RESUMEN
Amoebiasis is a disease caused by Entamoeba histolytica, affecting the large intestine of humans and occasionally leading to extra-intestinal lesions. Entamoeba dispar is another amoeba species considered commensal, although it has been identified in patients presenting with dysenteric and nondysenteric colitis, as well as amoebic liver abscess. Amoebic virulence factors are essential for the invasion and development of lesions. There is evidence showing that the association of enterobacteria with trophozoites contributes to increased gene expression of amoebic virulence factors. Enteropathogenic Escherichia coli is an important bacterium causing diarrhea, with high incidence rates in the world population, allowing it to interact with Entamoeba sp. in the same host. In this context, this study aims to evaluate the influence of enteropathogenic Escherichia coli on ACFN and ADO Entamoeba dispar strains by quantifying the gene expression of virulence factors, including galactose/N-acetyl-D-galactosamine-binding lectin, cysteine proteinase 2, and amoebapores A and C. Additionally, the study assesses the progression and morphological aspect of amoebic liver abscess and the profile of inflammatory cells. Our results demonstrated that the interaction between EPEC and ACFN Entamoeba dispar strains was able to increase the gene expression of virulence factors, as well as the lesion area and the activity of the inflammatory infiltrate. However, the association with the ADO strain did not influence the gene expression of virulence factors. Together, our findings indicate that the interaction between EPEC, ACFN, and ADO Entamoeba dispar strains resulted in differences in vitro and in vivo gene expression of Gal/GalNAc-binding lectin and CP2, in enzymatic activities of MPO, NAG, and EPO, and consequently, in the ability to cause lesions.
Asunto(s)
Entamoeba , Escherichia coli Enteropatógena , Factores de Virulencia , Escherichia coli Enteropatógena/patogenicidad , Escherichia coli Enteropatógena/genética , Entamoeba/patogenicidad , Entamoeba/genética , Entamoeba/fisiología , Factores de Virulencia/genética , Virulencia , Animales , Ratones , Absceso Hepático Amebiano/parasitología , Entamebiasis/parasitología , Humanos , Expresión GénicaRESUMEN
Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha-actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.
Asunto(s)
Entamoeba/fisiología , Fibronectinas/farmacología , GTP Fosfohidrolasas/metabolismo , Proteínas de Microfilamentos/metabolismo , Entamoeba/efectos de los fármacos , Entamoeba/ultraestructura , Entamoeba histolytica/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Microscopía Confocal , Proteínas Protozoarias/metabolismoRESUMEN
The rapid redistribution of surface antigen-antibody complexes in trophozoites of the human protozoan parasite Entamoeba histolytica, in a process known as capping, has been considered as a means of the parasite to evade the host immune response. So far, capping has been documented in the invasive E. histolytica, whereas the mobility of surface components in the non-invasive Entamoeba dispar is not known. E. dispar does not induce liver lesions in rodent experimental models, in contrast to the liver abscesses produced by E. histolytica in the same animal model. We have therefore analyzed the mobility of surface receptors to the lectin concanavalin A and of Rab11, a membrane-associated protein, in both species of Entamoebae by confocal fluorescence microscopy and transmission and scanning electron microscopy. The great majority of E. histolytica trophozoites became morphologically polarized through the formation of well-defined caps at the posterior pole of the parasite. Actin colocalized with the lectin caps. Antibodies against the membrane protein Rab 11 also produced capping. In striking contrast, in E. dispar, the mobility of concanavalin A surface receptors was restricted to the formation of irregular surface patches that did no progress to constitute well-defined caps. Also, anti-Rab 11 antibodies did not result in capping in E. dispar. Whether the failure of E. dispar to efficiently mobilize surface molecules in response to lectin or antibodies as shown in the present results is related to its non-invasive character represents an interesting hypothesis requiring further analysis.
Asunto(s)
Antígenos de Protozoos/metabolismo , Entamoeba/patogenicidad , Animales , Concanavalina A/metabolismo , Entamoeba/metabolismo , Entamoeba/fisiología , Proteínas de Unión al GTP/metabolismo , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía FluorescenteRESUMEN
Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.
