RESUMO
Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.
Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , Entamoeba/efeitos dos fármacos , Entamoeba/crescimento & desenvolvimento , Proteínas de Protozoários/antagonistas & inibidores , Esporos de Protozoários/efeitos dos fármacos , Esporos de Protozoários/crescimento & desenvolvimento , ATPases Transportadoras de Cálcio/análise , ATPases Transportadoras de Cálcio/genética , Calreticulina/análise , Inibidores Enzimáticos/metabolismo , Indóis/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Tapsigargina/metabolismo , Vesículas Transportadoras/químicaRESUMO
In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.
Assuntos
Actinas/metabolismo , Entamoeba/crescimento & desenvolvimento , Entamoeba/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/genética , Sequência de Aminoácidos , Animais , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Entamoeba/genética , Humanos , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Proteínas rab de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The reptilian parasite Entamoeba invadens is accepted as a model for the study of the Entamoeba encystation process. Here we describe the production and characterization of a mAb (B4F2), generated against a component of the E. invadens cyst wall. This mAb specifically recognizes a 48-kDa protein present in cytoplasmic vesicles of cells encysting for 24 h. In mature cysts (96 h), the antigen was detected on the cyst surface. By two-dimensional electrophoresis and mass spectrometry analysis, the B4F2 specific antigen was identified as enolase. Levels of enolase mRNA were increased in encysting cells and the B4F2 mAb was found to inhibit cyst formation. Therefore, these results strongly suggest a new role for enolase in E. invadens encystation, and the B4F2 mAb will be useful tool to study its role in the differentiation process.
Assuntos
Entamoeba/fisiologia , Fosfopiruvato Hidratase/fisiologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Western Blotting , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Entamoeba/enzimologia , Entamoeba/crescimento & desenvolvimento , Entamoeba/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Hibridomas , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Fosfopiruvato Hidratase/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trofozoítos/imunologia , Trofozoítos/fisiologiaRESUMO
The human large intestine can harbor two morphologically similar amoebae; the invasive Entamoeba histolytica and the non-invasive Entamoeba dispar. Whereas E. histolytica can produce intestinal and extra-intestinal lesions, E. dispar is present in non-symptomatic carriers. Although biochemical, genetic and proteomic studies have identified clear differences between these Entamoebae, it has become clear that several molecules, once assumed to be involved in tissue destruction, exist in both the virulent and the avirulent species. As surface molecules may play a role in invasion and could therefore determine which amoebae are invasive, we analyzed the glycoconjugate composition of E. histolytica and E. dispar using lectins. There was a significant difference between E. histolytica and E. dispar in the expression of glycoconjugates containing d-mannose and N-acetyl-alpha-D-galactosamine residues, but not between virulent and avirulent strains of E. histolytica. N-glycoconjugates with terminal alpha (1-3)-linked mannose residues participate in the adhesion and subsequent cytotoxicity of E. histolytica to cultured hamster hepatocytes. One of them probably is the Gal/GalNAc lectin.
Assuntos
Entamoeba histolytica/metabolismo , Entamoeba/metabolismo , Glicoconjugados/metabolismo , Acetilgalactosamina/análise , Animais , Adesão Celular , Células Cultivadas , Cricetinae , Entamoeba/classificação , Entamoeba/crescimento & desenvolvimento , Entamoeba/patogenicidade , Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba histolytica/patogenicidade , Entamebíase/parasitologia , Entamebíase/patologia , Glicoconjugados/química , Hepatócitos/parasitologia , Hepatócitos/patologia , Abscesso Hepático Amebiano/parasitologia , Abscesso Hepático Amebiano/patologia , Manose/análise , VirulênciaRESUMO
The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous D-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.
Assuntos
Entamoeba/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Esfingolipídeos/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Diglicerídeos/metabolismo , Entamoeba/citologia , Entamoeba/efeitos dos fármacos , Entamoeba/crescimento & desenvolvimento , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Esfingolipídeos/biossíntese , Esfingomielinas/biossíntese , Esfingosina/análogos & derivados , Esfingosina/antagonistas & inibidores , Esfingosina/biossíntese , Fatores de Tempo , beta-Alanina/análogos & derivados , beta-Alanina/farmacologiaRESUMO
Exposure to extremely low-frequency (ELF) electromagnetic fields appears to result in a number of important biological changes. In the present study, we evaluated the effects of 60 Hz sinusoidal magnetic fields (MF) at magnetic flux densities of 1.0, 1.5 and 2.0 mT on growth and differentiation of the protozoan Entamoeba invadens. We demonstrated an inhibitory growth effect when trophozoite cultures were exposed to 1.5 and 2.0 mT. Furthermore, we found that there was not a synergistic effect in cultures co-exposed to MF and Metronidazole, a cytotoxic drug against amoebic cells. In addition, MF exposure inhibited the encystation process of E. invadens.
