Tritrichomonas foetus: Ultrastructural localization of basic proteins and carbohydrates

EXPERIMENTAL

PARASITOLOGY

Tritrichomonas MARLENE Institute

de Biojkca,

54,

135-144 (1982)

foetus:

Ultrastructural Localization and Carbohydrates

BENCHIMOL, Universidade

CEZAR A. ELIAS,

of Basic Proteins

AND WANDERLEY

Federal do Rio de Janerio, Cidade Universitdria, 21.910, Rio de Janeiro, RJ, Brasil

DE SOUZA Ilha do FundBo,

(Accepted for publication 30 May 1980) BENCHIMOL, M., ELIAS, C. A., AND DE SOUZA, W. 1982. Tritrichomonas foetus: Ultrastructural localization of basic proteins and carbohydrates. Experimental Parasitology 54, 135- 144. The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (E-PTA) techniques were used to localize basic proteins at the ultrastructural level in the protozoa, Tritrichomonas foetus. The periodic acid-thiosemicarbazide-silver proteinate technique was used to localize carbohydrates. With the AS technique reaction product was seen only in the hydrogenosomes. With the E-PTA technique, reaction product was seen in the microtubules that form the basal bodies, flagella, pelta, and axostyle, in the costa, in the hydrogenosomes, and at the region of the recurrent flagellum’s adhesion to the cell body. Previous acetylation of the cells under conditions that block most free amino groups abolished (AS technique) or greatly reduced (E-PTA technique) staining. Carbohydrates were localized on the cell surface; in the membranes of the hydrogenosomes, Golgi complex, and cytoplasmic vesicles; and in the glycogen particles. The specificity of the AS and E-PTA techniques to detect basic proteins is based on observations made with T. foetus as well as with other cell types. INDEX DESCRIPTORS: Tritrichomonas foetus; Protozoa, parasitic; Basic proteins; Carbohydrates: Ultrastructure; Cytochemistry

INTRODUCTION

Two cytochemical methods associated with electron microscopy have been used to localize basic polypeptides: one uses phosphotungstic acid in alcoholic solutions (E-PTA) and the other uses ammoniacal silver (AS). Few papers have been published on cytochemical localization of basic polypeptides in protozoa. These studies would be of interest since basic proteins are involved in the control of cellular differentiation and regulation of gene function in eucaryotic cells. Moreover, recent studies show that they may play a role in the process of penetration of parasitic protozoa inside host cells (Kilejian 1976; de Souza and SoutoPadron 1978). We thought, therefore, that it would be of interest to investigate the localization of basic proteins in some

pathogenic protozoa. Previously we presented results related to some trypanosomatids (Souto-Padron and de Souza 1978, 1979) and in the sporozoon Toxoplasma gondii (de Souza and Souto-Padron 1978). In the present report we describe results obtained by using the AS and E-PTA methods to locate basic proteins in Tritrichomonas foetus, a pathogenic protozoon of the urogenital tract of cattle. In addition the specificity of both techniques is discussed in relation to controls performed and results obtained in other cell types. In order to analyze better the E-PTA method, carbohydrates were also detected using the periodic acid - thiosemicarbazide - silver proteinate method (Thiery 1967). MATERIALS

AND METHODS

Tritrichomonas foetus was isolated by Dr. H. Guida (EMBRAPA, Rio de Janeiro) from the urogenital tract

13.5 0014-4894/82/050135-10$02.0010 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved

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of a bull in the state of Rio de Janeiro, Brasil, and has been maintained by weekly transfers at room temperature (about 23 C) in a medium with the following composition (g/liter): liver hydrolysate, 25.0; D(f)glucose, 5.0; sodium chloride, 6.5; agar-agar, 0.5; and 10% inactivated and filter-sterilized fresh bovine serum. Cells were grown in 150 x 15-mm tubes containing 20 ml of medium. The inoculum consisted of 1 ml of 24-hr culture at 36.5 C. About 4.5 x lo6 cells/ml were inoculated per tube. The cells were cultivated for 24-28 hr at 36.5 C. A layer (about 1.5 cm) of paraffin oil was added to the culture tubes to keep the diffusion of oxygen low. Parasite cells were centrifuged at 15OOg for 10 min and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 2 hr at room temperature. After fixation they were washed in buffer, postfixed in 1% 0~0, in cacodylate buffer for 2 hr at room temperature, dehydrated in either acetone or ethanol, and embedded in Epon. Ultrathin sections were obtained with an LKB Ultratome III ultramicrotome and examined unstained or after staining with uranyl acetate or lead citrate or both, in an AEI EM6-B electron microscope. For detection of carbohydrates, ultrathin sections of cells fixed in glutaraldehyde and OsOl were collected on gold grids and treated with periodic acid (l%, 20 min at room temperature) to oxidize adjacent hydroxyl or o-amino alcohol groups into aldehydes. After rinsing in distilled water, aldehydes were condensed with thiosemicarbazide (1% thiosemicarbazide in 10% acetic acid for 48 hr at room temperature) to yield the corresponding thiosemicarbazones which are powerful reducing agents. Thus, after rinsing sequentially with 10, 5, and 1% acetic acid and with distilled water, the sections were exposed to silver proteinate (1%) for 30 min in the dark at room temperature. Sections were observed unstained (Thiery 1967). Controls were done by omission of one of the steps in the method. For detection of basic proteins, the phosphotungstic acid method (E-PTA) was employed as previously described (Gordon and Bensch 1968; Souto-Padron and de Souza 1978). After glutaraldehyde fixation the cells were dehydrated (without postlixation with 0~0~) in ethanol and incubated for 2 hr at room temperature in 2% PTA in absolute ethanol after which they were washed in ethanol and embedded in Epon. The ammoniacal silver (AS) method was used as described by MacRae and Meetz (1970). After fixation, the cells were thoroughly washed in distilled water and incubated for 5 min at room temperature in the AS solution. This solution was prepared just before use by gradual addition of 10% silver nitrate solution to concentrated ammonium hydroxide until a slight turbidity appeared and persisted. After incubation in this solution, the cells were washed in distilled water and placed for 5 min in a 3% formaldehyde solution, where a brown coloration appeared. After washing in distilled water they were postfixed in OsO,, dehydrated in

