W. de Souza

Interaction of Trypanosoma cruzi with macrophages in vitro: dissociation of the attachment and internalization phases by low temperature and cytochalasin B.

Chicken macrophages, obtained by cultivation of blood monocytes, were infected with epimastigote and bloodstream trypomastigote forms ofTrypanosoma cruzi strain Y. The percentage of macrophages containing parasites within parasitophorous vacuoles and of flagellates attached to cell surfaces was determined. By incubation of the macrophages at 4°C or in the presence of cytochalasin B it was possible to dissociate the attachment from the internalization phases in the process of infection of macrophages. Both treatments had a marked effect on the internalization of epimastigote and trypomastigote forms. Cytochalasin B treatment and placement of the macrophages at 4° C before infection inhibited this process by about 99 and 96%, respectively. These results suggest that endocytosis is the principal mechanism of internalization ofT. cruzi by macrophages. They show also that epimastigote and trypomastigote forms ofT. cruzi have a different rate of adhesion to the macrophage surface.

The fine structure of acidocalcisomes in Trypanosoma cruzi.

Trypanosoma cruzi survives in vertebrate and invertebrate hosts and has developed mechanisms that allow it to adapt to changes in the microenvironment such as temperature, pH, and ionic composition. Most of its calcium is concentrated in an organelle named the acidocalcisome, which is acidified by a (V-H+)-adenosine triphosphatase and has H+/Ca2+ countertransportation for calcium uptake. In this work, acidocalcisomes were examined using different transmission electron microscopy techniques. In thin sections of different stages, acidocalcisomes presented a circular shape with an electron-dense inclusion containing P3−, Ca2+, Na+, Mg2+, K+, and Zn2+. They could be distinguished from gold-labeled albumin-containing reservosomes in whole epimastigotes, and a morphometric analysis showed higher amounts of these organelles in amastigotes as compared with epimastigotes and trypomastigotes. It is possible that this variation in the amount of acidocalcisomes in the different evolutive stages could reflect adaptation mechanisms used by the parasite to survive and multiply in different environmental conditions.

Endoplasmic reticulum and Golgi-like elements in Entamoeba

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.

A stereological study of the differentiation process in Trypanosoma cruzi

When epimastigote forms ofTrypanosoma cruzi grown in a rich medium (LIT) are transferred to a simple, chemically defined medium (TAU3AAG, containing Ca2+, Mg2+, K+, Na+,l-proline,l-glutamate, andl-aspartate in phosphate buffer, they transform into trypomastigote forms. Morphometric analysis of transmission electron micrographs of thin sections of parasites collected at different steps of the transformation process showed that no changes occurred in the volume density of mitochondria and cytoplasmic vacuoles. However, a significant increase in the volume density of the kinetoplast DNA network as well as the lipid inclusions and a decrease in that of the reservosome (a special type of endosome) was observed. These observations suggest that during differentiation,T. cruzi accumulates lipids and uses molecules contained in the reservosome as its main energy source.

Fine structure of the biogenesis of Giardia lamblia encystation secretory vesicles

Synthesis, transport, and assembly of the extracellular cyst wall is the hallmark of Giardia lamblia encystation. Much is known of the biochemical pathways and their regulation. However, from a cell biology point of view, the biogenesis of the encystation specific vesicles (ESVs) that transport cyst wall proteins to the periphery of the cell is poorly understood. Therefore, we exploited a number of complementary ultrastructural approaches to test the hypothesis that the formation of ESVs utilizes a novel regulated secretory pathway. We analyzed parasites at different stages of encystation in vitro by electron microscopy of thin sections, freeze fracture replicas, and three-dimensional reconstruction from serial sections of cells fixed for cytochemical localization of the endoplasmic reticulum (ER) marker, glucose 6-phosphatase. We also used a stereological approach to determine the area occupied by the ER, clefts, ESVs, and cyst wall. Taken together, our kinetic data suggest that some ER cisternae first dilate to form clefts, which enlarge into the ESVs. Living non-encysting and early-encysting trophozoites were labeled around the periphery of both nuclei with C(6)-NBD-ceramide. At 18-21 h, outward migration of some ESVs frequently caused protrusions at the periphery of encysting trophozoites. The presence of lysosome-like peripheral vesicles between the ESV and plasma membrane of the cell was confirmed using acridine orange, an acidic compartment marker. Our data suggest that G. lamblia has a novel secretory pathway in which certain functions of the ER and Golgi co-localize spatially and temporally. These studies will increase understanding of the evolutionary appearance of regulated secretory pathways for assembly of a primitive extracellular matrix in an early diverging eukaryote.

Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells.

Giardia duodenalis is a parasitic protozoan that causes diarrhea and other symptoms which together constitute a disease known as giardiasis. Although the disease has been well defined, the mechanisms involving the establishment of the infection have not yet been fully elucidated. In this study, we show that after 24h of interaction between parasites and intestinal Caco-2 cells, there was an alteration of the paracellular permeability, as observed by an approximate 42% of reduction in the transepithelial electrical resistance and permeation to ruthenium red, which was concomitant with ultrastructural changes. Nevertheless, epithelium viability was not affected. We also demonstrate that there was no change in expression of junctional proteins (tight and adherens) but that the distribution of these proteins in Caco-2 cells after parasite adhesion was significantly altered, as observed via laser scanning confocal microscopy 3D reconstruction. The present work shows that adhesion of Giardia duodenalis trophozoites to intestinal cells in vitro induces disturbances of the tight, adherens and desmosomal junctions.