Asunto(s)
Pared Celular/ultraestructura , Entamoeba/ultraestructura , Esporas Protozoarias/ultraestructura , Permeabilidad de la Membrana Celular , Microscopía por Crioelectrón , Entamoeba/fisiología , Histocitoquímica , Microscopía Electrónica de Rastreo , Microscopía Electrónica de TransmisiónRESUMEN
The sequence of hepatic necrotic-inflammatory events produced by Entamoeba dispar are originally described in this work. For the first time were described in details the experimental lesions produced by E. dispar, as well as the distribution of the trophozoites detected by the immunohistochemistry. Animals experimentally infected with E. dispar presented necrosis, thrombosis and chronic granulomatous inflammation. Immunoreactive products derived from trofozoites were observed close or associated with trophozoites, epithelioid cells, leucocytes and hepatocytes. Few are the articles on the literature about virulence of E. dispar, which is approximately 9 times more frequent than to E. histolytica. Variation in the virulence is, therefore expected and signalizing the need of the continuity of studies with E. dispar strains from different places in the world. Taking into account that E. dispar is a closely related species to E. histolytica, these studies could determine new elements involved with E. histolytica pathogenesis, helping us to understand better the disease.
Asunto(s)
Entamoeba/fisiología , Entamebiasis/complicaciones , Parasitosis Hepáticas/patología , Hígado/patología , Animales , Cricetinae , Entamebiasis/inmunología , Entamebiasis/patología , Enfermedad Granulomatosa Crónica/patología , Inmunohistoquímica , Inflamación/patología , Necrosis/etiología , Necrosis/patología , Ratas , Trombosis/etiología , Trombosis/patologíaRESUMEN
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.
Asunto(s)
Entamoeba/fisiología , Fosfopiruvato Hidratasa/fisiología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Western Blotting , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Entamoeba/enzimología , Entamoeba/crecimiento & desarrollo , Entamoeba/inmunología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Hibridomas , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Fosfopiruvato Hidratasa/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofozoítos/inmunología , Trofozoítos/fisiologíaRESUMEN
In a 12-month longitudinal study, a cohort of Mexican HIV+/AIDS patients was checked several times for Entamoeba infection, with the parasites identified, as E. histolytica or E. dispar, using PCR. The polymorphic region of the parasites' chitinase genes was investigated by PCR, with the variation in amplicon sizes being used as a measure of the genetic variation among the isolates. The patients found infected with Entamoeba at the start of the study displayed varied patterns of infection clearance and re-infection. The analysis of the polymorphisms in the chitinase gene revealed seven polymorphic patterns in the E. histolytica isolates investigated and three in the E. dispar isolates. Many of the patients were each re-infected with Entamoeba at least once during the 12 months of follow-up. As seen in a previous study in Mexico, none of the E. histolytica-infected patients developed any clinical symptoms of invasive amoebiasis during the follow-up period. The results highlight the complexity of the host-parasite relationship in human amoebiasis.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/epidemiología , Entamoeba/fisiología , Entamebiasis/epidemiología , Infecciones por VIH/parasitología , Infecciones Oportunistas Relacionadas con el SIDA/parasitología , Adulto , Anciano , Animales , Quitinasas/genética , Entamoeba/enzimología , Entamoeba/genética , Entamebiasis/genética , Femenino , Infecciones por VIH/epidemiología , Seropositividad para VIH , VIH-1 , Interacciones Huésped-Parásitos , Humanos , Estudios Longitudinales , Masculino , México/epidemiología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Estadística como AsuntoRESUMEN
Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.
Asunto(s)
Endocitosis , Eucariontes , Animales , Entamoeba/metabolismo , Entamoeba/fisiología , Entamoeba/ultraestructura , Eucariontes/metabolismo , Eucariontes/fisiología , Eucariontes/ultraestructura , Giardia/metabolismo , Giardia/fisiología , Giardia/ultraestructura , Histocitoquímica , Leishmania/metabolismo , Leishmania/fisiología , Leishmania/ultraestructura , Microscopía Electrónica , Plasmodium/metabolismo , Plasmodium/fisiología , Plasmodium/ultraestructura , Trichomonas/metabolismo , Trichomonas/fisiología , Trichomonas/ultraestructura , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/fisiología , Trypanosoma brucei brucei/ultraestructura , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiología , Trypanosoma cruzi/ultraestructuraRESUMEN
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.