Assuntos
Campos Eletromagnéticos , Entamoeba/crescimento & desenvolvimento , Entamoeba/efeitos da radiação , Animais , Antiprotozoários/farmacologia , Relação Dose-Resposta à Radiação , Entamoeba/efeitos dos fármacos , Entamoeba/fisiologia , Dose Letal Mediana , Metronidazol/farmacologia , Distribuição AleatóriaRESUMO
In regions with high prevalence, Blastocystis hominis is frequently found in association with Entamoeba histolytica/E. dispar in xenic cultures. Its exacerbated growth is often superimposed on the growth of amebas, thus impeding the continuation of the amebas in the culture, within a few generations. The present study reports on the excellent efficacy (100%) of the antifungal agent miconazole in eliminating B. hominis from cultures of E. histolytica/E. dispar, thereby maintaining the integrity of the trophozoites of the amebas. Nystatin presented low efficacy (33.3%).
Assuntos
Antifúngicos/farmacologia , Blastocystis hominis/efeitos dos fármacos , Meios de Cultura , Entamoeba/crescimento & desenvolvimento , Miconazol/farmacologia , Animais , Entamoeba histolytica/crescimento & desenvolvimento , Testes de Sensibilidade MicrobianaRESUMO
In regions with high prevalence, Blastocystis hominis is frequently found in association with Entamoeba histolytica/E. dispar in xenic cultures. Its exacerbated growth is often superimposed on the growth of amebas, thus impeding the continuation of the amebas in the culture, within a few generations. The present study reports on the excellent efficacy (100 percent) of the antifungal agent miconazole in eliminating B. hominis from cultures of E. histolytica/E. dispar, thereby maintaining the integrity of the trophozoites of the amebas. Nystatin presented low efficacy (33.3 percent).
Em regiões de alta prevalência, Blastocystis hominis é freqüentemente encontrado em associação com Entamoeba histolytica/E. dispar em cultivos xênicos. Seu crescimento exacerbado se sobrepõe muitas vezes ao das amebas, impedindo a manutenção destas em cultura, dentro de poucas gerações. O presente estudo relata a excelente eficácia (100 por cento) do antifúngico miconazol na eliminação de B. hominis dos cultivos de E. histolytica/E. dispar, mantendo-se a integridade dos trofozoítos das amebas. A nistatina apresentou eficácia baixa (33,3 por cento).
Assuntos
Humanos , Animais , Antifúngicos/farmacologia , Blastocystis hominis/efeitos dos fármacos , Meios de Cultura , Entamoeba/crescimento & desenvolvimento , Miconazol/farmacologia , Entamoeba histolytica/crescimento & desenvolvimento , Testes de Sensibilidade MicrobianaRESUMO
Two xenic isolates and cloned cultures of Entamoeba dispar were submitted to monoxenization using Crithidia fasciculata as the associated organism. Growth in monoxenic cultivation and ability of xenic and monoxenic trophozoites to destroy VERO cells and produce lesions in hamster livers were compared to those of a virulent E. histolytica. Parental and cloned E. dispar under monoxenic cultivation showed a remarkable lower growth than the monoxenic E. histolytica and were avirulent in both in vivo and in vitro tests. When xenically cultured, trophozoites of E. dispar showed a moderate lytic activity against VERO cells (1.5 to 41.8% of destruction) but caused severe hepatic lesions in hamsters as those caused by the virulent E. histolytica (29 to 100% in prevalence and 0.86 to 4.00 in lesion degree). Although E. dispar has not been associated with invasive disease in men, the ability of xenic trophozoites to produce prominent tissue damage in experimental conditions has indicated that some strains have a considerable pathogenic potential when in presence of bacteria.
