RESUMO
Highly dynamic ribosomes, glycogen granules, thinly fibrillar material, and multiple membrane-bound vesicles are embedded in the matrix-rich cytoplasm of Entamoeba spp. trophozoites. The absence of a Golgi apparatus in these amoebae has been commonly accepted. Here we challenge this observation by incubating Entamoeba histolytica and Entamoeba dispar with monensin, an ionophore that produces swelling of the Golgi apparatus. We observe changes in the trophozoites through standard transmission electron microscopy, cryofixation and cryosubstitution, and analyze the label and expression of known resident proteins of the cis-GM130 and trans-TGN38 Golgi network through confocal microscopy and Western blot assays. Cryosubstitution and standard methods using the treatment, preserved membranous lamellae resembling Golgi components. GM130 and TGN38 Golgi antigens were found by immunoelectron, immunoblot, and co-localization by confocal microscopy using the reagent NBD C6-ceramide. Our results indicate that previously undetected Golgi apparatus components are present in the cytoplasm of E. histolytica and E. dispar.
Assuntos
Entamoeba histolytica , Entamoeba , Complexo de Golgi , Microscopia Confocal , Monensin/farmacologiaRESUMO
An association between varicella zoster virus (VZV) and multiple sclerosis (MS) has been reported in Mexican populations. The aim of this study was to compare the response of T cells from MS patients, during relapse and remission, to in vitro stimulation with VZV, adenovirus (AV) and Epstein-Barr virus (EBV). Proliferation and cytokine secretion of T cells from 29 relapsing-remitting MS patients and 38 healthy controls (HC) were analyzed by flow cytometry after stimulating with VZV, AV or EBV. IgG and IgM levels against VZV and EBV were quantified using Enzyme-Linked Immunosorbent Assay. Relapsing MS patients showed a higher percentage of responding CD4+ and CD8+ T cells against VZV compared to AV. In HC and remitting MS patients, proliferation of CD4+ T cells was higher when stimulated with VZV as compared to EBV. Moreover, T cells isolated from remitting patients secreted predominantly Th1 cytokines when cell cultures were stimulated with VZV. Finally, high concentration of anti-VZV IgG was found in sera from patients and controls. The results support previous studies of an VZV-MS association in the particular population studied and provide additional information about the possible role of this virus in the pathogenesis of MS.
Assuntos
Herpesvirus Humano 3/fisiologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/virologia , Linfócitos T/imunologia , Adenoviridae/fisiologia , Adulto , Anticorpos Antivirais/imunologia , Citocinas/metabolismo , Feminino , Herpesvirus Humano 3/imunologia , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/fisiologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/virologia , Recidiva , Indução de RemissãoRESUMO
Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.
Assuntos
Acanthamoeba castellanii/ultraestrutura , Balamuthia mandrillaris/ultraestrutura , Peroxinas/genética , Peroxissomos/ultraestrutura , Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Balamuthia mandrillaris/enzimologia , Balamuthia mandrillaris/genética , Catalase/metabolismo , Microscopia Eletrônica de Transmissão , Peroxinas/metabolismo , Peroxissomos/enzimologia , Peroxissomos/genética , FilogeniaRESUMO
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.
Assuntos
Entamoeba/fisiologia , Fibronectinas/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas dos Microfilamentos/metabolismo , Entamoeba/efeitos dos fármacos , Entamoeba/ultraestrutura , Entamoeba histolytica/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Microscopia Confocal , Proteínas de Protozoários/metabolismoRESUMO
The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.
