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    R E V I E W

    Wanderley de Souza

    Special organelles of some pathogenic protozoa

    Received: 30 May 2002 / Accepted: 3 July 2002 / Published online: 3 September 2002 Springer-Verlag 2002

    Abstract Parasitic protozoa comprise a large number ofspecies, some of which are agents of important diseases.They are also of interest from the point of view of cellbiology since they contain special organelles and struc-

    tures. This review analyses our present knowledge of (1)the glycosomes, found in members of the Kinetoplastidaorder, (2) the hydrogenosomes found in some anaerobicprotozoa, especially in trichomonads, (3) the acidocal-cisomes, recently described in several protozoa, and (4)structures and organelles participating in the endocyticpathway in trypanosomatids.

    Introduction

    The protozoan kingdom comprises a large number of

    species, including some which are agents of human andveterinary diseases such as malaria, leishmaniasis,Chagas disease, African trypanosomiasis, amebiasis,trichomoniasis, giardiasis, toxoplasmosis, coccidiosis,theileriosis, and babesiosis, to mention only the moreimportant. Some of these protozoa, as is the case ofTrichomonas, have a simple life cycle. For others, such asthe Apicomplexa (which includes Plasmodium, Toxopl-asma, Eimeria, etc), and some trypanosomatids, the lifecycle is relatively complex, displaying several develop-mental stages in the vertebrate host and, in some cases,in invertebrate hosts. These protozoa are also of interestfrom the point of view of cell biology, since they contain

    special cytoplasmic structures and organelles which havebeen studied in some detail in the last years and have

    provided new information of general biological interest.I will review here, from the perspective of cell biology,some of the important organelles found in a relativelysmall number of protozoa. These protozoa are, however,

    important since they are the causative agents of impor-tant diseases. Two main groups of organelles, hereconsidered in a wide sense, will be reviewed. They areclassified as organelles which are: (1) involved in meta-bolic pathways, and (2) involved in the endocytic path-way.

    Organelles involved in metabolic pathways

    The peroxisome (glycosome in trypanosomatids)

    Membrane-bounded cytoplasmic structures resembling

    those initially designated as microbodies and later asperoxisomes in mammalian cells have been described intrypanosomatids since the initial studies on their finestructure (reviews in Vickerman and Preston 1974; DeSouza 1984). The peroxisomes usually appear as spher-ical organelles with a diameter of about 0.7 lm and arerandomly distributed throughout the cell (Fig. 1). Insome cells, as is the case of Leptomonas samueli, theyappear as long structures which can reach a length of2.8 lm (Fig. 2) (Souto-Padron and De Souza 1982). Insome Phytomonas species, the peroxisomes may bepackaged arranged and even associated with cytoskeletalstructures (Figs. 3, 4) (Attias et al. 1988). For instance,

    in one isolate from the lactiferous plant Euphorbiahyssopifolia the glycosomes form two rows separated bythe presence of a bundle of filamentous structures(Fig. 3). In one isolate from Euphorbia characias, theglycosomes are located in such a way that they look likestacks of flattened disks (Fig. 4). Usually the glycosomesare distributed throughout the cells. However, in try-panosomatids which harbour an endosymbiont, theperoxisomes concentrate around the symbiont (Mottaet al. 1997). Usually the glycosome presents a homoge-nous and slightly dense matrix, although a crystalloid

    Parasitol Res (2002) 88: 10131025DOI 10.1007/s00436-002-0696-2

    W. de SouzaLaborato rio de Ultraestrutura Celular Hertha Meyer,Instituto de Biofsica Carlos Chagas Filho, CCS-Bloco G,Universidade Federal do Rio de Janeiro,Ilha do Fundao, 21949-900, Rio de Janeiro-RJ, BrazilE-mail: [email protected].: +55-21-22602364Fax: +55-21-212808193

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    core can be seen in some protozoa such as Trypanosomabrucei and Leishmania mexicana (Vickerman andPreston 1974).

    Peroxisomes have been defined as organelles that arebounded by a single membrane and that contain catalaseand H2O2-producing oxidases. Catalase has been used asan enzyme marker, easily localized using the alkalinediaminobenzidine medium, to identify an organelle as aperoxisome. The application of this approach in try-panosmatids led to the identification of two groups. Oneincludes digenetic trypanosomatids such as T. brucei,Trypanosoma cruzi and Leishmania in which no signifi-cant enzyme activity could be detected either by enzymeassay or by cytochemistry. The other includes the mo-nogenetic trypanosomatids such as Crithidia, Lepto-monas, etc, in which the enzyme is easily detected (Museand Roberts 1973; Souto-Padron and De Souza 1982).Even in the monogenetic organisms there is some vari-ation in the intensity of labeling of the glycosomes whenthe cells are incubated in the presence of the cytochem-ical medium designed for the detection of catalase ac-tivity. Labeling was much more intense in Crithia

    fasciculata than in L. samueli (Muse and Roberts 1973;Souto-Padron and De Souza 1982). Genes coding forcatalase have been found in Toxoplasma gondii(Kaaschand Joiner 2000; Ding et al. 2000). Catalase activitycould be detected using the diaminobenzidine techniquein an organelle described as a peroxisome in T. gondii(Kaasch and Joiner 2000). However, Ding et al. (2000)did not find evidence for the presence of catalase in amembrane bound organelle using an immunocyto-chemical approach, with antibodies generated againstsynthetic peptides made on the basis of informationfrom the genes coding for the enzyme, and cell frac-tionation. These authors suggested that the enzyme is

    located in the cytosol (Ding et al. 2000).The number of glycosomes and the area they occupy

    in the cytoplasm of trypanosomatids vary according tothe species and even to the developmental stage of thesame species. This was first shown in T. brucei, whichhas a large number of glycosomes in bloodstream forms,in which the mitochondrion is poorly developed. In theprocyclic form, with fewer glycosomes, the mitochond-rion is highly developed (Vickerman and Preston 1974;Tetley and Vickerman 1991). Opperdoes et al. (1984)estimated that the bloodstream form of T. brucei hasaround 230 glycosomes. In the case of other trypano-somatids around 50 glycosomes have been found