Assuntos
Meios de Cultura , Entamoeba/patogenicidade , Fígado/parasitologia , Animais , Chlorocebus aethiops , Cricetinae , Crithidia fasciculata , Entamoeba/crescimento & desenvolvimento , Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba histolytica/patogenicidade , Humanos , Fígado/patologia , Células Vero , VirulênciaRESUMO
Two xenic isolates and cloned cultures of Entamoeba dispar were submitted to monoxenization using Crithidia fasciculata as the associated organism. Growth in monoxenic cultivation and ability of xenic and monoxenic trophozoites to destroy VERO cells and produce lesions in hamster livers were compared to those of a virulent E. histolytica. Parental and cloned E. dispar under monoxenic cultivation showed a remarkable lower growth than the monoxenic E. histolytica and were avirulent in both in vivo and in vitro tests. When xenically cultured, trophozoites of E. dispar showed a moderate lytic activity against VERO cells (1.5 to 41.8 percent of destruction) but caused severe hepatic lesions in hamsters as those caused by the virulent E. histolytica (29 to 100 percent in prevalence and 0.86 to 4.00 in lesion degree). Although E. dispar has not been associated with invasive disease in men, the ability of xenic trophozoites to produce prominent tissue damage in experimental conditions has indicated that some strains have a considerable pathogenic potential when in presence of bacteria.
Dois isolados de Entamoeba dispar em cultivo polixênico e culturas clonadas deles obtidas foram submetidos à monoxenização utilizando Crithidia fasciculata como organismo associado. O crescimento em cultivo monoxênico dos isolados e clones, bem como sua capacidade de destruir células VERO (efeito citopático) e de produzir lesões hepáticas em hamster foram comparados a uma cepa virulenta de E. histolytica. Os trofozoítos de E. dispar em cultivo monoxênico apresentaram um crescimento nitidamente menor que o de E. histolytica e foram avirulentos tanto no teste in vivo quanto in vitro. Entretanto, isolados e clones de E. dispar em cultivo polixênico exibiram uma atividade lítica moderada sobre as células VERO (1,5 to 41,8 por cento de destruição) e causaram lesões hepáticas em hamster (29 a 100 por cento em prevalência e 0,86 a 4,00 no grau de lesão) tão extensas quanto aquelas causadas pela E. histolytica. Embora E. dispar não seja associada à doença invasiva no homem, a ocorrência de lesões teciduais significativas, causadas por trofozoítos em condições experimentais, indica que esta espécie pode apresentar potencial patogênico considerável quando em presença de bactérias intestinais.
Assuntos
Humanos , Animais , Cricetinae , Meios de Cultura , Entamoeba/patogenicidade , Fígado/parasitologia , Chlorocebus aethiops , Crithidia fasciculata , Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba histolytica/patogenicidade , Entamoeba/crescimento & desenvolvimento , Fígado/patologia , Células Vero , VirulênciaRESUMO
The current media for axenic cultivation of Entamoeba histolytica and Entamoeba invadens are supplemented with bovine or equine serum, which provides several essential nutrients to amoebas. Serum has also been considered an essential component in encystation media for E. invadens. A substitute of serum, PACSR has been described as an alternative for growth of E. histolytica and also maintains growth of E. invadens. When PACSR was used instead of serum for encystation of E. invadens the efficiency was the same as for serum. Our present data show that PACSR can support the growth and induction of encystation of E. invadens strain IP-1.
Assuntos
Aminoácidos/fisiologia , Meios de Cultura Livres de Soro/química , Entamoeba/fisiologia , Lipídeos/fisiologia , Animais , Entamoeba/crescimento & desenvolvimento , Entamoeba/ultraestrutura , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
A strain of Entamoeba dispar was characterized by clinical diagnosis, serological and electrophoretical isoenzyme analysis and by the polymorphism of a 482 bp genomic fragment analysis. The pathogenesis and virulence of this strain was investigated considering the experimental infection in hamster livers in association with the original intestinal microbiota. Liver lesions were observed in hamsters experimentally infected with trophozoites from xenic cultures, but not from the monoxenic cultures. Moreover, clones obtained from re-isolated strain Wil1R1 showed a distinct biological behavior. In fact, animals inoculated with Wil1R1ClB3 showed an intense acute inflammatory reaction with destructive focal hepatic lesions. These lesions were characterized as amebic abscesses. The association between bacteria and ameba has been fairly well studied because it affects the pathogenicity of the amebas and has important therapeutic implications. In this study, we demonstrated that E. dispar in association with the original microbiota is able to produce lesions in hamster liver in spite of its having been considered to be non-pathogenic in the hamster model. Based on these results we suggest that diagnosis of amebiasis needs to be made with more care and that clinical and therapeutical procedures need to be revised.