Assuntos
Giardia lamblia/química , Peroxissomos/química , Proteínas de Protozoários/isolamento & purificação , 3,3'-Diaminobenzidina/química , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Western Blotting , Cério/química , Coenzima A Ligases/imunologia , Coenzima A Ligases/metabolismo , Biologia Computacional , Imunofluorescência , Giardia lamblia/enzimologia , Giardia lamblia/imunologia , Giardia lamblia/ultraestrutura , Histocitoquímica , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Oxirredutases/metabolismo , Peroxinas/análise , Peroxinas/imunologia , Peroxissomos/enzimologia , Proteínas de Protozoários/análise , Coelhos , Coloração e RotulagemRESUMO
The virulence of various amoebic parasites has been correlated with the presence of electron-dense granules (EDGs) in the cytoplasm of trophozoites. Here, we report the finding by transmission electron microscopy of a large number of EDGs in a recent culture of Acanthamoeba culbertsoni, isolated from a severe case of human keratitis. When this isolate was maintained in culture for 6 mo, the granules almost disappeared. However, after induction of mice brain lesions with the long-term cultured isolate, recovered amoebas had abundant EDGs. Trophozoites of the original isolate, or those recovered from experimental lesions, secreted EDGs into the medium when incubated with MDCK cells. To analyze a possible cytotoxic effect the conditioned medium was incubated with MDCK monolayers. After 5 h, the media containing EDGs produced opening of the tight junctions; at 24 h, cell viability was compromised, and at 48 h most of the cells were detached from the monolayer. In contrast, trophozoites in long-term cultures did not release EDGs to the medium during incubation with MDCK cells, and the corresponding conditioned medium did not have any effect on MDCK monolayers. Our observations further support the hypothesis that EDGs play a role in the cytopathogenic mechanisms of A. culbertsoni.
Assuntos
Acanthamoeba/patogenicidade , Acanthamoeba/ultraestrutura , Amebíase/parasitologia , Ceratite/parasitologia , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/ultraestrutura , VirulênciaRESUMO
Three months after the World Health Organization declared the epidemic of Zika virus infections to be a Public Health Emergency of International Concern, we can look back at what we have learned and prospects for controlling the disease. Although Zika virus infections may explain many cases of brain damage in newborns, it may not be the only cause. We need a clear association between confirmed cases of Zika infections in pregnant women and microcephaly in newborns. Until we reach a firm conclusion, past experience with another virus that causes damage to newborns offers some hope. The development and almost universal use of rubella vaccine has all but eliminated the congenital rubella syndrome in the world. Rapid development of Zika virus vaccine might well do the same for this epidemic.
Assuntos
Infecção por Zika virus/prevenção & controle , Epidemias/prevenção & controle , Feminino , Humanos , Recém-Nascido , Microcefalia/etiologia , Microcefalia/prevenção & controle , Gravidez , Complicações Infecciosas na Gravidez/prevenção & controle , Rubéola (Sarampo Alemão)/prevenção & controle , Síndrome da Rubéola Congênita/prevenção & controle , Vacina contra Rubéola/uso terapêutico , Infecção por Zika virus/epidemiologiaRESUMO
Free-living amoebae (FLA) are widely distributed worldwide. Some genera included in this group act as opportunistic pathogens causing fatal encephalitis and Acanthamoeba keratitis (AK), a sight-threatening infection of the cornea associated with the use of soft contact lenses that could even end in blindness if an early diagnosis and treatment are not achieved. Furthermore, the numbers of AK cases keep rising worldwide mainly due to an increase of contact lens wearers and lack of hygiene in the maintenance of lenses and their cases. In Mexico, no cases of AK have been described so far although the isolation of other pathogenic FLA such as Naegleria fowleri and Balamuthia mandrillaris from both clinical and environmental sources has been reported. The present study reports two cases of Acanthamoeba keratitis diagnosed in two patients admitted to the Hospital "Luis Sánchez Bulnes" for Blindness Prevention in Mexico City, Mexico. Corneal scrapes and contact lenses were checked for the presence of Acanthamoeba strains in both patients. Strains were axenized after initial isolation to classify at the genotype level. After sequencing the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA (rDNA) gene of Acanthamoeba, genotype T3 and genotype T4 were identified in clinical case 1 and 2, respectively. To our knowledge, these are the first reported cases of AK in Mexico in the literature and the first description of Acanthamoeba genotypes T3 and T4 as causative agents of amoebic infection.
