Hydrogenosomes under microscopy

Tissue and Cell 41 (2009) 151–168

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Review

Hydrogenosomes under microscopy Marlene Benchimol ∗ Universidade Santa Úrsula, Laboratório de Ultraestrutura Celular, Rio de Janeiro, Brazil

a r t i c l e

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Article history: Received 30 November 2008 Received in revised form 4 January 2009 Accepted 13 January 2009 Available online 17 March 2009 Keywords: Hydrogenosome Trichomonas Electron microscopy

a b s t r a c t A hydrogenosome is a hydrogen-producing organelle, evolutionary related to mitochondria and is found in Parabasalia protozoa, certain chytrid fungi and certain ciliates. It displays similarities to and differences from mitochondria. Hydrogenosomes are spherical or slightly elongated organelles, although very elongated hydrogenosomes are also found. They measure from 200 nm to 1 ␮m, but under stress conditions can reach up to 2 ␮m. Hydrogenosomes are surrounded by two closely apposed membranes and present a granular matrix. Cardiolipin has been detected in their membranes, and frataxin, which is a conserved mitochondrial protein involved in iron metabolism, was also recently found. Hydrogenosomes have one or multiple peripheral vesicles, which incorporate calcium. The peripheral vesicle can be isolated from the hydrogenosomal matrix and can be considered as a distinct hydrogenosomal compartment. Dysfunctional hydrogenosomes can be removed by an autophagic process and further digested by lysosomes. Hydrogenosomes divide in three different ways, like mitochondria, by segmentation, partition and the heart form. They may divide at any phase of the cell cycle. Nucleoid or electron dense deposits found in hydrogenosomes can be considered artifacts or dysfunctional hydrogenosomes. The hydrogenosome does not contain a genome, although DNA has already been detected in one anaerobic ciliate. Hydrogenosomes can be considered as good drug targets since their metabolism is distinct from mitochondria. © 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Similarities to and differences from mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogenosome metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphological methods to study hydrogenosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell fractionation and isolated hydrogenosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Medical interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogenosome morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The different hydrogenosome shapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The hydrogenosome envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The peripheral vesicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The matrix of the hydrogenosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fungal hydrogenosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogenosome division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogenosome behavior in the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proximity with other cellular structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogenosomes and endoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunolabeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogenosome death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolutionary origin of the hydrogenosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The cardiolipin story . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The proteome of hydrogenosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Correspondence to: Rua Jornalista Orlando Dantas 59, Botafogo, Rio de Janeiro, RJ, Brazil, CEP 222-31-010. Tel.: +55 21 2237 0440; fax: +55 21 2237 0440. E-mail address: [email protected]. 0040-8166/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2009.01.001

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1. Introduction Hydrogenosomes are organelles found in anaerobic or microaerophila organisms, such as protozoa of the Trichomonadida Order (Figs. 1a, 2, 3, and 4a, b, e), in some anaerobic fungi (Figs. 1b, c 2f, and 4d) (Yarlett et al., 1986; Marvin-Sikkema et al., 1992; Benchimol et al., 1997), in rumen ciliates and flagellates (Fig. 4c, f) (Yarlett et al., 1981, 1984; Snyers et al., 1982) and also in some free-living ciliates (van Bruggen et al., 1984). The hydrogenosomecontaining microorganisms do not present typical mitochondria (Embley et al., 2003). Hydrogenosomes are not present in multicellular animals or plants or in other anaerobic protists, such as amoebas and giardias. The most extensive studies of this organelle have been carried out in the trichomonad species (Fig. 2). The hydrogenosome is a crucial organelle for this parasite, since it produces molecular hydrogen and ATP by oxidizing pyruvate or malate under anaerobic conditions (Müller, 1990) (Fig. 5). The oxidative decarboxylation of pyruvate in hydrogenosomes is coupled to ATP synthesis and linked to ferredoxin-mediated electron transport. This organelle has this name because it is able to produce molecular hydrogen (Lindmark and Müller, 1973; Müller, 1993) (Fig. 5). Hydrogenosomes contain enzymes that participate in the metabolism of pyruvate formed during glycolysis and thus form ATP (reviewed in Müller, 1993). Hydrogenosomes are best studied in cells like Tritrichomonas foetus (Fig. 2) and Trichomonas vaginalis (Figs. 1a and 6c), which are flagellated parasitic protists that inhabit the urogenital tract of cattle and humans, respectively. They often induce reproductive failure and are a cause of significant economic losses and social and health disturbances worldwide. In trichomonads, hydrogenosomes have been recognized by light microscopists for a long time as paraxostylar and paracostal granules, due to their proximity to the axostyle (bundle of microtubules) and the costa, a periodic proteinaceous structure (Fig. 2a, b). As a general rule, hydrogenosomes are spherical or slightly elongated organelles found in non-mitochondrial organisms (Figs. 2 and 6). In Trichomonas hydrogenosomes measure from 200 to 500 nm, but under drug treatment they can reach up to 2 ␮m (Fig. 18). 2. Similarities to and differences from mitochondria Several authors consider hydrogenosomes as modified mitochondria, since there are evidences of similarities, at least in trichomonads (Lahti et al., 1992; Johnson et al., 1993; Bui et al., 1996; Germot et al., 1996; Hrdy´ et al., 2004; Bradley et al., 1997;

Biagini et al., 1997; Häusler et al., 1997; Dyall et al., 2000; Embley et al., 1997, 2003; De Andrade Rosa et al., 2006; Dolezal et al., 2007). The origin of the hydrogenosome has been the subject of intense discussion since it also shares some structural and morphological features when compared to mitochondria, for example, it is enveloped by two membranes (Figs. 3 and 6) (Benchimol and De Souza, 1983), divides autonomously by fission (Fig. 7) (Benchimol et al., 1996b), imports proteins post-translationally (Johnson et al., 1993), produces ATP (Lindmark and Müller, 1973) and presents cardiolipin (Figs. 21 and 22) (De Andrade Rosa et al., 2006). In addition, targeting and translocation of proteins into hydrogenosomes presents similarities to mitochondrial protein import (Bradley et al., 1997) and a member of the mitochondrial carrier family was found in hydrogenosomes (Dyall et al., 2000). However, there is also evidence showing that hydrogenosomes and mitochondria may not be so close in terms of their origins. Some of these differences are: the lack of a genome, with a possible exception of hydrogenosomes from Nyctotherus ovalis (Akhmanova et al., 1998), the lack of a respiratory chain, cytochromes, the F0 –F1 ATPase, the tricarboxylic acid cycle and oxidative phosphorylation (Müller, 1990; Clemens and Johnson, 2000). Among the similarities between hydrogenosomes and mitochondria, we can cite: (1) both are surrounded by two membranes and present a granular matrix (Fig. 6a) (Benchimol and De Souza, 1983); (2) they divide in three different ways: segmentation, partition and the heart form (Fig. 7) (Benchimol et al., 1996b; Benchimol and Engelke, 2003); (3) they may divide at any phase of the cell cycle (Fig. 8) (Benchimol and Engelke, 2003); (4) both produce ATP (Lindmark and Müller, 1973) (Fig. 5); (5) both participate in the metabolism of pyruvate formed during glycolysis (Lindmark and Müller, 1973) (Fig. 5); (6) they are able to utilize oxygen as a terminal electron acceptor (Cerkasov et al., 1978); (7) both present a relationship with the endoplasmic reticulum (Fig. 9) (Benchimol, 2008a); (8) they incorporate calcium (Fig. 6a) (Benchimol and De Souza, 1983; Chapman et al., 1985); (9) they import proteins post-translationally (Bradley et al., 1997); (10) both present a beta-succinyl-coenzyme A synthetase, a soluble hydrogenosomal protein with an amino-terminal sequence that resembles mitochondrial presequences (Lahti et al., 1992); (11) both present a membrane targeting pathway, a member of the mitochondrial carrier family (Dyall et al., 2000); (12) both present Frataxin, a conserved mitochondrial protein (Dolezal et al., 2007); (13) both present a mitochondrial-type 70-kDa heat shock protein (Bui et al., 1996; Germot et al., 1996) and Cpn60 (Bui et al., 1996); (14) they contain the NADH dehydrogenase module of mitochondrial complex I (Hrdy´ et al., 2004); (15) hydrogenosomal proteins are synthesized on free polyribosomes (Lahti and Johnson, 1991); (16)