    (Soares and De Souza 1988). Morphometrical analysishas shown that glycosomes occupy an area of about 9.0and 2.4% in bloodstream trypomastigotes and procyclicforms of T. brucei, respectively. The mitochondrion oc-cupies an area of 3.3 and 19.5%, respectively (Bohringerand Hecker 1975). In the case of non-pathogenic try-panosomatids, mitochondria and glycosomes togetheroccupy from 9 to 19% of the total cell volume. In mostof the trypanosomatids, the mitochondrion occupies avolume of about 9% and the glycosomes of about 3%(Soares and De Souza 1988). In the case of L. samueli,

    however, glycosomes are very abundant, accounting forabout 8% of the cell volume (Souto-Padron et al. 1980).

    The influence of the growth conditions on the vol-ume occupied by the two organelles was studied inmore detail in Herpetomonas roitmani. When the cellswere grown in a medium containing glucose as thecarbon source, glycosomes and mitochondria corre-sponded to a cell volume of 8.9 and 5%, respectively.However, when grown in the presence of proline, gly-cosomes and mitochondria accounted for 2 and 13.5%of the cell volume, respectively (Faria e Silva et al.2000). These observations show that there is a balancein the volume occupied by mitochondria and glyco-somes in this species so that the total volume occupiedby the two organelles corresponds to about 14% of thetotal cell volume.

    A major contribution to the role played by the per-oxisomes in trypanosomatids came from the work ofOpperdoes and co-workers, initially with T. brucei andthen extended to other members of the Trypanosomat-idae. They found, using cell fractionation and bio-chemical studies, that the glycolytic enzymes involved in

    the conversion of glucose to 3-phosphoglycerate are lo-cated in the peroxisomes (reviewed in Opperdoes 1987).Based on these results, the term glycosome was sug-gested to designate the microbodies or peroxisomes oftrypanosomatids. An interesting observation was thatthe glycolytic enzymes isolated from the glycosomeshave a higher isoelectric point than similar enzymesfound in the cytosol of mammalian cells. This explainsthe intense labeling of glycosomes when trypanosomat-ids are stained with ethanolic phosphotungstic acid un-der conditions in which basic proteins are localized(Fig. 5) (Souto-Padron and De Souza 1979). Using thistechnique, the cells are fixed in glutaraldehyde only,

    dehydrated in ethanol and then incubated for a fewhours in the presence of an ethanolic solution of phos-photungstic acid before being embedded in a resin. Anelectrondense reaction product appears in some struc-tures, especially in the glycosomes.

    In addition to catalase, the peroxisomes of mamma-lian cells have more than 50 different enzymes involvedin different metabolic pathways, such as peroxide me-tabolism, b-oxidation of fatty acids, ether phospholipidsynthesis, etc. Evidence has been obtained indicatingthat, in addition to the metabolic routes describedabove, other metabolic pathways such as carbon dioxidefixation (Opperdoes and Cottem 1982), purine salvage,

    and pyrimidine biosynthesis de novo (reviews inOpperdoes 1987; Shih et al. 1998) also take place in theglycosomes of trypanosomatids, although they occur inthe cytosol of other cells.

    The glycosome does not possess a genome. Therefore,all of the proteins found in it are encoded by nucleargenes, translated on free ribosomes, and post-transla-tionally imported into the organelle. The uptake ofproteins into the glycosomes occurs within 5 min ofprotein synthesis and is due to the presence of severaltargeting signals (review in Parsons et al. 2001).

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    The hydrogenosome

    Rounded structures localized at the side of the axostyleand the costa were recognized in trichomonads usinglight microscopy and designated as paraxostylar orparacostal granules. The first electron microscopicstudies confirmed the localization of these granules andshowed that they were enveloped by a membrane sys-tem. However, it was only after biochemical studiesshowed that the organelle was involved in a metabolicprocess leading to the production of ATP and molecularhydrogen (which led to its designation as a hydrogeno-some), that several groups started to study it. The hy-drogenosome has been the subject of excellent reviewspublished in the last decade (Muller 1993; Benchimol

    1999b). Its functional role in the general metabolism oftrichomonads has been the subject of intense investiga-tion since the early description of the organelle (Muller1993, 1997; Martin and Muller 1998). Therefore, I willonly focus on those aspects of the hydrogenosome whichare considered of more relevance from the perspective ofthe present review.

    From the morphological point of view, the hydro-genosome appears in most of the cells as a sphericalor slightly elongated structure with a mean diameterof 0.5 lm (Figs. 6, 7), reaching 1.0 lm in dividing

    organelles. In some cells, as occurs in Monocercomonassp., very elongated hydrogenosomes, which may reach

    a length of 2.0 lm, can be observed (Fig. 8) (Benchi-mol 1999b). Most of the hydrogenosomes are locatedclose to the costa and the axostyle. However, they canalso be seen in all regions of the protozoan, even in themore posterior tip of the cell.

