RESUMO
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
Trypanosoma brucei glycosomes (microbodies containing nine enzymes involved in glycolysis) have been purified to near homogeneity from bloodstream-form trypomastigotes for the purpose of morphologic and biochemical analysis. Differential centrifugation followed by two isopycnic centrifugations in an isotonic Percoll and in a sucrose gradient, respectively, resulted in 12- to 13-fold purified glycosomes with an overall yield of 31%. These glycosomes appeared to be highly pure and contained less than 1% mitochondrial contamination as judged by morphometric and biochemical analyses. In intact cells, glycosomes displayed a remarkably homogeneous size distribution centered on an average diameter of 0.27 micron with a standard deviation of 0.03 micron. The size distribution of isolated glycosomes differed only slightly from that measured in intact cells. One T. brucei cell contained on average 230 glycosomes, representing 4.3% of the total cell volume. The glycosomes were surrounded by a single membrane and contained as phospholipids only phosphatidyl choline and phosphatidyl ethanolamine in a ratio of 2:1. The purified glycosomal fraction had a very low DNA content of 0.18 microgram/mg protein. No DNA molecules were observed that could not have been derived from contaminating mitochondrial or nuclear debris.
Assuntos
Microcorpos/ultraestrutura , Trypanosoma brucei brucei/ultraestrutura , Animais , Fracionamento Celular/métodos , DNA/análise , Glicólise , Microcorpos/enzimologia , Microscopia Eletrônica , Fosfolipídeos/análise , Ratos , Ratos Endogâmicos , Trypanosoma brucei brucei/enzimologiaRESUMO
The dense granules of the intracellular protozoan Toxoplasma gondii are secretory vesicles that play a major role in the structural modifications of the parasitophorous vacuole (PV) in which the parasite develops. The biogenesis of dense granules as well as the regulatory mechanisms controlling their specific exocytosis are still poorly understood. In this paper, we analyzed the secretory pathway of dense granule proteins (GRA proteins) in extracellular T. gondii through the effects of brefeldin A (BFA). Ultrastructural studies of BFA-treated parasites showed disassembly of the Golgi apparatus and accumulation of GRA proteins in a dilated vacuolar system connected to the nuclear envelope. BFA reversibly blocked the intracellular transport of the newly synthesized GRA proteins in a dose-dependent manner (blockade of 95% at 1 microg/ml of BFA). By contrast, discharge of GRA proteins from preformed dense granules was unaffected by BFA over a course of 60 min incubation. GRA protein secretion was dependent on incubation temperature as it only occurred above 26 degrees C and it could be stimulated by external factors. This stimulus might be provided by factor(s) present in the serum of the extracellular medium, as incubation of parasites in serum-free medium resulted in a dramatic decrease in protein secretion. Exocytosis can be restored in a dose-dependent fashion by serum addition (maximal stimulatory activity in the 30-200 kDa range) and was optimal at an extracellular pH of 6.5. Altogether, these results demonstrate that GRA proteins are exported through the Golgi apparatus via the classical secretory pathway and can be experimentally discharged from storage dense granules as regulated secretory proteins in response to specific stimulation, arguing in favor of a regulated component for dense granule exocytosis in T. gondii.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Exocitose , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Exocitose/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Concentração de Íons de Hidrogênio , Temperatura , Células Tumorais Cultivadas/parasitologia , Vacúolos/fisiologiaRESUMO
The growth of the culture-adapted procyclic forms of Trypanosoma brucei (procyclics) is accelerated by supplementation of the medium with low-density lipoprotein (LDL) particles. This effect can be attributed to receptor-mediated endocytosis of LDL, followed by utilization of lipids carried by the lipoproteins. Indeed, procyclics that normally contain ergosterol synthesized de novo, also incorporate exogenous cholesterol in their membranes. In turn, import of exogenous lipids down-regulates the isoprenoid biosynthetic machinery as measured by a approx. 3-fold decrease of [14C]acetate incorporation into sterols and a approx. 2-fold decrease of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, compared with cells grown in lipoprotein-depleted medium. Synvinolin, a specific inhibitor of HMG-CoA reductase that slows down the procyclic growth in vitro and decreases [14C]acetate incorporation into sterols, produces striking morphological modifications, including an arrest at cytokinesis and an extensive swelling of the kinetoplast-mitochondrion system. These cytotoxic effects are amplified in the absence of lipoprotein supply. In conclusion, procyclics may acquire sterols from both exogenous and endogenous sources. To a large extent, these two pathways compensate each other, illustrating adaptation of the parasites to survive in extremely different environments.
