Scanning Electron Microscopy of Attached Trophozoites of Pathogenic Entamoeba histolytica1

The surface morphology of Entamoeba histolytica trophozoites of HM 1:IMSS (axenic and monoxenic) and HK9 (axenic) strains cultured on plastic and MDCK cell substrates was examined using scanning electron microscopy (SEM). The conditions for processing trophozoites were determined by comparing the SEM observations with the morphology of living amebas examined by light microscopy. The most frequent surface differentiations in all the amebas observed with SEM were lobopodia. Round cytoplasmic projections were found in approximately half of the axenic amebas. Endocytic stomas and filopodia were more common in monoxenic cultures while the uroid was found in only 2–8% of all examined amebas. The basal surfaces of the trophozoites, involved in both attachment and cytolysis, showed no unusual features, except for the presence of a small number of short filopodia at the outer edge. No differences were found in the morphology of amebas grown on artificial and natural substrates. These observations demonstrate that there are significant quantitative differences in the surface morphology of cultured trophozoites of different strains of E. histolytica and that association with bacteria produces an increase in the relative number of surface specializations of the parasite.     

Microscopic observations on the filopodia of Entamoeba histolytica.

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter micrometer thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 micrometers in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called "surface-active lysosomes with trigger," "dendritic plasmalemmal extensions," and "extra-amebic vesicles" by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.     

The Ultrastructure of Trophozoites of Entamoeba histolytica with Particular Reference to Spherical Arrangements of Osmiophilic Cylindrical Bodies

SYNOPSIS. Trophozoites of Entamoeba histolytica were cultured under xenic, monoxenic and axenic conditions. Some of the monoxenically cultured trophozoites were grown in the presence of Bacteroides symbiosus and others in the presence of Trypanosoma cruzi (Mexican strain). Other trophozoites were obtained from experimentally produced amebic liver abscesses in hamsters. The trophozoites were centrifuged and prepared for study by electron microscopy. Acid phosphatase activity in the parasites was determined by cytochemical reactions.
The various elements of the trophozoites were described and special attention was given to the spherical arrangements of electrons-dense cylindrical units surrounding finely granular material. Their presence was independent of the strain studied and of the nutrient elements in the culture medium. The cylindrical units probably arise from endoplasmic reticulum elements and their possible function in the digestive processes and aggression mechanisms of the trophozoites is discussed.
Acid phosphatase activity was found in round non-branching intranuclear bodies in trophozoites cultured in various media. Whether these bodies represent part of the lysosomal system of the parasite is unknown.
In preliminary work on the action of some amebicidal drugs, a special arrangement of cytomembranes morphologically similar to a Golgi complex was frequently seen in trophozoites of E. histolytica.     

Microscopic Observations on the Filopodia of Entamoeba histolytica*

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter m? thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 pan in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called “surface-active lysosomes with trigger,”“dendritic plasmalemmal extensions,” and “extra-amebic vesicles” by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.     

Entamoeba dispar: infrastructure, Surface Properties and Cytopathic Effect

The cytological features of Entamoeba dispar , recently recognized by biochemical and molecular biology criteria as a distinct species, were compared to those of Entamoeba histolytica. When cultured under axenic conditions, living trophozoites of E. dispar strain SAW 760RR clone A were more elongated in form, had a single frontal pseudopodium, and showed a noticeable uroid. In sections of E. dispar trophozoites stained with Toluidine blue, characteristic areas of cytoplasmic metachromasia were seen due to the presence of large deposits of glycogen, seldom found in E. histolytica strain HM1:IMSS. Under the light microscope the periphery of the nucleus in E. dispar was lined by finer, more regularly distributed dense granules. With transmission electron microscopy the surface coat of E. dispar was noticeable thinner. In addition, E. dispar had a lower sensitivity to agglutinate with concanavalin A and a higher negative surface charge, measured by cellular microelectrophoresis. The cytopathic effect of E. dispar was much slower, analyzed by the gradual loss of transmural electrical resistance of MDCK epithelial cell monolayers mounted in Ussing chambers. Whereas in E. histolytica phagocytosis of epithelial cells plays an important role in its cytopathic effect, E. dispar trophozoites placed in contact with MDCK cells showed only rare evidence of phagocytosis. The results demonstrate that the morphology of E. dispar is different to that of E. histolytica , both at the light microscopical and the ultrastructural levels. In addition, they show that E. dispar in axenic culture has a moderate cytopathic effect on epithelial cell monolayers. However, when compared to E. histolytica, the in vitro lytic capacity of E. dispar is much slower and less intense.     

