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Anionic sites on Toxoplasma gondii tissue cyst wall: Expression, uptake and characterization
Micron 38 (2007) 651–658 www.elsevier.com/locate/micron
Anionic sites on Toxoplasma gondii tissue cyst wall: Expression, uptake and characterization Erick Vaz Guimara˜es a, Mariana Acquarone a, Laı´s de Carvalho b, Helene Santos Barbosa a,* a
Laborato´rio de Biologia Estrutural, Departamento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil b Laborato´rio de Cultura de Ce´lulas, Departamento Histologia e Embriologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, RJ, Brazil Received 17 July 2006; received in revised form 5 September 2006; accepted 7 September 2006
Abstract Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide, which causes widespread human and animal diseases. The need for new therapeutic agents along with the biology of these parasites has fueled a keen interest in the understanding of the nutrients acquisition by these parasites. Studies on the characterization of the T. gondii cyst wall as well as the contribution of the host cell to this formation have been little explored. The aim of this paper was to investigate the electric surface charge of the T. gondii tissue cysts by ultrastructural cytochemistry, through polycationic markers, employing ruthenium red (RR) and cationized ferritin (CF). Glycosaminoglycans revealed by RR were localized on the cyst wall as a homogeneous granular layer electrondense, all over its surface. The incubation of living tissue cysts with CF for 20 min at 4 8C followed by the increase of temperature to 37 8C indicated that T. gondii cyst wall is negatively charged and that occurs an incorporation of anionic sites by the cyst wall, through vesicles and tubules, and their posterior location in the cyst matrix. So, as to identify which group of molecules produces negative charge in the cyst wall, we used enzymes for cleavage on different types of molecules, demonstrating that the negative charge in the cyst wall is mainly produced by phospholipids. Our results, described in this work show, for the first time, the negativities of the cyst wall, the incorporation and the traffic of intracellular surface molecules by T. gondii cyst wall. Our model of study can give an important contribution to the knowledge of the biology and the processes involved in nutrients acquisition by bradyzoites living inside the cysts and, and also be applied as a target for the direct action of drugs against the cyst. # 2006 Elsevier Ltd. All rights reserved. Keywords: Toxoplasma gondii; Tissue cysts; Bradyzoites; Anionic sites; Enzymatic treatment
1. Introduction The electric charge on cell surface plays an important role in some cellular processes, including cell–cell interaction, cellular differentiation and endocytosis (van Oss, 1978; Spangenberg and Crawford, 1987; Mutsaers and Papadimitriou, 1988). Concerning the membranes composition of eukaryotic cells, it has been demonstrated that the possible candidates for producing negative charge in the membranes surface are mainly: the carboxylate and sulphate groups in the mucopolysaccharides acids; phosphate groups in phospholipids, and the carboxylate groups, widely distributed in glutamic and neuraminic acid (Weiss, 1969; Mehereshi, 1972). The
* Corresponding author. Tel.: +55 21 2598 4413; fax: +55 21 2260 4434. E-mail address: [email protected] (H.S. Barbosa). 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.09.002
interaction of surface charges between host cells and protozoa, together with other specific mechanisms, contributes to the internalization process of these organisms (Klotz, 1994; Kleffmann et al., 1998; Maruyama et al., 1998). In the case of Toxoplasma gondii tachyzoites, in spite of the absence of lectin bind sites on its surface (Sethi et al., 1977; Handman et al., 1980; Hoshino-Shimizu et al., 1980; Derouin et al., 1981), these forms have negative surface charge with an electrophoretic mobility (Cintra et al., 1986), similar to the protozoa that present ‘‘coat’’ of glycoconjugate as Trypanosoma cruzi (Souto-Padro´n et al., 1984). Proteases and glycosidases enzymatic studies developed with T. gondii tachyzoites showed that residues of sialic acid are not present on the surface of these parasites. The treatment with phospholipase C reduced the electrophoretic mobility of the parasites, indicating that phosphate groups must contribute to their negative surface charge (Cintra et al., 1986). Akaki et al.
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(2001), analyzing the surface charge of protozoan under atomic force microscopy, described that the area of initial contact between parasites, such as T. gondii, Leishmania amazonensis, Entamoeba histolytic and T. cruzi, and the host cell presents positive charge. The cyst wall is important to the maintenance and integrity of the parasite inside of the host cell for long periods and it has been accepted the hypothesis that it is produced by parasitophorous vacuole membrane modifications, after the invasion of the tachyzoites, and its interconversion to bradyzoites (reviewed in Weiss and Kim, 2000). The T. gondii cyst wall may limit the communication of the parasite with its host cells and also the antigen presentation to the host, contributing to the persistence of this intracellular parasite. The structure and function of the cyst wall components have been poorly characterized (Sethi et al., 1977; Meingassner et al., 1977; Derouin et al., 1981; Zhang et al., 2001). A basic property of eukaryotic cells is the endocytosis, which represents the capacity to incorporate extracellular fluids, molecules, solutes and particles to the inside of intracellular vesicles through different mechanisms. Information of endocytosis mechanisms by Apicomplexa parasites has started to be elucidated (Coppens et al., 2000; reviewed in Robibaro et al., 2001). The ultrastructural evidences of endocytosis in T. gondii indicated the micropore as the structure responsible for the nutrients incorporation, for both tachyzoites and bradyzoites forms (Nichols et al., 1994). Recently, receptor mediated endocytosis in T. gondii was described, through the bond and internalization of sulphated glycans similar to the heparin, while fluid-phase markers were incorporated by non-specific pinocytosis (Botero-Kleiven et al., 2001). Ultrastructural and functional evidences of an endocytic pathway in T. gondii have been described, in which acidic compartments were located in the rhoptries, suggesting that these organelles are related to lysosomes (Shaw et al., 1998). In addition, it has been demonstrated in tachyzoites, the existence of tubule-vesicle compartments associated with the Rab5, a molecular marker for early endosomes (Robibaro et al., 2001). The incorporation and traffic of intracellular nutrients in T. gondii are fields yet to be explored in detail such as in tachyzoite, bradyzoite and tissue cyst. With all the evidences above, the aim of this work is to verify ultrastructurally the tissue cysts surface electric charge, using cationized ferritin (CF) as a cationic marker, to analyze the dynamics of its incorporation and fate in the tissue cysts, besides the nature of anionogenic groups using enzymatic treatment.