Assuntos
Ceratite por Acanthamoeba/diagnóstico , Acanthamoeba/classificação , Encefalite/diagnóstico , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , Ceratite por Acanthamoeba/parasitologia , Adulto , Lentes de Contato/parasitologia , Córnea/parasitologia , DNA Ribossômico/genética , Encefalite/parasitologia , Feminino , Genótipo , Humanos , México , Análise de Sequência de DNA , Adulto JovemRESUMO
A serious accident experienced firsthand by a senior authority of the American medical establishment serves as a catalyst to comment with regards to the excellent attention provided at the intensive care facilities of "ivy league" hospitals, in contrast with the poor attention received by the patient in other institutions. On this article it is also highlighted the importance of the nurses' labor to the hospitalized patient.
Se resume la experiencia "en carne propia" de una de las grandes autoridades médicas de los Estados Unidos al sufrir un gravísimo accidente casero. Se comentan las excelencias de los cuidados intensivos en hospitales de tercer nivel, en contraste con la despersonalización y el desinterés que recibió el paciente en otras instituciones médicas. Se realza la importancia de la labor de las enfermeras en la atención al paciente hospitalizado.
Assuntos
Vértebras Cervicais/lesões , Hospitais de Ensino/normas , Médicos , Relações Profissional-Paciente , Qualidade da Assistência à Saúde , Fraturas da Coluna Vertebral/terapia , Idoso de 80 Anos ou mais , História do Século XXI , Humanos , Masculino , MassachusettsRESUMO
Entamoeba histolytica is the causative agent of human intestinal and liver amebiasis. The extraordinary phagocytic activity of E. histolytica trophozoites has been accepted as one of the virulence mechanisms responsible for their invasive capacity. The recognition of the noninvasive Entamoeba dispar as a different species has raised the question as to whether the lack of pathogenic potential of this ameba correlates with a limited phagocytic capacity. We have therefore compared the process of erythrophagocytosis in both species by means of light and video microscopy, hemoglobin measurement, and the estimation of reactive oxygen species (ROS). In the present study, we confirmed that E. dispar has lower erythrophagocytic capacity. We also observed by video microscopy a new event of erythrocyte opsonization-like in both species, being more characteristic in E. histolytica. Moreover, E. dispar showed a lower capacity to produce ROS compared with the invasive species and also showed a large population of amoebae that did not engulf any erythrocyte over time. Our results demonstrate that E. histolytica has a higher phagocytic capacity than E. dispar, including a higher rate of production of ROS in the course of ingesting red blood cells.
Assuntos
Entamoeba histolytica/citologia , Entamoeba/citologia , Eritrócitos/parasitologia , Fagocitose , Animais , Bovinos , Sistemas Computacionais , Hemoglobinas/metabolismo , Humanos , Microscopia de Vídeo , Óxidos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE: To describe the adhesion properties of Acanthamoeba castellanii trophozoites to silicone hydrogel contact lenses of first generation (lotrafilcon A), second generation (galyfilcon A), and third generation (comfilcon A) and correlate the results with their specific surface characteristics, time of interaction, and suspension media. METHODS: Qualitative and quantitative assessments of the adhesion of 200 trophozoites of A. castellanii on contact lenses in culture medium (Bacto Casitone) and isotonic saline (IS) at different time points (15 minutes and 6 hours) were determined. RESULTS: By scanning electron microscopy, A. castellanii trophozoites were observed firmly adhered to the surface of hydrogel lenses after 15 minutes of interaction. The surface of lotrafilcon A lenses on which amoebae adhere better (16.4±10.2 amoebae/lens section) is rough and folded, which increases the contact surface with trophozoites, allowing acanthopodia to attach firmly. Contrarily, galyfilcon A lenses have a smoother surface, and lower numbers of amoebae were observed adhered to these lenses (4.7±2.9 amoebae/lens section). Even fewer amoebae adhered to the smoother surface of the comfilcon A lens (2.2±1.7 amoebae/lens section). Trophozoites showed similar behavior in both Bacto Casitone medium and IS. CONCLUSION: A rough surface may contribute to better adhesion of amoebae to silicone hydrogel lenses. Although a reduced numbers of trophozoites adhered to smooth lenses, trophozoites are a risk factor for amoebic keratitis. Isotonic saline facilitated trophozoite survival, suggesting that homemade saline solutions may contribute to the persistence of trophozoites, especially when there is no proper hygiene regimen used with the contact lens cases.
