Micron 73 (2015) 28–35
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Micron journal homepage: www.elsevier.com/locate/micron
Changes in the structural organization of the cytoskeleton of Tritrichomonas foetus during trophozoite-pseudocyst transformation Ivone de Andrade Rosa a,b , Wanderley de Souza a,b,c , Marlene Benchimol a,b,c,d,∗ a
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Rio de Janeiro, Brazil Instituto Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Brazil d UNIGRANRIO – Universidade do Grande Rio, Caxias, Rio de Janeiro, Brazil b c
a r t i c l e
i n f o
Article history: Received 30 October 2014 Received in revised form 2 March 2015 Accepted 18 March 2015 Available online 26 March 2015 Keywords: Endoflagellar form Axostyle Costa High resolution scanning electron microscopy Protozoan Cytoskeleton
a b s t r a c t Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. It grows in axenic media as a trophozoite with a pear-shaped body, three anterior flagella, and one recurrent flagellum. However, under some well-controlled experimental conditions in vitro, as well as in vivo in infected bulls, the parasite acquires a spherical or elliptical shape, and the flagella are internalized but the cells do not display a cyst wall. This form, known as the endoflagellar or pseudocystic form, is viable, and can be transformed back to trophozoites with pear-shaped body. We used confocal laser scanning microscopy, and high resolution scanning electron microscopy to examine the changes that take place in the protozoan cytoskeleton during trophozoite-pseudocyst transformation. Results confirmed previous studies and added new structural information to the organization of cytoskeletal structures during the transformation process. We observed that changes take place in the pseudocysts’ axostyle and costa, which acquired a curved shape. In addition, the costa of multinucleated/polymastigont pseudocysts took variable conformations while curved. The costa accessory structure, as well as a network of filaments connecting this structure to the region where the recurrent flagellum associates to the protozoan body, was not seen in pseudocysts. In addition, the axostyle was fragmented during trophozoite-pseudocyst transformation. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The flagellated protist Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle (Rae and Crew, 2006). It usually presents a pear-like shape, known as a trophozoite, characterized by the presence of three anterior flagella and one recurrent flagellum. Under some well controlled experimental conditions, including changes in temperature (Granger et al., 2000; Pereira-Neves et al., 2012) or when the cytoskeleton is modified due to action of drugs such as colchicine (Madeiro da Costa and Benchimol, 2004), the trophozoite acquires a rounded or elliptical shape and internalizes the
Abbreviations: EFF, endoflagellar form; SEM, scanning electron microscopy; TEM, transmission electron microscopy; LSCM, laser scanning confocal microscopy; PBS, phosphate-buffered saline; BSA/PBS, bovine serum albumin/PBS. ∗ Corresponding author at: Laboratório de Ultraestrutura Celular, Universidade Federal do Rio de Janeiro-CCS-Bloco G, Ilha do Fundão, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 22370440; fax: +55 21 99853 2754. E-mail address: [email protected] (M. Benchimol). http://dx.doi.org/10.1016/j.micron.2015.03.008 0968-4328/© 2015 Elsevier Ltd. All rights reserved.
flagella, transforming into an endoflagellar form (EFF), also known as a pseudocyst because a cyst wall is not formed (Pereira-Neves et al., 2003). Initially, pseudocysts were considered to be a degenerative stage of the protozoan (Powell, 1936; Samuels, 1959). However, several studies have shown clearly that they are living cells able to undertake nuclear division to form multinucleated cells, able to interact with and provoke damage to host cells (Mariante et al., 2004; Pereira and Almeida, 1940; Pereira-Neves et al., 2003; Pereira-Neves and Benchimol, 2009). In addition, once stimuli to pseudocyst induction are removed they transform again into piriform trophozoites (Pereira-Neves and Benchimol, 2009). Clear evidence that pseudocysts form in vivo is their presence in naturally infected bulls (Pereira-Neves et al., 2011). During pseudocyst formation, several morphological changes occur in the cytoskeleton. Previous studies used light microscopy and conventional scanning (SEM) and transmission electron microscopy (TEM) to show that axial structures such as the axostyle and costa, assumed a curved shape (Mariante et al., 2004; PereiraNeves et al., 2003, 2011; Pereira-Neves and Benchimol, 2009). In this study we further characterize changes to the cytoskeleton using the laser scanning confocal microscopy (LSCM), and high
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Fig. 1. (a) Scanning electron microscopy of the parasites obtained after the pseudocyst induction, where the trophozoites assumed the spherical form and internalized the flagella. (b) Quantitative analyses of the parasites that were transformed into pseudocyst at the end the induction process.
resolution scanning electron microscopy, thus extending to the pseudocyst form previous studies carried out with trophozoites (de Andrade Rosa et al., 2013). 2. Material and methods 2.1. Cell culture The K strain of T. foetus was isolated by Dr. H. Guida (Embrapa, Rio de Janeiro, Brazil) from the urogenital tract of a bull, and it has been maintained in culture since the 1970s. Cells were cultivated in TYM Diamond’s medium (Diamond, 1957) supplemented with 10% fetal calf serum and grown for 24 h at 36.5 ◦ C. 2.2. Pseudocyst induction
2.5. Scanning electron microscopy Cytoskeletons were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), washed in PBS (pH 7.2), and post-fixed for 30 min in 1% OsO4 in 0.1 M phosphate buffer (pH 7.2), and dehydrated using an ascending series of ethanol washes, ending with 100% ethanol. They were then critical point-dried with CO2 using a Bal-Tec CPD 030 critical point dryer, and sputter-coated with a thin layer (1–2 nm) of chromium or carbon using a Leica EM SCD 500 or 005 sputter coater. The FEI Nova NanoLab 600 and Magellan field emission SEMs were used at accelerating voltages of 2 and 1 kV, respectively. For better identification of some structures, colors were added to some images using Adobe Photoshop color balance.
Cultures of T. foetus grown for 30 h at 37 ◦ C in TYM medium were cooled to 4 ◦ C for up to 4 h, without changing the medium. The pseudocyst formation was followed by phase contrast microscopy. To quantify pseudocysts, at least 100 cells were examined by scanning electron microscopy in three independent experiments in triplicate. The results were expressed in percentage.