2.2. Cysts isolation and purification The methodology used was adapted and modified from that described by Freyre (1995) and Popiel et al. (1996). Mice were killed in CO2 chamber, and the brains were surgically removed under aseptic conditions. After immersion at 4 8C in phosphate buffered saline (PBS), the brains were washed in the same buffer to remove blood cells, fragmented with the aid of scissors, and macerated in PBS by successive passages of the fragments using 18-23 G needles. After that, 20 ml of the total suspension were placed between slide/coverslip (24 mm 32 mm), and the total number of cysts was determined in the total area of the coverslip using light microscopy. The procedures with animals were carried out in accordance with the guidelines established by the Fundac¸a˜o Oswaldo CruzFIOCRUZ, Committee of Ethics for the Use of Animals, by license CEUA 0229-04. Tissue suspension containing cysts was filtered with cell dissociation sieve-tissue grinder kit (Sigma Chemical Co.) to remove small tissue fragments and cellular debris. Then, the suspension was centrifuge at 400 g for 10 min and the pellet was resuspended in Eagle’s medium supplemented with 25% dextran (about 1 brain per 2.5 ml of final solution). After centrifugation at 2200 g for 10 min, the pellet containing the cysts was recovered and resuspended in PBS, centrifuged at 400 g for 10 min to remove the dextran solution, and then diluted in the same buffer. The determination of the cysts number was carried out as described above. 2.3. Detection and fate of anionic sites The CF was used to verify the presence of anionic sites on cysts wall surface and its fate in tissue cyst. After isolation, the cysts were incubated with 200 mg/ml of CF in PBS, pH 7.2, for 20 min at 4 8C. The intracellular traffic of anionic sites was monitored by the posterior incubation of the cysts for 1–48 h at 37 8C. Then, the cysts were washed three times in PBS for the removal of ferritin particles that did not adhere to the surface of the cysts wall. The material was fixed in 2.5% of glutaraldehyde (GA) buffered in 0.1 M of sodium cacodylate buffer containing 3.5% of sucrose, pH 7.2, for 1 h at 4 8C. The samples were then washed in the same buffer three times for 10 min each and postfixed for 1 h at 4 8C in 1% of osmium tetroxide (OsO4) in 0.1 M sodium cacodylate buffer, rinsed, dehydrated and embedded in Epoxy resin. Thin sections were stained with uranyl acetate and lead citrate, and then examined under a Zeiss EM10C transmission electron microscope.
2. Materials and methods
2.4. Ruthenium red (Luft, 1971)
2.1. Parasites
In these assays, the tissue cysts, after isolation and purification, were fixed in 2.5% GA in 0.1 M sodium cacodylate buffer containing 0.02% of ruthenium red, pH 7.2, for 1 h at 4 8C. After the fixation, the cysts were washed in 0.1 M of the same buffer two times for 10 min each and postfixed in 1% OsO4 containing 0.02% of ruthenium red in 0.1 M of sodium cacodylate buffer for 30 min at 4 8C. The cysts were
T. gondii tissue cysts of ME-49 strain (Type II) were used. The parasites were maintained in C57BL/6 female mice, weighing about 12–18 g each, and inoculated intraperitoneally with about 30 cysts/animal. After 4–8 weeks, the tissue cysts were isolated from the brain, as described below.
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then washed three times in 0.1 M of sodium cacodylate buffer containing 0.01% of ruthenium red for 10 min each; dehydrated and embedded in Epoxy resin as described above.
The cysts were then washed three times in the same buffer and processed for observation under transmission electron microscopy as described above.
2.5. Enzymatic treatments of tissue cysts
3. Results
The cysts were treated with the following enzymes: phospholipase A2 (PLA2) from Naja mossambica mossambica (10, 50 and 100 mg/ml); phospholipase C (PLAC) type IX from Clostridium perfringens (0.2, 0.5, 1 and 2 U/ml) and Neuraminidase from Vibrio cholerae (0.2 and 0.5 U/ml) diluted in 0.85% NaCl solution, pH 7.4, during 30 and 60 min at 37 8C; protease type XIV from Streptomyces griseus and trypsin from porcine pancreas (1, 10, 100 and 500 mg/ml) diluted in 0.85% NaCl solution, pH 7.4, during 5 min at 37 8C. After the treatments, the material was washed in 0.85% NaCl solution three times and incubated with 200 mg/ml of CF diluted in the same medium, pH 7.4, for 20 min at 4 8C. The experimental control was kept by incubating tissue cysts with only 200 mg/ml of CF for 20 min at 4 8C without any enzymatic treatment.
3.1. Detection and fate of anionic sites The cationized ferritin (CF) is a polycationic derivative electrondense of native ferritin ionized at physiological pH that allows experiments with living cells. The CF was used in our studies as a marker of surface anionic sites of tissue cysts. The incubation of the living tissue cysts with CF for 20 min at 4 8C and subsequent elevation of temperature to 37 8C for 1 h, revealed the distribution of the CF in patches (Fig. 1) and also as a fine particle layer with uniform distribution on the cyst wall (Fig. 2), indicating that T. gondii cyst wall is negatively charged. In these conditions invaginations of the cyst wall were filled with the tracer (Figs. 1 and 2). Vesicles of different diameters and tubules
Figs. 1–3. Anionic sites in T. gondii tissue cysts using cationized ferritin (CF). Fig. 1. Incubation of the tissue cysts for 20 min at 4 8C and subsequent elevation of temperature to 37 8C for 1 h, showing formation of clusters of CF on the surface of the CW (arrowhead), internalization of CF particles through vesicles (V) and tubules (T) from the cyst wall (CW). Particles were found in invaginations of the CW (arrow), in tubules and vesicles in the cystic matrix (*) below the CW. Fig. 2. Tissue cysts showing a vesicle with CF particles near the apical complex (AC) of intracystic parasites. Fig. 3. High magnification allows to observe details of vesicles (V) and tubules (T) containing CF particles in the cyst matrix next to the granular region. Bars: 0.5 mm.
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containing ferritin particles were localized right below the granular region (Fig. 3), in the cystic matrix (Figs. 2 and 3) and next to or in direct contact with the membrane of intracystic bradyzoites (Figs. 2 and 4–6). 3.2. Ruthenium red The ultrastructural analysis of glycosaminoglycans in thin sections of T. gondii tissue cysts after incubation with the
ruthenium red showed a fine granular electrondense layer all over surface and in the invagination of the cyst wall (Fig. 7). Small vesicles filled with the marker, located right below the internal face of the membrane and also in the granular region, are transversal cuts of membrane invaginations containing ruthenium red (Fig. 7). The intracystic matrix and the parasites did not present marking, since it is a result of the well known property that ruthenium red do not penetrate in whole membranes.
Figs. 4–6. Anionic sites in T. gondii tissue cysts using CF. Figs. 4 and 5. In detail, vesicles (V) of different sizes and tubule (T) containing the label are observed near the bradyzoityes membrane (Bz). Fig. 6. After 2 h of FC incubation at 37 8C, it was possible to find vesicles containing ferritin very near and also adhered to intracystic parasites. Bars: 0.5 mm.
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Fig. 7. Detection of glycosaminoglycans in T. gondii tissue cysts incubated with ruthenium red (RR). Revelation of glycosaminoglycans on the T. gondii cysts membrane after incubation with RR. This image shows detail of the membrane of the cyst, the invaginations with particles of ruthenium red (arrow) and transversal cuts of these invaginations making visible the vesicles containing the marker (arrowhead). CW: cyst wall. Bar: 0.5 mm.