Further studies on the organization of the hydrogenosome in Tritrichomonas foetus

The fine structure of the hydrogenosome of Tritrichomonas foetus was analysed using different approaches: routine transmission electron microscopy, quick-freezing techniques followed by freeze-fracture, deep-etching and freeze-substitution, cryo-ultramicrotomy, serial sectioning followed by three-dimensional (3D) reconstruction and cytochemical detection of carbohydrates, Ca++ and phosphates. The presence of two closely apposed unit membranes surrounding the hydrogenosome, as well as its internal vesicle, was shown both in thin sections of well-preserved cells and in freeze-fracture replicas. Analysis of light micrographs, thin serial sections used to 3-D reconstruction and freeze-fracture replicas, show that the hydrogenosome of T. foetus resembles a sphere, but presents a protusion towards the cytoplasm. The vesicle varies in size from organelle to organelle and represent about 8.5% of the volume of the organelle. Based on the fact that the vesicle (a) presents a distinct morphological appearance from the hydrogenosome matrix, (b) was the main site of Ca++-accumulation, (c) presents phosphatase activity and (d) its membrane presents N-acetyl-glucosamine-containing glycoconjugates, as revealed by incubation of cryosections in the presence of gold-labeled WGA, we conclude that it represents a specialized sub-compartment of the hydrogenosome. Freeze-fracture followed by deep etching showed the presence of large number of particles, probably correspondent to macromolecules, within the hydrogenosomal matrix. These structures were not randomly distributed.

Identification of sialic acids on the cell surface of hyphae and conidia of the human pathogen Fonsecaea pedrosoi.

Sialic acids were characterized on the cell surface of conidia and hyphae of Fonsecaea pedrosoi, one of the agents of chromoblastomycosis. Neuraminidase-treated conidia had a reduced negative electrophoretic mobility and, in comparison with untreated cells, bound fewer particles of colloidal iron hydroxide and of cationized ferritin. Sialic acid residues in conidia are linked to galactopyranosyl units as indicated by the increased reactivity of neuraminidase-treated cells with peanut agglutinin. N-acetylneuraminic acid was the only derivative found in the mycelium whereas conidia contained both N-glycolyl- and N-acetylneuraminic acids.

ROLE OF THE HEAD IN THE ULTRASTRUCTURAL MIDGUT ORGANIZATION IN RHODNIUS PROLIXUS LARVAE : EVIDENCE FROM HEAD TRANSPLANTATION EXPERIMENTS AND ECDYSONE THERAPY

Studies on the effects of decapitation, head transplantation and ecdysone therapy on the ultrastructural organization of the midgut in 5th-instar larvae of Rhodnius prolixus, were carried out. Control insects had a typical and significant organization of the epithelial cells (mainly microvilli, extracellular membrane layers and basal portion of the epithelial cells) of the midgut (stomach and intestine) during the entire period of the experiment. However, the host larvae, when decapitated 1 day after feeding, demonstrated significant changes in the ultrastructural organization of the epithelial cells of these compartments. In converse experiments, head transplantations from untreated donors 4-5 days after feeding into headless larvae sustained the ultrastructural organization of the epithelial cells in the midgut. Oral therapy with ecdysone (5 &mgr;g/mL of blood meal) in decapitated insects significantly reversed the altered organization of the stomach and intestine. These results point to a brain factor, possibly the prothoracicotropic hormone (PTTH) which stimulates ecdysteroid production in the prothoracic glands, may be a factor responsible, directly or indirectly, for the midgut cell organization in R. prolixus.

Surface charge of resident, elicited, and activated mouse peritoneal macrophages.

The surface charpa of resident, thioglycollate‐elicited, and Trypanosoma cruzi‐ectivated mouse peritoneal macrophages was analyzed using cell electrophoresis. AN macrophages had a net negative surface charge. Activated macrophages had a lower zeta potential and a higher isoelectrophoretic point than resident and elicited macrophages. The populations of resident, elicited, and activated macrophages were heterogeneous in terms of surface charge. The analysis of the effect of the pH of the solution in which the macrophages were suspended on their cellular electrophoretic mobility (EPM) indicated that their surface contained both positively and negatively charged dissociating groups. The contribution of sialic acid residuos to the surface change was determined by analyzing the effect of neuraminidase treatment on the EPM of the cells. Activated mecrophages possessed more sialic add residuos exposed on their surface, and sensitive to the neuraminidase from Clostridium perfrigens, than resident and elicited macrophages. Treatment of the cells with the neuraminidase from Vibrio cholerae, however, reduced the surface charge of all macrophages in about the same extent. Macrophages had their mean EPM reduced when Incubated in the presence of Ca++, suggesting that some cell surface anionogenic sites have Ca++‐binding capacity.