Fig. 1. (a) Tritrichomonas foetus under light microscopy, DIC (differential interferential contrast). The hydrogenosomes (H) are seen as small granules along the cell axostyle. Bar = 10 ␮m (Benchimol, unpublished). (b, c) Neocallimastix frontalis. Two forms of the life cycle of this fungus as seen by DIC (differential interferential contrast). Both forms present hydrogenosomes (arrows). (b). Fungus spore from N. frontalis: sexual and asexual reproduction of the fungi is commonly via spores, often produced on specialized structures or in fruiting bodies. Bar = 2 ␮m (Benchimol, unpublished). (c) This figure shows the multicellular filaments called hyphae forming a mycelium. Bar = 50 ␮m (Benchimol, unpublished).

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Fig. 2. General view of hydrogenosomes. Tritrichomonas foetus by transmission electron microscopy (Fig. 1a) and a diagram (Fig. 1b). Note that hydrogenosomes (H) are preferentially located along the axostyle (Ax) and costa (C). AF, anterior flagella; BB, basal bodies; ER, endoplasmic reticulum; F, parabasal filament; G, Golgi; GL, glycogen granules; L, lysosomes; N, nucleus; Nu, nucleolus, P, pelta; R, recurrent flagellum; UM, undulating membrane; V, vacuoles. Bar = 500 nm. (Fig. 1a, Benchimol, unpublished; Fig. 1b, From Benchimol, 2004).

and both present cardiolipin (Figs. 21 and 22) (De Andrade Rosa et al., 2006). In ciliate hydrogenosomes other similarities to mitochondria have been described, such as: (1) presence of internal membranes which look like mitochondria (Fenchel and Finlay, 1995); (2) these hydrogenosomes are also calcium stores (Biagini et al., 1997) and (3) they display a membrane potential (Biagini et al., 1997). However, there are differences, such as: (1) presence of hydrogenase; (2) production of molecular hydrogen; (3) absence of genetic material, at least in trichomonas (Clemens and Johnson, 2000); (4) lack a respiratory chain and cytochromes (Lloyd et al., 1979b); (5) absence of the F0 –F1 ATPase (Lloyd et al., 1979a); (6) absence of the tricarboxylic acid cycle (Müller, 1993); (7) lack of oxidative phosphorylation (Müller, 1990); (8) lack of sensitivity to metabolic inhibitors such as rotenone and cyanide (Cerkasov et al., 1978) and (9) presence of peripheral vesicles (Figs. 3b and 6a, c) (Benchimol et al., 1996a; Benchimol, 2008a).

(CoA) to form acetyl–CoA; this latter product is converted to acetate and the energy of the thioesther bond is conserved in two successive steps, resulting in substrate level phosphorylation. Oxidative pyruvate decarboxylation in T. vaginalis, differently from the other eukaryotic cells, is catalyzed by pyruvate:ferredoxin oxidoreductase, an enzyme found in several bacteria and in a number of microorganisms. Electrons released from pyruvate are transferred to ferredoxin, a low molecular weight electron carrier protein. The ferredoxin represents the key electron transport component of the hydrogenosome and once reduced by pyruvate:ferredoxin oxidoreductase it is reoxidized with protons as terminal electrons acceptors through the action of a hydrogenase and thus producing molecular hydrogen. This process

3. Hydrogenosome metabolism The trichomonad metabolism starts in the cytosol, where glycolysis takes place; various intermediates of glycolysis give rise to intermediate products, such as pyruvate, which corresponds to the classical glycolysis pathway observed in other eukaryotic cells. Malate can also be produced and like pyruvate, enters into the hydrogenosome (Fig. 5). Within the hydrogenosome, pyruvate is oxidatively decarboxylated to acetyl coenzyme A and CO2. During pyruvate decarboxylation electrons are transferred primarily to protons with H2 formation and in this reaction CO2 is liberated and the acyl moiety is transferred to coenzyme A

Fig. 3. (a, b) Transmission electron microscopy of two hydrogenosomes of T. foetus under different plane sections. The hydrogenosome double membranes are easily visualized (arrow in a) encircling the whole organelle. (b) This figure shows a hydrogenosome with a peripheral vesicle (PV). Note its contents. Bar = 100 nm (Benchimol, unpublished).

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Fig. 4. General view of different cells that harbor hydrogenosomes. (a) Trichomonas augusta. This protozoan was obtained from the rectum of Rana pipiens. The hydrogenosomes (H) are heart-shaped, which could indicate a division process. Bar = 200 nm. (Benchimol, unpublished); (b) Tetratrichomonas sp. The hydrogenosomes (H) are clustered and elongated. Bar = 300 nm. (Benchimol, unpublished); (c) Ciliate protist from Rumen. Bar = 300 nm. (Benchimol, unpublished); (d) Neocallimastix frontalis. This fungus presents hydrogenosomes distributed in the cytoplasm, without any preferential location. Bar = 400 nm. (Benchimol, unpublished). W, cell wall; (e) Monocercomonas sp. as seen after the Thiéry technique. Note the elongated hydrogenosomes, which have their membranes stained, indicative of carbohydrates. Bar = 100 nm. (from Diniz and Benchimol, 1998); (f) Flagellate protist from Rumen. The hydrogenosomes (H) are seen in clusters and in large numbers. Bar = 100 nm (Benchimol, unpublished).