3.3. Enzymatic treatments of tissue cysts In order to identify which group of molecules is responsible for the negative charge of the cyst wall, we cleaved the surface molecules enzymatically. The cyst control, incubated with CF (Fig. 8) and after treatment with PLAC, maintained the labeling on the cyst surface (Fig. 9). The incubation of tissue cysts with PLA2 (10 and 50 mg/ml) for 30 min at 37 8C revealed that this was the only enzyme that diminished the expression with CF. Cysts showed a decrease of the CF distribution when treated with 10 mg/ml of PLA2 (Figs. 10 and 11), when compared with the control (Fig. 8) that was incubated only with CF. No significant decrease in the expression of anionic sites in the cyst wall was observed when other enzymes (neuraminidase, protease type XIV, trypsin) were employed (data not showed). Depending on the concentrations of the enzymes, the cysts breached, turning impracticable the analysis of the anionic sites expression. In a same cyst, the surface presented CF followed by areas without any marking with the CF (Fig. 10). The higher magnification of Fig. 10 shows the cyst wall area label with CF (Fig. 11). No label was observed using 50 mg/ml of enzyme (Fig. 12). Our results showed that the groups of molecules that are probably producing negative charge in the cyst wall are the phospholipids. 4. Discussion Our results with ultrastructural cytochemistry using CF and ruthenium red clearly demonstrated that the T. gondii cyst wall is negatively charged. Studies on the plasma membrane of protozoa have suggested that its components play an essential role in pathogenic processes, interacting with molecules of the surface of the host cells, activating the invasion process (Pimenta and De Souza, 1983; De Carvalho et al., 1985; Cintra et al., 1986; Kleffmann et al., 1998; Akaki et al., 2001). Thus, the analysis of the surface molecules of the pathogens and their target cells is primordial for the better knowledge of this interaction. There are few studies about the surface charge of the T. gondii and the molecules which are responsible for the origin of these charges (De Carvalho and De Souza, 1990; Akaki et al., 2001; Stumbo et al., 2002). Locksley et al. (1982) described that the tachyzoite surface has a negative charge and Cintra et al. (1986) demonstrated that this charge is sensible to phospholipase C, suggesting that phosphate groups contribute to its surface charge. The characterization of the surface
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components of bradyzoites and tissue cysts has been little explored, despite the high relevance of both in the course of chronic toxoplasmosis (reviewed in Dubey et al., 1998; Weiss and Kim, 2000). The formation of the cyst wall and the constitution of its matrix are events that follow the differentiation of bradyzoites and these walls have been considered as results of the modification of the parasitophorous vacuole membrane that surrounds the parasite within the host cell (Gross et al., 1995; Bohne et al., 1996, 1999; Weiss and Kim, 2000). Our results demonstrated the presence of negative electric charge on the surface of tissue cysts, using two cationic markers: (i) CF that presents electrostatic binds to the negative charge molecules and (ii) ruthenium red, an inorganic marker, in which atoms of oxygen, ruthenium and amine all together in known configuration as complex amine, present high affinity with polyanionic substances, such as the glycosaminoglycans. The positive reaction to ruthenium red in Plasmodium berghei merozoites surface and in T. gondii tachyzoites was described by Seed et al. (1974) and Cintra et al. (1986), respectively. Considering the absence of binding to lectins on the surface of P. berghei and T. gondii, Cintra et al. (1986) suggested that ruthenium red linkage is only an electrostatic interaction of the polycationic marker with the parasite surface containing anionic components whose surface electric charge could be given by anionic phospholipids. Such a conclusion cannot be inferred from tissue cysts, since the groups of Sethi et al. (1977), Derouin et al. (1981) and Zhang et al. (2001) demonstrated, yet with conflicting results, residues of carbohydrates on tissue cysts surface, although residues of sialic acid have not been demonstrated, nor has the presence of glycosaminoglycans in the composition of cyst wall been investigated. We are demonstrating for the first time the negative charge of the cyst wall, using two markers that present different properties to bind the anionic groups on the surface of tissue cysts of T. gondii. The nature of the membrane components of these cysts that produce this negativity remains an open question, but our results with the enzymatic treatment of tissue cysts denote the participation of phospholipids on the anionic nature of the cyst wall. Amongst all the enzymes used in the treatment of the tissue cysts, only phospholipase A2 was efficient in reducing the anionic sites of the surface of these cysts. PLA2 is a family of lipolytic enzymes involved in the phospholipid digestion, remodeling of cell membranes and host defense. They cleave the fatty acyl bond at the sn-2 position of membrane glycerophospholipids to generate unsaturated fatty acids and lysophospholipids. In mammals, this activity results in the release of arachidonic acid from the membrane phospholipids, which is the starting material for the generation of a wide range of biologically active lipid mediators, including prostaglandins, leukotrienes, thromboxanes and prostacyclins (reviewed in Valentin and Lambeau, 2000; East and Isacke, 2002). There is no literature on studies about the lipids composition of the cyst wall. Our results suggest that the negative electric charge of the cyst is given partially or totally by some groups of the fatty acids tail that bind to carbon 2 of the glycerol structure. These conclusions are due to the absence or decrease of CF in
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Figs. 8–12. Enzymatic treatment in T. gondii tissue cysts. Fig. 8. Experimental control without enzymatic treatment showing the CW labeled with CF over the surface. Fig. 9. Cysts treated with 1 U/ml phospholipase C maintain the labeling for CF. Fig. 10. Treatment of the cysts with 10 mg/ml phospholipase A2 presenting for 30 min decrease in the labeling of the anionic sites on CW after incubation with CF. Bradyzoites (Bz). Fig. 11. High magnification of cyst of Fig. 10 showing the region containing CF particles adhered to the cyst wall membrane. Fig. 12. Cyst treated with 50 mg/ml phospholipase A2 showing absence of labeling of the anionic sites on CW when post-incubated with CF. Bar: 0.5 mm.