Assuntos
Acanthamoeba castellanii/isolamento & purificação , Lentes de Contato Hidrofílicas/microbiologia , Hidrogéis , Elastômeros de Silicone , Microscopia Eletrônica de Varredura , Propriedades de Superfície , Fatores de Tempo , TrofozoítosRESUMO
The morphological analysis of the cytopathic effect on MDCK cell monolayers and hamster cornea and qualitative and quantitative analyses of conditioned medium and proteases were evaluated and compared between two strains of Acanthamoeba genotype T4. Further than highlighting the biological differences found between both strains, the most important observation in this study was the fact that proteases both in total extracts and in conditioned medium are apparently not determinant in tissue destruction. An interestingly finding was that no lysis of corneal tissue was observed as it was previously suggested. These results, together with previous studies, allow us to conclude that the invasion and disruption of corneal tissue is performed by the penetration of the amoebae through cell junctions, either by the action of proteases promoting cellular separation but not by their destruction and/or a mechanical effect exerted by amoebae. Therefore, contact-dependent mechanisms in Acanthamoeba pathogenesis are more relevant than it has been previously considered. This is supported because the phagocytosis of recently detached cells as well as those attached to the corneal epithelium leads to the modification of the cellular architecture facilitating the migration and destruction of deeper layers of the corneal epithelium.
Assuntos
Acanthamoeba , Amebíase , Epitélio Corneano , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba/enzimologia , Acanthamoeba/patogenicidade , Acanthamoeba/ultraestrutura , Amebíase/enzimologia , Amebíase/patologia , Animais , Cricetinae , Cães , Epitélio Corneano/metabolismo , Epitélio Corneano/parasitologia , Epitélio Corneano/ultraestrutura , Junções Intercelulares/metabolismo , Junções Intercelulares/parasitologia , Junções Intercelulares/ultraestrutura , Células Madin Darby de Rim Canino , Masculino , MesocricetusRESUMO
During Acanthamoeba castellanii trophozoite-cysts differentiation, four morphological stages were identified by scanning electron microscopy: trophozoite, precyst, immature cysts, and mature cysts. Fluorescence microscopy reveals the presence of small cumulus of actin in the cytoplasm of precysts after treatment with rhodamine phalloidin. By the contrary, in mature cysts, fluorescence was not observed. However, when excystation was induced, large fluorescent patches were present. By transmission electron microscopy, encysting amebas showed small cytoplasmic vesicles containing fibrillar material, surrounded by a narrow area of thin fibrils. Similar appearance was observed in pseudopods and phagocytic invaginations. In addition, large aggregates of rod-shape elements, similar to the chromatoid bodies, described in other amebas, were present in the cytoplasm. These cysts presented large areas with orange fluorescence after treatment with acridine orange.
Assuntos
Acanthamoeba castellanii/ultraestrutura , Esporos de Protozoários/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de FluorescênciaRESUMO
Observations on cultured Acanthamoeba royreba trophozoites and in vitro cytopathogenicity of this amoeba are described. In culture, amoebae were active, pleomorphic and moved on the substrate by producing endocytic structures and emitting slight cytoplasmic microprojections from the cell surface. These projections were formed by hyaline cytoplasm and they were related to motion structures such as acanthopodia and lamellipodia, in which actin provides a framework that allows rapid changes in morphology. In the cytoplasm abundant vacuoles of different size and content were seen. By means of electron microscopy, it was possible to observe the compact fibrogranular appearance of the cytoplasm, along with the main cellular organelles such as the Golgi complex, the endoplasmic reticulum, digestive vacuoles, mitochondria and contractile vacuoles. Incubation of MDCK epithelial cell monolayers with conditioned medium did not produce a significant structural damage to the monolayer, even after 24h of incubation. When the trophozoites were incubated with the target cells the monolayer exhibited a clear injury created by the amoebae, which produced focal damage. Nevertheless, the rest of the monolayer appeared to remain intact, suggesting that a contact-dependent interaction is necessary to damage the target cells. These observations demonstrate the low invasive capacity of this amoeba.