is coupled to ATP synthesis, which is linked to ferredoxinmediated electron transport. In addition to protons being reduced to molecular hydrogen by the action of hydrogenase, acetate, ATP and CO2 are also liberated (Fig. 5). According to Müller (1990) no electron transport occurs across the hydrogenosomes membranes. 4. Morphological methods to study hydrogenosomes Several approaches have been used to better visualize the morphological aspects of the hydrogenosome structures. When transmission electron microscopy (Figs. 2–4 and 6–9), as well the field emission scanning EM (FESEM) (Fig. 6e) were used, detailed views of the organelle were seen. The use of cytochemistry (Figs. 10–12), cell fractionation (Fig. 13), high-voltage EM (Fig. 14a), immunocytochemistry (Fig. 14d), fluorescent compounds (Fig. 15) and 3D reconstruction (Fig. 16) has added further information. In addition, freezing methods have allowed the visualization of protein distribu-

tion in cell membranes and better organelle preservation (Figs. 14b and 17). 5. Cell fractionation and isolated hydrogenosomes The hydrogenosome has been isolated using cell fractionation techniques (Díaz and De Souza, 1997; Benchimol, 2000). However, alterations induced by the fractionation technique were demonstrated (Benchimol, 2000). The morphology of freshly isolated hydrogenosomes was compared with those found in whole cells of T. foetus and examined using different methods. It was shown that after fractionation procedures the hydrogenosome ultrastructure had been changed and the isolated hydrogenosome presented patchwork-like structures, rosettes, and the inner hydrogenosomal membrane had been displaced (Benchimol, 2000). Alterations to the intramembrane particle distribution were reported as seen by freeze-fracture and alterations in the density of hydrogenosome, suggesting that the morphology of this organelle can be changed

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Fig. 5. Metabolic pyruvate metabolism of T. foetus in hydrogenosomes. The process starts in the cytosol, where glycolysis takes place. Various intermediates of the glycolysis give rise to intermediate products, such as pyruvate, which corresponds to the classical glycolysis pathway. Malate can also be produced and enters in the hydrogenosome. Pyruvate is oxidatively decarboxylated and the electrons are transferred primarily to protons with H2 formation. Oxidative pyruvate decarboxylation in trichomonas, differently from the other eukaryotic cells, is catalyzed by a different enzyme, pyruvate:ferredoxin oxidoreductase, an enzyme found in several bacteria and in a number of microorganisms. Under anaerobic conditions, hydrogenase catalyzes the formation of molecular hydrogen through the reduction of protons. However, when the drug metronidazole is used, it replaces protons as the acceptor of electrons donated by ferredoxin. ATP, CO2 , and acetate are also produced. Fd, ferredoxin; PEP, phosphoenolpyruvate.

as a consequence of cell fractionation or as an expression of its metabolic state (Benchimol, 2000). 6. Medical interest T. vaginalis is a protozoan parasite that provokes vaginitis and an acute inflammatory discharge. The parasite infects the urogenital tract in humans displaying high tissue and host specificity and it is responsible for the most prevalent non-viral sexually transmitted disease (Petrin et al., 1998). Another parasite, T. foetus is a cattle urogenital parasite, with similar importance to T. vaginalis. Both parasites present hydrogenosomes, but not mitochondria. Accordingly hydrogenosomes can be considered an excellent drug target (Benchimol, 2008b) since its metabolic pathway is distinct from that found in mitochondria. Thus, medicines against hydrogenosomes probably will not affect the host-cell, as occurs in trichomonad treatment. Nowadays, the main drug used against trichomonads is metronidazole (Fig. 5), although other drugs such as ␤-lapachone, colchicine, taxol, nocodazole, griseofulvin, cytochalasins and hydroxyurea among others have been used in trichomonad basic research studies (Fig. 18). All these drugs provoked cell modifications, such as: (1) flagella internalization forming pseudocysts; (2) dysfunctional hydrogenosomes (Figs. 18 and 19); (3) hydrogenosomes with abnormal sizes and shapes and with an electron dense deposit called nucleoid (Fig. 19) and (4) intense autophagy in which hydrogenosomes are removed and further digested by lysosomes (Fig. 20). Among the drugs affecting hydrogenosomes, metronidazole has a special place, since it is the drug of choice in trichomoniasis chemotherapy because it inhibits multiplication and kills trichomonads. Metronidazole and related 5-nitroimidazoles are the only drugs available for the treatment of human urogenital trichomoniasis caused by the protozoan parasite T. vaginalis. In the 1950s, a strain of Streptomyces that was able to produce a nitroimidazole derivative presenting an anti-trichomonal activity, was discovered. It was named metronidazole and it is used under the commercial name of Flagyl. Metronidazole is administered in an inactive form and enters the cells by passive diffusion (Kulda et

al., 1993) and it is reduced to its cytotoxic radical anion within the hydrogenosome (Fig. 5). It is well-known that oxidative decarboxylation of pyruvate, which is coupled to ATP synthesis and which is also linked to ferredoxin-mediated electron transport, occurs in the hydrogenosome. This pathway is responsible for metabolic activation of 5-nitroimidazole drugs, such as metronidazole. Electron transport components in the organelle, pyruvate:ferredoxin oxidoreductase and ferredoxin, donate a single electron to the drug, converting it to a nitroso cytotoxic free radical which is cytotoxic (Fig. 5). In spite of the variety of nitroimidazoles available worldwide, metronidazole remains the most frequently used and has become the standard for T. vaginalis infections. However, one important problem concerning the use of metronidazole and other derivatives are the drug-resistant trichomonads. In addition to drug-resistance, metronidazole may lead to adverse symptoms in humans, and pregnant women cannot use it. Thus, searching for new medicines for the treatment of trichomoniasis is crucial. A reduced transcription of the ferredoxin gene in metronidazole-resistant T. vaginalis has been demonstrated (Quon et al., 1992). This group showed data correlating drug resistance with altered regulation of ferredoxin gene transcription, in which a reduction in gene transcription resulted in decreased intracellular levels of ferredoxin. This, in turn, could play a role in metronidazole resistance by decreasing the ability of the cell to activate the drug. A novel pathway involved in the drug activation within the hydrogenosome has been described (Hrdy´ et al., 2005). This group showed that trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from the hydrogenosomes. In addition, Land et al., 2001 observed a reduction or loss of multiple hydrogenosomal proteins associated with hydrogenosome metabolism in highly drug-resistant trichomonas. These authors proposed that this reduction would reduce or eliminate the ability of the parasite to activate the drug. They also found that the resistant strains presented cells 20% smaller in size and hydrogenosomes with approximately one-third the size of those cells which were drug-sensitive.

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Fig. 6. Routine preparation of hydrogenosomes (H) in different organisms: (a, b, e) Tritrichomonas foetus; (c) Trichomonas vaginalis, (e) Monocercomonas sp. (f) the anaerobic fungus Neocallimastix frontalis. Note that all hydrogenosomes are enveloped by a double membrane (arrows). In T. foetus (a, b, e), the hydrogenosomes are spherical and present a single peripheral vesicle (asterisk), whereas in T. vaginalis (e) several peripheral vesicles are seen surrounding the organelle (asterisks). In Monocercomonas (d) the hydrogenosome is very elongated. (a, c, d, f) Here are samples observed in TEM, whereas in (b) the cell was quick-frozen by slam-freezing and submitted to freeze-etching technique. Notice that the peripheral vesicle is distinct form the rest of the organelle. (e) This figure presents a T. foetus hydrogenosome observed under high resolution scanning electron microscopy (FESEM). CW, cell wall, R, endoplasmic reticulum; N, nucleus, V, vesicle. Bar = 50 nm. (a–c, e, Benchimol, unpublished); (b) taken from Benchimol, 2000; (a, f) from Benchimol, 2008c; (c) Benchimol, unpublished; (d) Taken from Diniz and Benchimol, 1998; (e) from Benchimol, 2008c; (f) taken from Benchimol et al., 1997).

