versión impresa ISSN 0716-0720
Parasitol. día v.25 n.3-4 Santiago jul. 2001
On the ultrastructure of Trichomonas vaginalis:
cytoskeleton, endocytosis and hydrogenosomes
*Cátedra de Parasitología Clínica Departamento de Biología, Bioquímica y Farmacia Universidad Nacional del Sur San Juan 6708000-Bahía Blanca Argentina. E-mail: firstname.lastname@example.org
**Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) - CONICET/UNS C.C. 857 Camino La Carrindanga km 7 F8000FWB - Bahía Blanca Argentina.
This paper is focused on the study of the ultrastructure of Trichomonas vaginalis in liquid cultures. Its cytoskeleton, the morphology of its hydrogenosomes, and endocytosis phenomena have been observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For the present study, the traditional techniques for SEM and TEM have been slightly modified for the processing of this flagellate. Following our experiments, it can be concluded that: 1. The modified techniques are adequate for the ultrastructural study of this protozoon. 2. There are no mitochondria in T. vaginalis. 3. T. vaginalis might use micropinocytosis processes related to coated vesicles as a habitual endo- and exocytosis mechanism, while phagocytosis is observed for major vesicles. 4. As to the cytoskeleton, microtubules are numerous and display different structures, which are analyzed in this paper. 5. Many hydrogenosomes are found in the cytoplasm of T. vaginalis underneath the undulating membrane and along the axostyle, each with electron-dense deposits in the manner of «operculums.»
Key words: Trichomonas vaginalis, SEM, TEM, ultrastructure, hidrogenosomas.
Trichomonas vaginalis1 is a flagellate, protozoon parasite in human vagina and urethra. This parasite can be found in asymptomatic carriers as well as in patients suffering from urethritis and vaginitis; the latter are pathologies in which this microorganism proves to be responsible for several reported lesions.
T. vaginalis is an ovoid or pyriform parasite, 7 to 30 mm long (average length) and 5 to 15 mm wide. It has four anterior flagella and a recurrent fifth one accompanying its undulating membrane, a nucleus, hydrogenosomes2, an axostyle which runs the length of the parasite, a glycogen-rich cytoplasm, a variety of vacuoles (including lysosomes), and numerous micro-tubules with varied structures3-7.
The purpose of this study is to carry out a structural analysis of T. vaginalis paying particular attention to its cytoskeleton, hydrogeno-somes, and the endocytosis phenomena observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
MATERIALS AND METHODS
Parasite Isolates: Several cultures (OXOID CM 161, Hampshire, England) with T. vaginalis were used. Isolates were obtained from adult women carrying the flagellate protozoon. The medium containing the parasites was incubated at 37ºC for 72 hs.
Concentration and fixation of parasites: Parasites were concentrated by centrifugation for 3 min at 1,500 rpm. Sediments were fixed with 1% glutaraldehyde in 0.05 M phosphate buffer pH 7.2. Fixation for SEM and TEM processing was performed at 4º C.
Scanning Electron Microscopy processing (SEM): After fixation, the material was washed in 0.05 M phosphate buffer pH 7.2. It was subsequently dehydrated in increasing concentrations of acetone following then with this methodology: samples were put into cylinders (1cm long and 0.50 cm of diameter) made of Eppendorf tubes. Millipore filters (pore No 5) were used to cover the cylinder edges and were firmly fixed by means of rings made up from the caps of the Eppendorf tubes. The samples thus encapsulated were washed, dehydrated and taken to critical point. Filters containing the adhered material were then removed and subsequently coated with gold in a sputter coater model 3 PELCO8,9. Observations were carried out with a JEOL 35 CP electron scanning microscope at 6KV. Electron micrographs were taken on a VP-120 Kodak Electron Image-film.
Transmission Electron Microscopy processing (TEM): The prefixed material was washed in 0.05 M phosphate buffer pH 7.2 and then postfixed in 1% OsO4 in the same buffer for 2 hs. It was subsequently washed twice in the same buffer. A further washing in 0.05 M maleate buffer pH 5 was carried out. The material was then put in a solution of 0.5% uranyl acetate in maleate buffer for 60 min10. It was subsequently washed in this buffer and then dehydrated in increasingly graded ethanol. Washings were carried out by centrifugation for 3 min at 1,500 rpm using a 2036 ROLCO centrifuge (Argentina).