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cysts treated with PLA2. The occurrence of cysts with discrete marker after the enzymatic treatment can be a consequence of a partial access of the enzyme to some cysts. However, it is little probable, since the cysts remained under constant agitation during the treatment. Another hypothesis that can be considered is that the fatty acids tail of carbon 1 bind to glycerol own similar cleaved tail for PLA2, also producing negative charge, that would only be completely eliminated by treating the cysts with PLA1 and/or B. The presence of cysts with marked and not marked regions can be in consequence of a heterogeneous distribution of the glycerolphospholipids after treatment where not all the negative charge was eliminated, or of the heterogeneous distribution of other molecules that also give negative charge to the cyst wall. We demonstrated ultrastructurally, using CF that negatively charged molecules present in the cystic wall are incorporated by tubules and vesicles formed from the membrane that delimits the cyst wall and its posterior localization in the matrix. Previous works about the ultrastructure of the cyst wall had described the presence of these structures in the granular region and matrix of the cysts, being its origin unknown (Wanko et al., 1962; Matsubayashi and Akao, 1963; Jacobs, 1967). Mehlhorn and Frenkel (1980), through the ultrastructural analysis of the cyst wall in the skeletal muscle, suggested that the vesicles found in the granular region could be derived from the cyst membrane. We confirmed the origin of these structures from the membrane, supported by the first demonstration of the traffic of a marker (CF) of the cyst membrane and its intracystic fate. Additionally, we verified the presence of vesicles, neighbor or close to the bradyzoites plasma membrane. There are no evidences of the fusion of these vesicles and the parasites membrane, but we can suggest that it could be one of the pathways of nutrients originating from the host cell. We also did not find any evidence of the incorporation of vesicles containing cationized ferritin through the micropore, as described by Nichols et al. (1994), when they observed vesicles and cellular debris in micropores of intracystic bradyzoites. Our observations, showing the cyst wall through its delimiting membrane with many invaginations which provably enlarged the cyst surface area, facilitating the exchange of materials between the intracystic parasites and cytosol of the host cell, are in accordance with Jones et al. (1986), Ferguson and Hutchison (1987) and Nichols et al. (1994). The nutrients required by bradyzoites confined inside the cyst wall may be necessary for its maintenance, multiplication and the synthesis of amylopectin granules (Guimara˜es et al., 2003; Dzierszinski et al., 2004). The amylopectin is synthesized in the bradyzoite, suggesting that the biogenesis of these complex carbohydrate structures may be of key importance in the bradyzoite differentiation and persistence of cysts during host infection by T. gondii (Coppin et al., 2003). Tachyzoites in the interior of parasitophorous vacuole develop a vesicle-tubular network that seems to be partly contiguous to the parasite plasma membrane and to form connections to the parasitophorous vacuole membrane (Sibley et al., 1986, 1995; Halonen et al., 1996). This network, due to its large surface area, could play an
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important role in the exchange of solutes between the intracellular parasite and its host (Sibley et al., 1995; Labruyere et al., 1999; reviewed in Saliba and Kira, 2001). The argument that the study of the endocytosis in bradyzoites is limited in virtue of the damages caused to the parasites during the processes of their isolation from tissue cysts by mechanic or enzymatic digestion (Nichols et al., 1994) deserves to be reviewed for two reasons: first, the methods of isolation of bradyzoites from more recent tissue cysts and the bioassays in mice have demonstrated ‘‘in vivo’’ infectivity, at least minimizing the effect of the processes on the viability of the parasites (Freyre, 1995; Popiel et al., 1996); second, the endocytosis by bradyzoites can be potentially explored through experimental protocols, using whole cysts. Our model can give an important contribution to the knowledge of the biology and the processes involved in the nutrients acquisition by cysts and bradyzoites, and be also applied as a target for the direct action of drugs against of T. gondii cyst. Acknowledgements Support was provided by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa Estrate´gico de Apoio a` Pesquisa em Sau´de—PAPES IV and Instituto Oswaldo Cruz-Fiocruz. References Akaki, M., Nakano, Y., Nagayasu, E., Nagakura, K., Kawai, S., Aikawa, M., 2001. Invasive forms of Toxoplasma gondii, Leishmania amazonensis and Trypanosoma cruzi have a positive charge at their contact site with host cells. Parasitol. Res. 87, 193–197. Bohne, W., Holpert, M., Gross, U., 1999. Stage differentiation of the protozoan parasite Toxoplasma gondii. Immunobiology 201, 248–254. Bohne, W., Parmley, S.F., Yang, S., Gross, U., 1996. Bradyzoite-specific genes. Curr. Top. Microbiol. Immunol. 219, 81–91. Botero-Kleiven, S., Fernandez, V., Lindh, J., Richter-Dahlfors, A., von Euler, A., Wahlgren, M., 2001. Receptor-mediated endocytosis in an apicomplexan parasite (Toxoplasma gondii). Exp. Parasitol. 98, 134–144. Cintra, W.M., Silva-Filho, F.C., De Souza, W., 1986. The surface charge of Toxoplasma gondii: a cytochemical and electrophoretic study. J. Submicrosc. Cytol. 18, 773–781. Coppens, I., Sinai, A.P., Joiner, K.A., 2000. Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition. J. Cell Biol. 149, 167–180. Coppin, A., Dzierszinski, F., Legrand, S., Mortuaire, M., Ferguson, D., Tomavo, S., 2003. Developmentally regulated biosynthesis of carbohydrate and storage polysaccharide during differentiation and tissue cyst formation in Toxoplasma gondii. Biochimie 85, 353–361. De Carvalho, T.U., Souto-Padroon, T., De Souza, W., 1985. Trypanosoma cruzi: surface charge and freeze-fracture of amastigotes. Exp. Parasitol. 59, 12–23. De Carvalho, L., De Souza, W., 1990. Internalization of surface anionic sites and phagosome-lysosome fusion during interaction of Toxoplasma gondii with macrophages. Eur. J. Cell Biol. 51, 211–219. Derouin, F., Beauvais, B., Lariviere, M., Guillot, J., 1981. Binding of fluorescein-labelled lectins on trophozoites and cysts of 3 strains of Toxoplasma gondii. C. R. Seances Soc. Biol. Fil. 175, 761–768. Dubey, J.P., Lindsay, D.S., Speer, C.A., 1998. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin. Microbiol. Rev. 11, 267–299.
