscopia de Fluorescência , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxoplasma/genéticaRESUMO
Apicomplexan parasites--including the causative agents of malaria (Plasmodium sp.) and toxoplasmosis (Toxoplasma gondii)--harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear-encoded apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast-deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence-activated cell sorting, but die in the subsequent host cell, confirming the 'delayed death' phenotype previously described pharmacologically, and validating the apicoplast as essential for parasite viability.
Assuntos
Plastídeos/genética , Toxoplasma/genética , Animais , Eucariotos/genética , Genes de Protozoários , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Microscopia Eletrônica , Mutação , Fenótipo , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Simbiose/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/microbiologiaRESUMO
Lactacystin, a specific inhibitor of proteasomes in eukaryotic cells, did not block parasite entry or the establishment of the parasitophorous vacuole, but did inhibit parasite growth and daughter cell budding, as well as DNA synthesis. Two other proteasome inhibitors, MG-132 and proteasome inhibitor 1, also blocked parasite growth and intracellular development. Adding lactacystin to established, dividing parasites, rapidly blocked parasite growth and daughter cell budding at all stages in the process. Pre-treating host cells with lactacystin did not block parasite entry or development. Moreover, under the conditions used, the host cells appeared not to be adversely affected indicating that host cell proteasome activity was not essential for parasite entry or development. Concomitant with these effects on parasite growth and division were morphological changes in the parasite including the appearance of whorls of ER-derived membranes presumably related to the failure to breakdown misfolded proteins. These changes were specific to lactacystin and were not seen in parasites treated with other protease inhibitors. Although the ER-derived structures resembled autophagic bodies, similar structures could not be induced by serum starvation nor did the membranous whorls acidify or undergo morphological changes consistent with autophagosomal maturation. These results highlight the possible role of proteasome activity in Toxoplasma in intracellular development and the regulation of parasite replication. However, how the dividing parasite recycles its organelles and the functional relationship between any lysosomal autophagic pathway and proteasomes in the parasite remains unresolved.
Assuntos
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Leupeptinas/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/parasitologia , Animais , Cisteína Endopeptidases/farmacologia , DNA de Protozoário/biossíntese , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Humanos , Microscopia Eletrônica , Complexos Multienzimáticos/farmacologia , Complexo de Endopeptidases do Proteassoma , Toxoplasma/efeitos dos fármacos , Toxoplasma/ultraestrutura , Vacúolos/efeitos dos fármacosRESUMO
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
Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.
Assuntos
Plasmodium falciparum/metabolismo , Proteínas de Protozoários/fisiologia , Animais , Transporte Biológico , Marcação de Genes , Vacinas Antimaláricas , Organelas/metabolismo , Fragmentos de Peptídeos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/genética , Frações Subcelulares/química , VirulênciaRESUMO
Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gamma phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.
Assuntos
Adenosina Quinase/química , Adenosina Quinase/metabolismo , Trifosfato de Adenosina/análogos & derivados , Adenosina/metabolismo , Toxoplasma/enzimologia , Adenosina Quinase/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Ânions/metabolismo , Antiprotozoários/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Catálise , Sequência Conservada , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Pró-Fármacos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Tubercidina/análogos & derivados , Tubercidina/química , Tubercidina/metabolismo , Água/metabolismoRESUMO
We have used drugs to examine the role(s) of the actin and microtubule cytoskeletons in the intracellular growth and replication of the intracellular protozoan parasite, Toxoplasma gondii. By using a 5 minute infection period and adding the drugs shortly after entry we can treat parasites at the start of intracellular development and 6-8 hours prior to the onset of daughter cell budding. Using this approach we found, somewhat surprisingly, that reagents that perturb the actin cytoskeleton in different ways (cytochalasin D, latrunculin A and jasplakinolide) had little effect on parasite replication although they had the expected effects on the host cells. These actin inhibitors did, however, disrupt the orderly turnover of the mother cell organelles leading to the formation of a large residual body at the posterior end of each pair of budding parasites. Treating established parasite cultures with the actin inhibitors blocked ionophore-induced egression of tachyzoites from the host cells, demonstrating that intracellular parasites were susceptible to the effects of these inhibitors. In contrast, the anti-microtubule drugs oryzalin and taxol, and to a much lesser extent nocodazole, which affect microtubule dynamics in different ways, blocked parasite replication by disrupting the normal assembly of the apical conoid and the microtubule inner membrane complex (IMC) in the budding daughter parasites. Centrosome replication and assembly of intranuclear spindles, however, occurred normally. Thus, daughter cell budding per se is dependent primarily on the parasite microtubule system and does not require a dynamic actin cytoskeleton, although disruption of actin dynamics causes problems in the turnover of parasite organelles.
Assuntos
Actinas/fisiologia , Microtúbulos/fisiologia , Sulfanilamidas , Toxoplasma/citologia , Toxoplasma/crescimento & desenvolvimento , Actinas/antagonistas & inibidores , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Calcimicina/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/parasitologia , Citoesqueleto/ultraestrutura , Dinitrobenzenos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/parasitologia , Fibroblastos/ultraestrutura , Inibidores do Crescimento/farmacologia , Humanos , Ionóforos/antagonistas & inibidores , Ionóforos/farmacologia , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Organelas/efeitos dos fármacos , Organelas/fisiologia , Organelas/ultraestrutura , Paclitaxel/farmacologia , Tiazóis/farmacologia , Tiazolidinas , Toxoplasma/efeitos dos fármacos , Toxoplasma/ultraestruturaRESUMO
Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gi phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.
Assuntos
Adenosina Quinase/química , Adenosina Quinase/metabolismo , Adenosina/metabolismo , Toxoplasma/enzimologia , Adenosina Quinase/antagonistas & inibidores , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Ânions/metabolismo , Antiprotozoários/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Sequência de Bases , Sítios de Ligação , Catálise , Sequência Conservada , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Pró-Fármacos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tubercidina/análogos & derivados , Tubercidina/química , Tubercidina/metabolismo , Água/metabolismoRESUMO
Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.