The samples were then embedded in spurr resina at room temperature and polymerized at 60º C for 12 h. Ultrafine sections were contrasted in uranyl acetate and lead citrate11. Observations were made with a JEOL 100 transmission electron microscope at 80KV. Electron micrographs were taken on a SO-163 Kodak Electron Image-film.
1. GENERAL MORPHOLOGY
SEM shows that T. vaginalis has an ovoid shape (Figure. 1), 6 to 28 mm long and 5 to 12 mm wide, with four free anterior flagella (Figure 2) and a recurrent fifth one. This fifth flagellum accompanies the undulating membrane and is adhered to the mid-portion of one of its faces (Figure 3).
|Figure. 1. Ovoid conformation of T. vaginalis by SEM. Flagella (F) and undulating membrane (UM) are seen. Cocoid structures can be observed on the flagellum (C) (X9,000). Figure. 2. SEM micrograph of T. vaginalis. Four flagella (F) emerging from the anterior region; undulating membrane (UM) and part of the axostyle (Ax) can be observed in the posterior region (X8,600). Figure. 3. Higher magnification of Fig 2. Flagella (F); the recurrent flagellum (rF) accompanies the undulating membrane (X15,000).|
The fine structure of T. vaginalis was studied by TEM. In Figure 4, glycogen granules, vacuoles and hydrogenosomes are easily distinguishable following a longitudinal ordering. A more detailed image of the nucleus is shown in Figure 5.
Figure 6 shows the presence of 0.1-to-0.3 mm invaginations having the same morpho-logical characteristics and electronic density as those of coated pits. Figure 7 shows these invaginations at a higher magnification. The range of these invaginations was between 0.1 and 0.9 µm. In addition to demonstrating the existence of receptor-mediated endocytosis, we could also prove that, for larger structures, T. vaginalis uses phagocytosis. This is shown in Figure 8, in which the flagellate is seen phagocyting a bacterium.
|Figure. 4. Ultrastructure of the T. vaginalis by TEM. The plasma membrane, the undulating membrane and its flagellum appear well preserved. In the posterior region of the cytoplasm, the nucleus (N), hydrogenosomes (H), glycogen granules and vacuoles (X6,700) are observed. Figure. 5. T. vaginalis nucleus at a higher magnification. The perinuclear cisterna is observed as being surrounded by a second cisterna (X20,000). Endocytosis. Figure 6. Invagination of the plasma membrane having the same electron dense characteristics as those of coated vesicles (CV) (X40,000). Figure. 7. Coated vesicles at a higher magnification showing a clathrine net (Cl) on the cytoplasmatic side and an electron-dense component within the clathrine net (X120,000). Phagocytosis. Figure. 8. A bacterium being phagocyted by T. vaginalis (X50,000) is shown.|
Figure 9 shows the anterior part of the protozoon with the basal bodies of its five flagella surrounded by the pelta. The «microtubules», fencing the costa in a «palisade-like» conformation, can be also observed. Figure 10 shows another section of the anterior region of the parasite where two basal bodies, ciliary roots, a necklace of microtubules belonging to the pelta, part of the costa and two of the anterior flagella longitudinally cut, can be all visualized. Figure 11 shows an anterior section of the flagellum where the four basal bodies, part of the pelta, and the microtubular axostyle adjacent to the basal bodies can be observed. Parabasal electron-dense fibers can also be observed at both sides. At a higher magnification (Figure. 12) the axostyle shows microtubules alterning with electron-dense «eyelash-like» areas separating the microtubules from one another. Figure 13 shows a transversal cut of the flagellum with its microtubules organized in nine peripheral pairs and a central pair; the membrane is seen well preserved. Figure 14 shows a transversal cut of the parasite undulating membrane where the recurrent flagellum is adhered to the upper third of the membrane. The undulating membrane can be visualized as a cytoplasmic prolongation. Figure 15 shows a transversal section of the costa with its microtubules tied to each other in a «palisade-like» conformation; there are no visible spaces among each microtubule.