    Morphological studies have shown that cytoplasmicstructures such as the glycogen particles, microtubules,and profiles of the endoplasmic reticulum establish anintimate contact with the hydrogenosomes.

    The morphogenesis of the hydrogenosome has beenstudied in several species. Two distinct types of organelledivision have been reported. In the first type, designatedas the partition process, division begins with the invag-

    ination of the inner hydrogenosomal membrane whichforms a transverse septum separating the hydrogeno-some matrix into two compartments (Fig. 9). In thesecond, designated the segmentation process, there is aninitial growth of the organelle which then becomeselongated, with the subsequent appearance of a con-striction zone in its central portion (Fig. 10). Micro-fibrilar structures seem to help the furrowing process.

    The hydrogenosome is surrounded by two closelyapposed unit membranes (Fig. 11) (Benchimol andDe Souza 1983). These can be easily observed in all

    Figs. 15. Different views ofglycosomes (G) found in try-panosomatids. Most of the gly-cosomes are spherical, althoughlong structures can be seen(Fig. 2). In most of the casesthey are scattered throughoutthe cytoplasm. However, theycan be organized in parallelarrays (Figs. 3, 4). Basic pro-teins can be detected in the

    glycosomal matrix using theethanolic phosphotungstic acidtechnique (Fig. 5). However,they can be organized in paral-lel arrays (Figs. 3 and 4), withfilamentous structures inbetween (arrow in Fig. 3). mMitochondrion; g glycosome, nnucleus

    Fig. 1. Bloodstream trypomas-tigote form of Trypanosomabrucei, 40,000

    Fig. 2. Leptomonas samueli,40,000. (After Souto-Padronand De Souza 1982)

    Fig. 3. Phytomonas, 25,000.(After Attias et al. 1988)

    Fig. 4. Phytomonas, 20,000.(After Attias et al. 1988)

    Fig. 5. Herpetomonas samu-elpessoai, 38,000. (AfterSouto-Padron and De Souza1979)

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    organisms containing the organelle when they are fixedin aldehyde solutions containing calcium chloride. In-vaginations of the hydrogenosomal membrane forminginner compartments are occasionally seen. Using theperiodic acid-thiosemicarbazide-silver proteinate tech-

    nique applied to thin sections, reaction products, indi-cative of the presence of carbohydrates, are localized inthe hydrogenosomal membrane. The portion of themembrane which encloses the peripheral vesicle of thehydrogenosome is heavily labeled with wheat germ ag-glutinin (Benchimol et al. 1996a), thus suggesting thepresence of N-acetyl-glucosamine. Freeze-fracture stud-ies have shown the presence of intramembranous parti-cles, which correspond to membrane integral proteins,on both fracture faces of the two hydrogenosomalmembranes (Fig. 12). Differences in the density of the

    particles in the various membranes were reported. Deepetching views of quick frozen cells revealed the presenceof complexes of intramembranous particles in the matrix

    of the organelle (Fig. 13). These complexes may corre-spond to metabolic sites made of proteins with enzy-matic activity.

    The hydrogenosome has been purified using cellfractionation techniques and was shown to containseveral protein bands, with those of 120, 66, 60, 59, 48,45 and 35 kDa as the major ones (Morgado Diaz andDe Souza 1997).

    One characteristic feature of most of the hydro-genosomes is the presence of structures designated asperipheral vesicles. In some species, only one or twovesicles are seen. In others, as is the case of Trichomonasvaginalis, several peripheral vesicles are found (M.

    Benchimol, pers. comm.). The vesicle corresponds toabout 8% of the volume of the hydrogenosome. Usuallythe vesicle is flat, however, its size and shape may varysignificantly. When the cells are fixed in solutions con-taining calcium, an electrondense product is seen withinthe vesicle. The results obtained using X-ray micro-analysis (Chapman et al. 1985), electron spectroscopicimaging (De Souza and Benchimol 1988) and removal ofthe dense material by incubation of thin sections in thepresence of EGTA (Benchimol and De Souza, 1983)indicate that the peripheral vesicle is a site for the ac-cumulation of calcium. The vesicle could be purifiedfrom a hydrogenosomal fraction which was solubilized

    with Triton X-100. Further treatment with proteinase Ksolubilized the matrix components and left a pure frac-tion containing structures with the same morphology asthe vesicles, thus suggesting the existence of a highcontent of membrane-associated proteins. SDS-PAGEanalysis of the isolated vesicles showed that theyconcentrate proteins of 66, 45, and 32 kDa (MorgadoDiaz and De Souza 1997). The 45 kDa protein is aglycoprotein rich in N-acetylglucosamine.