2.6. Morphometric analyses
2.3. Cytoskeleton preparation
2.7. Statistical analyses
Using 10 L 0.01% poly-l-lysine (mol wt 150,000–300,000, Sigma, USA) on a glass coverslip and allowed to stand for at least 15 min, then rinsed in distilled water after which a 20 L sample of living cells (107 cells/mL) was added. After 15 min, samples were rinsed in phosphate-buffered saline (PBS), pH 7.2, to remove nonadherent cells. Cytoskeletons were prepared by treating with 2% Triton X-100 and 2% NP-40 in a modified buffer previously designed for cytoskeleton preservation (IC buffer: 10 mM Tris Base, 2 mM EDTA, 2 mM DTT, 2 mM MgSO4 , 150 mM KCl, 30% glycerol, pH 7.4) for 30 min or 1 h (Palm et al., 2005).
Statistical comparisons were performed using the paired t test, and P values less than 0.0001 were considered to be statistically significant.
2.4. Immunofluorescence microscopy The cytoskeleton of both piriform trophozoites and pseudocysts were fixed for 1 h with 4% freshly prepared formaldehyde in phosphate buffer (0.1 M, pH 8.0). Samples were allowed to adhere to glass coverslips previously coated with poly-l-lysine, and cells were permeabilized with 0.1% Triton X-100. The free aldehyde groups were quenched using 50 mM NH4 Cl and 3% bovine serum albumin/PBS (BSA/PBS). Samples were then labeled with monoclonal antibody anti--tubulin (Amersham Bioscience, USA), and diluted 1:10 in 1% BSA/PBS. The coverslips were incubated for 1 h with an Alexa Fluor 488-conjugated anti-mouse antibody diluted 1:100 in BSA/PBS. Finally, the coverslips were washed and examined using a Leica TCS SP5 confocal microscope equipped with an Argon 488 laser emission bandwidth filter.
The areas and lengths of thirty axostyles of both forms were measured using iTEM software, as well the lengths of thirty costae of both forms of T. foetus. All results were analyzed using the GraphPad Prism 4.
3. Results 3.1. Pseudocyst induction In previous studies it has been established that incubation of trophozoites at low temperature is a highly efficient and reproducible method to induce the transformation of the pear-shaped parasites into pseudocysts. We used this well-established methodology in the present study and we obtained around 80% of pseudocysts in the cultures (Fig. 1). 3.2. Localization of microtubules Using a monoclonal antibody that recognizes -tubulin, we observed that cytoskeletal changes occur during the transformation from piriform to pseudocyst. In piriform parasites, the three anterior flagella and recurrent flagellum are externalized. A typical axial axostyle was also observed by immunolabeling with the -tubulin-recognizing antibody (Fig. 2) confirming previous observations (Lopes et al., 2001). Intense fluorescence was seen where the microtubule sheet turns upon itself to form the axostylar trunk (Fig. 2). Using LSCM it was possible to observe labeling of
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Fig. 2. Detection of microtubules in the piriform trophozoites of T. foetus by immunofluorescence microscopy. Note that the axostyle (Ax) appears as an axial structure, and the fluorescence intensity is higher in the axostylar trunk. The three anterior flagella (AF) and one recurrent flagellum (RF) are externalized and labeled.
Fig. 3. Detection of microtubules in T. foetus pseudocysts by immunofluorescence microscopy. The cells present a curved axostyle (Ax) and the flagella (F) are internalized (b, c; e, f and h, i). Some cells present a small region of the curved axostyle, where labeling was not detected (arrow; e, f), and duplicated axostyles indicate that the pseudocyst is in the process of division (arrowhead; h, i).
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Fig. 4. Localization of microtubules in trophozoites (a) and in pseudocysts (b, c) of T. foetus after extraction of the plasma membrane by immunofluorescence microscopy. (a) An intense labeling is observed in the anterior flagella (AF), pelta (arrowhead), recurrent flagellum (RF) and axostylar trunk (Ax). (b) One population of pseudocysts presents a curved axostyle with intense labeling at the ends, whereas other cells exhibit labeling only in the anterior region of the axostyle (c).
the pelta, the internalized flagella, and a curved axostyle in pseudocysts (Fig. 3). Other differences observed in pseudocysts were: (a) the presence of intense fluorescence in the anterior region of the axostyle (Fig. 3b); (b) a short region where the axostyle appeared curved, but not labeled (Fig. 3e), and (c) pseudocysts with duplicated axostyles, indicating that a mitotic process took place (Fig. 3c). When the plasma membrane of the protozoan was removed by detergent extraction, it was then possible to identify the labeled pelta localized in the anterior region of the cell (Fig. 4a–c). One population of pseudocysts presented a curved axostyle with intense labeling at the ends (Fig. 4d–f). However, other cells presented labeling only in the anterior region (Fig. 4g–i), suggesting that the axostyle may undergo antigenic modification during protozoan transformation. 3.3. Ultrastructural changes in pseudocysts The cytoskeleton of the trophozoites was visualized using high resolution scanning electron microscopy of detergent-extracted cells under conditions where the shape of the cells was not changed during membrane extraction. Our observations confirm and extend
previous studies (Benchimol, 2005; de Andrade Rosa et al., 2013). As observed by LSCM, that flagella were externalized and the axial axostyle presented a region where the microtubules turned upon themselves. In addition, the axostyle presented a concave region, where a network of filaments surrounded the nucleus, visible by high resolution SEM (Fig. 5). It is important to point out that the microtubules of the pelta and the axostyle could easily be distinguished (Fig. 5). The costa was also observed as an axial structure as was the recently described accessory filament along the costa (de Andrade Rosa et al., 2013). A network of 12.5 ± 1.2 nm thick filaments were seen connecting the periphery of the costa accessory filament to the region where the recurrent flagellum attaches to the protozoan cell body, forming the undulating membrane (Fig. 5). High resolution SEM of the pseudocyst cytoskeleton showed that the nucleus was positioned in the center of the axostyle, the pelta involved the periflagellar canal, and the costa was curved, allowing this axial structure follow the shape of the rounded cell (Fig. 6). Interestingly, we observed that the axostyle was smaller and had an irregular edge in the multinucleated pseudocyst (Figs. 7 and 8), suggesting a fragmentation of this structure. This observation was supported by quantitative and morphometric
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Fig. 6. Cytoskeleton of the pseudocyst as seen by high resolution SEM. Note that the axostyle (Ax) is seen around the nucleus (N) as well the flagella (F) and the costa (C) displays a curved appearance.