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Fig. 7. Hydrogenosomes under division. Views of the segmentation process (a–d, j) of a dividing hydrogenosome (H). (a–b). The organelle is elongated showing a constriction in the central region. Note that in (a), a membranous profile seems to strangle the organelle (asterisk). Bar = 50 nm. ((a), Benchimol, unpublished). (b) Filaments are seen in the dividing region (arrows). Bar = 50 nm (From Benchimol et al., 1996b). (c) A hydrogenosome at end of the segmentation process. A septum (arrow) is seen separating the organelle into two compartments. Bar = 50 nm (Benchimol, unpublished). (d) Freeze-fracture of a T. foetus hydrogenosome showing the segmentation process. Arrows point to a linear array of particles at the septum constriction. Bar = 50 nm (From Benchimol et al., 1996b). (e–g) Thin section of three hydrogenosomes (H) dividing via the partition process. The hydrogenosome (H) becomes larger and an invagination of the inner hydrogenosomal membrane is observed, gradually dividing the hydrogenosomal matrix into two compartments. (e) This figure shows that initially the inner membrane separates the hydrogenosome (H) into two compartments, but they are still joined by the outer hydrogenosomal membrane (arrowhead). (e) Bar = 50 nm (from Benchimol et al., 1996b). (f) Bar = 50 nm (Benchimol, unpublished). (g) The peripheral vesicles are seen at opposite directions. Bar = 50 nm (Benchimol et al., 1996b). (h, i) T. foetus showing two hydrogenosomes in the process of heart division. Bar = 50 nm (From Benchimol and Engelke, 2003). (j) Freeze-etching of a T. foetus hydrogenosome under segmentation division after quick freezing of living cells. Arrows point to a linear array of particles at the septum constriction. Bar = 50 nm (From Benchimol and Engelke, 2003).

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Fig. 8. General aspect of T. foetus in interphase (a) and under division (b–d). Notice that in the interphase the hydrogenosomes are aligned on the costa and axostyle (a), whereas during the division they are close to the nucleus (b–d). Bar = 300 nm. (a) Taken from Benchimol, 2008c; (b, c) Taken from Benchimol and Engelke, 2003; (d) Taken from Ribeiro et al., 2000).

When drugs interfering in polyamine metabolism were used (Reis et al., 1999) contradictory results were obtained. Whereas Reis et al. (1999) claimed that when a putrescine analogue such as the 1, 4-diamino-2-butanone (DAB) was used, the growth of the parasite cultures was significantly inhibited and hydrogenosomes were progressively degraded, giving rise to large vesicles that displayed immunoreactive material when an antibody to beta-succinylcoenzyme A synthase, a hydrogenosomal enzyme, was tested. This

group published that inhibition of T. foetus ornithine decarboxylase (ODC) resulted in growth arrest, destruction of hydrogenosomes and decreased amounts of hydrogenosomal enzymes (Reis et al., 1999). However, Garcia et al. (2005) found quite different results since DAB-treated trichomonads did not reveal any adverse effects on the number and integrity of hydrogenosomes. Research into new drugs is in course in order to replace metronidazole as the main medicine, since it can provoke collateral effects and resistance.

Fig. 9. Hydrogenosomes and the endoplasmic reticulum. Thin sections of T. foetus showing that profiles of endoplasmic reticulum (ER) are seen in continuity (arrow) to the hydrogenosomes (H). Bar = 100 nm. (a) from Benchimol, 2008a; (b) From Benchimol, 2001; (c) Benchimol, unpublished; (d) from Benchimol, 1999.

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Fig. 10. Cytochemistry in hydrogenosomes. (a) T. foetus after cytochemistry for calcium using potassium pyroantimonate showing an intense labeling in the hydrogenosomes matrix (arrows) and in the Golgi (G). N, nucleus. Bar = 300 nm (from Benchimol et al., 1982b). (b) High magnification of hydrogenosomes (H) after fixation in rich calcium fixative, showing an intense positive reaction as seen by the calcium deposits. Bar = 100 nm. (Benchimol, unpublished). (c). Immuno-electron microscopy showing detection of malic enzyme in the hydrogenosomes (H) of T. vaginalis. The hydrogenosomal matrix is intensely labeled with gold particles. Bar = 100 nm (Benchimol, unpublished). (d) Electron microscopy of T. vaginalis treated with the drug BCECF, DAB and UV-illuminated. The hydrogenosomes show high electron density after illumination. H, hydrogenosome. Bar = 300 nm (Benchimol, unpublished). (for technical details see Scott et al., 1998). (e) T. foetus submitted to the postformaldeyde in the ammoniacal silver method. Silver particles are seen only in the hydrogenosomes (H). N, nucleus, Ax, axostyle. Bar = 300 nm (from Benchimol et al., 1982a). (f) Tritrichomonas foetus submitted to cytochemistry for basic proteins (ethanolic phosphotungstic acid, PTA), showing positive reaction in the hydrogenosomes matrix (H), in the axostyle (Ax), costa (C), basal bodies microtubules (bb), flagella (F), and pelta (P). Bar = 500 nm (from Benchimol et al., 1982a).

7. Hydrogenosome morphology All hydrogenosomes described till today are enveloped by a double membrane (Figs. 3 and 6a, f) (Benchimol et al., 1996a; van der Giezen et al., 1997; Benchimol et al., 1997). They present a granular matrix (Figs. 3 and 6a, b, e, f) and in trichomonads, one (Figs. Fig. 33b and 6a) or several peripheral vesicles (Fig. 6c), which store calcium (Fig. 6a) (Benchimol and De Souza, 1983). Hydrogenosomes present an average diameter of 300 nm (Fig. 6a–c, e), but may reach 2 ␮m in Monocercomonas sp. (Diniz and Benchimol, 1998) (Fig. 6d). Comparative ultrastructural studies by TEM on control (untreated trichomonads) (Figs. Fig. 11, 2a, 3, 4a, and 6a–e) with those cells treated with metronidazole, hydroxyurea, cytochalasin, fibronectin, hydrogen peroxide and others, revealed modifications in shape and size of the hydrogenosomes (Fig. 18) in the latter group (Benchimol, 1999; Benchimol, 2001; Ribeiro et al., 2002a; Mariante et al., 2003). 8. The different hydrogenosome shapes In trichomonads (Figs. 1a, 2, 3, 4a, and 5a–c) (Benchimol et al., 1996a), in some fungi such as Neocallimastix frontalis (Figs. 1b, c, 4d and 6f) (Yarlett et al., 1986; Benchimol et al., 1997; van der Giezen et al., 1997), in rumen ciliates and flagellates (Fig. 4c, f) (Yarlett et al., 1981, 1984) and in free-living ciliates, (Finlay and Fenchel, 1989) hydrogenosomes are spherical (Figs. 3, 8, 10, 12, 13, 14 and 15) or slightly elongated granules. However, in some cells, hydrogenosomes are not spherical but are very elongated structures (Fig. 6d),

such as those found in Monocercomonas sp (Diniz and Benchimol, 1998).