Entamoeba histolytica: ultrastructure of trophozoites recovered from experimental liver lesions

Ultrastructural studies on Entamoeba histolytica have been carried out mostly with trophozoites cultured for many years. Under these conditions, the availability of nutrients and the absence of environmental stimuli may switch off some phenotypic characteristics of the parasite. As a result, virulence of E. histolytica diminishes with prolonged culture passages, and the ability to form cysts disappears in axenically maintained trophozoites. The present analysis by transmission electron microscopy of trophozoites recovered from experimental amebic liver lesions in hamsters revealed two types of cytoplasmic changes. On the one hand, the number of peripheral electron dense granules significantly increased in amebas obtained from liver lesions 15 min and 6h after inoculation. On the other hand, large cytoplasmic vesicles with a microfibrillar content appeared in trophozoites cultured from 72 or 96 h hepatic lesions. With fluorescence microscopy, a chitin-like material was identified in these vesicles by reactivity with calcofluor M2R. Ultrastructurally, these cytoplasmic components resemble the encystation vesicles of Entamoeba invadens and Giardia lamblia. The release of large amounts of electron dense granules, known to contain collagenase activity, probably contributes to degrade extracellular matrix components during tissue invasion. In addition, under the conditions mentioned above, amebas form encystation-like vesicles in an incomplete process of differentiation into cysts, which are the resistant form of the parasite.     

Characterization of a cytochalasin D-resistant mutant of Entamoeba histolytica

Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 microM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 microM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Abstract Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. THe immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica . The monoclonal antibody belonged to IgG₁ isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant ( P < 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant ( P < 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant ( P < 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. The immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica. The monoclonal antibody belonged to IgG1 isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant (P less than 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant (P less than 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant (P less than 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Characterization of a Cytochalasin D-Resistant Mutant of Entamoeba histolytica

Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 μM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 μM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas.     

An experimental model for amoebic abscess production in the cheek pouch of the Syrian golden hamster, Mesocricetus auratus

A new experimental model was developed in hamsters for amoebic abscess caused by Entamoeba histolytica. E. histolytica trophozoites were cultured in a liquid axenic medium, and then injected intradermally into the cheek pouch of the Syrian golden hamster, Mesocricetus auratus. Inoculation consistently resulted in abscess formation at the site in 20 of 22 (91%) study animals. The amoebic nature of the abscesses was confirmed by light microscopy and histopathologic examination. Abscess formation was maximal at day 12 post-inoculation. Potential applications of this simple and reliable model include further elucidation of the pathogenesis of invasive amoebiasis, studies of the host response to amoebae, and in vivo evaluation of chemotherapeutic agents that show in vitro efficacy against E. histolytica.     