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Meingassner, J.G., Matthaei, C., Teutsch, H.F., Sasse, D., 1977. Histochemistry of the carbohydrate metabolism in cysts of Toxoplasma gondii. Z. Parasitenkd. 51, 219–228. Mutsaers, S.E., Papadimitriou, J.M., 1988. Surface charge of macrophages and their interaction with charged particles. J. Leukoc. Biol. 44, 17–26. Nichols, B.A., Chiappino, M.L., Pavesio, C.E., 1994. Endocytosis at the micropore of Toxoplasma gondii. Parasitol. Res. 80, 91–98. Pimenta, P.F., De Souza, W., 1983. Leishmania mexicana amazonensis: surface charge of amastigote and promastigote forms. Exp. Parasitol. 56, 194– 206. Popiel, I., Gold, M.C., Booth, K.S., 1996. Quantification of Toxoplasma gondii bradyzoites. J. Parasitol. 82, 330–332. Robibaro, B., Hoppe, H.C., Yang, M., Coppens, I., Ngo, H.M., Stedman, T.T., Paprotka, K., Joiner, K.A., 2001. Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii. Int. J. Parasitol. 31, 1343–1353. Saliba, K.J., Kira, K., 2001. Nutrient acquisition by intracellular apicomplexan parasites: staying in for dinner. Int. J. Parasitol. 31, 1321–1330. Seed, T.M., Aikawa, M., Sterling, C., Rabbege, J., 1974. Surface properties of extracellular malaria parasites: morphological and cytochemical study. Infect. Immun. 9, 750–761. Sethi, K.K., Rahman, A., Pelster, B., Brandis, H., 1977. Search for the presence of lectin-binding sites on Toxoplasma gondii. J. Parasitol. 63, 1076–1080. Shaw, M.K., Roos, D.S., Tilney, L.G., 1998. Acidic compartments and rhoptry formation in Toxoplasma gondii. Parasitology 117, 435–443. Sibley, L.D., Krahenbuhl, J.L., Adams, G.M., Weidner, E., 1986. Toxoplasma modifies macrophage phagosomes by secretion of a vesicular network rich in surface proteins. J. Cell Biol. 103, 867–874. Sibley, L.D., Niesman, I.R., Parmley, S.F., Cesbron-Delauw, M., 1995. Regulated secretion of multi-lamellar vesicles leads to formation of a tubulovesicular network in host-cell vacuoles occupied by Toxoplasma gondii. J. Cell Sci. 108, 1669–1677. Souto-Padro´n, T., De Carvalho, T.U., Chiari, E., De Souza, W., 1984. Further studies on the cell surface charge of Trypanosoma cruzi. Acta Trop. 41, 215– 225. Spangenberg, P., Crawford, N., 1987. Surface membrane electrokinetic properties of polymorphonuclear leucocytes: subpopulation heterogeneity and phagocytic competence. J. Cell Biochem. 34, 259–268. Stumbo, A.C., Barbosa, H.S., Carvalho, T.M., Porto, L.C., Carvalho, L., 2002. Anionic sites, fucose residues and class I human leukocyte antigen fate during interaction of Toxoplasma gondii with endothelial cells. Mem. Inst. Oswaldo Cruz 97, 517–522. Valentin, E., Lambeau, G., 2000. Increasing molecular diversity of secreted phospholipases A(2) and their receptors and binding proteins. Biochim. Biophys. Acta 1488, 59–70. van Oss, C.J., 1978. Phagocytosis as a surface phenomenon. Annu. Rev. Microbiol. 32, 19–39. Wanko, T., Jacobs, L., Gavin, M.A., 1962. Electron microscope study of Toxoplasma cysts in mouse brain. J. Protozool. 9, 235–242. Weiss, L.M., 1969. The cell periphery. Int. Rev. Cytol. 26, 63–105. Weiss, L.M., Kim, K., 2000. The development and biology of bradyzoites of Toxoplasma gondii. Front. Biosci. 5, D391–D405. Zhang, Y.W., Halonen, S.K., Ma, Y.F., Wittner, M., Weiss, L.M., 2001. Initial characterization of CST1, a Toxoplasma gondii cyst wall glycoprotein. Infect. Immun. 69, 501–507.
Anionic sites on Toxoplasma gondii tissue cyst wall: Expression, uptake and characterization Erick Vaz Guimara˜es a, Mariana Acquarone a, Laı´s de Carvalho b, Helene Santos Barbosa a,* a
Laborato´rio de Biologia Estrutural, Departamento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil b Laborato´rio de Cultura de Ce´lulas, Departamento Histologia e Embriologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, RJ, Brazil Received 17 July 2006; received in revised form 5 September 2006; accepted 7 September 2006
Abstract Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide, which causes widespread human and animal diseases. The need for new therapeutic agents along with the biology of these parasites has fueled a keen interest in the understanding of the nutrients acquisition by these parasites. Studies on the characterization of the T. gondii cyst wall as well as the contribution of the host cell to this formation have been little explored. The aim of this paper was to investigate the electric surface charge of the T. gondii tissue cysts by ultrastructural cytochemistry, through polycationic markers, employing ruthenium red (RR) and cationized ferritin (CF). Glycosaminoglycans revealed by RR were localized on the cyst wall as a homogeneous granular layer electrondense, all over its surface. The incubation of living tissue cysts with CF for 20 min at 4 8C followed by the increase of temperature to 37 8C indicated that T. gondii cyst wall is negatively charged and that occurs an incorporation of anionic sites by the cyst wall, through vesicles and tubules, and their posterior location in the cyst matrix. So, as to identify which group of molecules produces negative charge in the cyst wall, we used enzymes for cleavage on different types of molecules, demonstrating that the negative charge in the cyst wall is mainly produced by phospholipids. Our results, described in this work show, for the first time, the negativities of the cyst wall, the incorporation and the traffic of intracellular surface molecules by T. gondii cyst wall. Our model of study can give an important contribution to the knowledge of the biology and the processes involved in nutrients acquisition by bradyzoites living inside the cysts and, and also be applied as a target for the direct action of drugs against the cyst. # 2006 Elsevier Ltd. All rights reserved. Keywords: Toxoplasma gondii; Tissue cysts; Bradyzoites; Anionic sites; Enzymatic treatment
1. Introduction The electric charge on cell surface plays an important role in some cellular processes, including cell–cell interaction, cellular differentiation and endocytosis (van Oss, 1978; Spangenberg and Crawford, 1987; Mutsaers and Papadimitriou, 1988). Concerning the membranes composition of eukaryotic cells, it has been demonstrated that the possible candidates for producing negative charge in the membranes surface are mainly: the carboxylate and sulphate groups in the mucopolysaccharides acids; phosphate groups in phospholipids, and the carboxylate groups, widely distributed in glutamic and neuraminic acid (Weiss, 1969; Mehereshi, 1972). The
* Corresponding author. Tel.: +55 21 2598 4413; fax: +55 21 2260 4434. E-mail address: [email protected] (H.S. Barbosa). 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.09.002
interaction of surface charges between host cells and protozoa, together with other specific mechanisms, contributes to the internalization process of these organisms (Klotz, 1994; Kleffmann et al., 1998; Maruyama et al., 1998). In the case of Toxoplasma gondii tachyzoites, in spite of the absence of lectin bind sites on its surface (Sethi et al., 1977; Handman et al., 1980; Hoshino-Shimizu et al., 1980; Derouin et al., 1981), these forms have negative surface charge with an electrophoretic mobility (Cintra et al., 1986), similar to the protozoa that present ‘‘coat’’ of glycoconjugate as Trypanosoma cruzi (Souto-Padro´n et al., 1984). Proteases and glycosidases enzymatic studies developed with T. gondii tachyzoites showed that residues of sialic acid are not present on the surface of these parasites. The treatment with phospholipase C reduced the electrophoretic mobility of the parasites, indicating that phosphate groups must contribute to their negative surface charge (Cintra et al., 1986). Akaki et al.
