Springer-Verlag 1997
Parasitol Res (1997) 83: 252–256
ORIGINAL PAPER
E.J.T. de Melo · W. de Souza
A cytochemistry study of the inner membrane complex of the pellicle of tachyzoites of Toxoplasma gondii
Received: 20 May 1996/Accepted: 6 September 1996
Abstract Confocal laser scanning microscopy and transmission electron microscopy were used to study the inner membrane complex of tachyzoites of Toxoplasma gondii. DiOC6, a lipophilic cationic fluorescent dye used to visualize the endoplasmic reticulum of eukaryotic cells, labeled cytoplasmic structures in a reticulated pattern and the periphery of the nucleus of the host cell. Intracellular and extracellular tachyzoites were stained. Observation of several focal planes showed labeling of the most peripheral region of the protozoan. Reaction product was observed in the outer nuclear membrane, in profiles of the endoplasmic reticulum, and in the inner membrane complex of tachyzoites subjected to the KI-OsO4 technique. Taken together, these observations suggest that the inner membrane complex may represent a specialized region of the endoplasmic reticulum of tachyzoites of T. gondii.
Introduction Toxoplasma gondii is a cosmopolitan parasitic protozoan with the capacity to invade virtually all nucleated cells in vertebrate hosts (Jones et al. 1972; Werk 1985; Morisaki et al. 1995). Within a modified endocytic va-
E.J.T. de Melo · W. de Souza (&) Laborato´rio de Biologia Celular e Tecidual Centro de Biocieˆncias e Biotecnologia (CBB) Universidade Estadual do Norte Fluminense. Av. Alberto Lamego, 2000, Campos dos Goytacazes, Rio de Janeiro, RJ, Brasil, Tel: +55-0247-263713; Fax: +55-247-263714; email:
[email protected] E.J.T. de Melo · W. de Souza Laborato´rio de Ultraestrutura Celular Hertha Meyer, Inst. Biofı´sica Carlos Chagas, Filho Universidade Federal do Rio de Janeiro, Ilha do Funda˜o, Rio de Janeiro, Brasil
cuole known as the parasitophorous vacuole (PV) it multiplies as a tachyzoite form with a generation time of 5–10 h until the host cell has been completely destroyed. The tachyzoite presents a group of structures located at its anterior pole that play an important role in parasite development and survival. These structures include rhoptries, dense granules, and micronemes (Dubremetz and Schwartzman1993; Dubremetz et al. 1993; Joiner 1993). Previous studies have analyzed in some detail the fine structure of the tachyzoite (Sheffield and Melton 1968). Its surface is covered by a complex pellicle formed by an outer plasma membrane, an intermediate membrane, and an inner membrane. The intermediate and inner membranes are closely apposed, constituting the inner pellicle complex (Sheffield and Melton 1968; Pfefferkorn 1990). Freeze-fracture studies have shown that the four fracture faces identified in the inner membrane complex present differences in the distribution of intramembranous particles, which at some regions are not randomly distributed but organized in special domains (Cintra and De Souza 1985). Previous studies have shown that the inner membrane complex does not surround the whole tachyzoite. Interruptions occur at the apical and the micropore regions. Evidence exists that they form plates that are connected to each other (Porchet and Torpier 1977). The nature of the inner membrane complex has not been determined. There is no biochemical marker to identify it. Observations made in other cells, especially in trypanosomatids, have shown that profiles of the endoplasmic reticulum irradiate from a more central portion of the cytoplasm toward the cell periphery, forming submembranous cisternae (Pimenta and De Souza 1983). To test the hypothesis that the inner membrane complex could be part of a specialized portion of the endoplasmic reticulum, we used two approches: (1) labeling of the tachyzoites with DiOC6, a fluorescent dye that has been shown to label the endoplasmic reticulum of living cells (Terasaki et al. 1984),
253
followed by observation in a confocal laser scanning microscope, and (2) incubation of previously fixed parasites in the presence of potassium iodide followed by postfixation with osmium tetroxide, which has been shown by transmission electron microscopy to impregnate the cisternae of the endoplasmic reticulum (Locke and Huie 1983; Figueiredo and Soares 1995). The results obtained suggest that the inner membrane complex may be a highly specialized portion of the endoplasmic reticulum of tachyzoites of T. gondii.