Interaction between pathogenic amebas and fibronectin: substrate degradation and changes in cytoskeleton organization

Invasion of human tissues by the parasitic protozoan Entamoeba histolytica is a multistep process involving, as a first step, the recognition of surface molecules on target tissues by the amebas or trophozoites. This initial contact is followed by the release of proteolytic and other activities that lyse target cells and degrade the extracellular matrix. In other parasitic diseases, as well as in certain cancers, the interaction of invasive organisms or cells with fibronectin (FN) through specific receptors has been shown to be the initial step in target cell recognition. Interaction with FN triggers the release of proteolytic activities necessary for the effector cell migration and invasion. Here, we describe the specific interaction of Entamoeba histolytica trophozoites with FN, and identify a 37-kD membrane peptide as the putative receptor for FN. The interaction between the parasite and FN leads to a response reaction that includes the secretion of proteases that degrade the bound FN and the rearrangement of amebic actin into "adhesion plates" at sites of contact with FN-coated surfaces. The kinetics of the interaction was determined by measuring the binding of soluble 125I-FN to the trophozoites and visualization of the bound protein using specific antibodies. Degradation of FN was measured by gel electrophoresis and the release of radioactivity into the incubation medium. Focal degradation of FN was visualized as black spots under the trophozoites at contact sites with fluorescent FN. We conclude that the interaction of E. histolytica with FN occurs through a specific surface receptor. The interaction promotes amebic cytoskeleton changes and release of proteases from the parasite. The binding and degradation of extracellular matrix components may facilitate the migration and penetration of amebas into tissues, causing the lesions seen in human hosts.     

Entamoeba shows reversible variation in ploidy under different growth conditions and between life cycle phases

Under axenic growth conditions, trophozoites of Entamoeba histolytica contain heterogenous amounts of DNA due to the presence of both multiple nuclei and different amounts of DNA in individual nuclei. In order to establish if the DNA content and the observed heterogeneity is maintained during different growth conditions, we have compared E. histolytica cells growing in xenic and axenic cultures. Our results show that the nuclear DNA content of E. histolytica trophozoites growing in axenic cultures is at least 10 fold higher than in xenic cultures. Re-association of axenic cultures with their bacterial flora led to a reduction of DNA content to the original xenic values. Thus switching between xenic and axenic growth conditions was accompanied by significant changes in the nuclear DNA content of this parasite. Changes in DNA content during encystation-excystation were studied in the related reptilian parasite E. invadens. During excystation of E. invadens cysts, it was observed that the nuclear DNA content increased approximately 40 fold following emergence of trophozoites in axenic cultures. Based on the observed large changes in nuclear size and DNA content, and the minor differences in relative abundance of representative protein coding sequences, rDNA and tRNA sequences, it appears that gain or loss of whole genome copies may be occurring during changes in the growth conditions. Our studies demonstrate the inherent plasticity and dynamic nature of the Entamoeba genome in at least two species.     

Reactivity of Monoclonal Antibodies to Species-Specific Antigens of Entamoeba histolytica

Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica -like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica -like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia , and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis     

Vahlkampfia sp: structural observations of cultured trophozoites

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.     

Structural bases of the cytolytic mechanisms of Entamoeba histolytica

The cellular bases of the powerful cytolytic activity of the human protozoan parasite Entamoeba histolytica were explored by studying the effect of the virulent strain HM1:IMSS on epithelial monolayers of MDCK cells using a combination of time-lapse microcinematography and transmission and scanning electron microscopy. Early alterations of the epithelial cell membranes were detected by measuring changes in the transepithelial electrical resistance of MDCK monolayers mounted in Ussing chambers. The aggressive mechanism of E. histolytica trophozoites was found to be a complex, multifactorial phenomenon that included hit-and-run damage to the plasma membrane of effector cells mediated through contact, phagocytosis of lysed or apparently intact, but detached, MDCK cells, and intracellular degradation of ingested cells. Following contact with amebas, the epithelial monolayers showed a pronounced lowering of transepithelial resistance, opening of tight junctions, distortion of microvilli, surface blebbing, and the presence of minute focal discontinuities in the plasma membrane. There was no evidence of amebic exocytosis, membrane fusion, or junction formation between the parasite and host plasma membranes. Although modifications in the epithelial cell membranes usually preceded lysis, the cytolytic activity of the parasite did not exclusively involve damage to the plasma membrane of the cultured host cells but also was mediated by avid phagocytosis, the displacement and separation of neighboring cells by means of pseudopodial activity, and the "pinching-off" of the peripheral cytoplasm of epithelial cells.     