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(2001), analyzing the surface charge of protozoan under atomic force microscopy, described that the area of initial contact between parasites, such as T. gondii, Leishmania amazonensis, Entamoeba histolytic and T. cruzi, and the host cell presents positive charge. The cyst wall is important to the maintenance and integrity of the parasite inside of the host cell for long periods and it has been accepted the hypothesis that it is produced by parasitophorous vacuole membrane modifications, after the invasion of the tachyzoites, and its interconversion to bradyzoites (reviewed in Weiss and Kim, 2000). The T. gondii cyst wall may limit the communication of the parasite with its host cells and also the antigen presentation to the host, contributing to the persistence of this intracellular parasite. The structure and function of the cyst wall components have been poorly characterized (Sethi et al., 1977; Meingassner et al., 1977; Derouin et al., 1981; Zhang et al., 2001). A basic property of eukaryotic cells is the endocytosis, which represents the capacity to incorporate extracellular fluids, molecules, solutes and particles to the inside of intracellular vesicles through different mechanisms. Information of endocytosis mechanisms by Apicomplexa parasites has started to be elucidated (Coppens et al., 2000; reviewed in Robibaro et al., 2001). The ultrastructural evidences of endocytosis in T. gondii indicated the micropore as the structure responsible for the nutrients incorporation, for both tachyzoites and bradyzoites forms (Nichols et al., 1994). Recently, receptor mediated endocytosis in T. gondii was described, through the bond and internalization of sulphated glycans similar to the heparin, while fluid-phase markers were incorporated by non-specific pinocytosis (Botero-Kleiven et al., 2001). Ultrastructural and functional evidences of an endocytic pathway in T. gondii have been described, in which acidic compartments were located in the rhoptries, suggesting that these organelles are related to lysosomes (Shaw et al., 1998). In addition, it has been demonstrated in tachyzoites, the existence of tubule-vesicle compartments associated with the Rab5, a molecular marker for early endosomes (Robibaro et al., 2001). The incorporation and traffic of intracellular nutrients in T. gondii are fields yet to be explored in detail such as in tachyzoite, bradyzoite and tissue cyst. With all the evidences above, the aim of this work is to verify ultrastructurally the tissue cysts surface electric charge, using cationized ferritin (CF) as a cationic marker, to analyze the dynamics of its incorporation and fate in the tissue cysts, besides the nature of anionogenic groups using enzymatic treatment.
2.2. Cysts isolation and purification The methodology used was adapted and modified from that described by Freyre (1995) and Popiel et al. (1996). Mice were killed in CO2 chamber, and the brains were surgically removed under aseptic conditions. After immersion at 4 8C in phosphate buffered saline (PBS), the brains were washed in the same buffer to remove blood cells, fragmented with the aid of scissors, and macerated in PBS by successive passages of the fragments using 18-23 G needles. After that, 20 ml of the total suspension were placed between slide/coverslip (24 mm 32 mm), and the total number of cysts was determined in the total area of the coverslip using light microscopy. The procedures with animals were carried out in accordance with the guidelines established by the Fundac¸a˜o Oswaldo CruzFIOCRUZ, Committee of Ethics for the Use of Animals, by license CEUA 0229-04. Tissue suspension containing cysts was filtered with cell dissociation sieve-tissue grinder kit (Sigma Chemical Co.) to remove small tissue fragments and cellular debris. Then, the suspension was centrifuge at 400 g for 10 min and the pellet was resuspended in Eagle’s medium supplemented with 25% dextran (about 1 brain per 2.5 ml of final solution). After centrifugation at 2200 g for 10 min, the pellet containing the cysts was recovered and resuspended in PBS, centrifuged at 400 g for 10 min to remove the dextran solution, and then diluted in the same buffer. The determination of the cysts number was carried out as described above. 2.3. Detection and fate of anionic sites The CF was used to verify the presence of anionic sites on cysts wall surface and its fate in tissue cyst. After isolation, the cysts were incubated with 200 mg/ml of CF in PBS, pH 7.2, for 20 min at 4 8C. The intracellular traffic of anionic sites was monitored by the posterior incubation of the cysts for 1–48 h at 37 8C. Then, the cysts were washed three times in PBS for the removal of ferritin particles that did not adhere to the surface of the cysts wall. The material was fixed in 2.5% of glutaraldehyde (GA) buffered in 0.1 M of sodium cacodylate buffer containing 3.5% of sucrose, pH 7.2, for 1 h at 4 8C. The samples were then washed in the same buffer three times for 10 min each and postfixed for 1 h at 4 8C in 1% of osmium tetroxide (OsO4) in 0.1 M sodium cacodylate buffer, rinsed, dehydrated and embedded in Epoxy resin. Thin sections were stained with uranyl acetate and lead citrate, and then examined under a Zeiss EM10C transmission electron microscope.
2. Materials and methods
2.4. Ruthenium red (Luft, 1971)
2.1. Parasites
In these assays, the tissue cysts, after isolation and purification, were fixed in 2.5% GA in 0.1 M sodium cacodylate buffer containing 0.02% of ruthenium red, pH 7.2, for 1 h at 4 8C. After the fixation, the cysts were washed in 0.1 M of the same buffer two times for 10 min each and postfixed in 1% OsO4 containing 0.02% of ruthenium red in 0.1 M of sodium cacodylate buffer for 30 min at 4 8C. The cysts were
T. gondii tissue cysts of ME-49 strain (Type II) were used. The parasites were maintained in C57BL/6 female mice, weighing about 12–18 g each, and inoculated intraperitoneally with about 30 cysts/animal. After 4–8 weeks, the tissue cysts were isolated from the brain, as described below.
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then washed three times in 0.1 M of sodium cacodylate buffer containing 0.01% of ruthenium red for 10 min each; dehydrated and embedded in Epoxy resin as described above.
The cysts were then washed three times in the same buffer and processed for observation under transmission electron microscopy as described above.
2.5. Enzymatic treatments of tissue cysts
3. Results
The cysts were treated with the following enzymes: phospholipase A2 (PLA2) from Naja mossambica mossambica (10, 50 and 100 mg/ml); phospholipase C (PLAC) type IX from Clostridium perfringens (0.2, 0.5, 1 and 2 U/ml) and Neuraminidase from Vibrio cholerae (0.2 and 0.5 U/ml) diluted in 0.85% NaCl solution, pH 7.4, during 30 and 60 min at 37 8C; protease type XIV from Streptomyces griseus and trypsin from porcine pancreas (1, 10, 100 and 500 mg/ml) diluted in 0.85% NaCl solution, pH 7.4, during 5 min at 37 8C. After the treatments, the material was washed in 0.85% NaCl solution three times and incubated with 200 mg/ml of CF diluted in the same medium, pH 7.4, for 20 min at 4 8C. The experimental control was kept by incubating tissue cysts with only 200 mg/ml of CF for 20 min at 4 8C without any enzymatic treatment.
3.1. Detection and fate of anionic sites The cationized ferritin (CF) is a polycationic derivative electrondense of native ferritin ionized at physiological pH that allows experiments with living cells. The CF was used in our studies as a marker of surface anionic sites of tissue cysts. The incubation of the living tissue cysts with CF for 20 min at 4 8C and subsequent elevation of temperature to 37 8C for 1 h, revealed the distribution of the CF in patches (Fig. 1) and also as a fine particle layer with uniform distribution on the cyst wall (Fig. 2), indicating that T. gondii cyst wall is negatively charged. In these conditions invaginations of the cyst wall were filled with the tracer (Figs. 1 and 2). Vesicles of different diameters and tubules
Figs. 1–3. Anionic sites in T. gondii tissue cysts using cationized ferritin (CF). Fig. 1. Incubation of the tissue cysts for 20 min at 4 8C and subsequent elevation of temperature to 37 8C for 1 h, showing formation of clusters of CF on the surface of the CW (arrowhead), internalization of CF particles through vesicles (V) and tubules (T) from the cyst wall (CW). Particles were found in invaginations of the CW (arrow), in tubules and vesicles in the cystic matrix (*) below the CW. Fig. 2. Tissue cysts showing a vesicle with CF particles near the apical complex (AC) of intracystic parasites. Fig. 3. High magnification allows to observe details of vesicles (V) and tubules (T) containing CF particles in the cyst matrix next to the granular region. Bars: 0.5 mm.