    The hydrogenosomal matrix usually appears granularand homogeneous, occasionally with a dense amorphous

    Figs. 611. Different views of the hydrogenosomes observed inTritrichomonas foetus. Usually the hydrogenosome is sphericalalthough long organelles can be seen in Monocercomonas sp.(Fig. 8). Figures 6 and 7 show the distribution of the organelles asseen in thin sections and in whole-extracted cells, respectively.Figures 9 and 10 show two different types of division of theorganelle. Figure 11 clearly shows the presence of two membranesenveloping the organelle

    Fig. 6. Tritrichomonas foetus, 8,000. (Courtesy of M. Benchimol)

    Fig. 7. Tritrichomonas foetus, 6,000. (Coutesy of M Benchimol)

    Fig. 8. Monocercomonas sp., 50,000. (After Benchimol 1999b)

    Fig. 9. Tritrichomonas foetus, 70,000. (After Benchimol et al.1996b)

    Fig. 10. Tritrichomonas foetus, 22,000. (After Benchimol et al.1996b)

    Fig. 11. Tritrichomonas foetus, 42,000. (After Benchimol andDe Souza 1983)

    b

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    or crystalline core. The granular nature of the matrix isbest visualized in replicas of quick frozen, freeze-frac-tured, deep-etched and rotary replicated examples inwhich particles with a mean diameter of 6 nm are ob-served (Fig. 13). Large particles with a mean diameter of20 nm and showing a certain orientation can also beseen. When the cells are incubated in the presence ofcytochemical media designed for the localization of ba-sic proteins or calcium, reaction products can be seenthroughout the matrix (Fig. 14). In the case of calciumlocalization, the reaction product is also seen within theperipheral vesicle (review in Benchimol 1999b).

    The hydrogenosome does not contain nucleic acids.The hydrogenosomal proteins are synthesized in freecytoplasmic ribosomes and post-translationally insertedinto the organelle via leader sequences which are absentfrom the mature protein and which are similar to

    mitochondrial presequences (Lahti and Johnson 1991;Johnson et al. 1993).

    As previously discussed, glycoconjugates are found inthe hydrogenosomal membrane, especially in the mem-brane lining the peripheral vesicle. At present, we do nothave a clear explanation for this observation. One pos-sibility is the fusion of vesicles derived from the endo-plasmic reticulum with the hydrogenosome duringgrowth of the organelle (Benchimol et al. 1996a). Theclose proximity of the endoplasmic reticulum to thehydrogenosome is also observed during the process ofautophagy of the organelle, as characterized byBenchimol (1999a).

    The acidocalcisome

    Since the beginning of the 20th century, microscopistshave observed the presence of metachromatic granules,designated as volutin granules, in microorganismsstained with basic dyes (Meyer 1904). In protozoa, thisstructure has received several designations such as res-

    ervoir of metabolic products, pigment bodies, osmio-philic granules, polyphosphate granules or volutingranules (Anderson and Ellis 1965). Some studies haveshown that certain vacuoles contain a linear polymer oforthophosphate residues linked by high-energy phos-phoanhydre bonds, forming so called polyphosphates,or to contain pyrophosphate (review in Docampo andMoreno 1999). Biochemical studies have indicated thepresence of phosphate-containing compounds in severaltrypanosomatids, however their subcellular localizationwas not well established (Janakidevi et al. 1965; Blum1989).

    On the other hand, electron microscopic studies have

    shown the presence of at least four types of electron-dense structures in the cytoplasm of trypanosomatids.One is homogeneous, is not membrane bounded, doesnot present electron-lucent areas, and may be consti-tuted of lipids since it is not visualized as dense granulesin cells not fixed with osmium tetroxide. A second typeof lipid granule is membrane bounded and described inCrithidia deanei (Soares et al. 1987). A third type ofgranule seems to be involved with iron metabolism as itaccumulates hemo derivatives. For instance, the largedense granule found in Trypanosoma cyclopis dependson the presence of hemoglobin in the culture medium.Peroxidase activity can be detected in these granules

    (Carvalho et al. 1979). The fourth type of dense granuleis found in all trypanosomatids and will be the subject ofthe comments below. It has been suggested that this typeof dense structure contains polyphosphates, although nochemical evidence for their presence has been presented(Vickerman and Tetley 1977; Williams and McLaren1981). In any case, structures designated as electron-dense granules, volutin granules or inclusion vacuoleshave been considered for many years as part of thestructure of trypanosomatids, but without anyspecial relevance to cell physiology. The use of X-ray

    Fig. 12. Freeze-fracture view of the outer (O) and inner (I)membranes lining the hydrogenosome of Tritrichomonas foetus.The peripheral vesicle is indicated (arrow). 60,000. (Courtesy ofM. Benchimol)

    Fig. 13. Deep etch view of hydrogenosomes (H) of T. foetus. Insome of them complexes of particles are seen within the matrix(large arrow). The peripheral vesicle is also indicated (small arrow).60,000. (Courtesy of M. Benchimol)

    Fig. 14. Thin section of T. foetus submitted to the silverammoniacal technique designed for the detection of basic proteins.Reaction product is seen in the hydrogenosome matrix (H).30,000. (Courtesy of M. Benchimol)

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    microanalysis, with which it is possible to correlate animage seen in the transmission electron microscope withelement composition, showed the presence of Ca, Mg,Na, Zn and Fe in the cytoplasmic electron-dense gran-ules of trypanosomatids (Carvalho and De Souza 1977;Vickerman and Tetley 1977; Williams and McLaren1981; Dvorak et al. 1988; LeFurgey et al. 1990).

    From the morphological point of view, the structuresnow designated as acidocalcisomes have been observedsince the first observations of thin sections of trypano-somatids by electron microscopy. They are membranebounded structures which contains an electron-densecontent. The amount of dense material varies accordingto the procedures used to process the samples for elec-tron microscopy. In routine procedures, part of thedense material may be removed, leaving a thin densering below the membrane (Fig. 15). An electron-denseproduct is seen within the acidocalcisomes of cells fixedin the presence of potassium pyroantimonate, whichprecipitates calcium (Lu et al. 1998). The whole materialis better preserved in cells fixed using the high-pressurefreezing method followed by freeze-substitution where

    all acidocalcisomes appear as organelles completely fil-led with electron-dense material (Miranda et al. 2000).Frozen sections have a similar appearance (Scott et al.1997; Miranda et al. 2000). The best way to obtain ageneral view of all acidocalcisomes is to allow the wholecell to dry on carbon and formvar-coated grids in atransmission electron microscope, especially if it isequipped with an energy filter so that electron spectro-scopic images can be obtained (Fig. 16) (Miranda et al.2000).