9. The hydrogenosome envelope The double membranes that surround hydrogenosomes are very thin, presenting a thickness of only 6 nm and are very closely apposed to each other (Fig. 6a). As a general rule no space is observed between the two membranes (Benchimol and De Souza, 1983; Benchimol et al., 1996a). CaCl2 in the fixation solution, as well as the use of potassium ferricyanide together with reduced osmium, is important for good visualization of the two membranes (Benchimol and De Souza, 1983; Benchimol et al., 1996a) (Figs. 3 and 6a, f). When the thin-sections obtained for visualization in TEM are thicker than 60 nm the two membranes are hardly visualized. Freeze-etch preparations (Figs. 6b and 14b) allowed a better visualization of hydrogenosome matrix and in some cases filaments were seen emanating from the hydrogenosomes (Figs. 6b and 14b) to cytoskeletal structures in trichomonas such as the cytoskeleton, axostyle and costa structures (Benchimol et al., 2000). The two hydrogenosomal membranes, which were cytochemically detected when the Thiéry technique (periodic acid–thiosemicarbazide–silver proteinate technique) was used (Fig. 12) (Benchimol et al., 1996a), presented carbohydrates. In addition, the use of gold-labeled lectins, such as WGA (Fig. 13b) showed that the membrane of the peripheral vesicle compartment is intensely labeled indicating the presence of N-acetyl-glucosamine (Fig. 13b) (Benchimol and Bernardino, 2002).

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Fig. 11. Enzymes in hydrogenosomes. Cytochemistry for enzymes such as: glucose-6-phosphatase (a, d), IDP-diphosphatase (b); Ca2+ -ATPase (c); acid phosphatase (e). Note that the ER is labeled, as well the hydrogenosome peripheral vesicles (arrows). G, Golgi, H, hydrogenosome, L, lysosome, N, nucleus. (a, b) Bar = 300 nm; (c–e) Bar = 200 nm. (a–c) Taken from Benchimol, 2008a; (d) taken from Benchimol, unpublished; (e) taken from Benchimol, 2008a.

Freeze-fracture showed a different number and distribution of intramembranous particles in the two hydrogenosome membranes (Benchimol et al., 1996a; Benchimol, 2001). Four fracture faces were identified: two concave faces representing the P faces of the outer and the inner membranes and two convex faces representing the E faces of the two membranes (Fig. 17). The P and E faces of the outer membrane were frequently found. Although no quantitative analyzes were carried out the P face seems to have a higher particle density than the E face. The E face of the inner membrane was observed only in a few cases (Benchimol et al., 1996a). Special arrangements of intramembranous particles (rosettes) were found only when isolated hydrogenosomes were freeze-fractured after cell fractionation (not shown) (Benchimol, 2000). Invaginations of the hydrogenosome membrane were observed in trichomonads under drug stress, delimitating inner compartments (Fig. 18). Some of the compartments had the same morphology and electron density as the hydrogenosomal matrix while others had a lower density and presented tubular structures (Benchimol et al., 1996a; Benchimol, 2001). Ciliate hydrogenosomes in Metopus contortus and Cyclidium porcatum present internal membranes and look like mitochondria (Fenchel and Finlay, 1995). In addition, these hydrogenosomes are also calcium stores and they display membrane potential, which are similar to features found in mitochondria (Biagini et al., 1997). 10. The peripheral vesicle When present, the peripheral vesicle is considered as a distinct compartment (Fig. 13b, c), since its content and morphological appearance is quite different from the rest of the organelle (Fig. 6b) (Díaz and De Souza, 1997; Benchimol et al., 1993; Benchimol et al., 1996a; Benchimol, 2008a). T. foetus hydrogeno-

somes present one or two peripheral vesicles (Figs. 3b and 6a), whereas T. vaginalis hydrogenosomes exhibit several vesicles at the organelle periphery (Fig. 6c). Thus, the number of these compartments can be useful in taxonomic studies (Benchimol, unpublished). Díaz and De Souza (1997) were able to purify a hydrogenosomal fraction (Fig. 13a) and then another hydrogenosomal sub-fraction containing only the peripheral vesicles (Fig. 13c), showing that it is a distinct compartment. The isolated peripheral vesicles maintained their flattened morphology, suggesting that each individual vesicle has its own inherent structural framework (Fig. 13c). SDS-PAGE showed that proteins of 66, 45 and 32 kDa were localized in the peripheral vesicle (Díaz and De Souza, 1997). Western blot analysis revealed the presence of glycoproteins, a major one being 45 kDa in the peripheral vesicle of the hydrogenosome. When studied by freeze-fracture or freeze-etching, the peripheral vesicle shows a smooth surface (Fig. 6b) and presents pores with 20 nm in diameter with a density of 7/␮m2 when observed after freeze-etching (Fig. 6b). The hydrogenosome peripheral vesicle varies in size and electron density. Morphometric analysis showed that it represents 8.6% of the whole organelle in T. foetus. The peripheral vesicle is surrounded along its full extension by two closely apposed unit membranes. In some preparations the peripheral vesicle looks empty (Fig. 12), but it depends on the fixation procedure. Studies have demonstrated that the peripheral vesicle is a compartment which stores calcium (Fig. 6a, c) and other cations such as magnesium (Benchimol and De Souza, 1983; Chapman et al., 1985; Ribeiro et al., 2001). The peripheral compartment is occupied by electron-dense reaction products after various cytochemical detections such as for calcium (Fig. 6a, c), phosphatases such as glucose-6-phosphatase (Fig. 11a, d), IDP-diphosphatase (Fig. 11b), acid phosphatase (Fig. 11e) and also Ca2+ -ATPase (Fig. 11c) among others (Benchimol, 2008a; Queiroz et al., 1991).

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Fig. 12. Carbohydrates in hydrogenosomes (H) after the Thiéry technique. The hydrogenosomal membranes, including the membrane that surrounds the peripheral vesicle (PV) are positive for carbohydrates. The glycogen granules (GL) are well labeled. Bar = 100 nm (Benchimol, unpublished).