Asunto(s)
Campos Electromagnéticos , Entamoeba/crecimiento & desarrollo , Entamoeba/efectos de la radiación , Animales , Antiprotozoarios/farmacología , Relación Dosis-Respuesta en la Radiación , Entamoeba/efectos de los fármacos , Entamoeba/fisiología , Dosificación Letal Mediana , Metronidazol/farmacología , Distribución AleatoriaRESUMEN
Field emission scanning electron microscopy (FE-SEM) provides a range of strategies for investigating the structural organization of biological systems, varying from isolated macromolecules to tissue organization and whole organisms. This review covers some of the results so far obtained using FE-SEM observation and various protocols of sample fixation to analyze the structural organization of parasitic protozoa and their interaction with host cells. The employment of FE-SEM can be broadened through the use of gold-labeled molecules or tracers, gradual extraction by detergents, and cleavage techniques. These analyses provide significant contributions to the characterization of these organisms concerning ultrastructure, cytoskeleton, motility and intracellular behavior.
Asunto(s)
Eucariontes/fisiología , Eucariontes/ultraestructura , Riñón/parasitología , Microscopía Electrónica de Rastreo/instrumentación , Microscopía Electrónica de Rastreo/métodos , Neutrófilos/parasitología , Animales , Línea Celular , Citoesqueleto/ultraestructura , Entamoeba/fisiología , Entamoeba/ultraestructura , Eucariontes/clasificación , Interacciones Huésped-Parásitos , Humanos , Riñón/citología , Leishmania/fisiología , Leishmania/ultraestructura , Toxoplasma/fisiología , Toxoplasma/ultraestructura , Trypanosoma cruzi/fisiología , Trypanosoma cruzi/ultraestructuraRESUMEN
Processos de secreção celular desempenham papel relevante na biologia e no ciclo de vida de protozoários patogênicos. A presente revisão analisa, sob uma perspectiva de biologia celular, o processo de secreção em (a) micronemas, roptrias e grânulos densos encontrados em membros do grupo Apicomplexa, onde essas estruturas participam da penetração do protozoário no interior da célula hospedeira, na sua sobrevivência intravacuolar e no posterior egresso da célula hospedeira, (b) a fenda de Maurer, encontrada em Plasmodium, uma estrutura envolvida na secreção de proteínas sintetizadas pelo protozoário intravacuolar e transportada, através de vesículas, para a superfície do eritrócito, (c) a secreção de macromoléculas na bolsa flagelar de tripanosomatídeos, e (d) a secreção de proteínas que fazem parte da parede cística de Giardia e Entamoeba e que se concentram nas vesículas de encistamento.
Asunto(s)
Animales , Eucariontes , Microtúbulos , Orgánulos , Proteínas Protozoarias , Vesículas Secretoras , Apicomplexa/citología , Apicomplexa/fisiología , Eucariontes , Entamoeba/citología , Entamoeba/fisiología , Giardia/citología , Giardia/fisiología , Microtúbulos/fisiología , Orgánulos/fisiología , Proteínas Protozoarias/fisiología , Vesículas Secretoras/fisiología , Trypanosomatina/citología , Trypanosomatina/fisiologíaRESUMEN
Secretory processes play an important role on the biology and life cycles of parasitic protozoa. This review focus on basic aspects, from a cell biology perspective, of the secretion of (a) micronemes, rhoptries and dense granules in members of the Apicomplexa group, where these organelles are involved in the process of protozoan penetration into the host cell, survival within the parasitophorous vacuole and subsequent egress from the host cell, (b) the Maurer's cleft in Plasmodium, a structure involved in the secretion of proteins synthesized by the intravacuolar parasite and transported through vesicles to the erythrocyte surface, (c) the secretion of macromolecules into the flagellar pocket of trypanosomatids, and (d) the secretion of proteins which make the cyst wall of Giardia and Entamoeba, with the formation of encystation vesicles.
Asunto(s)
Eucariontes/metabolismo , Microtúbulos/metabolismo , Orgánulos/metabolismo , Proteínas Protozoarias/metabolismo , Vesículas Secretoras/metabolismo , Animales , Apicomplexa/citología , Apicomplexa/fisiología , Entamoeba/citología , Entamoeba/fisiología , Eucariontes/citología , Eucariontes/fisiología , Giardia/citología , Giardia/fisiología , Microtúbulos/fisiología , Orgánulos/fisiología , Proteínas Protozoarias/fisiología , Vesículas Secretoras/fisiología , Trypanosomatina/citología , Trypanosomatina/fisiologíaRESUMEN
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.