    Acidocalcisomes usually appear as spherical struc-tures with an average diameter (SD) of 20090 nm.They can be observed in all portions of the cell, although

    they are preferentially located at the cell periphery. Inthe epimastigotes of T. cruzi, they are especially con-centrated in the middle portion of the body, althoughsome are also seen in the portion of the cell body asso-ciated with the flagellum. In trypomastigotes, acidocal-cisomes are preferentially localized in the anteriorsection. Although usually randomly distributed in somecells, they were seen in amastigotes in an aligned orga-nization, suggesting an interaction with cytoskeletalcomponents of the cell. Close contact between theacidocalcisome and the nucleus, lipid inclusions,mitochondria and sub-pellicular microtubules has beenobserved.

    The number of acidocalcisomes varies from species tospecies and even in the various developmental stages ofthe same species. A morphometrical study carried out onT. cruzi showed that amastigote forms possessed greaternumbers of acidocalcisomes, which occupyed a largervolume of the cell, than epimastigotes or trypomastig-otes (Miranda et al. 2000).

    A major breakthrough in the study of the cytoplasmicvacuoles of trypanosomatids took place in 1994 when

    Vercesi et al. (1994) suggested that the cytoplasmicvacuoles contained a very high Ca2+ concentration anda Ca2+-H+ translocating ATPase activity and desig-nated them as acidocalcisomes. This conclusion wasbased on previous studies carried out on trypanoso-matids, the slime mold Dictiostelium discoideum, mam-malian cells as well as experimental data from T. brucei.Following the initial observations carried out inT. brucei, Docampo and co-workers extended theirstudies and have shown the presence of acidocalcisomesin other trypanosomatids, such as T. cruzi (Docampoet al. 1995) and Leishmania amazonensis (Lu et al. 1997),and in other protozoa such as T. gondii (Moreno and

    Zhong 1996) and Plasmodium falciparum (Luo et al.1999; Marchesini et al. 2000). This organelle wasrecently characterized in Chlamydomonas reinhardtii(Ruiz et al. 2002) and in Dictiostelium discoideum(Marchesini et al. 2002).

    The first indication that the acidocalcisome is anacidic organelle comes from studies which show that thedense inclusion vacuoles found in the amastigotes ofL. amazonensis accumulate the weak base 3-(2,4-dini-troanilino)-3-amino-N-methyldipropylamine (DAMP),which could be localized using an immunocytochemical

    Figs. 15, 16. Visualization of the acidocalcisome in a thin sectionof conventionally processed cells for electron microscopy (Fig. 15)and in whole cells observed in a transmission electron microscopeequipped with an energy filter (Fig. 16). In thin sections most of theacidocalcisomes appear as a membrane bounded vacuole contain-ing an electron dense material at its periphery (arrow). Bothmicrographs are from Trypanosoma cruzi

    Fig. 15. 120,000. (After Miranda et al. 2000)

    Fig. 16. 15,000. (After Miranda et al. 2000)

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    approach (Antoine et al. 1988). Subsequently, it wasshown that a similar structure found in procyclic formsofT. brucei became larger when the cells were cultivatedin the presence of chloroquine, an acidotropic drug(Coppens et al. 1993). Later, it was shown by fluores-cence microscopy that vacuoles of varying size found inT. brucei and T. cruzi were labeled with acridine orangeand that the accumulation of this dye was sensitive tobafilomycin A, nigericin, and NH4Cl (Vercesi et al. 1994;Docampo et al. 1995). In the case of the epimastigotes ofT. cruzi, it is important to keep in mind that anotheracidic organelle, the reservosome, also concentrates ac-ridine orange (Soares et al. 1992). The exact pH of theacidocalcisome has not yet been determined.

    The matrix of the acidocalcisome has mainly beenanalysed in terms of its elemental composition basedprimarily on analytical methods associated with elec-tron microscopy. In these experiments, the elementcontent was compared between the inner portion of theorganelle and other portions of the cell, such as thecytoplasm. The following elements were shown to beconcentrated in the acidocalcisome: P, Mg, Ca, Na and

    Zn, and very little Cl, K and S (Dvorak et al. 1988;LeFurgey et al. 1990; Scott et al. 1997; Miranda et al.2000). The low content of S suggests a low proteinscontent. It is important to point out that care shouldbe taken in the interpretation of microanalytical data,especially if fixed cells are used. It is well known thatfixation changes the permeability of cell membranes tosome ions. Electron energy loss spectroscopy revealedthe presence of P and O, suggesting the presence ofcarbohydrates (Scott et al.1997). Recent studies haveshown that the phosphorus observed in the acidocal-cisomes of T. cruzi is in the forms of PPi, long-chainand short-chain polyphosphates (Urbina et al. 1999;

    Ruiz et al. 2001). In addition, PPi seems to be the mostabundant high energy phosphate present in T. cruzi(Urbina et al. 1999) and T. gondii (Rodrigues et al.1999).