Assuntos
Esteróis/metabolismo , Trypanosoma brucei brucei/metabolismo , Adaptação Fisiológica , Animais , Meios de Cultura , Endocitose , Inibidores Enzimáticos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases , Metabolismo dos Lipídeos , Lipoproteínas LDL/metabolismo , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Microscopia Eletrônica , Sinvastatina , Esteróis/biossíntese , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.
Assuntos
Metabolismo dos Carboidratos , Microcorpos/metabolismo , Plantas/parasitologia , Trypanosomatina/metabolismo , Animais , Glucose/metabolismo , Mitocôndrias/metabolismo , Fosfofrutoquinase-1/metabolismo , Piruvato Quinase/metabolismo , Amido/metabolismo , Trypanosomatina/enzimologia , Trypanosomatina/ultraestruturaRESUMO
Activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the key enzyme in the biosynthesis of steroids and polyisoprenoids in mammalian cells, has been detected in both the bloodstream form and the culture-adapted procyclic form of Trypanosoma brucei (3.7 +/- 0.6 and 12.7 +/- 1.8 pmol mevalonate produced min-1 (mg cell protein)-1, respectively). The enzyme activity is enriched 6-fold in microsomal fractions. Several competitive inhibitors of mammalian HMG-CoA reductase, including synvinolin (simvastatin), inhibit the multiplication of both forms of trypanosome in vitro (IC50, approx. 25-50 microM after 2-3 days). This growth inhibition is potentiated by agents interfering with the exogenous supply of cholesterol, such as antibodies blocking the low-density lipoprotein (LDL) receptor, or 5 microM chloroquine. Conversely, growth inhibition by synvinolin can be largely reverted either by 300 nM LDL or by products of the mevalonate pathway, such as 20 mM mevalonate and in procyclics by 100 microM squalene or cholesterol. In procyclics, low concentrations of synvinolin selectively inhibit the incorporation of [14C]acetate into sterols, but not into fatty acids. These results argue for a critical role in trypanosomes of a mevalonate pathway, that is involved in the biosynthesis of sterol and probably of other metabolites. The HMG-CoA reductase activity is decreased 2-fold in procyclics incubated with 4 mM mevalonate and increased 2-fold in the presence of 2.5 microM synvinolin. Synvinolin also upregulates LDL binding up to 4-fold. These data suggest that HMG-CoA reductase and LDL receptor expression are regulated in T. brucei as in mammalian cells, to ensure sterol homeostasis.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Trypanosoma brucei brucei/enzimologia , Acetatos/metabolismo , Animais , Inibidores de Hidroximetilglutaril-CoA Redutases , Lipoproteínas LDL/metabolismo , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Ratos , Ratos Wistar , Sinvastatina , Frações Subcelulares/enzimologiaRESUMO
In the bloodstream form of Trypanosoma brucei, specific receptors mediate the endocytosis of host low-density lipoprotein particles. We have explored the fate of ligand and receptor with a biochemical approach, using inhibitors of the endocytotic apparatus. The weak base chloroquine rapidly accumulates in trypanosomes, its uptake is prevented by the proton ionophore monensin, and it induces the swelling of intracellular vacuoles, indicating that their content is acidic. Cell-associated LDL is rapidly degraded into intermediately sized fragments and TCA-soluble material that can be recovered in cell extracts and extracellular medium. Chloroquine, leupeptin and E64, but not PMSF, efficiently prevent LDL proteolysis, suggesting that degradation occurs in those acidic compartment(s) and is mediated by thiol-protease(s). Both monensin and chloroquine decrease the number of LDL receptors exposed at the cell surface, a phenomenon amplified in the presence of LDL. This provides indirect evidence that internalised LDL receptors are recycled at a rate which is slowed down by receptor occupancy and by agents that impair acidification of the endocytic organelles. Finally, chloroquine decreases by half the growth rate of procyclic trypanosomes in vitro at 5 micrograms ml-1. At 40 mg kg-1 per day, it also slows down the increase in parasitaemia and prolongs the survival time of infected mice by up to 2 days.