Fig. 5. T. foetus trophozoites visualized by high resolution SEM. An overview of the cytoskeleton of the parasite which was artificially colored to better show the different structures. Notice the pelta (P), the axial axostyle (Ax) with its axostylar trunk showing helicoidal arranged microtubules, and a region of the nucleus supported by a network of filaments (Nt). The costa (C) is seen with the accessory filament (FC), and fibrils (F) are found connecting the costa to the recurrent flagellum (RF). One parabasal filament (PF) is also observed as well the three anterior flagella (AF).
analyses, which showed that the 63.3% ± 4.7 of axostyles (Fig. 9a) of multinucleated pseudocysts presented a length (3.1 ± 0.27 m), that were one-fourth of those seen in the piriform trophozoites (13.9 ± 1.3 m). Similar results were obtained when the area occupied by the axostyle was estimated; the piriform cells displayed axostyles occupying an area of 14.830 ± 1.02 m2 whereas in the pseudocysts was 4.963 ± 0.712 m2 (Fig. 10a, b). The costa also underwent morphological modifications in the multinucleated pseudocysts. Using high resolution SEM, it was possible to observe that this structure displayed a hooked or U shape (Fig. 8) and was localized around the axostyle (Fig. 8a–d). The curved costa (seen in 71% ± 2.65 of the cells and shown in Fig. 9) could present several shapes, but its size did not vary significantly (Fig. 10c). Unexpectedly, the accessory filament, as well the filament network connecting the costa to the recurrent flagellum region, was no longer observed in the pseudocyst cytoskeleton. 4. Discussion The role of the pseudocyst in cell cycle is still discussed, however this form has been observed in natural conditions in different species of the trichomonads. It has been shown that pseudocysts of Tritrichomonas muris differentiate during the passage through the colon of the mice, are eliminated in the feces are considered to be infective agents of murine trichomonosis (Lipman et al.,
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Fig. 7. High resolution SEM of the cytoskeleton of a multinucleated pseudocyst of T. foetus. (a) Fragmented axostyles (Ax) are seen and the costae (C) are curved. (b, c) Higher magnification of the curved costa.
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1999; Stachan et al., 1984). The infectivity of the pseudocysts has been observed in Trichomonas vaginalis, where a significant amount of pseudocysts were detected in discharge from urethra (Małyszko and Januszko, 1991). In addition, pseudocysts inoculated intra-vaginally demonstrated ability to induce trichomonosis in mice (Hussein and Atwa, 2008). Recently, the possible role of the pseudocysts in exacerbating cervical cancer was suggested in a study performed with parasites isolated from cervical patients with symptomatic trichomoniasis, including patients with cervical neoplasia (Afzan and Suresh, 2012). In T. foetus, pseudocysts were the predominant form observed in preputial samples (Pereira-Neves et al., 2011) and it has been shown that they can be transformed back to trophozoites and exert a cytotoxic effect, and may represent a resistant developmental stage formed under unfavorable conditions (Pereira-Neves et al., 2012). As a result, attention to this stage has increased along with the need to obtain further information on its behavior, ultrastructure, and role during the life cycle of T. foetus. In addition, further studies in experimentally infected animals are necessary to establish a relationship between its presence and the trichomonosis disease. Previous studies have demonstrated that the transformation of a polarized trophozoite into the rounded pseudocyst form requires cytoskeleton modifications such as flagella internalization (Granger et al., 2000) and changes in the shape of the axostyle and costa (PereiraNeves et al., 2003). However, studies performed thus far have not revealed the three dimensional organization of the pseudocyst cytoskeleton. Using a new generation of scanning electron microscopes that achieve a resolution of about 0.8 nm, details of cell structures and their relationships to each other have been obtained. Using this approach, we recently provided detailed information about the structural organization of the piriform parasites of the T. foetus where new structures were described (de Andrade Rosa et al., 2013). Using the same approach, we confirmed in the present report that in contrast to the piriform cells, which display an axial axostyle, the pseudocyst presents a curved axostyle as previously suggested (Boggild et al., 2002; Mariante et al., 2004; Pereira-Neves et al., 2003), or even a fragmented axostyle, as observed in multinucleated pseudocysts. This information is in close agreement with our observation by LSCM of cells labeled for -tubulin, where pseudocyst cytoskeletons display an intense labeling only in the anterior region. A similar result was reported using TEM of the intestinal trichomonad cysts of Trichomitus batrachorum, where the axostyles missed their extremities (Brugerolle, 1973; Samuels, 1957). Together, the available data strongly suggest that during trophozoite-pseudocyst transformation, changes take place in the axostyle that lead to its fragmentation, explaining the labeling pattern observed by LSCM as well as the images obtained by high resolution SEM. It is possible, as previously suggested, that changes take place on the exposition of epitopes of the axostyle tubulin, thus explaining the variation in the fluorescence labeling pattern observed in pseudocysts during mitosis (Pereira-Neves et al., 2003). In addition, a distinct tubulin-labeling pattern was found when piriform and pseudocyst form of T. muris was compared (Boggild et al., 2002). The authors suggested that the difference could be due to post-translational modifications on tubulin (Boggild et al., 2002). Moreover, a proteomic study performed with three different forms of the T. vaginalis (piriform, pseudocyst and ameboid cells) demonstrated differences in protein expression, including cytoskeleton proteins (Yeh et al., 2013). The serine/threonine protein phosphatases 2A (PP2A) are highly conserved in all eukaryotes and among other functions is associated with remarkable changes in cytoskeleton remodeling (Sontag and Sontag, 2006). In addition, other findings suggested that PP2As act as native regulators of low-temperature responses of the cytoskeleton and cold stress responses in other organisms (País et al., 2009), thus it would be worth the study of the role of PP2A in response
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Fig. 8. Organization of the cytoskeleton of the pseudocyst as visualized by high resolution SEM. (a–d) Note the fragmented axostyles (Ax) and the presence of a curved costa (C) around one of the axostyles (a, b). A U-shaped costa (c, d) is observed.