    The membrane of the acidocalcisome is about 6 nmthick. It has not yet been isolated so that there is littleinformation on its proteins and no information about itslipid content. The use of an immunochemical (immu-noblotting, immunoprecipitation and immunocyto-chemistry) approach showed the presence of thefollowing enzymes in the membrane of the acidocalsio-mes of trypanosomatids: (1) a vacuolar H+-ATPase(Benchimol et al. 1998; Moreno et al. 1998); (2) a Ca2+-

    H+

    -translocating ATPase whose gene was cloned, se-quenced and expressed. Antibodies generated againstthe protein product of the gene (Tca1) labeled themembrane of the acidocalcisome as well as the plasmamembrane of T. cruzi (Lu et al. 1998); (3) a vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) was identified and localized in the membrane ofthe acidocalcisome and in the plasma membrane oftrypanosomatids and Apicomplexa using antibodiesrecognizing the enzyme found in plants (Scott et al.1998; Rodrigues et al. 1999; Lu et al. 1999).

    Excellent recent reviews have analysed the biochem-ical and functional properties of the acidocalcisome(Docampo and Moreno 1999, 2001), therefore thesepoints will not be discussed in the present review.

    Organelles involved in the endocytic pathway

    Pathogenic protozoa live in environments such as thebloodstream, within the host cells, in the intercellularspace, in the lumen or in association with the surface ofcells lining the digestive tract of their hosts. Most of themdo not have organelles involved in the storage of meta-bolic products. Their nutrition takes place both throughthe direct transport of single molecules through theplasma membrane or through a process of endocytosis.This latter process will be reviewed here for the try-panosomatids.

    The flagellar pocket of trypanosomatids

    All trypanosomatids have a region known as the fla-gellar pocket, which appears as a depression found inthe anterior region of the cell from which the flagellumemerges. It is formed by an invagination of the plasmamembrane which establishes a direct continuity withthe membrane of the flagellum (Figs. 18, 19). As themembranes lining the cell body and the flagellum es-tablish physical contact with each other at the point ofemergence of the flagellum, the pocket can be consid-ered as a special extracellular compartment which is insome way isolated from the extracellular medium. In-deed, the use of cytochemical labels has shown thatmacromolecules and large particles added to the culture

    medium may be excluded from the pocket. Morpho-logical and cytochemical studies have shown that thejunction established between the two membranes re-sembles a hemi-desmosome since the aggregation ofintramembranous particles, as seen in freeze-fracturereplicas, and the presence of dense material below themembrane were observed (Souto-Padron and De Souza1979).

    There are several lines of evidence showing that theflagellar pocket is a highly specialized region of thesurface of trypanosomatids: (1) it is the only regionwhich does not present the layer of microtubules, knownas sub-pellicular microtubules, closely associated with

    the membrane (reviews in Webster and Russel 1993;Landfear and Ignatushchenko 2001; McConville et al.2002); (2) the membrane lining the pocket, which cor-responds to 0.4% to 3% of the total cell surface, differsboth from the membrane lining the cell body and theflagellar membrane in terms of the distribution of in-tramembranous particles, and the localization of pro-teins, including some enzymes (reviews in De Souza1989; Landfear and Ignatushchenko 2001); (3) there ismuch morphological and cytochemical evidence show-ing that the pocket is the place where intense endocytic

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    and exocytic activity takes place. The incubation oftrypanosomatids in the presence of labeled macromole-cules revealed intense labeling of the pocket and theincorporation of the molecules through small endocyticvesicles formed in the pocket and then internalized intothe cytoplasm of the protozoan. Studies carried outmainly with T. brucei have shown that the flagellarpocket is the only site of internalization of macromole-cules by the protozoan. It has been shown that receptorsfor LDL (Coppens et al. 1987, 1988, 1991) and trans-ferrin (Webster 1989) are localized in the membranelining the pocket, facilitating the incorporation of thesemacromolecules by the protozoan. The macromoleculesare then incorporated via small uncoated and coatedvesicles (Langreth and Balber 1975; Webster 1989). A77 kDa protein associated with the coat was character-ized in T. brucei (Shapiro and Webster 1989; Websterand Shapiro 1990). More recently, clathrin heavy chainwas characterized and showed to be localized in theflagellar pocket, in a tubular system and in the trans-Golgi network of T. brucei (Morgan et al. 2001). Afterbudding from the pocket, the vesicles move into a tu-

    bular network compartment, which resembles the earlyendosome found in mammalian cells, localized betweenthe flagellar pocket and the nucleus, before fusing withround-shaped lysosome-like organelles (Webster 1989).On the other hand, when trypanosomatids are incubatedin the presence of drugs which stimulate exocytic activitya large number of vesicles are seen within the flagellarpocket. In addition several macromolecules with enzymeactivity can be detected within the pocket, as is the casewith acid phosphatase which appears as a filamentousstructure composed of a 100 kDa phosphoglycoproteinwith non-covalently associated proteo high molecularweight phosphoglycan. In addition there is also a fibrous

    material consisting of complex phosphoglycans (Stierhofet al. 1994); (4) in those trypanosomatids which have acontractile vacuole, the central vacuole, to which thereticular components of the spongiome converge, es-tablishes contact with the membrane lining the flagellarpocket and probably fusion between the membraneslining the two compartments occurs in order to releasethe water accumulated in the vacuole (Linder andStaehelin 1979). The central portion of the spongiomeaccumulates liquid collected by a network of tubuleswhich are in contact with it.