Materials and methods Parasites Tachyzoites from the virulent RH strain of Toxoplasma gondii were maintained by intraperitoneal passage in Swiss mice and were collected in Ringer’s solution at pH 7.2 at 48–72 h after infection. The ascites fluid obtained from infected mice was centrifuged at 200 g for 7–10 min at room temperature to remove cells and debris. The supernatant, which contained the parasites, was collected and centrifuged at 1,000 g for 7–10 min. The pellet obtained was washed two or three times with phosphate-buffered saline solution (PBS, pH 7.2) and resuspended to a density of 106 parasites/ml in 199 medium without fetal calf serum. The parasites were used within 30–40 min of their removal from the mouse peritoneal cavity, and the viability was evaluated using a dye-exclusion test with trypan blue.
Electron microscopy Vero cells were plated in culture flasks, cultivated as described above, and allowed to interact with the parasites. After interaction the cultures were washed with PBS and fixed for 1 h at room temperature in a solution containing 1% glutaraldehyde, 4% paraformaldehyde, 5 mM CaCl2 , and 5% sucrose in 0.1 M cacodylate buffer (pH 7.2). Then they were washed with cacodylate buffer supplemented with 5% sucrose and postfixed for 1 h in a solution containing 1% OsO4, 0.8% potassium ferrocyanide, and 5 mM CaCl2 in 0.1 M cacodylate buffer (pH 7.2). The cells were rinsed with cacodylate buffer, dehydrated in acetone, and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and then observed with a Zeiss 902 electron microscope. For the osmium tetroxide-potassium iodide technique the cells were plated in culture flasks, cultivated as described above, and allowed to interact with the parasites. After interaction the cultures were washed with PBS and fixed for 30 min at room temperature in a solution containing 2.5% glutaraldehyde and 5% sucrose in 0.1 M cacodylate buffer (pH 7.2). Then the cells were washed with potassium iodide (1% KI) solution in 0.1 M cacodylate buffer (pH 7.2) and postfixed in a dark environment for 24–48 h at 28° C in a solution containing 1% OsO4/1%KI in 0.1 M cacodylate buffer (pH 7.2). Subsequently the cells were rinsed with a 1% KI solution, dehydrated in acetone, and embedded in Epon. Ultrathin sections were observed by transmission electron microscopy after being either left unstained or briefly stained for 5 min with uranyl acetate. As a control, some cells were postfixed in the absence of potassium iodide. The sections were observed with Zeiss EM912 and EM902 electron microscopes.
Results and discussion Host cell Vero cells (kidney fibroblasts of the African green monkey) were maintained in Falcon plastic flasks containing 199 medium supplemented with 4% fetal calf serum and were passed by trypsinization when the cell density approached that of a confluent monolayer. At 1 day before their use in the experiments, approximately 2 × 105 Vero cells were placed on Linbro tissue plates that contained a round sterile coverslip or were plated into 25-cm2 flasks (3–5 × 105 cells/flask) and maintained at 37 °C overnight in an atmosphere containing 5% CO2. Host-cell/parasite interaction Parasites suspended in 199 medium were incubated for 1 h in the presence of Vero cells using a 5:1 parasite/host-cell ratio. After incubation the cells were washed twice with PBS to remove extracellular parasites, incubated for periods varying from 24 to 48 h at 37 °C, and processed for fluorescence or electron microscopy as described below. Confocal microscopy For visualization of the endoplasmic reticulum, DiOC6 (3-3′-dihexyloacarbocyanine iodide) staining was employed. The cells grown on coverslips (control or infected) were fixed for 2–3 min at room temperature in 0.25% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Cells were stained with a 2 to 5-lg/ml concentration of the fluorescent dye in cacodylate buffer for 30 s at room temperature. After being rinsed three times, the coverslips were examined in a Zeiss confocal laser scanning microscope (CLSM) using a 488-nm argon laser. Photographs were taken from the monitor on Tri-X (ASA 100) film using the automatic exposure control of the Zeiss camera. The Tri-X film was developed for 5 min in Kodak HC 110 (dilution B).