Entamoeba histolytica:Identification of Functional Gsand GiProteins as Possible Signal Transduction Elements in the Interaction of Trophozoites with Fibronectin

Soid-Raggi, L. G., Torres-Maárquez, M. E., and Meza, I. 1998.Entamoeba histolytica:Identification of functional G_sand G_iproteins as possible signal transduction elements in the interaction of trophozoites with fibronectin.Experimental Parasitology90, 262–269. Trophozoites ofEntamoeba histolyticaadhere to several components of the extracellular matrix. Binding is mediated by specific receptors identified in the parasite surface. Interaction of trophozoites with FN induces the formation of special adhesion structures that are dynamic cytoskeleton membrane complexes and facilitate both adhesion and substrate degradation. The process requires activation of signaling pathways in which PLC, IP₃, Ca²⁻, and PKC participate. These observations, and recent experiments showing increments in cAMP in the trophozoites during the interaction with FN, suggest that FN receptors in the amebic surface could be coupled to G-proteins. We report here that trophozoite plasma membrane peptides of 92, 49, 42, 37, and 21 kDa are ADP-ribosylated byVibrio choleraeandBordetella pertussistoxins. Three of them are also recognized by antibodies prepared against the α-subunit of G_s-and G_i-proteins. Adenylyl cyclase activity detected in isolated membranes was strongly stimulated by treatment with the toxins. Forskolin (an agonist of the enzyme) and FN also induced increments in the enzymatic activity. Live amebas incubated with the toxins showed enhanced adhesion to FN substrates and a striking reorganization of polymerized actin. The actin rearrangement is reminiscent of the one induced by either forskolin or dibutyril cyclic AMP treatment. Our present data show the presence and the functionality of G_s- and G_i-like proteins and their apparent activation duringin vitrointeraction of amebas with FN and complement previous observations indicating the operation of signal transduction mechanisms inE. histolytica.     

Protease activity in Giardia duodenalis trophozoites of axenic strains isolated from symptomatic and asymptomatic patients

We have examined by gelatin-SDS-PAGE the protease activity in cell lysates of Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic patient (BTU-11) and an asymptomatic carrier (BTU-10), and the reference strain Portland 1 (P1). The proteolysis band patterns showed differences among strains isolated from asymptomatic and symptomatic individuals. The lysate of the strain BTU-10, showed only five hydrolysis bands, while a greater number of bands (10-11 bands) was seen in strains BTU-11 and P1. The protease activity in all lysates was inhibited by cysteine (E-64 and iodoacetamide) and serine proteases (TPCK and TLCK) inhibitors, but not by PMSF and EDTA. In general, the results revealed protease activities in G. duodenalis trophozoites of Brazilian axenic strains and the predominance of cysteine proteinases. It should be stressed the inter-strain difference in hydrolysis band patterns observed between strains isolated from symptomatic patients and the strain obtained from an asymptomatic carrier.     

Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica.

Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa.     

Pathogenic and non-pathogenic Entamoeba: pore formation and hemolytic activity

Pore-forming activity in planar lipid bilayers and liposomes of extracts from differentially pathogenic Entamoeba and the capacity of trophozoites and subcellular fractions to lyse human red blood cells (hrbc) were investigated. In all amebas studied, the two activities paralleled each other. They were high in E. histolytica irrespective of the virulence of the particular strain, but low in non-pathogenic E. histolytica-like amebas of human origin as well as in E. invadens, which is pathogenic for reptiles, and in E. moshkovskii isolated from sewage. We conclude that the capacities to insert pores and to lyse are not sufficient for virulence although they may be necessary. The subcellular distribution of the hemolytic activity of E. histolytica and its sensitivity to a variety of inhibitors and activators differ from those of other known amebic cytotoxic activities including pore formation. Therefore, there may be an additional constituent of E. histolytica involved in the cytotoxicity of the parasite.