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containing ferritin particles were localized right below the granular region (Fig. 3), in the cystic matrix (Figs. 2 and 3) and next to or in direct contact with the membrane of intracystic bradyzoites (Figs. 2 and 4–6). 3.2. Ruthenium red The ultrastructural analysis of glycosaminoglycans in thin sections of T. gondii tissue cysts after incubation with the
ruthenium red showed a fine granular electrondense layer all over surface and in the invagination of the cyst wall (Fig. 7). Small vesicles filled with the marker, located right below the internal face of the membrane and also in the granular region, are transversal cuts of membrane invaginations containing ruthenium red (Fig. 7). The intracystic matrix and the parasites did not present marking, since it is a result of the well known property that ruthenium red do not penetrate in whole membranes.
Figs. 4–6. Anionic sites in T. gondii tissue cysts using CF. Figs. 4 and 5. In detail, vesicles (V) of different sizes and tubule (T) containing the label are observed near the bradyzoityes membrane (Bz). Fig. 6. After 2 h of FC incubation at 37 8C, it was possible to find vesicles containing ferritin very near and also adhered to intracystic parasites. Bars: 0.5 mm.
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Fig. 7. Detection of glycosaminoglycans in T. gondii tissue cysts incubated with ruthenium red (RR). Revelation of glycosaminoglycans on the T. gondii cysts membrane after incubation with RR. This image shows detail of the membrane of the cyst, the invaginations with particles of ruthenium red (arrow) and transversal cuts of these invaginations making visible the vesicles containing the marker (arrowhead). CW: cyst wall. Bar: 0.5 mm.
3.3. Enzymatic treatments of tissue cysts In order to identify which group of molecules is responsible for the negative charge of the cyst wall, we cleaved the surface molecules enzymatically. The cyst control, incubated with CF (Fig. 8) and after treatment with PLAC, maintained the labeling on the cyst surface (Fig. 9). The incubation of tissue cysts with PLA2 (10 and 50 mg/ml) for 30 min at 37 8C revealed that this was the only enzyme that diminished the expression with CF. Cysts showed a decrease of the CF distribution when treated with 10 mg/ml of PLA2 (Figs. 10 and 11), when compared with the control (Fig. 8) that was incubated only with CF. No significant decrease in the expression of anionic sites in the cyst wall was observed when other enzymes (neuraminidase, protease type XIV, trypsin) were employed (data not showed). Depending on the concentrations of the enzymes, the cysts breached, turning impracticable the analysis of the anionic sites expression. In a same cyst, the surface presented CF followed by areas without any marking with the CF (Fig. 10). The higher magnification of Fig. 10 shows the cyst wall area label with CF (Fig. 11). No label was observed using 50 mg/ml of enzyme (Fig. 12). Our results showed that the groups of molecules that are probably producing negative charge in the cyst wall are the phospholipids. 4. Discussion Our results with ultrastructural cytochemistry using CF and ruthenium red clearly demonstrated that the T. gondii cyst wall is negatively charged. Studies on the plasma membrane of protozoa have suggested that its components play an essential role in pathogenic processes, interacting with molecules of the surface of the host cells, activating the invasion process (Pimenta and De Souza, 1983; De Carvalho et al., 1985; Cintra et al., 1986; Kleffmann et al., 1998; Akaki et al., 2001). Thus, the analysis of the surface molecules of the pathogens and their target cells is primordial for the better knowledge of this interaction. There are few studies about the surface charge of the T. gondii and the molecules which are responsible for the origin of these charges (De Carvalho and De Souza, 1990; Akaki et al., 2001; Stumbo et al., 2002). Locksley et al. (1982) described that the tachyzoite surface has a negative charge and Cintra et al. (1986) demonstrated that this charge is sensible to phospholipase C, suggesting that phosphate groups contribute to its surface charge. The characterization of the surface
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components of bradyzoites and tissue cysts has been little explored, despite the high relevance of both in the course of chronic toxoplasmosis (reviewed in Dubey et al., 1998; Weiss and Kim, 2000). The formation of the cyst wall and the constitution of its matrix are events that follow the differentiation of bradyzoites and these walls have been considered as results of the modification of the parasitophorous vacuole membrane that surrounds the parasite within the host cell (Gross et al., 1995; Bohne et al., 1996, 1999; Weiss and Kim, 2000). Our results demonstrated the presence of negative electric charge on the surface of tissue cysts, using two cationic markers: (i) CF that presents electrostatic binds to the negative charge molecules and (ii) ruthenium red, an inorganic marker, in which atoms of oxygen, ruthenium and amine all together in known configuration as complex amine, present high affinity with polyanionic substances, such as the glycosaminoglycans. The positive reaction to ruthenium red in Plasmodium berghei merozoites surface and in T. gondii tachyzoites was described by Seed et al. (1974) and Cintra et al. (1986), respectively. Considering the absence of binding to lectins on the surface of P. berghei and T. gondii, Cintra et al. (1986) suggested that ruthenium red linkage is only an electrostatic interaction of the polycationic marker with the parasite surface containing anionic components whose surface electric charge could be given by anionic phospholipids. Such a conclusion cannot be inferred from tissue cysts, since the groups of Sethi et al. (1977), Derouin et al. (1981) and Zhang et al. (2001) demonstrated, yet with conflicting results, residues of carbohydrates on tissue cysts surface, although residues of sialic acid have not been demonstrated, nor has the presence of glycosaminoglycans in the composition of cyst wall been investigated. We are demonstrating for the first time the negative charge of the cyst wall, using two markers that present different properties to bind the anionic groups on the surface of tissue cysts of T. gondii. The nature of the membrane components of these cysts that produce this negativity remains an open question, but our results with the enzymatic treatment of tissue cysts denote the participation of phospholipids on the anionic nature of the cyst wall. Amongst all the enzymes used in the treatment of the tissue cysts, only phospholipase A2 was efficient in reducing the anionic sites of the surface of these cysts. PLA2 is a family of lipolytic enzymes involved in the phospholipid digestion, remodeling of cell membranes and host defense. They cleave the fatty acyl bond at the sn-2 position of membrane glycerophospholipids to generate unsaturated fatty acids and lysophospholipids. In mammals, this activity results in the release of arachidonic acid from the membrane phospholipids, which is the starting material for the generation of a wide range of biologically active lipid mediators, including prostaglandins, leukotrienes, thromboxanes and prostacyclins (reviewed in Valentin and Lambeau, 2000; East and Isacke, 2002). There is no literature on studies about the lipids composition of the cyst wall. Our results suggest that the negative electric charge of the cyst is given partially or totally by some groups of the fatty acids tail that bind to carbon 2 of the glycerol structure. These conclusions are due to the absence or decrease of CF in
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Figs. 8–12. Enzymatic treatment in T. gondii tissue cysts. Fig. 8. Experimental control without enzymatic treatment showing the CW labeled with CF over the surface. Fig. 9. Cysts treated with 1 U/ml phospholipase C maintain the labeling for CF. Fig. 10. Treatment of the cysts with 10 mg/ml phospholipase A2 presenting for 30 min decrease in the labeling of the anionic sites on CW after incubation with CF. Bradyzoites (Bz). Fig. 11. High magnification of cyst of Fig. 10 showing the region containing CF particles adhered to the cyst wall membrane. Fig. 12. Cyst treated with 50 mg/ml phospholipase A2 showing absence of labeling of the anionic sites on CW when post-incubated with CF. Bar: 0.5 mm.