    The cytostome and the reservosome in T. cruzi

    Micrographs are shown in Figs. 19, 20, 21, 22, 23, 24,25. The epimastigote stage of members of the Trypano-soma genus belonging to the Schyzotrypanum sub-genus,such as T. cruzi, Trypanosoma vespertilionis andTry-panosoma dionisii, has a flagellar pocket which differs inshape form those found in the other developmentalstages. In the promastigote and ophistomastigote forms,the pocket is symmetrical due to the fact that the fla-gellum emerges from the central portion of the anterior

    region, while in the epimastigote and trypomastigoteforms the flagellum emerges laterally.

    In the case of the epimastigote and amastigote formsof members of the subgenus Schizotrypanum, there is ahighly specialized structure known as the cytostome-cytopharynx complex. This appears as a funnel-shapedstructure formed due to a deep invagination of theplasma membrane which may reach the nuclear region(Fig. 22). The opening of this complex, which is knownas the cytostome, may reach a diameter of 0.3 lm and issignificantly smaller in the deeper portion of the cyto-pharynx. The sub-pellicular microtubules follow the in-vagination of the plasma membrane. There is aspecialized region of the membrane lining the parasitewhich starts in the opening of the cytostome and pro-jects towards the flagellar pocket region. Freeze-fracturestudies have shown that this area is delimited from theother portions of the plasma membrane by a palisade-like array of closely associated particles (Figs. 19, 20, 21)(De Souza et al. 1978). The specialized region has veryfew intramembranous particles, as seen in freeze-fracturereplicas. However, its surface is very rugous, as observed

    in fracture-flip replicas which show the actual surface ofthe membrane lining the cytostome (Fig. 20). This ismost probably due to the presence of a large number ofmacromolecules associated with the cell surface in thatregion but which are not inserted in the membrane bi-layer so that they can not be observed in freeze-fracturereplicas. Indeed, the use of cytochemical techniques thatreveal surface glycoconjugates, as is the case of ruthe-nium red or the localization of concanavalin A-bindingsites using concanavalin A-peroxidase, have shown thatthe surface coat of T. cruziis thicker in the region liningthe cytostome.

    When epimastigotes are incubated in the presence of

    gold-labeled macromolecules such as transferrin, LDL,etc, they initially bind to the cytostome region and thenare internalized via endocytic vesicles which are formedat the bottom of the cytopharynx (Fig. 22). Recentmorphometric studies have shown that, in the case ofepimastigote forms of T. cruzi, about 85% of the goldparticles seen were associated with the cytostome (Porto-Carreiro et al. 2000) rather than with the flagellarpocket, as occurs with T. bruceiin which the particles areseen within the flagellar pocket. This observation makesT. cruzi the cell presenting the most polarized system ofendocytic activity described.

    Following binding to the cytostome, macromolecules

    are rapidly internalized via the cytopharynx and appearin small endocytic vesicles which bud from the deepestregion of this structure. Subsequently, these vesicles fuseto each other to form tubular structures which can beobserved in the most central portion of the protozoan.Incubation of the parasites in the presence of ammoni-um chloride or chloroquine, which are known to blockvesicle fusion by sequestering protons from the cell cy-toplasm thus hindering proton pumping into the endo-cytic compartments, facilitates the observation of anintricate, branched network of tubular structures

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    containing the gold-labeled macromolecules (Fig. 23)(Porto-Carreiro et al. 2000). A similar structure, ex-tending from the flagellar pocket to the posterior end of

    the cell, has been observed in Leishmania (Weise et al.2000; Ghedin et al. 2001; Mullin et al. 2001). In the caseofT. cruzi, the macromolecules are later concentrated instructures known as the reservosome (Fig. 24). Eachepimastigote form presents several reservosomes, mainlylocalized in the posterior region of the cell. Although itsmorphology can vary according to the growth condi-tions and the parasite strain, usually it is a sphericalorganelle with a mean diameter of 0.7 lm, surroundedby a unit membrane. The matrix of the reservosome is

    slightly dense and has some inclusions. Cytochemicalstudies have shown that the matrix is mainly made up ofproteins and that the inclusions contain lipids (Soaresand De Souza 1988). The organelle was designated asreservosome because it accumulates all macromoleculesingested by the parasite through an endocytic process,and it gradually disappears when epimastigotes are in-cubated in a poor culture medium, conditions whichtrigger the process of transformation of the non infectiveepimastigote into infective trypomastigote forms(Bonaldo et al. 1988; Soares and De Souza 1988).

    Most of the endocytic compartments of T. cruzi areacidic as indicated by labeling with acridine orange, afluorescent probe widely used to monitor pH gradientsacross membranes. Labeling of the tubular intermedi-ate compartment and of the reservosomes was observed(Porto Carreiro et al. 2000). The determination of thepH of the organelle using the DAMP technique indi-cated a value of pH 6.0, suggesting that the reservo-some corresponds to a pre-lysosomal compartment. Noacid phosphatase activity can be detected in theorganelle.

    One characteristic feature of the reservosome inT. cruzi is that it accumulates a large amount of cruzi-pain, the major cysteine proteinase found in the cell

    (Cazzulo et al. 1997). Although glycosylated, thisproteins does not possess mannose-6-phosphate residues(Cazzulo et al. 1990). No mannose-6-phosphate recep-tors could be immunocytochemically detected in T. cruzi(Soares et al. 1992). Therefore, another not yet charac-terized intracellular route is used to deliver cruzipain tothe endosomal system.