|Cytoskeleton. Figure 9. Electron micrograph showing the cytoplasm and its five basal bodies (BB) surrounded by the pelta (P); a portion of the costa (C) can be also observed. (X14,000). Figure 10. T. vaginalis anterior section showing the birth of four anterior flagella (F); a longitudinal cut of two of the flagella shows peripheral microtubules and the central pair in their interior. One of the basal bodies (BB) can be observed distinctively showing its nine triplets of peripheral microtubules and no central pair. Ciliary rootlets (CR) are observed as emerging from these microtubules. The cytoplasm shows microtubules belonging to the pelta (P) (X27,000). Figure 11. Tangential cut of the anterior section of the parasite showing the transverselly-sectioned microtubules of the axostyle (Ax), the pelta (P) and the four basal bodies (X24,0 00). Figure 12. Axostyle (Ax) shown in Fig 11, now at a higher magnification (X50,000). Figure 13. Transversal cut of a flagellum showing nine pairs of peripheral microtubules and the central pair (X67,000). Figure 14. Transversal cut of the undulating membrane (UM) being accompanied by the recurrent flagellum (X27,000). Figure 15. Longitudinal section of the costa (C) showing the «palisade-like» conformation displayed by its microtubules (X27,000).|
Figures 16, and 17 show hydrogenosomes, which are slightly electron-dense spherical organelles with a diameter of 0.5-microns. These membrane-coated organelles are mostly located near the costa, as if detaching themselves from the nucleus and following the axostyle towards the posterior part of the parasite (Figure. 4), and have a completely electron-dense sort of «cap» or «operculum.» On the other hand, the «cap» in Figure 18 is empty, as if its contents had been removed.
Hydrogenosomes. Figure 16. Hydrogenosomes (H) with electron-dense (X 40,000) «caps» or «operculums» (arrow). Figure 17. Hydrogenosome (H) with a «cap» or «operculum» having a low electron-dense content. Figure 18. Hydrogenosome with an empty «cap.»
The following observations have resulted from this ultrastructure study:
1. In the samples analysed the endoplasmic reticulum can be distinctively visualized generally surrounding the nucleus. In this respect, it should be reminded that our material remained in culture medium for 48 h prior to fixation.
Our observations of the ultrastructure of T. vaginalis agree with others authors4 whom refer to the absence of mitochondria in the flagellate cytoplasm. But another investigator12, on the contrary, refers to the presence of mitochondria in the flagellate cytoplasm. However, we think it relevant to consider the inclusion of this parasite within the sub-order 1. PARABA-SILIDEA13 according to which, one of the most distinctive features is precisely the absence of mitochondria14.
2. The frequency with which we have found coated vesicles, previously reported5, suggests that T. vaginalis might use the micropinocytosis process associated to coated vesicles as a usual selective receptor-mediated endo- and exocytosis mechanism. Some authors have suggested, by biochemical analyses, the presence of receptors for HDL in T. vaginalis15. This micropinocytosis process is well documented by ourselves16. Other authors have concluded that there may be a relationship between these vesicles and the parasite virulence5. It seems pertinent to relate these coated vesicles with other findings by the authors18,19, in particular those concerning proteins with an apparent molecular weight between 14 and 60 kDa that appear to be excreted by the parasite, agreeing with observations by Alderete17. Moreover, considering 1) that iron appears to be essential for the regulation of the parasite's adherence to epithelial cells 20; 2) that this element could be incorporated through hemoglobin or transferrin, which are particularly abundant during menstruation; and 3) that, in assays carried out by the authors using human anti-hemoglobin and anti-transferrin monoclonal antibodies (not shown in this study), hemoglobin was found attached to the flagellate underneath the undulating membrane, we can conclude that, at least concerning iron incorporation, T. vaginalis seems to use receptor-mediated endocytosis.
In this paper, bacterium phagocytosis by the protozoon is reported in agreement with other authors´observations4,5,21.