Assuntos
Lipoproteínas LDL/metabolismo , Trypanosoma brucei brucei/metabolismo , Ácidos/metabolismo , Animais , Cloroquina/farmacocinética , Cloroquina/farmacologia , Endocitose , Microscopia Eletrônica , Ratos , Ratos Wistar , Receptores de LDL/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/ultraestrutura , Tripanossomíase Africana/parasitologia , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestruturaRESUMO
We have previously shown that an antiserum raised against the 86-kDa fragment of the low-density lipoprotein-receptor (LDL-receptor) of bloodstream Trypanosoma brucei brucei shows extensive cross-reactivity with the mammalian LDL-receptor. Here we report on the production and characterisation of 30 monoclonal antibodies (mAbs) raised against the same 86-kDa fragment of the T. b. brucei LDL-receptor. Of these, only 8 mAbs recognise in an ELISA test the purified (presumably intact) 145-kDa LDL-receptor. Seven of them also recognise the LDL-receptors isolated from rat and rabbit, whereas one mAb (1A9) is specific for the trypanosome LDL-receptor. A pool of several mAbs inhibits by 90% the specific binding of 125I-LDL to trypanosomes at 4 degrees C, but does not interfere with binding of 125I-LDL to rat fibroblasts. 125I-mAb 1A9 is efficiently taken up by T. b. brucei at 30 degrees C and its uptake is inhibited by an excess of unlabelled LDL particles, indicating that mAb 1A9 follows the LDL-receptor pathway. Uptake of 125I-mAb 1A9 by rat fibroblasts is less efficient and is not significantly reduced by an excess of unlabelled LDL. MAb 1A9 as well as other pooled mAbs activate rabbit complement, leading to lysis of trypanosomes in vitro. We conclude that the T. b. brucei LDL-receptor contains at least one specific epitope that is accessible on live cells to antibodies and which can activate the complement system.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Epitopos/imunologia , Receptores de LDL/imunologia , Trypanosoma brucei brucei/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antiprotozoários/farmacologia , Antígenos de Superfície , Ativação do Complemento/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Lipoproteínas LDL/metabolismo , Fragmentos de Peptídeos/imunologia , RatosRESUMO
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
In plasma, a significant part of suramin circulates in tight association with low-density lipoproteins (LDL). At therapeutically obtainable concentrations (100 microM) of suramin, about 85% of the total amount of the drug was bound to proteins, approximately 15% of which was bound to LDL. The molar ratio of suramin bound to LDL in serum was 7.5. The capacity of the high-affinity binding sites of LDL were 6.6 x 10(6) M-1, both in Tris buffer and in ultrafiltrate of serum. Suramin (100 microM) decreased the uptake of host LDL through receptor-mediated endocytosis by Trypanosoma brucei, with approximately 50%. LDL served as the only carrier for suramin uptake. Serum albumin, another important carrier for suramin in blood, was not able to promote suramin uptake, neither was delipidified plasma. The suramin taken up by T. brucei was recovered, in part, in the lysosomal fractions. It is suggested that deprivation of the parasite from cholesterol and phospholipids by an inhibition of the uptake of LDL, contributes to the mode of action of suramin, in addition to the many other effects that the drug may exert on the parasite. The toxic side-effects of suramin on the host are discussed in the light of its association with circulating lipoproteins.