Assuntos
Disenteria Amebiana/parasitologia , Entamoeba/patogenicidade , Abscesso Hepático Amebiano/parasitologia , Animais , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Infecções Bacterianas/complicações , Brasil , Técnicas de Cocultura , Cricetinae , Crithidia fasciculata/patogenicidade , Disenteria Amebiana/microbiologia , Entamoeba/genética , Entamoeba/crescimento & desenvolvimento , Entamoeba/isolamento & purificação , Entamoeba/metabolismo , Fezes/parasitologia , Humanos , Fígado/microbiologia , Fígado/parasitologia , Fígado/patologia , Abscesso Hepático Amebiano/microbiologia , Abscesso Hepático Amebiano/patologia , Mesocricetus , Polimorfismo Genético , Infecções Protozoárias em Animais/complicações , VirulênciaAssuntos
Cafeína/farmacologia , Diferenciação Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Entamoeba/efeitos dos fármacos , Epinefrina/farmacologia , Fluoreto de Sódio/farmacologia , Teofilina/farmacologia , Animais , Entamoeba/citologia , Entamoeba/crescimento & desenvolvimento , Entamoeba/metabolismoRESUMO
The cytoplasm of Entamoeba is characterized by the presence of a large number of vesicles of different size and shape. Previous electron microscopic studies have not clearly revealed the presence of the endoplasmic reticulum and the Golgi complex. In the present study two approaches were used aimed at the identification of these two structures in trophozoites of Entamoeba moshkovskii and Entamoeba histolytica: (a) cytochemical techniques associated with transmission electron microscopy, such as osmium tetroxide-zinc iodide, localization of glucose-6-phosphatase and thiaminopyro-phosphatase, and (b) labeling of the structures with the fluorescent dyes DiOC6 and C6-NBD ceramide followed by visualization of the labeled cells by confocal laser scanning microscopy. Our observations suggest that some of the cytoplasmic vacuoles may correspond to components of the endoplasmic reticulum and the Golgi complex of Entamoeba.
Assuntos
Retículo Endoplasmático/ultraestrutura , Entamoeba/ultraestrutura , Complexo de Golgi/ultraestrutura , Animais , Retículo Endoplasmático/enzimologia , Entamoeba/enzimologia , Entamoeba/crescimento & desenvolvimento , Entamoeba histolytica/enzimologia , Entamoeba histolytica/ultraestrutura , Corantes Fluorescentes , Glucose-6-Fosfatase/metabolismo , Complexo de Golgi/enzimologia , Histocitoquímica , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Tiamina Pirofosfatase/metabolismo , Vacúolos/enzimologia , Vacúolos/ultraestruturaAssuntos
Antígenos de Protozoários/imunologia , Portador Sadio/imunologia , Entamoeba histolytica/imunologia , Entamoeba/imunologia , Antígenos HLA/análise , Adolescente , Adulto , Animais , Portador Sadio/parasitologia , Estudos de Casos e Controles , Entamoeba/crescimento & desenvolvimento , Entamoeba histolytica/crescimento & desenvolvimento , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeAssuntos
Portador Sadio/tratamento farmacológico , Entamoeba histolytica/isolamento & purificação , Entamoeba/isolamento & purificação , Entamebíase/tratamento farmacológico , Animais , Portador Sadio/economia , Portador Sadio/epidemiologia , Portador Sadio/parasitologia , Análise Custo-Benefício , Entamoeba/crescimento & desenvolvimento , Entamoeba histolytica/crescimento & desenvolvimento , Entamebíase/economia , Entamebíase/epidemiologia , Entamebíase/parasitologia , Humanos , México/epidemiologia , PrevalênciaRESUMO
PEHPS medium, developed for zxenic cultivation of Entamoeba histolytica and E. invadens, was also capable of supporting the growth of a Trichomonas vaginalis strain, with an inoculum of 1 to 100 trichomonads/ml. The lorithmic growth phase in PEHPS or in TYI-S-33 medium lasted 72 h; yield (3.33 ñ 0.56 x 10 a the 6 trichomonads/ml), duplication time (4.27 h), number of duplications (16.85), or increase ratio (33,328) in PEHPS medium showed no significant differences with those obtained in TYI-S33 under similar culture conditions. Accordingly, PEHPS medium might be used for the axenic cultivation of T. vaginalis