Fig. 9. Quantitative analyses of the cytoskeleton alterations in parasite transformed into pseudocyst. (a) Cytoskeletons with short axostyles (**P < 0.005) and (b) with curved costa (***P < 0.0005).
to low temperature stress and in pseudocyst formation in trichomonads. Our observations using high resolution SEM clearly show fragmentation of the axostyle in pseudocysts. Such fragmentation was not observed in previous analysis of thin sections by TEM where only a thin slice of the cell was examined. In contrast, high resolution SEM of the cytoskeleton preparations allows a 3D view of the entire cell. The ventral disk of trophozoites of G. intestinalis, a large microtubule-containing structure, also breaks during the
encystation process and the fragments are stored in the cyst and reassembled during the excystation process when new trophozoites are formed (Sheffield and Bjorvat, 1977). Further studies are necessary to clarify the mechanisms involved on the process of fragmentation of these complex cytoskeleton structures. We also observed that the costa of the pseudocyst displayed morphological alterations during trophozoite-pseudocyst transformation. The most evident one was the conversion from a straight to a variable shape. Furthermore, we verified that the accessory
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Fig. 10. Morphometric analyses of the length (a) and area (b) of the axostyle and length of the costa (c) of piriform and pseudocysts of T. foetus. ***P < 0.001.
filament and the fibrils network previously described in piriform parasites were not visualized in pseudocysts. Because these structures seem to be involved in the establishment of connections of the costa with the recurrent flagellum attachment area, their disappearance during the transformation process was expected, once the recurrent flagellum is internalized, as previously shown (PereiraNeves et al., 2003). In conclusion, our present study using confocal laser scanning microscopy and high resolution SEM confirmed previous observations made with fluorescence microscopy and add new information for a better understanding of changes that take place in the cytoskeleton of T. foetus during the transformation of trophozoites into pseudocysts. Examples include (a) axostyle fragmentation, (b) curving of the costa, especially in multinucleated cells where the costa acquires variable conformations, (c) loss of the filamentous network which connects the costa to recurrent flagellum, and (d) loss of the accessory filament observed in the costa of trophozoites. Acknowledgements This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundac¸ão Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), Programa de Núcleos de Excelência (PRONEX), Instituto Nacional de Metrologia, Qualidade e Tecnologia (Inmetro) and Associac¸ão Universitária Santa Úrsula (AUSU). The authors thank Luis Sérgio de Araújo Cordeiro Júnior and Ricardo Chaves Vilela for technical support. References Afzan, M.Y., Suresh, K., 2012. Pseudocyst forms of Trichomonas vaginalis from cervical neoplasia. Parasitol. Res. 111, 371–381. Benchimol, M., 2005. New ultrastructural observations on the skeletal matrix of Tritrichomonas foetus. Parasitol. Res. 97, 408–416. Boggild, A.K., Sundermann, C.A., Estridge, B.H., 2002. Localization of posttranslationally modified alpha-tubulin and pseudocyst formation in tritrichomonads. Parasitol. Res. 88, 468–474. Brugerolle, G., 1973. Sur l’existence de vrais kystes ches les Trichomonadines intestinalis. Ultrastructure des kystes de Trichomitus batrachorum Petry 1852, Trichomitus sanguisugae Alexeieff 1911, et Monocercomonas tipulae Mackinnon 1910. C. R. Acad. Sci. Paris Ser. D 277, 2193–2196. de Andrade Rosa, I., de Souza, W., Benchimol, M., 2013. High-resolution scanning electron microscopy of the cytoskeleton of Tritrichomonas foetus. J. Struct. Biol. 183, 412–418.
Diamond, L.S., 1957. The establishment of various trichomonads of animals and man in axenic cultures. J. Parasitol. 43, 488–490. Granger, B.L., Warwood, S.J., Benchimol, M., de Souza, W., 2000. Transient invagination of flagella by Tritrichomonas foetus. Parasitol. Res. 86, 699–709. Hussein, E.M., Atwa, M.M., 2008. Infectivity of Trichomonas vaginalis pseudocysts inoculated intra-vaginally in mice. J. Egypt. Soc. Parasitol. 38, 749–762. Lipman, N.S., Lampen, N., Nguyen, H.T., 1999. Identification of pseudocysts of Tritrichomonas muris in Armenian hamsters and their transmission to mice. Lab. Anim. Sci. 49, 313–315. Lopes, L.C., Ribeiro, K.C., Benchimol, M., 2001. Immunolocalization of tubulin isoforms and post-translational modifications in the protists Tritrichomonas foetus and Trichomonas vaginalis. Histochem. Cell Biol. 116, 17–29. Madeiro da Costa, R.F., Benchimol, M., 2004. The effect of drugs on cell structure of Tritrichomonas foetus. Parasitol. Res. 92, 159–170. Małyszko, E., Januszko, T., 1991. Detection of Trichomonas vaginalis in men. Pol. Tyg. Lek. 46, 997–999. Mariante, R.M., Lopes, L.C., Benchimol, M., 2004. Tritrichomonas foetus pseudocysts adhere to vaginal epithelial cells in a contact-dependent manner. Parasitol. Res. 92, 303–312. ˜ País, S.M., Téllez-Inón, M.T., Capiati, D.A., 2009. Serine/threonine protein phosphatases type 2A and their roles in stress signaling. Plant Signal. Behav. 4, 1013–1015. Palm, D., Weiland, M., McArthur, A.G., Winiecka-Krusnell, J., Cipriano, M.J., Birkeland, S.R., Pacocha, S.E., Davids, B., Gillin, F., Linder, E., Svärd, S., 2005. Developmental changes in the adhesive disk during Giardia differentiation. Mol. Biochem. Parasitol. 141, 199–207. Pereira, C., Almeida, W.F., 1940. Sobre a verdadeira natureza das “formas ameboides”, dos pretensos “cistos” e formas “degenerativas” no gênero “Trichomonas Donne, 1836”. Arch. Inst. Biol. 11, 347–366. Pereira-Neves, A., Ribeiro, K.C., Benchimol, M., 2003. Pseudocysts in trichomonads – new insights. Protist 154, 313–329. Pereira-Neves, A., Benchimol, M., 2009. Tritrichomonas foetus budding from multinucleated pseudocysts. Protist 160, 536–551. Pereira-Neves, A., Campero, C.M., Martínez, A., Benchimol, M., 2011. Identification of Tritrichomonas foetus pseudocysts in fresh preputial secretion sample from bulls. Vet. Parasitol. 