Assuntos
Lipoproteínas LDL/metabolismo , Suramina/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Quimioterapia Combinada , Lipoproteínas LDL/sangue , Ligação Proteica , Suramina/farmacologiaRESUMO
Bloodstream forms of Trypanosoma brucei do not synthesize sterols de novo and therefore cannot survive in medium devoid of lipoproteins. Growth of parasites is essentially supported by receptor-mediated endocytosis of low-density lipoproteins (LDLs), which carry phospholipids and cholesteryl esters. These lipids are released from internalized LDL after apoprotein B-100 is degraded by acidic thiol-proteases in the endolysosomal apparatus and then metabolized, as in mammalian cells. The LDL receptor is recycled and its expression is regulated by the sterol stores. Documented pharmacological and immunological interferences with LDL receptor-mediated lipid supply to the bloodstream forms are summarized, and the potential for new approaches to fight against these parasites is evaluated. In contrast to bloodstream forms, cultured procyclic forms can acquire sterols from both exogenous (lipoprotein endocytosis) and endogenous (biosynthesis of ergosterol) sources. The rate-limiting steps of both endocytosis (surface LDL receptor expression) and biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase activity) are regulated by the cellular content of sterol. These two pathways thus complement each other to yield a balanced sterol supply, which demonstrates adaptative capacities to survive in totally different environments and fine regulatory mechanisms of sterol homeostasis.
Assuntos
Esteróis/metabolismo , Trypanosoma brucei brucei/fisiologia , Adaptação Biológica , Animais , Transporte Biológico , Endocitose , Homeostase , Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismoRESUMO
Cells of Leishmania donovani in co-culture with Trypanosoma brucei, were severely affected in their growth, resulting in swelling and subsequent lysis. Similar effects were also observed when Crithidia luciliae or Phytomonas sp. were co-cultured with T. brucei. Direct contact between the cells under investigation and T. brucei was necessary because T. brucei did not hamper the growth of the other trypanosomatids, when separated by a filter with 0.2 microns pore size. Examination of this phenomenon at the ultrastructural level, in a co-culture of L. donovani and T. brucei, suggests that the plasma membrane permeability is increased in the former, as a result of a close cellular contact between the two cell types.
Assuntos
Leishmania donovani/crescimento & desenvolvimento , Trypanosoma brucei brucei/crescimento & desenvolvimento , Animais , Adesão Celular , Crithidia/crescimento & desenvolvimento , Meios de Cultura , Leishmania donovani/ultraestrutura , Microscopia Eletrônica , Trypanosoma brucei brucei/ultraestrutura , Trypanosomatina/crescimento & desenvolvimentoRESUMO
Lesions caused by Leishmania amazonensis normally heal, but relapses occur due to parasite persistence in host tissues. It has been proposed that infection of fibroblasts plays an important role in this process by providing the parasites with a safe haven in which to replicate. However, most previous studies have focused on the entry of Leishmania into macrophages, a process mediated by serum opsonins. To gain insight into a possible role of nonopsonic entry in the intracellular persistence of amastigotes, we examined the invasion of Chinese hamster ovary (CHO) cells. Amastigotes entered CHO cells by a cytochalasin D, genistein, wortmannin, and 2,3-butanedione monoxime-sensitive pathway and replicated within phagolysosomes. However, unlike most phagocytic processes described to date, amastigote internalization in CHO cells involved activation of the GTPases Rho and Cdc42 but not Rac-1. When uptake was mediated by fibronectin or when amastigotes were opsonized with immunoglobulin G and internalized by Fc receptor-expressing CHO cells, Rac-1 activation was restored and found to be required for parasite internalization. Given the essential role of Rac in assembly of the respiratory burst oxidase, invasion through this nonopsonic, Rac-1-independent pathway may play a central role in the intracellular survival of Leishmania in immune hosts.