In addition, the hydrogenosome peripheral vesicle has been shown to be the site of zinc accumulation (Benchimol et al., 1993) which has been associated to male resistance to trichomoniasis and to calcium regulation (Benchimol and De Souza, 1983; De Souza and Benchimol, 1988; Chapman et al., 1985). A report demonstrated the elemental composition of the hydrogenosome using cryofixed samples in a comparative microanalytical study (energy dispersive X-ray analysis and electron spectroscopic imaging) of hydrogenosomes of T. foetus (Ribeiro et al., 2001). The study included the elemental composition and the mapping of calcium, phosphorus and oxygen. The presence of aluminum and cobalt ions in the hydrogenosomal vesicle was established in this article. 11. The matrix of the hydrogenosome The hydrogenosome matrix is homogeneous, presenting a granular appearance, which is different from the cytoplasmic matrix (Figs. 2, 3, 4, 6, 7, 8 and 9). It has been described as homogeneously granular, occasionally presenting a dense amorphous or crystalline core, also known as nucleoid (Fig. 19) (Honigberg and Brugerolle, 1990). Observations indicated that this core is not a usual structure, appearing however either when the protozoa are incubated in the presence of drugs or when good fixation is not achieved (Benchimol et al., 1996a). The electrondense core is frequently seen in not-well-preserved cells, a situation in which the hydrogenosome proteins could coagulate and precipitate, leading to the formation of the core (Fig. 19). In healthy or

well-preserved cells (Figs. 2, 3, 4, 6, 7 and 8) it is very unusual to find this electrondense amorphous core and thus it is no longer considered as a hydrogenosomal structure (Benchimol et al., 1996a). The hydrogenosome matrix exhibited positive labeling when calcium cytochemistry such as potassium pyroantimonate (Fig. 10a) (Benchimol et al., 1982b) or fixation in rich calcium fixative was performed, showing an intense positive reaction seen as calcium deposits (Fig. 10b). In addition, immunolabeling of malic enzyme (Fig. 10c) or cytochemical detection of basic proteins such as ammoniacal silver and PTA (phosphotungstic acid) (Fig. 10e, f) revealed a positive reaction (Benchimol et al., 1982a). Correlative microscopy was applied when T. vaginalis was treated with the drug BCECF, DAB and the cells were subsequently submitted to UV light illumination in the light microscope. Afterwards, the cells that presented positive reaction in the hydrogenosomes were selected and then they were prepared for TEM observations. With this method the hydrogenosomes exhibited a high electron density (Fig. 10d) (Scott et al., 1998). The granular structure of the hydrogenosome matrix may be clearly visualized in replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells (Figs. 6b and 14b). When the fracture plane exposed the internal portion of the hydrogenosome matrix a large number of particles were seen. Most of them had a diameter of 6 nm. Some, however, were larger, with a diameter of 20 nm. These particles were not randomly distributed. A certain orientation in their array was noted (Fig. 14b).

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12. Fungal hydrogenosomes The hydrogenosomes of the anaerobic fungus Neocallimastix frontalis are round or elongated structures, always enveloped by two distinct, but tightly apposed membranes (Figs. 1a, b, 4d, and 6f) (van der Giezen et al., 1997; Benchimol et al., 1997). Images of this organelle division (Benchimol et al., 1997) demonstrated a sharp similarity to the hydrogenosomes of trichomonad protozoa. These observations suggest that hydrogenosomes are homologous organelles in unrelated species weakening the hypothesis of a polyphyletic origin and reinforcing the hypothesis that fungal and trichomonad hydrogenosomes are derived from an ancestral endosymbiont (van der Giezen et al., 1997; Benchimol et al., 1997). 13. Hydrogenosome division

Fig. 13. Isolated hydrogenosomes. (a) Hydrogenosomes isolated by Percoll-sucrose density centrifugation. The hydrogenosomes are seen as spherical organelles presenting a peripheral vesicle. Bar = 400 nm. (b) T. foetus cryosection labeled with gold-conjugated WGA. The membrane lining the peripheral vesicle of the hydrogenosome (H), but not other portions of the organelle, is labeled with gold particles (arrows). (c) Hydrogenosome peripheral vesicles (arrowheads) isolated from a pure hydrogenosome fraction, show that they form a distinct sub-compartment from the hydrogenosomal matrix (M). (a) from Benchimol, unpublished). (b) from Benchimol et al., 1996a. (c) Bar = 100 nm (courtesy of Dr. José Andrés Morgado Díaz).

Hydrogenosomes, as almost all other organelles, grow by proliferation of preexisting organelles. Thus, each daughter cell will receive a complete set of organelles during cell division. Morphological evidence was presented showing that trichomonad hydrogenosomes, like mitochondria, may divide by three distinct processes: (1) segmentation (Fig. 7a–d, j), (2) partition (Fig. 7e–g) and heart-form (Fig. 7h, i). In the segmentation process, the hydrogenosome grows, becoming elongated with the appearance of a constriction in the central portion (Fig. 7a–d, j) (Benchimol et al., 1996b). Microfibrillar structures (Fig. 7b) appear to help the furrowing process, ending with a total fission of the organelle. In the partition process, rounded hydrogenosomes, in a bulky form, are further separated by a membranous internal septum (Fig. 7e–g). The division begins by an invagination of the inner hydrogenosome membrane, forming a transversal septum, separating the organelle matrix into two compartments (Benchimol et al., 1996b). A necklace of intramembranous particles delimiting the outer hydrogenosomal membrane in the region of organelle division was observed by freeze-etching (Fig. 7d, j). In the hydrogenosome heart-shaped process (Benchimol and Engelke, 2003), the organelle gradually presents a membrane invagination on one side, leading to the organelle division (Fig. 7h, i). In this case, the hydrogenosome grows anteriorly and the process of division starts at one of the organelle poles, which becomes larger

Fig. 14. Association of hydrogenosomes and other cell structures. (a) The plasma membrane of T. foetus was removed and the preparation was critical point dried and observed in a high voltage electron microscopy with 1000 kV of electron acceleration. Note that hydrogenosomes (H) are preferentially located along the axostyle (Ax) and costa (C). F, anterior flagella; RF, recurrent flagellum; N, nucleus; P, pelta. Bar = 1 ␮m (from Benchimol, 2008c). (b) Freeze-etching after quick-freezing by slam-freezing and rotatory shadowing shows a hydrogenosome (H) in close association with cytoskeletal structures, probably microtubules and with the endoplasmic reticulum (ER). Bar = 50 nm (from Benchimol, unpublished). (c) Thin section of hydrogenosomes showing their close proximity with microtubules (MT). N, nucleus. (from Benchimol, 2008c). Bar = 200 nm. (d) Immunocytochemistry using a monoclonal anti-tubulin antibody in a Unicryl thin-section of T. foetus. Notice that the microtubules (MT) are labeled with gold particles, thus evidencing the proximity of the hydrogenosomes and microtubules. Bar = 50 nm (Benchimol, unpublished).