175, 1–8. Pereira-Neves, A., Nascimento, L.F., Benchimol, M., 2012. Cytotoxic effects exerted by Tritrichomonas foetus pseudocysts. Protist 163, 529–543. Powell, W.N., 1936. Trichomonas vaginalis DONNÉ, 1836: its morphologic characteristic mitosis and specific identity. Am. J. Hyg. 24, 145–169. Rae, D.O., Crew, J.E., 2006. Tritrichomonas foetus. Vet. Clin. North Am. Food Anim. Pract. 22, 595–611. Samuels, R., 1957. Studies of Tritrichomonas batachorum (perty) 2. Normal mitosis and morphogenesis. Trans. Am. Microsc. Soc. 76, 295–307. Samuels, R., 1959. Studies of Tritrichomonas batachorum 3: abnormal mitosis and morphogenesis. Trans. Am. Microsc. Soc. 78, 49–65. Sheffield, H.G., Bjorvat, B., 1977. Ultrastructure of the cyst of Giardia lamblia. Am. J. Trop. Med. Hyg. 26, 23–30. Sontag, J.M., Sontag, E., 2006. Regulation of cell adhesion by PP2A and SV40 small tumor antigen: an important link to cell transformation. Cell Mol. Life Sci. 63, 2979–2991. Stachan, R., Nicol, C., Kunstyr, I., 1984. Heterogeneity of Tritrichomonas muris pseudocysts. Protistologica, 157–163. Yeh, Y.M., Huang, K.Y., Richie, Gan, R.C., Huang, H.D., Wang, T.C., Tang, P., 2013. Phosphoproteome profiling of the sexually transmitted pathogen Trichomonas vaginalis. J. Microbiol. Immunol. Infect. 46, 366–373.
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Micron journal homepage: www.elsevier.com/locate/micron
Changes in the structural organization of the cytoskeleton of Tritrichomonas foetus during trophozoite-pseudocyst transformation Ivone de Andrade Rosa a,b , Wanderley de Souza a,b,c , Marlene Benchimol a,b,c,d,∗ a
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Rio de Janeiro, Brazil Instituto Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Brazil d UNIGRANRIO – Universidade do Grande Rio, Caxias, Rio de Janeiro, Brazil b c
a r t i c l e
i n f o
Article history: Received 30 October 2014 Received in revised form 2 March 2015 Accepted 18 March 2015 Available online 26 March 2015 Keywords: Endoflagellar form Axostyle Costa High resolution scanning electron microscopy Protozoan Cytoskeleton
a b s t r a c t Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. It grows in axenic media as a trophozoite with a pear-shaped body, three anterior flagella, and one recurrent flagellum. However, under some well-controlled experimental conditions in vitro, as well as in vivo in infected bulls, the parasite acquires a spherical or elliptical shape, and the flagella are internalized but the cells do not display a cyst wall. This form, known as the endoflagellar or pseudocystic form, is viable, and can be transformed back to trophozoites with pear-shaped body. We used confocal laser scanning microscopy, and high resolution scanning electron microscopy to examine the changes that take place in the protozoan cytoskeleton during trophozoite-pseudocyst transformation. Results confirmed previous studies and added new structural information to the organization of cytoskeletal structures during the transformation process. We observed that changes take place in the pseudocysts’ axostyle and costa, which acquired a curved shape. In addition, the costa of multinucleated/polymastigont pseudocysts took variable conformations while curved. The costa accessory structure, as well as a network of filaments connecting this structure to the region where the recurrent flagellum associates to the protozoan body, was not seen in pseudocysts. In addition, the axostyle was fragmented during trophozoite-pseudocyst transformation. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The flagellated protist Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle (Rae and Crew, 2006). It usually presents a pear-like shape, known as a trophozoite, characterized by the presence of three anterior flagella and one recurrent flagellum. Under some well controlled experimental conditions, including changes in temperature (Granger et al., 2000; Pereira-Neves et al., 2012) or when the cytoskeleton is modified due to action of drugs such as colchicine (Madeiro da Costa and Benchimol, 2004), the trophozoite acquires a rounded or elliptical shape and internalizes the
Abbreviations: EFF, endoflagellar form; SEM, scanning electron microscopy; TEM, transmission electron microscopy; LSCM, laser scanning confocal microscopy; PBS, phosphate-buffered saline; BSA/PBS, bovine serum albumin/PBS. ∗ Corresponding author at: Laboratório de Ultraestrutura Celular, Universidade Federal do Rio de Janeiro-CCS-Bloco G, Ilha do Fundão, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 22370440; fax: +55 21 99853 2754. E-mail address: [email protected] (M. Benchimol). http://dx.doi.org/10.1016/j.micron.2015.03.008 0968-4328/© 2015 Elsevier Ltd. All rights reserved.
flagella, transforming into an endoflagellar form (EFF), also known as a pseudocyst because a cyst wall is not formed (Pereira-Neves et al., 2003). Initially, pseudocysts were considered to be a degenerative stage of the protozoan (Powell, 1936; Samuels, 1959). However, several studies have shown clearly that they are living cells able to undertake nuclear division to form multinucleated cells, able to interact with and provoke damage to host cells (Mariante et al., 2004; Pereira and Almeida, 1940; Pereira-Neves et al., 2003; Pereira-Neves and Benchimol, 2009). In addition, once stimuli to pseudocyst induction are removed they transform again into piriform trophozoites (Pereira-Neves and Benchimol, 2009). Clear evidence that pseudocysts form in vivo is their presence in naturally infected bulls (Pereira-Neves et al., 2011). During pseudocyst formation, several morphological changes occur in the cytoskeleton. Previous studies used light microscopy and conventional scanning (SEM) and transmission electron microscopy (TEM) to show that axial structures such as the axostyle and costa, assumed a curved shape (Mariante et al., 2004; PereiraNeves et al., 2003, 2011; Pereira-Neves and Benchimol, 2009). In this study we further characterize changes to the cytoskeleton using the laser scanning confocal microscopy (LSCM), and high
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Fig. 1. (a) Scanning electron microscopy of the parasites obtained after the pseudocyst induction, where the trophozoites assumed the spherical form and internalized the flagella. (b) Quantitative analyses of the parasites that were transformed into pseudocyst at the end the induction process.