Assuntos
Leishmania mexicana/imunologia , Proteínas Opsonizantes/imunologia , Fagocitose/imunologia , Transdução de Sinais/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Células CHO , Cricetinae , Ativação Enzimática , Fibronectinas/imunologia , Imunoglobulina G/imunologia , Lisossomos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína cdc42 de Ligação ao GTP/imunologiaRESUMO
In contrast to mammalian cells, bloodstream forms of Trypanosoma brucei show no activity for fatty acid and sterol synthesis and critically depend on plasma low density lipoprotein (LDL) particles for their rapid growth. We report here that these parasites acquire such lipids by receptor-mediated endocytosis of LDL, subsequent lysosomal degradation of apoprotein B-LDL, and utilization of these lipids. Uptake of LDL-associated [3H]sphingomyelin and of LDL-associated [3H]cholesteryl oleate paralleled each other, and that of 125I-apoprotein B-LDL showed saturation and could be inhibited by unlabeled LDL or by anti-LDL receptor antibodies. Metabolism of lipids carried by LDL was abolished by chloroquine and by the thiol protease inhibitor, leupeptin. Sphingomyelin was cleaved by an acid sphingomyelinase to yield ceramide, which was itself split up into sphingosine and fatty acids. The latter were further incorporated into phosphatidylcholine, triacylglycerols, or cholesteryl esters. Similarly, cholesteryl oleate was hydrolyzed by an acid lipase to yield free cholesterol, which was reesterified with fatty acids, presumably in the cytosol. Like free cholesterol, LDL provided substrate for cholesterol esterification. In the culture-adapted procyclic form of T. brucei, which is capable of sterol synthesis, exogenous LDL-cholesterol rather than endogenously synthesized sterol was utilized for sterol esterification. Interference with exogenous supply of lipids via receptor-mediated endocytosis of LDL should be explored to fight against trypanosomiasis.
Assuntos
Lipoproteínas LDL/sangue , Trypanosoma brucei brucei/metabolismo , Animais , Transporte Biológico , Ésteres do Colesterol/metabolismo , LDL-Colesterol/metabolismo , Humanos , Cinética , Lipase/metabolismo , Lipoproteínas LDL/isolamento & purificação , Fosfatidilcolinas/metabolismo , Ratos , Ratos Wistar , Esfingomielinas/metabolismo , Fatores de Tempo , Triglicerídeos/metabolismo , TrítioRESUMO
The trypanosome LDL receptor has been isolated from bloodstream form and cultured insect-stage trypanosomes as a protein of Mr 145,000, using a rapid purification procedure in the presence of a cocktail of protease inhibitors, whereas previously a polypeptide of Mr 86,000 was purified as the LDL receptor. Both the 145,000 and the 86,000 polypeptides are glycosylated and recognized by a monospecific antibody raised against the 86,000 species. This antibody inhibits LDL binding to the intact trypanosomes, to the isolated 145,000 receptor and to the 86,000 species. Hence, the previously isolated 86,000 polypeptide is a degradation product probably representing the cleaved-off ectodomain of the trypanosome LDL receptor.
Assuntos
Glicoproteínas/isolamento & purificação , Fígado/metabolismo , Receptores de LDL/isolamento & purificação , Trypanosoma brucei brucei/metabolismo , Animais , Anticorpos Monoclonais , Western Blotting , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/metabolismo , Humanos , Lipoproteínas LDL/isolamento & purificação , Lipoproteínas LDL/metabolismo , Peso Molecular , Ratos , Ratos Endogâmicos , Receptores de LDL/metabolismoRESUMO
Using a direct immunofluorescent technique and monoclonal antibody, chlamydia trachomatis was detected in the conjunctival smears of 13 out of 47 patients presenting with conjunctivitis or keratoconjunctivitis (an incidence of 28%). Two patients presented with acute symptoms of few days duration, whereas the condition was chronic and of long duration in 11 patients. Conjunctival changes noted were upper and lower palpebral conjunctival follicles and papillae (11 patients), chemosis (5 patients), upper tarsal scar (2 patients) and pseudomembrane (one patient). Corneal involvement was detected in 9 patients and was manifested as micropannus (6 patients), multiple small epithelial punctate stains (3 patients), extensive pannus affecting the upper third of the cornea (2 patients), subepithelial punctate infiltrates similar to that of adenovirus infection (2 patients) and upper limbal follicles (one patient). Identification of chlamydia trachomatis in conjunctival smears by use of the monoclonal antibody is a simple, rapid and reliable laboratory procedure.