The examination of thin sections of tachyzoites of Toxoplasma gondii by transmission electron microscopy showed that they are covered by a typical plasma membrane. About 18 nm below it the inner membrane complex was formed by two closely apposed 65-nmthick membranes. No space was seen between the two membranes (Figs. 1, 2). Tachyzoites fixed in glutaraldehyde and subsequently postfixed in an osmium tetroxide solution containing potassium iodide showed deposition of reaction product both in the cisternae of the endoplasmic reticulum, including the nuclear envelope, and associated with the inner membrane complex but not with the outer plasma membrane (Figs. 3–5). No such reaction product was visualized when potassium iodide was omitted from the postfixation solution (data not shown). Although the exact mechanism of staining with osmium tetroxide-potassium iodide is not known, it has been suggested that it is partially due to the presence of disulfide bridges in proteins that form a reducing environment (for a review see Locke and Huie 1983). Several reports have described staining of the endoplasmic reticulum and, in some cells, of some cisternae of the Golgi complex and even secretion granules (Locke and Huie 1983; Figueiredo and Soares 1995) when the cells have been postfixed in a KI-containing osmium solution. It is noteworthy that we did not observe staining of the Golgi complex or of structures such as rhoptries, micronemes, and dense granules, which are usually considered as secretory structures used by the
254
Figs. 1,2 Transmission electron microscopy of thin sections of intracellular tachyzoites of Toxoplasma gondii. Intravacuolar tachyzoites present a complex surface system formed by a plasma membrane (long arrows) and an inner membrane complex (short arrows). (R Rhoptries, N nucleus, C conoid, T tachyzoite) Fig. 1 × 2,400. Fig. 2 × 7,200 Figs. 3–5 Cells subjected to the
potassium iodide-osmium tetroxide technique. Reaction product is seen in profiles of the endoplasmic reticulum, including the nuclear envelope (Fig. 3), and in the inner membrane complex (Figs. 4, 5). (N nucleus, E endoplasmic reticulum, T tachyzoite) Fig. 3 × 10,000. Fig. 4 × 9,100. Fig. 5 × 35,000
parasite during its penetration into the host cells (Cesbron-Delauw and Capron 1993). When we observed intracellular tachyzoites, it was clear that deposition of reaction product occurred mainly in the inner membrane complex rather than in the conventional profiles of the endoplasmic reticulum. A second approach for characterization of the inner membrane complex was the staining of previously fixed
cells with DiOC6, a lipophilic cationic dye that has been used to label the endoplasmic reticulum of mammalian cells (Terasaki 1989). The cytoplasm of uninfected cells showed a reticulated labeling pattern (Fig. 6), as has previously been described for other cells (Terasaki et al. 1984). Intense labeling of free tachyzoites was observed (Figs. 7–9), mainly concentrated at the perinuclear region and in the most peripheral portion of the protozoan.
255
Figs. 6–12 Confocal laser scanning microscopy of Vero cells and tachyzoites of T. gondii with the fluorescent probe DiOC6. Uninfected host cells show the organization of the endoplasmic reticulum (Fig. 6, star) in a characteristic pattern. Extracellular and intravacuolar tachyzoites show an intense surface fluorescence, as can be seen in two different focal planes (Figs. 6–9). The intravacuolar tachyzoites
present also display stain (Figs. 9,10: arrows). Figs. 11,12 Interferential and fluorescence microscopy, respectively, of the same cell. Intravacuolar tachyzoites (rosaceous structure in the parasitophorous vacuole, V ) and nuclear membranes are visible. (T Tachyzoites, N nucleus, V parasitophorous vacuole) Fig. 6 × 1,500. Fig. 7 × 4,200. Fig. 8 4,200. Fig. 9 × 6,300. Fig. 10 × 4,200. Fig. 11, 12 × 1,800
256