resolution scanning electron microscopy, thus extending to the pseudocyst form previous studies carried out with trophozoites (de Andrade Rosa et al., 2013). 2. Material and methods 2.1. Cell culture The K strain of T. foetus was isolated by Dr. H. Guida (Embrapa, Rio de Janeiro, Brazil) from the urogenital tract of a bull, and it has been maintained in culture since the 1970s. Cells were cultivated in TYM Diamond’s medium (Diamond, 1957) supplemented with 10% fetal calf serum and grown for 24 h at 36.5 ◦ C. 2.2. Pseudocyst induction
2.5. Scanning electron microscopy Cytoskeletons were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), washed in PBS (pH 7.2), and post-fixed for 30 min in 1% OsO4 in 0.1 M phosphate buffer (pH 7.2), and dehydrated using an ascending series of ethanol washes, ending with 100% ethanol. They were then critical point-dried with CO2 using a Bal-Tec CPD 030 critical point dryer, and sputter-coated with a thin layer (1–2 nm) of chromium or carbon using a Leica EM SCD 500 or 005 sputter coater. The FEI Nova NanoLab 600 and Magellan field emission SEMs were used at accelerating voltages of 2 and 1 kV, respectively. For better identification of some structures, colors were added to some images using Adobe Photoshop color balance.
Cultures of T. foetus grown for 30 h at 37 ◦ C in TYM medium were cooled to 4 ◦ C for up to 4 h, without changing the medium. The pseudocyst formation was followed by phase contrast microscopy. To quantify pseudocysts, at least 100 cells were examined by scanning electron microscopy in three independent experiments in triplicate. The results were expressed in percentage.
2.6. Morphometric analyses
2.3. Cytoskeleton preparation
2.7. Statistical analyses
Using 10 L 0.01% poly-l-lysine (mol wt 150,000–300,000, Sigma, USA) on a glass coverslip and allowed to stand for at least 15 min, then rinsed in distilled water after which a 20 L sample of living cells (107 cells/mL) was added. After 15 min, samples were rinsed in phosphate-buffered saline (PBS), pH 7.2, to remove nonadherent cells. Cytoskeletons were prepared by treating with 2% Triton X-100 and 2% NP-40 in a modified buffer previously designed for cytoskeleton preservation (IC buffer: 10 mM Tris Base, 2 mM EDTA, 2 mM DTT, 2 mM MgSO4 , 150 mM KCl, 30% glycerol, pH 7.4) for 30 min or 1 h (Palm et al., 2005).
Statistical comparisons were performed using the paired t test, and P values less than 0.0001 were considered to be statistically significant.
2.4. Immunofluorescence microscopy The cytoskeleton of both piriform trophozoites and pseudocysts were fixed for 1 h with 4% freshly prepared formaldehyde in phosphate buffer (0.1 M, pH 8.0). Samples were allowed to adhere to glass coverslips previously coated with poly-l-lysine, and cells were permeabilized with 0.1% Triton X-100. The free aldehyde groups were quenched using 50 mM NH4 Cl and 3% bovine serum albumin/PBS (BSA/PBS). Samples were then labeled with monoclonal antibody anti--tubulin (Amersham Bioscience, USA), and diluted 1:10 in 1% BSA/PBS. The coverslips were incubated for 1 h with an Alexa Fluor 488-conjugated anti-mouse antibody diluted 1:100 in BSA/PBS. Finally, the coverslips were washed and examined using a Leica TCS SP5 confocal microscope equipped with an Argon 488 laser emission bandwidth filter.
The areas and lengths of thirty axostyles of both forms were measured using iTEM software, as well the lengths of thirty costae of both forms of T. foetus. All results were analyzed using the GraphPad Prism 4.
3. Results 3.1. Pseudocyst induction In previous studies it has been established that incubation of trophozoites at low temperature is a highly efficient and reproducible method to induce the transformation of the pear-shaped parasites into pseudocysts. We used this well-established methodology in the present study and we obtained around 80% of pseudocysts in the cultures (Fig. 1). 3.2. Localization of microtubules Using a monoclonal antibody that recognizes -tubulin, we observed that cytoskeletal changes occur during the transformation from piriform to pseudocyst. In piriform parasites, the three anterior flagella and recurrent flagellum are externalized. A typical axial axostyle was also observed by immunolabeling with the -tubulin-recognizing antibody (Fig. 2) confirming previous observations (Lopes et al., 2001). Intense fluorescence was seen where the microtubule sheet turns upon itself to form the axostylar trunk (Fig. 2). Using LSCM it was possible to observe labeling of
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Fig. 2. Detection of microtubules in the piriform trophozoites of T. foetus by immunofluorescence microscopy. Note that the axostyle (Ax) appears as an axial structure, and the fluorescence intensity is higher in the axostylar trunk. The three anterior flagella (AF) and one recurrent flagellum (RF) are externalized and labeled.
Fig. 3. Detection of microtubules in T. foetus pseudocysts by immunofluorescence microscopy. The cells present a curved axostyle (Ax) and the flagella (F) are internalized (b, c; e, f and h, i). Some cells present a small region of the curved axostyle, where labeling was not detected (arrow; e, f), and duplicated axostyles indicate that the pseudocyst is in the process of division (arrowhead; h, i).