Assuntos
Infecções por Chlamydia , Ceratoconjuntivite/etiologia , Doença Aguda , Adolescente , Adulto , Doença Crônica , Túnica Conjuntiva/patologia , Córnea/patologia , Feminino , Humanos , Ceratoconjuntivite/complicações , Ceratoconjuntivite/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
The slender bloodstream form of Trypanosoma brucei shows receptor-mediated endocytosis of low density lipoprotein (LDL) particles of its hosts. We have purified the LDL receptor of this species nearly to homogeneity (about 1000-fold purification) and obtained monospecific polyclonal antibodies against it. As analyzed by NaDodSO4/polyacrylamide gel electrophoresis, the purified receptor consists of a single subunit, with an apparent molecular mass of 86 kDa. Its isoelectric point is 5.9. On the average, each cell exposes 52,000 copies of low-affinity receptors (Kd of 250 nM) and 1800 copies of high-affinity receptors (Kd of 5.7 nM). According to indirect en bloc immunolabeling of fixed parasites, the receptor appears to be localized to the flagellar pocket membrane and the flagellar membrane and to be completely absent from the rest of the pericellular membrane. LDL is required for optimal growth of the trypanosome in vitro: cell growth can be inhibited either by removal of LDL from the culture medium or by antibodies against the purified LDL receptors. In both cases, growth is restored by the addition of excess LDL.
Assuntos
Receptores de LDL/isolamento & purificação , Trypanosoma brucei brucei/análise , Animais , Anticorpos , Eletroforese em Gel de Poliacrilamida , Endocitose , Cinética , Lipoproteínas LDL/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
The rapid growth of Trypanosoma brucei brucei in the blood and tissue fluids of vertebrates requires the receptor-mediated endocytosis of LDL from the host (Coppens et al. 1987; Gillett and Owen 1987) and is slowed by a monospecific rabbit antiserum against the purified LDL receptor of the parasite. We have used this antiserum in combination with several well-characterized antigenic variants (originating from stock 427: MITat 1.1a, 1.3a, 1.4a, 1.5a, 1.5d, 1.8b) to examine whether the LDL receptor of T. b. brucei is a stable surface antigen, common to all parasite variants despite antigenic variation of the major surface glycoprotein, and whether it is immunologically distinct from the LDL receptor of the host. At low concentrations, binding at 4 degrees C of rat LDL to several variants of T. b. brucei and to isolated rat hepatocytes was inhibited to a similar extent by the antiserum. In double immunodiffusion, a single precipitation line was observed, showing continuity between the extracts of all variants as well as between that of trypanosomes and of mammalian tissues. In Western blots of trypanosome extracts, the LDL receptor was strongly labeled as a single band of Mr 145,000, whereas with a rat liver extract, a single band of similar electrophoretic mobility was weakly labeled at a high concentration of the antiserum. In conclusion, the LDL receptor occurred in all variants of T. b. brucei, was a stable surface antigen despite variation of the major surface glycoprotein, and displayed biochemical and immunological similarities with the LDL receptor of the rat host.
Assuntos
Variação Antigênica , Antígenos de Protozoários/imunologia , Receptores de LDL/imunologia , Trypanosoma brucei brucei/imunologia , Animais , Western Blotting , Reações Cruzadas , Imunodifusão , Masculino , Ratos , Ratos EndogâmicosRESUMO
The uptake of various host plasma proteins by the bloodstream form of Trypanosoma brucei was studied both biochemically, using radiolabeled proteins, and with the electron microscope, using colloidal gold particles as molecular tracers onto which plasma proteins had been adsorbed. Total plasma proteins and serum albumin were taken up by a mechanism of fluid endocytosis with low clearance (0.1 microliter [mg cell protein]-1 h-1), while low-density lipoprotein (LDL) and transferrin were taken up by a receptor-mediated process with a clearance of two to three orders of magnitude higher than that of serum albumin. Binding prior to uptake of LDL and transferrin was saturable, depended on the presence of Ca2+, and the labeled ligand could be displaced by the homologous but not by heterologous protein. Binding of gold-labeled proteins was seen only to the membrane of the flagellar pocket and not elsewhere on the plasma membrane. After 1 h of incubation at 30 degrees C with gold-labeled LDL and transferrin, labeled cellular structures represented respectively half and one-third of the total volume of all single-membrane bounded endocytotic and electron-dense vacuoles within the cell.