3. As shown in the figures included in this paper, in this parasite there are numerous microtubules oriented in several directions, displaying different structures that are likely to play different roles. The parasite costa is a «palisade-like» configuration of thick «microtubules» with no free room among each other. At a low magnification, this phenomenon is sometimes observed as thin structures between each «microtubule». But at a higher magnification, «microtubules» are visualized as being tied to each other forming a compact structure of a considerable apparent resistance. In the anterior part of the axostyle, it can be clearly observed that there are distinct eyelash-like electron-dense areas separating microtubules from one another. Some authors22,23 have already reported important observations about these thin structures. Juliano et al22 have studied the organization of these microtubules in the interface and their modifications during mitosis by indirect immunofluorescence using monospecific antibodies against sheep brain tubulin. They have also reported on the presence of tubuline in flagella, axostyle and pelta. Schwartzman and Krug23 have demonstrated the specificity of this tubular protein using anti-(-tubulin specific antibodies and have shown how two of seven monocolonal specific antibodies for mammalian (-tubulin recognized the axostyle, costa and flagella. Batista et al24 have stated that in addition to the flagellar microtubules, those from the pelta-axostyle system represent stable microtubules containing acetylated-(-tubulin. As to the fibrous structures in zooflagellate Protozoa, particularly the T. vaginalis costa, Eyden25 has observed that this parasite resembles a rod-like fibre and that it shows a more complex set of striation patterns. Both the trepomonad fibre and the trichomonad costa reveal fine longitudinal filaments in high resolution electron micrographs, which in the latter appear to have a zig-zag pattern. As to the axonemes shown by the authors, no differences with the characteristic microtubular morphology of flagella have been found. With respect to the undulating membrane, the authors have observed that, as it occurs in other Trichomonads, the recurrent flagellum accompanies the undulating membrane in its third-outer portion without reaching its border, as typically happens in Trypanosomas12.
4. In hydrogenosomes26, which are fundamental compartments for the energy metabolism of eucaryotes27, and as shown in Figure 16, the presence of electron-dense «caps» or «operculums» is noteworthy. Some of these are empty, suggesting that they had discharged their content into the cytoplasm or consumed it completely (Figure 18) or partially (Figure. 17). These «caps» could be calcium28 or magnesium29 deposits, and their function in connection to the flagellate remains unknown, although they could be related with the activation of a high-molecular-weight ATPase (300,000 to 400,000Da)29. However, based on experiments performed previously30, it is our view that they could be related with the phenomena of cytoadherence of the parasite to cells in the vaginal epithelium, as shown in Figure 1. Hydrogenosomes would play a significant role in the metabolism of anaerobic microorganisms such as T. vaginalis31.
5. The practicability and simplicity of SEM processing techniques9 facilitated the obtention of the above-mentioned results. As to TEM processing, the difference with other traditional techniques applied to protozoa, lies mainly in that for the present research an uranyl acetate in maleate buffer pH 5 treatment was used prior to dehydration in order to increase the contrasting effect.
El presente estudio está referido a la ultraestructura de Trichomonas vaginalis, cultivadas en medio líquido. Su citoesqueleto, fenómenos de endocitosis y la morfología de sus hidrogenosomas fueron observados por microscopía electrónica de barrido y de transmisión. Las técnicas clásicas para el procesamiento al SEM y TEM de este flagelado, fueron discreta y sutilmente modificadas por nosotros. Como resultado de nuestras experiencias se concluye que: 1. Las técnicas modificadas empleadas son adecuadas para el estudio morfológico y ultraestructural de este Protozoo. 2. Al TEM su citoplasma no muestra mitocondrias. 3. Utiliza los fenómenos de micropinocitosis asociados con vesículas con cubierta como mecanismo habitual de endo y exocitosis selectiva, mientras que para partículas mayores la fagocitosis es frecuentemente vista. 4. Con referencia al citoesqueleto, los microtúbulos que recorren el parásito son numerosos, conformando estructuras diversas, las cuales son analizadas en este trabajo. 5. Posee numerosos hidrogenosomas alineados debajo de la membrana ondulante y a lo largo del axostylo, mostrando importantes depósitos electrón-denso a modo de «opérculo» en cada uno.
Acknowledgements. The skillfull technical assistance in the use of electron microscopes by the Centro Regional de Investigaciones Básicas y Aplicadas de Bahía Blanca (CRIBABB) is gratefully acknowledged. This work was supported by a 1994 ZB 11 grant to the authors from the Secretaría de Ciencia y Técnica, Universidad Nacional del Sur, Bahía Blanca, Argentina.
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