When infected cells were analyzed we observed labeling of (a) the cytoplasm of the host cell, especially arround the parasitophorous vacuole (Fig. 10); (b) the membrane lining the parasitophorous vacuole; and (c) the intravacuolar tachyzoites (Figs. 10–12). In the latter case it was possible to obtain a focal place where the labeling pattern was such that we could individualize each tachyzoite (Figs. 10–12). We interpret these images as the result of intense labeling of a peripheral structure, probably the inner membrane complex. The intense fluorescence seen around the parasitophorous vacuole membrane may have resulted from the concentration of endoplasmic reticulum elements at this region. Previous freeze-fracture studies have shown that the inner membrane complex significantly differs from the outer membrane, which corresponds to the protozoan plasma membrane. Differences include the presence of very few, if any, protuberances indicative of the presence of cholesterol as detected using the polyenic antibiotic filipin (Cintra and De Souza 1985) and a special pattern of distribution of intramembrane particles (Porchet and Torpier 1977; Cintra and De Souza 1985). These observations suggest that the inner membrane complex, which is formed by two closely apposed unit membranes, is a specialized structure of tachyzoites of T. gondii as well as other members of the phylum Apicomplexa. Our present observations suggest that it is in some way associated with the endoplasmic reticulum, since it could be labeled by DiOC6 (as seen by confocal fluorescence microscopy) and by osmium tetroxide-potassium iodide (as vizualized by transmission electron microscopy), which identify the endoplasmic reticulum of mammalian cells. Acknowledgements The authors thank Dr. Tecia U de Carvalho for suggestions in the preparation of this manuscript. This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Financiadora de Estudos e Projetos (FINEP), and Fundac¸a˜o Estadual do Norte Fluminense (FENORTE).
References Cesbron-Delauw MF, Capron A (1993) Excreted/secreted antigens of Toxoplasma gondii – their origin and role in the host-parasite interaction. Res Immunol 144: 41–45 Cintra WM, De Souza W (1985) Distribution of intramembranous particles and filipin-sterol complexes in the cell membranes of Toxoplasma gondii. Eur J Cell Biol 37: 63–69 Dubremetz JF, Schwartzman JD (1993) Subcellular organelles of Toxoplasma gondii and host cell invasion. Res Immunol 144: 31–33 Dubremetz JF, Achbarou A, Bermudes D, Joiner K (1993) Kinetics and pattern of organelle exocytosis during Toxoplasma gondii/host cell interaction. Parasitol Res 79: 402–408 Figueiredo RCBQ, Soares MJ (1995) The Golgi complex of Tryapanosoma cruzi epimastigote forms. J Submicrosc Cytol Pathol 27: 209–215 Joiner K (1993) Cell entry by Toxoplasma gondii: all paths do not lead to success. Res Immunol 144: 34–38 Jones TC, Yeh I, Hirsch JG (1972) The interaction between Toxoplasma gondii and mammalian cells. J Exp Med 136: 1157–1172 Locke M, Huie P (1983) The mystery of the unstained Golgi complex cisternae. J Histochem Cytochem 31: 1019–1083 Morisaki JH, Heuser JE, Sibley D (1995) Invasion of Toxoplasma gondii occurs by active penetration of host cell. J Cell Sci 108: 2457–2464 Pfefferkorn W (1990) Cell biology of Toxoplasma gondii. In: Wyler DJ (ed) Modern parasite biology. Freeman and Company, New York, pp 26–50 Pimenta PFP, De Souza W (1983) Fine structure and cytochemistry of the endoplasmic reticulum and its association with the plasma membrane of Leishmania mexicana amazonensis. J Submicrosc Cytol 17: 413–419 ´ tude du germe infectieux de SarcoPorchet E, Torpier G (1977) E cystis tenella et Toxoplasma gondii par la technique du cryode´capage. Z Parasitenkd 54: 101–124 Sheffield HG, Melton ML (1968) The fine structure and reproduction of Toxoplasma gondii. J Parasitol 54: 209–226 Terasaki M (1989) Fluorescent labeling of endoplasmic reticulum. In: Wang Y, Taylor DL (eds) Fluorescence microscopy of living cells in culture. Academic Press, New York, pp 125–136 Terasaki M, Song J, Wong JR, Weiss JR, Chen LB (1984) Localization of endoplasmic reticulum in living and glutaraldehydefixed cells with fluorescent dyes. Cell 38: 101–108 Werk R (1985) How does Toxoplasma gondii enter host cells? Rev Infect Dis 7: 449–457