Assuntos
Ceratite por Acanthamoeba/parasitologia , Acanthamoeba/citologia , Acanthamoeba/classificação , Acanthamoeba/patogenicidade , Acanthamoeba/ultraestrutura , Animais , Cultura Axênica , Encéfalo/parasitologia , Meios de Cultivo Condicionados , Cães , Humanos , Pulmão/parasitologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Contraste de FaseRESUMO
Research in Mexico has had an important advance, including health research. However, despite great efforts to include medical research in decision making, it is necessary to strengthen existing actions and programs, generating new research agendas in health.
Assuntos
Pesquisa Biomédica , Pesquisa Biomédica/normas , Humanos , MéxicoRESUMO
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.
Assuntos
Antígenos de Protozoários/metabolismo , Entamoeba/patogenicidade , Animais , Concanavalina A/metabolismo , Entamoeba/metabolismo , Entamoeba/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia de FluorescênciaRESUMO
Some structural observations on cultured Vahlkampfia sp. trophozoites are reported. Trophozoites are active and pleomorphic, producing large cell protrusions related to locomotion such as lamellipodia, filopodia and endocytic structures formed by hyaline cytoplasm, in which actin provides a framework that allows rapid changes in morphology. As observed by transmission electron microscopy, the cytoplasm is highly granular masking some cell organelles and the major cytoplasmic membrane systems. The structure of cell organelles such as the nucleus, endoplasmic reticulum, and digestive vacuoles is described. A common finding was the presence of 50 nm electron-dense round granules that are not limited by a membrane and that appear scattered in the cytoplasm, and whose function remains unknown. Apparently, the cell reserve material is glycogen, since complete trophozoites were positive to Schiff periodic-acid technique.
Assuntos
Amebíase/parasitologia , Ceratite/parasitologia , Schizopyrenida/ultraestrutura , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Polissacarídeos/análise , Schizopyrenida/citologia , Schizopyrenida/crescimento & desenvolvimento , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/ultraestruturaRESUMO
Electron dense granules (EDGs) were identified by transmission electron microscopy in Entamoeba histolytica trophozoites recovered from hamster liver lesions. Abundant granules were present in trophozoites recovered after 15 min of liver inoculation. Variation in the size and morphology of these EDGs was also observed. Numerous granules were present in the plasma membrane when these parasites were incubated for 5 min with MDCK monolayers. Release of these EDGs was suggested by the presence of granules in contact with the surface of the target cell plasma membrane. Parasite phagocytic invaginations were observed after 10 min of parasite-monolayer interaction. In these structures, scarce granules were seen. Granules secretion was corroborated by obtaining of a pellet of these small structures from the incubation of trophozoites with collagen supernatant. Collagenase and gellatinase activity of this pellet was identified in SDS-PAGE gels. EDGs were also present in amebic hamster liver lesions. Our observations corroborate that these granules are secreted and suggest that may participate in the cytopathic effect of E. histolytica both in vitro and in vivo.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Entamoeba histolytica/ultraestrutura , Animais , Cultura Axênica , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/parasitologia , Colágeno/metabolismo , Colagenases/metabolismo , Cricetinae , Cães , Eletroforese em Gel de Poliacrilamida , Entamoeba histolytica/enzimologia , Ativação Enzimática , Gelatinases/metabolismo , Interações Hospedeiro-Parasita , Fígado/parasitologia , Fígado/patologia , Abscesso Hepático Amebiano/parasitologia , Abscesso Hepático Amebiano/patologia , Masculino , Fagocitose , Proteólise , Fatores de Tempo , Trofozoítos/enzimologia , Trofozoítos/ultraestruturaRESUMO
Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Naegleria fowleri/enzimologia , Naegleria fowleri/crescimento & desenvolvimento , Fosfopiruvato Hidratase/metabolismo , Proteínas de Protozoários/metabolismo , Naegleria fowleri/genética , Fosfopiruvato Hidratase/genética , Proteínas de Protozoários/genéticaRESUMO
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.