Identification of a Large Pre-Lysosomal Compartment in the Pathogenic Protozoon Trypanosoma Cruzi

Journal of Cell Science 102, 157-167 (1992) 157 Printed in Great Britain © The Company of Biologists Limited 1992 Identification of a large pre-lysosomal compartment in the pathogenic protozoon Trypanosoma cruzi MAURILIO J. SOARES1'2, THAIS SOUTO-PADR6N1 and WANDERLEY DE SOUZA1* 1 Departamento de Parasitologia e Bioflsica Celular, Institute/ de Biofisica Carlos Chagas Filho, UFRJ, 21949 Rio de Janeiro, RJ, Brazil 2Departamento de Ultraestrutura e Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, 20001 Rio de Janeiro, RJ, Brazil •Author for correspondence Summary Epimastigote forms of the pathogenic parasite Trypano- from Lowicryl-embedded cells demonstrated that the soma cruzi were used to study the endocytic process in a reservosomes are acidic compartments (pH 6.0, as protozoon. These elongated unicellular organisms are shown using DAMP as a pH probe) with no acid highly polarized cells: endocytosis occurs only at the phosphatase or typical lysosome-associated membrane anterior region through the cytostome and the flagellar proteins (LAMP 1, LAMP 2 and lgp 120), but rich in pocket membrane, areas of the plasma membrane where cysteine proteinase. These data suggest that the reservo- the cell cytoskeleton, formed by sub-peUicular micro- some is a pre-lysosomal compartment. Since cysteine tubules, is absent. When the cells were incubated at 4°C proteinase of T. cruzi contains no phosphorylated or 28°C with gold-labeled transferrin, fixed and pro- mannose residues and the cation-independent mannose cessed for routine transmission electron microscopy our 6-phosphate receptor could not be immunocytochemi- observations show that this ligand initially binds to the cally detected hi the reservosomes, it is possible that cytostome and the membrane lining the flagellar pocket lysosomal enzymes hi the epimastigote forms of T. cruzi and is subsequently ingested through a clathrtn-indepen- are targeted to compartments related to the endocytic dent receptor-mediated endocytotic process, with for- pathway through a mechanism different from that which mation of uncoated pits and vesicles. Ingested complexes occurs hi other eukaryotic cells. are carried in uncoated vesicles to the reservosomes, large membrane-bound organelles found mostly at the Key words: endocytosis, Trypanosoma cruzi, posterior end of the cell. Immunocytochemical data immunocytochemistry, pre-lysosomal compartment. Introduction movement, the ingested material to an inner late endosomal compartment (Matteoni and Kreis, 1987; Eukaryotic cells continuously ingest macromolecular Griffiths et al. 1989; Gruenberg et al. 1989). It has been complexes from the extracellular environment through recently suggested that late endosomes correspond to invaginations of the plasma membrane, either by non- pre-lysosomal compartments (PLCs) adjacent to, but selective fluid-phase pinocytosis or by concentrative functionally distinct from, the compartments of the receptor-mediated endocytosis, where the efficiency of frans-Golgi network (Griffiths et al. 1988). Although the process is greatly enhanced due to receptors present lysosomal enzymes could be detected in the PLCs, they on the cell surface that bind to specific ligand molecules, are both structurally and functionally distinct from the leading to their selective uptake. dense lysosomes, which, it has been suggested, either Recent studies on the endocytic pathway of eukary- bud off or mature from the late endosomes (Geuze et otic cells led to the proposition that distinct, pre- al. 1988; Griffiths et al. 1988; Griffiths et al. 1990). Such existing, cytoplasmic compartments are involved in the a vesicle shuttle model differs from the earlier matu- endocytic process and in the recycling of the plasma ration model, which assumes that no permanent membrane (Schmid et al. 1988; Gruenberg and Howel, endosomal compartments exist (reviewed by Hubbard, 1989; Gruenberg et al. 1989; Kornfeld and Mellman, 1989). 1989): ingested particles are first delivered to a The main goal of our work was to analyze the peripheral, tubulo-vesicular, early endosomal compart- structures involved in the endocytic process of epimasti- ment (Geuze et al. 1983; Mueller and Hubbard, 1986; gote forms of the protozoon Trypanosoma cruzi, a Schmid et al. 1988). Carrier vesicles bud off from this pathogenic uniflagellate organism that causes Chagas' structure and transport, by microtubule-dependent disease, which affects about 8 million people in South 158 M. J. Soares and others America. This parasite presents three morphologically antibodies, were obtained through Dr. Gareth Griffiths distinct forms: in the final vertebrate host, the protozoa (European Molecular Biology Laboratories, Heidelberg, can be found either intracellularly (dividing amasti- Federal Republic of Germany). gotes) or free-swimming in the blood (non-dividing Bovine transferrin was purchased from Sigma Chemical Co (Saint Louis, MO, USA). Gold particles 15 nm in diameter trypomastigotes); in the digestive tract of the invert- were prepared according to Frens (1973) and conjugated to ebrate hosts (triatomid blood-sucking bugs), the para- transferrin as described by Bendayan et al. (1987). sites appear mostly as the multiplying epimastigote form. Epimastigotes can be easily maintained in vitro in Parasites axenic culture media, and therefore have been largely Five-day-old culture forms (epimastigotes) of the T. cruzi Y used to study the cell biology of this parasite (reviewed strain were used. Parasites were in the mid log phase of by De Souza, 1984). growth, when the number of reservosomes was maximal (Paulin et al. 1983; Soares, unpublished observations). The Data on fluid-phase pinocytosis of peroxidase (De C Souza et al. 1978) and on receptor-mediated endo- cells were kept at 28 C in Warren's liquid medium (Warren, cytosis of gold-labeled albumin, peroxidase, transferrin 1960) supplemented with 10% fetal calf serum. and LDL (Soares and De Souza, 1991) by T. cruzi Ingestion of transferrin-gold showed that the ingested material entered the cells Parasites were collected by centrifugation at 1,500 g for 5 through the cytostome and/or the flagellar pocket minutes, washed in phosphate buffered saline (PBS), pH 7.2, region. In the epimastigote forms, the proteins accumu- and then incubated for 30 minutes at 4 or 28CC in the presence lated in a cytoplasmic organelle, previously designated of a gold-labeled transferrin solution diluted 1:5 in PBS as the reservosome (Soares and De Souza, 1988). (absorbance at 520 nm=0.5). The cells were then washed in Stereological measurements suggested that these nutri- PBS, fixed for 2 hours in a solution containing 1.5% ents are degraded during metacyclogenesis, a process glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate by which non-infective epimastigotes are transformed buffer, pH 7.2, washed in this same buffer and then post-fixed into infective trypomastigotes (Soares et al. 1989). In for 1 hour with 1% osmium tetroxide/0.8% potassium addition, recent studies have shown that the most ferricyanide in cacodylate buffer, pH 7.2, containing 5 mM important T. cruzi antigens, which are being used for calcium chloride. Thereafter, the cells were washed in cacodylate buffer, dehydrated in a graded ethanol series and diagnosis of Chagas' disease, are located in the embedded in Epon. Ultra-thin sections were stained with reservosomes (Krieger et al. 1990; Goldenberg et al. uranyl acetate and lead citrate, and observed by transmission unpublished data). electron microscopy. In this work we used gold-labeled transferrin to define more clearly the compartments involved in the Cytochemical detection of acid phosphatase activity endocytic pathway of a pathogenic protozoon. Our Parasites were collected by centrifugation, washed in PBS, C observations confirm previous studies showing that briefly fixed for 20 minutes at 4 C with 1% glutaraldehyde in trypanosomatids are highly polarized in terms of 0.1 M cacodylate buffer, pH 7.2, washed in 0.1 M cacodylate endocytic activity, and that transferrin concentrates in buffer, pH 7.2, and in 0.1 M Tris-maleate buffer, pH 5.0, and then incubated for 1 hour at 28°C in a medium containing 2 the reservosome, an acidic compartment rich in cys- mM cerium chloride, 5% sucrose, 0.1 M Tris-acetate buffer, teine proteinase, but which does not contain acid pH 5.0, and 2 mM cytidine 5'-monophosphate or 2 mM phosphatase or other lysosomal membrane proteins. sodium /S-glycerophosphate as substrate (Robinson and Karnovsky, 1983; Robinson, 1985). The cells were then washed in Tris-maleate and cacodylate buffers, refixed with Materials and methods 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer, post- fixed in 1% osmium tetroxide, dehydrated in a graded acetone Materials series and embedded in Epon. As a control, some cells were DAMP [3-(2,4-dinitroanihno)-3'-amino-7V-methyldipropyl- incubated in medium without substrate. Ultra-thin sections amine] and a monoclonal mouse antibody directed against were observed unstained or briefly stained with lead citrate by DNP (dinitrophenol) were obtained from Dr. R. G. W. transmission electron microscopy. Anderson (University of Texas, Dallas, TE, USA). Poly- clonal rabbit antibody against T. cruzi cysteine proteinase Lowicryl embedding for immunocytochemistry (Campetella et al. 1990) was obtained from Dr. J. J. Cazzulo For the localization of antigens, cells were collected by (Instituto de Investigations Bioqufmicas Fundati6n Campo- centrifugation at 1,500 g, washed in PBS and then fixed for 2 mar,

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