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cysts treated with PLA2. The occurrence of cysts with discrete marker after the enzymatic treatment can be a consequence of a partial access of the enzyme to some cysts. However, it is little probable, since the cysts remained under constant agitation during the treatment. Another hypothesis that can be considered is that the fatty acids tail of carbon 1 bind to glycerol own similar cleaved tail for PLA2, also producing negative charge, that would only be completely eliminated by treating the cysts with PLA1 and/or B. The presence of cysts with marked and not marked regions can be in consequence of a heterogeneous distribution of the glycerolphospholipids after treatment where not all the negative charge was eliminated, or of the heterogeneous distribution of other molecules that also give negative charge to the cyst wall. We demonstrated ultrastructurally, using CF that negatively charged molecules present in the cystic wall are incorporated by tubules and vesicles formed from the membrane that delimits the cyst wall and its posterior localization in the matrix. Previous works about the ultrastructure of the cyst wall had described the presence of these structures in the granular region and matrix of the cysts, being its origin unknown (Wanko et al., 1962; Matsubayashi and Akao, 1963; Jacobs, 1967). Mehlhorn and Frenkel (1980), through the ultrastructural analysis of the cyst wall in the skeletal muscle, suggested that the vesicles found in the granular region could be derived from the cyst membrane. We confirmed the origin of these structures from the membrane, supported by the first demonstration of the traffic of a marker (CF) of the cyst membrane and its intracystic fate. Additionally, we verified the presence of vesicles, neighbor or close to the bradyzoites plasma membrane. There are no evidences of the fusion of these vesicles and the parasites membrane, but we can suggest that it could be one of the pathways of nutrients originating from the host cell. We also did not find any evidence of the incorporation of vesicles containing cationized ferritin through the micropore, as described by Nichols et al. (1994), when they observed vesicles and cellular debris in micropores of intracystic bradyzoites. Our observations, showing the cyst wall through its delimiting membrane with many invaginations which provably enlarged the cyst surface area, facilitating the exchange of materials between the intracystic parasites and cytosol of the host cell, are in accordance with Jones et al. (1986), Ferguson and Hutchison (1987) and Nichols et al. (1994). The nutrients required by bradyzoites confined inside the cyst wall may be necessary for its maintenance, multiplication and the synthesis of amylopectin granules (Guimara˜es et al., 2003; Dzierszinski et al., 2004). The amylopectin is synthesized in the bradyzoite, suggesting that the biogenesis of these complex carbohydrate structures may be of key importance in the bradyzoite differentiation and persistence of cysts during host infection by T. gondii (Coppin et al., 2003). Tachyzoites in the interior of parasitophorous vacuole develop a vesicle-tubular network that seems to be partly contiguous to the parasite plasma membrane and to form connections to the parasitophorous vacuole membrane (Sibley et al., 1986, 1995; Halonen et al., 1996). This network, due to its large surface area, could play an
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important role in the exchange of solutes between the intracellular parasite and its host (Sibley et al., 1995; Labruyere et al., 1999; reviewed in Saliba and Kira, 2001). The argument that the study of the endocytosis in bradyzoites is limited in virtue of the damages caused to the parasites during the processes of their isolation from tissue cysts by mechanic or enzymatic digestion (Nichols et al., 1994) deserves to be reviewed for two reasons: first, the methods of isolation of bradyzoites from more recent tissue cysts and the bioassays in mice have demonstrated ‘‘in vivo’’ infectivity, at least minimizing the effect of the processes on the viability of the parasites (Freyre, 1995; Popiel et al., 1996); second, the endocytosis by bradyzoites can be potentially explored through experimental protocols, using whole cysts. Our model can give an important contribution to the knowledge of the biology and the processes involved in the nutrients acquisition by cysts and bradyzoites, and be also applied as a target for the direct action of drugs against of T. gondii cyst. Acknowledgements Support was provided by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa Estrate´gico de Apoio a` Pesquisa em Sau´de—PAPES IV and Instituto Oswaldo Cruz-Fiocruz. References Akaki, M., Nakano, Y., Nagayasu, E., Nagakura, K., Kawai, S., Aikawa, M., 2001. Invasive forms of Toxoplasma gondii, Leishmania amazonensis and Trypanosoma cruzi have a positive charge at their contact site with host cells. Parasitol. Res. 87, 193–197. Bohne, W., Holpert, M., Gross, U., 1999. Stage differentiation of the protozoan parasite Toxoplasma gondii. Immunobiology 201, 248–254. Bohne, W., Parmley, S.F., Yang, S., Gross, U., 1996. Bradyzoite-specific genes. Curr. Top. Microbiol. Immunol. 219, 81–91. Botero-Kleiven, S., Fernandez, V., Lindh, J., Richter-Dahlfors, A., von Euler, A., Wahlgren, M., 2001. Receptor-mediated endocytosis in an apicomplexan parasite (Toxoplasma gondii). Exp. Parasitol. 98, 134–144. Cintra, W.M., Silva-Filho, F.C., De Souza, W., 1986. The surface charge of Toxoplasma gondii: a cytochemical and electrophoretic study. J. Submicrosc. Cytol. 18, 773–781. Coppens, I., Sinai, A.P., Joiner, K.A., 2000. Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition. J. Cell Biol. 149, 167–180. Coppin, A., Dzierszinski, F., Legrand, S., Mortuaire, M., Ferguson, D., Tomavo, S., 2003. Developmentally regulated biosynthesis of carbohydrate and storage polysaccharide during differentiation and tissue cyst formation in Toxoplasma gondii. Biochimie 85, 353–361. De Carvalho, T.U., Souto-Padroon, T., De Souza, W., 1985. Trypanosoma cruzi: surface charge and freeze-fracture of amastigotes. Exp. Parasitol. 59, 12–23. De Carvalho, L., De Souza, W., 1990. Internalization of surface anionic sites and phagosome-lysosome fusion during interaction of Toxoplasma gondii with macrophages. Eur. J. Cell Biol. 51, 211–219. Derouin, F., Beauvais, B., Lariviere, M., Guillot, J., 1981. Binding of fluorescein-labelled lectins on trophozoites and cysts of 3 strains of Toxoplasma gondii. C. R. Seances Soc. Biol. Fil. 175, 761–768. Dubey, J.P., Lindsay, D.S., Speer, C.A., 1998. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin. Microbiol. Rev. 11, 267–299.
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