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Fig. 4. Localization of microtubules in trophozoites (a) and in pseudocysts (b, c) of T. foetus after extraction of the plasma membrane by immunofluorescence microscopy. (a) An intense labeling is observed in the anterior flagella (AF), pelta (arrowhead), recurrent flagellum (RF) and axostylar trunk (Ax). (b) One population of pseudocysts presents a curved axostyle with intense labeling at the ends, whereas other cells exhibit labeling only in the anterior region of the axostyle (c).
the pelta, the internalized flagella, and a curved axostyle in pseudocysts (Fig. 3). Other differences observed in pseudocysts were: (a) the presence of intense fluorescence in the anterior region of the axostyle (Fig. 3b); (b) a short region where the axostyle appeared curved, but not labeled (Fig. 3e), and (c) pseudocysts with duplicated axostyles, indicating that a mitotic process took place (Fig. 3c). When the plasma membrane of the protozoan was removed by detergent extraction, it was then possible to identify the labeled pelta localized in the anterior region of the cell (Fig. 4a–c). One population of pseudocysts presented a curved axostyle with intense labeling at the ends (Fig. 4d–f). However, other cells presented labeling only in the anterior region (Fig. 4g–i), suggesting that the axostyle may undergo antigenic modification during protozoan transformation. 3.3. Ultrastructural changes in pseudocysts The cytoskeleton of the trophozoites was visualized using high resolution scanning electron microscopy of detergent-extracted cells under conditions where the shape of the cells was not changed during membrane extraction. Our observations confirm and extend
previous studies (Benchimol, 2005; de Andrade Rosa et al., 2013). As observed by LSCM, that flagella were externalized and the axial axostyle presented a region where the microtubules turned upon themselves. In addition, the axostyle presented a concave region, where a network of filaments surrounded the nucleus, visible by high resolution SEM (Fig. 5). It is important to point out that the microtubules of the pelta and the axostyle could easily be distinguished (Fig. 5). The costa was also observed as an axial structure as was the recently described accessory filament along the costa (de Andrade Rosa et al., 2013). A network of 12.5 ± 1.2 nm thick filaments were seen connecting the periphery of the costa accessory filament to the region where the recurrent flagellum attaches to the protozoan cell body, forming the undulating membrane (Fig. 5). High resolution SEM of the pseudocyst cytoskeleton showed that the nucleus was positioned in the center of the axostyle, the pelta involved the periflagellar canal, and the costa was curved, allowing this axial structure follow the shape of the rounded cell (Fig. 6). Interestingly, we observed that the axostyle was smaller and had an irregular edge in the multinucleated pseudocyst (Figs. 7 and 8), suggesting a fragmentation of this structure. This observation was supported by quantitative and morphometric
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Fig. 6. Cytoskeleton of the pseudocyst as seen by high resolution SEM. Note that the axostyle (Ax) is seen around the nucleus (N) as well the flagella (F) and the costa (C) displays a curved appearance.
Fig. 5. T. foetus trophozoites visualized by high resolution SEM. An overview of the cytoskeleton of the parasite which was artificially colored to better show the different structures. Notice the pelta (P), the axial axostyle (Ax) with its axostylar trunk showing helicoidal arranged microtubules, and a region of the nucleus supported by a network of filaments (Nt). The costa (C) is seen with the accessory filament (FC), and fibrils (F) are found connecting the costa to the recurrent flagellum (RF). One parabasal filament (PF) is also observed as well the three anterior flagella (AF).
analyses, which showed that the 63.3% ± 4.7 of axostyles (Fig. 9a) of multinucleated pseudocysts presented a length (3.1 ± 0.27 m), that were one-fourth of those seen in the piriform trophozoites (13.9 ± 1.3 m). Similar results were obtained when the area occupied by the axostyle was estimated; the piriform cells displayed axostyles occupying an area of 14.830 ± 1.02 m2 whereas in the pseudocysts was 4.963 ± 0.712 m2 (Fig. 10a, b). The costa also underwent morphological modifications in the multinucleated pseudocysts. Using high resolution SEM, it was possible to observe that this structure displayed a hooked or U shape (Fig. 8) and was localized around the axostyle (Fig. 8a–d). The curved costa (seen in 71% ± 2.65 of the cells and shown in Fig. 9) could present several shapes, but its size did not vary significantly (Fig. 10c). Unexpectedly, the accessory filament, as well the filament network connecting the costa to the recurrent flagellum region, was no longer observed in the pseudocyst cytoskeleton. 4. Discussion The role of the pseudocyst in cell cycle is still discussed, however this form has been observed in natural conditions in different species of the trichomonads. It has been shown that pseudocysts of Tritrichomonas muris differentiate during the passage through the colon of the mice, are eliminated in the feces are considered to be infective agents of murine trichomonosis (Lipman et al.,
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Fig. 7. High resolution SEM of the cytoskeleton of a multinucleated pseudocyst of T. foetus. (a) Fragmented axostyles (Ax) are seen and the costae (C) are curved. (b, c) Higher magnification of the curved costa.