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Fig. 15. Hydrogenosomes labeled with Mytotracker, are seen by fluorescence microscopy as brilliant spots. Bar = 10 ␮m (Benchimol, unpublished).

than the remaining organelle. Gradually, the membrane at this pole is seen invaginating. Although the organelle division begins with an inward furrowing, no septum is formed. The heart-shaped process is also distinct from segmentation, since in this process an elongation of the organelle occurs first, giving a sausage-shape and a progressive attenuation of its mid-region. In the heart-shaped process, the organelle neither forms a septum nor elongates and in this way is considered a new organellar division process. The most common form of division observed in T. foetus was the segmentation process, whereas the partition is the most unusual division process observed in this protist. However, in the hydrogenosomes of the fungus N. frontalis partition was the most common division process observed (Benchimol, unpublished). On the other hand, in Trichomonas augusta the heart shape was the most frequently observed division process (Benchimol, unpublished). It is important to point out that all three forms of division

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could be found during any phase of the cell cycle and that the segmentation and heart form could be observed in the same cell. In addition, the organellar division is not synchronized (Benchimol and Engelke, 2003). Interestingly, these three modes of division and timing were also described previously in mitochondria (Tandler and Hoppel, 1973). The mitochondrial division process has been compared with the division in bacteria, since the mitochondrial inner membrane are likely to be of bacterial descent. In bacteria an FtsZ ring adheres to the inside of the bacterial membrane and constricts it to mediate division. The use of FtsZ homologues (van der Bliek, 2000) and dynamin has been shown to participate in mitochondrial division (Koch et al., 2005). However, these proteins have not yet been studied in dividing hydrogenosomes. In hydrogenosomes, the process of division of the inner membrane is unknown, whereas the division of the outer membrane appears to be mediated by membranous profiles, probably of endoplasmic reticulum origin. These structures could aid the hydrogenosome division or participate in another function, such as providing membranes for hydrogenosome growth. Lahti and Johnson (1991) have shown that hydrogenosomal proteins in T. vaginalis are synthesized on free polyribosomes, released into the cytoplasm and subsequently translated into the organelle. Proteins that are sorted into organelles and that multiply by fission are invariably made on free ribosomes (Gasser et al., 1982).

14. Hydrogenosome behavior in the cell cycle Dividing hydrogenosomes can be found in all phases of the cell cycle, which is similar to mitochondria (Suzuki et al., 1994). They have even been observed during the mitotic process (Benchimol and Engelke, 2003). During the interphase, the hydrogenosomes in trichomonads are distributed mainly along the axostyle and costa (Figs. 2a and 8a) and at mitosis onset the hydrogenosomes migrate close to the nucleus (Fig. 8c). During karyokinesis, the trichomonas hydrogenosomes follow the axostyle (Fig. 8b–d) (Benchimol and Engelke, 2003; Ribeiro et al., 2000) which could aid the organellar distribution among the daughter-cells.

Fig. 16. Three-dimensional reconstruction. 3D computer model showing a view of T. foetus under mitosis obtained from serial thin-sections. The hydrogenosomes (H, blue) are located among the profiles of the endoplasmic reticulum (violet, ER), and lysosomes (L, yellow) (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article). The proximity and continuity of the hydrogenosomes and ER can be depicted. Ax, axostyle; M, plasma membrane; N, nucleus; S, spindle. Bar = 100 nm (taken from Ribeiro et al., 2002b).

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Fig. 17. Freeze-fracture images of hydrogenosomes from T. foetus. (a) This figure shows a fractured-cell showing a prominent Golgi (G) with several lamellae and fenestrae, as well as profiles of endoplasmic reticulum (ER) in close proximity (arrows) with hydrogenosomes (H). Bar = 200 nm. (b, c) Hydrogenosomes (H) in close proximity with the endoplasmic reticulum (ER) observed by freeze-fracture. Note in (b) the double membrane (arrow) presenting different distribution of intramembranous particles in both membranes; (c) a row of intramembranous particles (arrow) can be seen in the hydrogenosome membrane. Bar = 50 nm. (a, b) Taken from Benchimol et al., 1996a; (c) Taken from Benchimol, unpublished).

15. Proximity with other cellular structures Hydrogenosomes are close to the glycogen granules, microtubules of the axostyle and cytoskeleton and endoplasmic reticulum. Glycogen particles although distributed throughout the protozoan are concentrated in the region where hydrogenosomes are located (Figs. 2a and 12). Delicate bridges were observed connecting the outer hydrogenosomal membrane to the microtubules of the axostyle (Fig. 14a, d) or cytoskeleton (Fig. 14b, c). These bridges could explain the hydrogenosome aligment along the axostyle. 16. Hydrogenosomes and endoplasmic reticulum An intimate association of the hydrogenosome with the endoplasmic reticulum (ER) has been described (Benchimol et al., 1996a; Benchimol, 1999). Possibly ER provides new membranes for hydrogenosome growth, since this organelle enlarges before its division. The ER could participate by providing the membrane lipids. Three-dimensional reconstruction of a T. foetus in division (Fig. 16) and in interphase (Benchimol, 2008a) demonstrated a close association of the membranous profiles of the ER and hydrogenosomes, in a similar way to mitochondria and peroxisomes (Franke and Kartenbeck, 1971). The continuities or close associations between hydrogenosomes and the rough or smooth endoplasmic reticulum are often observed (Figs. 9a, d, 11, 12, 14b, 16 and 17a). Some hydrogenosomes present membranous cisternae projecting to the cytoplasm, conferring bizarre images of the organelle (Fig. 9b, c). In some cases, the hydrogenosomal outer membrane displayed attached granules, similar to ribosomes, when cells were treated with cytochalasin B (Benchimol, 2000). Data indicated that certain mitochondrial phospholipids were formed in ER and then transferred to the mitochondrion (Jungalwala and Dawson, 1970). Since the hydrogenosome has been considered a modified mitochondria (Embley et al., 1997) and presents several similarities to this organelle it has been suggested that at least the peripheral vesicle of the hydrogenosomes originated from the ER. The hydrogenosome presents some characteristics common to the ER (Benchimol, 2008a). A micro-analytical study using energy dispersive X-ray analysis, 3-D reconstruction and cytochemistry of the hydrogenosome peripheral vesicle was performed and compared with the analysis using the same technology of the endoplasmic reticulum and the nuclear envelope of T. foetus. The authors compared both structures using: (1) the detection of ER enzymes by cytochemistry, such as glucose-6-phosphatase,

IDPase, acid phosphatase and Ca2+ -ATPase; (2) elemental composition by X-Ray microanalysis and the mapping of calcium, phosphorus and oxygen in both ER and hydrogenosome peripheral vesicle; (3) freeze-fracture; (4) TEM of routine and cryofixed cells by high-pressure freezing and freeze-substitution; (5) 3-D reconstruction, (6) monoclonal antibody anti-trichomonads ER; and (7) other cytochemical techniques that detect ER, such as the ZIO and lectins (Benchimol, 2008a). They found a similar composition of the tested enzymes and other elements present in the ER when compared with the hydrogenosome peripheral vesicle. So, like mitochondria, hydrogenosomes present relationships with the ER, especially the peripheral vesicle. In trichomonads, calcium storage takes place in hydrogenosomes and endoplasmic reticulum and more recently in the Golgi complex (Almeida et al., 2003). The detection of a Ca2+ -ATPase in the hydrogenosome peripheral vesicle and also in the ER suggests an interesting similarity between these two structures. 17. Immunolabeling Hydrogenosomes can be labeled when specific antihydrogenosomal protein antibodies are used, such as an anti-malic enzyme (Fig. 10c) or other proteins found in the hydrogenosomal matrix. Interestingly, similar labeling is also observed when the antibody anti-AP65, an anti-adhesin protein, is used. Alderete et al. (2001) explained this result as an example of molecular mimicry and functional diversity. 18. Hydrogenosome death T. foetus under serum deprivation, drug treatment (hydroxyurea, zinc sulfate, griseofulvin, cytoskeleton affecting drugs, such as nocodazole, colchicine and cytochalasin) (Fig. 18) (Madeiro and Benchimol, 2004; Benchimol et al., 1996a; Ribeiro et al., 2002a) and also under normal conditions presents signs of cell death such as apoptosis (Mariante et al., 2003, 2006) and autophagy (Fig. 20) (Benchimol, 1999). An inactive hydrogenosome can be promptly recognized by a dense deposit in its matrix, known in early literature as nucleoid (Fig. 19). Autophagy occurs in cells where hydrogenosomes or other cell structures are old, or need to be removed. In autophagy, the first event observed is the cisternae of the rough endoplasmic reticulum surrounding and enclosing the altered hydrogenosome, forming an isolation membrane (Fig. 20a), and afterwards an autophagic vacuole (Fig. 20b). Lysosomes fuse with the autophagic vacuole forming a degradative structure, the autophagosome (Fig. 20c). Hydrogenosomes are thus completely degraded, as occurs in some drug treatments (Benchimol, 1999).