Asunto(s)
Aminoácidos/fisiología , Medio de Cultivo Libre de Suero/química , Entamoeba/fisiología , Lípidos/fisiología , Animales , Entamoeba/crecimiento & desarrollo , Entamoeba/ultraestructura , Microscopía Electrónica , Microscopía FluorescenteRESUMEN
In the life cycle of Entamoeba species, the cyst and all the processes associated to it have been poorly studied. Entamoeba invadens, a serpent's parasite, has been commonly accepted as a model for the study of encystation and excystation. Here we analyzed through scanning and transmission electron microscopy the in vitro morphological differentiation of both processes. During encystation, the formation of an irregular net of fibrillar material on the surface of precysts was observed. In thin sections of cryofixed and cryosubstituted specimens, abundant vacuoles containing a microfibrillar material of similar appearance to the structural components of the cyst wall were found in the cytoplasm. Assays with a calcofluor probe on cryosections of encysting trophozoites and precysts showed the presence of fluorescent circular cytoplasmic structures. In the cyst stage, the fluorescence was located on the surface. During excystation, the detachment of the metacyst from the cyst wall was observed through scanning electron microscopy. Metacysts endocyting amorphous material which may correspond to cyst wall residues were commonly found. By transmission electron microscopy the formation of a crescent-shaped space between the plasma membrane and the cyst wall was observed. Abundant small electrondense bodies were found in the cytoplasm. Many of them were in close apposition to the plasma membrane and frequently some of them were seen projecting towards this newly formed space. Our results suggest that the microfibrillar content of the vacuoles corresponds to the cyst wall material, that the electrondense bodies may be involved in the excystation process, and that part of the cyst wall residues may be endocyted by the parasite.
Asunto(s)
Entamoeba/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Animales , Entamoeba/fisiologíaRESUMEN
Entamoeba histolytica is the pathogenic species of Entamoeba that causes amoebic dysentery and other invasive disease. The morphologically similar species, E. dispar, is non-pathogenic and accounts for about 90% of the previously estimated 500 million E. histolytica infections world-wide. Because of the recent redefinition of E. histolytica and E. dispar, and the limited number of drugs available to treat amoebiasis, a new approach to treatment of individuals carrying these parasites is necessary. A meeting of eminent scientists has recently agreed that on no account should prophylaxis against amoebiasis be given, and no treatment without symptoms should be administered. The expense of treating asymptomatic individuals, both monetary and at the risk of over-use of precious drugs, does not appear to be justified. It would seem wise that we preserve currently effective anti-amoebic drugs and avoid the development of drug-resistant E. histolytica.
Asunto(s)
Entamoeba/clasificación , Entamebiasis/parasitología , Amebicidas/administración & dosificación , Amebicidas/uso terapéutico , Animales , Contraindicaciones , Reservorios de Enfermedades , Entamoeba/patogenicidad , Entamoeba/fisiología , Entamoeba histolytica/clasificación , Entamoeba histolytica/patogenicidad , Entamoeba histolytica/fisiología , Entamebiasis/tratamiento farmacológico , Entamebiasis/epidemiología , Entamebiasis/transmisión , Entamebiasis/veterinaria , Heces/parasitología , Humanos , Mamíferos/parasitología , Especificidad de la Especie , VirulenciaRESUMEN
The membrane potential in Entamoeba is an important driving force for the uptake of substrates. In Entamoeba invadens PZ a membrane potential of -36 mV was obtained when Nernst equation was applied to the distribution at equilibrium of 86Rb+ in the presence of valinomycin. This could explain the levels of accumulation of up to 4 times found for positively charged substrates. Membrane potential was diminished by depolarizing conditions (high external K+ concentration in the presence of valinomycin). Moreover, we recorded continuously the membrane potential of Entamoeba invadens PZ and Entamoeba histolytica HM1 using the fluorescent lipophilic cation diisopropylthiodicarbocyanine. It was found that the uptake of this cation by the amoebae was fast in both species, conditions that modify the membrane potential (hyperpolarization and depolarization) produced changes in the fluorescence of the dye in agreement with its reported capability to detect variations in membrane potential. It can be concluded that these microorganisms have a membrane potential negative inside them.