    Studies carried out in mammalian and yeast cellshave shown that several small GTPases, known as Rabproteins, are involved in the vesicle traffic in the

    Figs. 17, 18, 19. Different views of the flagellar pocket (FP) oftrypanosomatids as seen in thin section (Fig. 17) and in a freeze-fracture replica (Fig. 18). Vesicles can be seen within the pocket(Fig. 18). Other structures such as the Golgi complex (G),kinetoplast (K), endosymbiont (E), virus-like particles (V), nucleus(N), mitochondria (M) and flagellum (F) are indicated

    Fig. 17. Crithidia desouzai, 20,000. (After Soares et al. 1989)

    Fig. 18. Herpetomonas samulepessoai, 18,000. (After De Souzaet al. 1979)

    Figs. 19, 20, 21. Conventional freeze-fracture (Fig. 19) and frac-ture flip (Fig. 21) showing the cytostome (Cy). This region

    significantly differs from other regions of the cell surface in termsof the organization and density of the intramembranous particles(Fig. 19), rugosity of the actual cell surface (Fig. 20) and intensityof labeling with concanavalin coupled to colloidal gold particles(Fig. 21)

    Fig. 19. 25,000

    Fig. 20. 35,000. (After Pimenta et al. 1989)

    Fig. 21. Label-fracture of the epimastigote form of T. cruzi.20,000. (After Pimenta et al. 1989)

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    cytoplasm (Novick and Zerial 1997). Several Rabproteins have been characterized and localized in welldefined compartments of the endocytic and exocyticpathways. Rab 1 and Rab 2 are found in the endo-plasmic reticulum-Golgi complex intermediate com-partment; Rab 3A is found in synaptic vesicles andchromafin granules; Rab 4 is found in early endosomes;Rab 5 has been found in the plasma membrane, inchlatrin-coated vesicles and early endosomes; Rab 7 andRab 9 are found in late endosomes. Rab 7-like geneshave already been characterized in T. brucei, Leishmaniaand T. gondii(review in Field et al. 1998, 1999). A Rab 7homologue, present as a single gene, was detected in T.cruzi (Leal et al. 2000) and the corresponding proteinwas localized by immunocytochemistry in the Golgicomplex rather than in the endosomes (Araripe et al.2002). Rab 11 has been localized in the Golgi complex of

    mammalian cells (Chen et al. 1998; Zeng et al. 1999)while in T. cruzi it was localized in the reservosomewhich, as indicated previously, corresponds to a lateendosome (Mendonca et al. 2000). Studies carried outon T. brucei characterized a Rab2 which was shown tobe localized in the endoplasmic reticulum (Field et al.1999) and Rab31, formerly designated as Trab7p,localized in the Golgi complex (Field et al. 2000).

    The megasome in Leishmania

    Amastigote forms of Leishmania parasites of the mexi-cana complex contain abundant membrane boundedelectron-dense structures in the cytoplasm, which vary insize, number and appearance and have been designatedas megasomes (Alexander and Vickerman 1975) due totheir large size, which can be as great as the nucleus(Figs. 25, 26). The matrix of the organelle is not hom-ogenous, presenting dense inclusions and vesicles. Cy-tochemical studies have shown that the megasomescontain acid phosphatase and cysteine proteinase

    (Antoine et al. 1988; Duboise et al. 1994; Pupkins et al.1996) and therefore correspond to typical lysosomes.Indeed it has been shown to be acidic organelle (Antoineet al. 1988) and susceptible to proteinase inhibitors(Antoine et al. 1989; Galva o-Quintao et al. 1990).Megasomes are not observed in promastigote forms,appearing only during the process of transformation ofthese forms into amastigotes. The mechanism of tar-geting cysteine proteinase to megasomes has been thesubject of investigation. Phosphorylated mannoseresidues do not exist in Leishmania cysteine proteinase.The involvement of a predomain region of the

    Figs. 2224. Different phases of the process of ingestion of gold-labeled transferrin by epimastigote forms of Trypanosoma cruzi.Initially the protein is found in the opening of the cytostome

    (Fig. 22). Then, it can be seen in a tubular structure, bettervisualized in whole cells (Fig. 23). Subsequently the protein isaccumulated in the reservosomes (Fig. 24)

    Fig. 22. 15,000. (After Porto Carreiro et al. 2000);

    Fig. 23. 90,000. (After Porto Carreiro et al. 2000)

    Fig. 24. 25,000. (After Porto Carreiro et al. 2000)

    Figs. 25, 26. Megasomes (arrows) found in amastigote forms ofL. amazonensis. They appear as spherical organelles of variablesize. The organelle shown in Fig 25 is larger than the protozoannucleus (N). K Kinetoplast; L lipid droplet; M mitochondrion; FPflagellar pocket. 22,000. (After Ueda Nakamura et al. 2001)

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    macromolecule in targeting megasomes is possible(Huete-Perez et al. 1999; Boukai et al. 2000).

    Morphometrical analysis has shown that megasomesare more abundant in intracellular amastigotes than inamastigotes growing in axenic cultures (Coombs et al.1986; Bates et al. 1992; Pral et al. 1993; Ueda-Nakamuraet al. 2001). They may account for 315% of the totalcell volume.

    Acknowledgements The work carried out in the authors labora-tory has been supported by Programa de Nucleos de Excelencia(PRONEX), Conselho Nacional de Desenvolvimento Cientfico eTecnolo gico (CNPq), Fundacao Carlos Chagas Filho de Apoio a`Pesquisa do Estado do Rio de Janeiro (FAPERJ), National Insti-tutes of Health (NIH) and the European Community. I thank mycolleague M. Benchimol for supplying micrographs.

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