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Fig. 18. Gallery of abnormal hydrogenosomes. When cells are submitted to stress conditions, such as incubation with fibronectin (c, j) (Benchimol, 2001) or other drugs such as metronidazole (a), colchicines, cytochalasin (b) or hydroxyurea (Madeiro and Benchimol, 2004), the hydrogenosomes present very abnormal shapes and sizes even reaching 2 ␮m. Note that the hydrogenosomes (H) are not spherical as in the routine preparations. They are very large and present internal compartments and abnormal peripheral vesicles. Enlarged vesicles and internal membranes are also seen (asterisks). Bar = 150 nm.

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Fig. 19. Nucleoids. Thin sections of T. foetus showing hydrogenosomes (H) after treatment with different drugs. An electrondense material is seen aggregated in the hydrogenosome matrix, which was described as nucleoid (arrows) in the early literature. The presence of this dense deposit indicates that this hydrogenosome is metabolically inactive. Bar = 200 nm. (a) Taken from Benchimol, 2008c, (b, c) Taken from Benchimol, unpublished).

Fig. 20. Hydrogenosomes in process of autophagy. T. foetus presenting views of hydrogenosomes in the process of autophagy (a, b) and posterior digestion in a digestive vacuole (DV), as seen in (c). (a) Thin section of a hydrogenosome (H) enclosed by the rough endoplasmic reticulum (ER). Bar = 50 nm. (b) Observation of a double-layered autophagic vacuole (arrows) containing an intact hydrogenosome (H) after high-pressure freezing and freeze-substitution. Arrows point to the double membrane of the enclosing vacuole. Bar = 100 nm. (a, b) Taken from Benchimol, 1999). (c) Hydrogenosomes (H) in a digestive vacuole (DV) are seen inside a vacuolar lysosome-like structure, suggesting a digestive process of an altered hydrogenosome. Bar = 300 nm (from Benchimol, 2008b).

19. Evolutionary origin of the hydrogenosome

20. The cardiolipin story

The evolutionary origins of the hydrogenosome remain in debate because, unlike mitochondria or plastids, they lack an associated genome, with a possible exception of the findings of Akhmanova et al. (1998) in Nyctotherus ovalis. Different hypotheses have been proposed for the origin of the trichomonad hydrogenosome: an independent endosymbiosis of an anaerobic eubacterium with a eukaryotic host or the conversion of an established mitochondrion adapted to an anaerobic lifestyle (Müller, 1993). Nowadays, hydrogenosomes are considered mitochondrionrelated organelles. One hypothesis is that hydrogenosomes could be derived from primitive endosymbiotic association, related to the origin of mitochondria. Thus, it has been proposed that hydrogenosomes could be biochemically modified mitochondria (Embley et al., 1997, 2003).

Cardiolipin (CLP) is almost exclusively present in membranes designed to generate an electrochemical potential gradient for ATP synthesis, including the mitochondrial inner membrane and bacterial plasma membrane (Koshkin and Greenberg, 2002). In eukaryotes it is the only lipid that is synthesized in the mitochondrion where it remains throughout the life of the cell (Haines and Dencher, 2002). Thus, the presence of CLP in hydrogenosomes is an additional argument for the hypothesis of a closer proximity of this organelle to mitochondria and its symbiotic origin. A controversy has been open on this topic since a first study by Cerkasovová et al., who demonstrated, in 1976, the presence of cardiolipin in hydrogenosomes of T. vaginalis and T. foetus. However, this evidence was later denied in 1982 by Paltauf and Meingassner who claimed for the absence of this molecule in hydrogenosomes. Recently,

Fig. 21. Localization of cardiolipin in hydrogenosomes by fluorescence using nonyl-acridine orange, in T. foetus. Note a positive staining in the hydrogenosomes. (a) DIC; (b) fluorescence; (c) overlay. Bar = 1 ␮m (De Andrade Rosa and Benchimol, unpublished).

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167

Acknowledgements This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), PRONEX (Programa de Núcleo de Excelência), FAPERJ (Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro), and AUSU (Associac¸ão Universitária Santa Úrsula). References

Fig. 22. TLC for cardiolipin detection. Thin-layer chromatograph of lipid extracts. Lanes: H, hydrogenosomal fraction of T. foetus; Tf, cell homogenate of T. foetus; Std, commercial cardiolipin standard; CLP (De Andrade Rosa and Benchimol, unpublished).

De Andrade Rosa et al. (2006) using more sensitive approaches such as HPLC and MALDI-TOF mass spectrometry clearly demonstrated that CLP is present in the hydrogenosomes from T. foetus (Figs. 21 and 22). This group also used 10-N-nonyl-acridine orange, a fluorescent chemical which interacts with cardiolipin (Petit et al., 1992) (Fig. 21) (De Andrade Rosa and Benchimol, unpublished. All these findings straighten the proposal for the endosymbiotic origin of hydrogenosomes and their similarity to mitochondria. 21. The proteome of hydrogenosomes The research on hydrogenosome proteomics is very important to our understanding of hydrogenosome functions and evolution. Recently, some groups were investigating the hydrogenosome proteome. For proteome analysis the complete genome sequence of the cells must be known and they must be grown axenically to cell densities that allow cell fractionation and hydrogenosome purification in order to obtain enough material for proteomic analyses in 2D electrophoresis and mass spectrometry. Thus, the only cell harboring hydrogenosomes that covers these prerequisites is T. vaginalis, which recently had its genome sequence published (Carlton et al., 2007). Partial proteome analysis identified 61 proteins, 55 of which had known functions (Henze, 2008). Purified hydrogenosomes from T. vaginalis and T. foetus have their proteins analyzed by 2D electrophoresis and mass spectrometry (Henze, 2008). This author estimated that the proteome of T. vaginalis consists of at least 200 proteins, much less than the mitochondria. Studies of the trichomonad proteomes are in course in several laboratories and there will be news about this interesting organelle shortly.

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