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1999; Stachan et al., 1984). The infectivity of the pseudocysts has been observed in Trichomonas vaginalis, where a significant amount of pseudocysts were detected in discharge from urethra (Małyszko and Januszko, 1991). In addition, pseudocysts inoculated intra-vaginally demonstrated ability to induce trichomonosis in mice (Hussein and Atwa, 2008). Recently, the possible role of the pseudocysts in exacerbating cervical cancer was suggested in a study performed with parasites isolated from cervical patients with symptomatic trichomoniasis, including patients with cervical neoplasia (Afzan and Suresh, 2012). In T. foetus, pseudocysts were the predominant form observed in preputial samples (Pereira-Neves et al., 2011) and it has been shown that they can be transformed back to trophozoites and exert a cytotoxic effect, and may represent a resistant developmental stage formed under unfavorable conditions (Pereira-Neves et al., 2012). As a result, attention to this stage has increased along with the need to obtain further information on its behavior, ultrastructure, and role during the life cycle of T. foetus. In addition, further studies in experimentally infected animals are necessary to establish a relationship between its presence and the trichomonosis disease. Previous studies have demonstrated that the transformation of a polarized trophozoite into the rounded pseudocyst form requires cytoskeleton modifications such as flagella internalization (Granger et al., 2000) and changes in the shape of the axostyle and costa (PereiraNeves et al., 2003). However, studies performed thus far have not revealed the three dimensional organization of the pseudocyst cytoskeleton. Using a new generation of scanning electron microscopes that achieve a resolution of about 0.8 nm, details of cell structures and their relationships to each other have been obtained. Using this approach, we recently provided detailed information about the structural organization of the piriform parasites of the T. foetus where new structures were described (de Andrade Rosa et al., 2013). Using the same approach, we confirmed in the present report that in contrast to the piriform cells, which display an axial axostyle, the pseudocyst presents a curved axostyle as previously suggested (Boggild et al., 2002; Mariante et al., 2004; Pereira-Neves et al., 2003), or even a fragmented axostyle, as observed in multinucleated pseudocysts. This information is in close agreement with our observation by LSCM of cells labeled for -tubulin, where pseudocyst cytoskeletons display an intense labeling only in the anterior region. A similar result was reported using TEM of the intestinal trichomonad cysts of Trichomitus batrachorum, where the axostyles missed their extremities (Brugerolle, 1973; Samuels, 1957). Together, the available data strongly suggest that during trophozoite-pseudocyst transformation, changes take place in the axostyle that lead to its fragmentation, explaining the labeling pattern observed by LSCM as well as the images obtained by high resolution SEM. It is possible, as previously suggested, that changes take place on the exposition of epitopes of the axostyle tubulin, thus explaining the variation in the fluorescence labeling pattern observed in pseudocysts during mitosis (Pereira-Neves et al., 2003). In addition, a distinct tubulin-labeling pattern was found when piriform and pseudocyst form of T. muris was compared (Boggild et al., 2002). The authors suggested that the difference could be due to post-translational modifications on tubulin (Boggild et al., 2002). Moreover, a proteomic study performed with three different forms of the T. vaginalis (piriform, pseudocyst and ameboid cells) demonstrated differences in protein expression, including cytoskeleton proteins (Yeh et al., 2013). The serine/threonine protein phosphatases 2A (PP2A) are highly conserved in all eukaryotes and among other functions is associated with remarkable changes in cytoskeleton remodeling (Sontag and Sontag, 2006). In addition, other findings suggested that PP2As act as native regulators of low-temperature responses of the cytoskeleton and cold stress responses in other organisms (País et al., 2009), thus it would be worth the study of the role of PP2A in response
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Fig. 8. Organization of the cytoskeleton of the pseudocyst as visualized by high resolution SEM. (a–d) Note the fragmented axostyles (Ax) and the presence of a curved costa (C) around one of the axostyles (a, b). A U-shaped costa (c, d) is observed.
Fig. 9. Quantitative analyses of the cytoskeleton alterations in parasite transformed into pseudocyst. (a) Cytoskeletons with short axostyles (**P < 0.005) and (b) with curved costa (***P < 0.0005).
to low temperature stress and in pseudocyst formation in trichomonads. Our observations using high resolution SEM clearly show fragmentation of the axostyle in pseudocysts. Such fragmentation was not observed in previous analysis of thin sections by TEM where only a thin slice of the cell was examined. In contrast, high resolution SEM of the cytoskeleton preparations allows a 3D view of the entire cell. The ventral disk of trophozoites of G. intestinalis, a large microtubule-containing structure, also breaks during the
encystation process and the fragments are stored in the cyst and reassembled during the excystation process when new trophozoites are formed (Sheffield and Bjorvat, 1977). Further studies are necessary to clarify the mechanisms involved on the process of fragmentation of these complex cytoskeleton structures. We also observed that the costa of the pseudocyst displayed morphological alterations during trophozoite-pseudocyst transformation. The most evident one was the conversion from a straight to a variable shape. Furthermore, we verified that the accessory
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Fig. 10. Morphometric analyses of the length (a) and area (b) of the axostyle and length of the costa (c) of piriform and pseudocysts of T. foetus. ***P < 0.001.
filament and the fibrils network previously described in piriform parasites were not visualized in pseudocysts. Because these structures seem to be involved in the establishment of connections of the costa with the recurrent flagellum attachment area, their disappearance during the transformation process was expected, once the recurrent flagellum is internalized, as previously shown (PereiraNeves et al., 2003). In conclusion, our present study using confocal laser scanning microscopy and high resolution SEM confirmed previous observations made with fluorescence microscopy and add new information for a better understanding of changes that take place in the cytoskeleton of T. foetus during the transformation of trophozoites into pseudocysts. Examples include (a) axostyle fragmentation, (b) curving of the costa, especially in multinucleated cells where the costa acquires variable conformations, (c) loss of the filamentous network which connects the costa to recurrent flagellum, and (d) loss of the accessory filament observed in the costa of trophozoites. Acknowledgements This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundac¸ão Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), Programa de Núcleos de Excelência (PRONEX), Instituto Nacional de Metrologia, Qualidade e Tecnologia (Inmetro) and Associac¸ão Universitária Santa Úrsula (AUSU). The authors thank Luis Sérgio de Araújo Cordeiro Júnior and Ricardo Chaves Vilela for technical support. References Afzan, M.Y., Suresh, K., 2012. Pseudocyst forms of Trichomonas vaginalis from cervical neoplasia. Parasitol. Res. 111, 371–381. Benchimol, M., 2005. New ultrastructural observations on the skeletal matrix of Tritrichomonas foetus. Parasitol. Res. 97, 408–416. Boggild, A.K., Sundermann, C.A., Estridge, B.H., 2002. Localization of posttranslationally modified alpha-tubulin and pseudocyst formation in tritrichomonads. Parasitol. Res. 88, 468–474. Brugerolle, G., 1973. Sur l’existence de vrais kystes ches les Trichomonadines intestinalis. Ultrastructure des kystes de Trichomitus batrachorum Petry 1852, Trichomitus sanguisugae Alexeieff 1911, et Monocercomonas tipulae Mackinnon 1910. C. R. Acad. Sci. Paris Ser. D 277, 2193–2196. de Andrade Rosa, I., de Souza, W., Benchimol, M., 2013. High-resolution scanning electron microscopy of the cytoskeleton of Tritrichomonas foetus. J. Struct. Biol. 183, 412–418.
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