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ethanol, and embedded in Epon. Some glutaraldehydefixed cells were acetylated by reaction with 10% acetic anhydride in pyridine for 90 min at 37 C before either the E-PTA or the AS treatment. Control cells were incubated under the same conditions in pyridine only. RESULTS

The general structure of Tritrichomonas foetus has been described in detail by Honigberg et al. (1971). Figure 1 shows some of the structures such as basal body, nucleus, costa, hydrogenosome, cytoplasmic vacuoles, and recurrent flagellum. The periodic acid-thiosemicarbazidesilver proteinate method (Thiery 1967) was used to detect carbohydrates in thin sections of Epon-embedded cells. In controls where either the periodic acid oxidation step or the thiosemicarbazide incubation were omitted there was no reaction product. Reaction product is seen mainly in many particles preferentially localized near the axostyle and which correspond to glycogen particles (Fig. 2). They have a diameter of 27 nm and are composed of 3-nm subunits. The glycogen particles react even with a short incubation time (30 min) in thiosemicarbazide. With longer incubation periods (24-48 hr) reaction product is associated with the plasma membrane which surrounds the whole body of the parasite as well as the flagella. The membranes of vesicles, food vacuoles, and the Golgi complex also show reaction product. The membrane of the hydrogenosomes shows a weak, but positive, reaction. The matrix of the hydrogenosomes shows a weak, but positive, reaction. The matrix of the hydrogenosomes and the microtubules which form the basal bodies, flagella, axostyle, and pelta do not react. The costa also does not show any reaction product (Fig. 2). For detection of basic proteins, the treatment of the cells with ethanolic phosphotungstic acid (E-PTA) gives a homogeneous electron-dense reaction product with some structures of T. foetus. After application of this technique, the cells appear well preserved. However, since they are

Tritrichomonasfoetus:

CARBOHYDRATES

AND BASIC PROTEINS

137

FIG. 1. General aspect of Tri’trichomonas foe&s. Ultrathin section stained with uranyl acetate and lead citrate. BB, basal body; C, costa; N, nucleus; RF, recurrent flagellum. The small arrow indicates the lateral arcuate system which surrounds the costa. x 12,000. Bar = 1 pm.

not postfixed with osmium tetroxide there is no good contrast in the cellular structures, except in those which react with EPTA. We have observed that when thin sections of E-PTA-treated cells are stained with lead citrate there is a precipitation of lead on structures which react with PTA. Since this precipitation improves the contrast, we observed either nonstained or leadstained sections. All hydrogenosomes show an intense reaction (Fig. 4). In some of them, the reaction is less intense in the central portion, probably reflecting difficulties in the penetration of the PTA. The cytoplasmic ribosomes show a weak reaction. The microtubules which form the flagella, basal bodies, pelta, and axostyle show an intense

reaction (Figs. 3, 4). The costa also reacts strongly showing the presence of a very dense band intercalated by a less dense one in the center of which there is a dense line. The lateral arcuate system which surrounds the costa does not react. The clockwise curving rootlet filaments associated with the basal bodies also react (Figs. 3, 4). Filaments radiating from the peripheral doublet microtubules of flagellum number 2 (following the numeration found in the diagram presented by Honigberg et al. (1971) are seen. Electron-transparent areas are seen in the cytoplasm, mainly near the axostyle, and may represent sites in which glycogen particles are located (Fig. 4). Reaction product is seen at the region of

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FIG. 2. Tritrichomonas foetus submitted to the periodic acid-thiosemicarbazide-silver proteinate technique for detection of carbohydrates. Ultrathin section unstained. Reaction product is seen on the cell surface, in the membranes of the Golgi complex (G), in the membrane of cytoplasmic vacuoles (star), in the membrane of hydrogenosomes (H) and in the glycogen particles (Cl). The microtubules which form the axostyle (arrowhead) and costa (C) did not show reaction. ~37,000. Bar = 0.5 pm.

adherence of the recurrent flagellum to the cell body. The reaction is restricted to a layer localized at the inner face of the cell body plasma membrane, any reaction appearing in the flagellum (except the axonemal microtubules). It appears as a very dense line from which some projections

radiate perpendicularly toward the cell, with a periodicity of about 16 nm. There is also a second plate which does not show reaction product (Fig. 4). Exposure of the cell suspension to 10% acetic anhydride in pyridine for 90 min greatly reduced the E-PTA staining (Fig. 5).

FIGS. 3, 4. Trirrichomonas foetus submitted to the ethanolic phosphotungstic acid (E-PTA) technique. Reaction product is seen in the microtubules which form the basal bodies (BB), flagella (F), axostyle, and pelta. The costa (C) and the hydrogenosomes (H) react strongly. Reaction product was also seen in filaments radiating from the peripheral doublet microtubules of flagellum number 2 (curved arrow in Fig. 3) and the cytoplasmic portion of the recurrent flagellum (RF)-cell body adhesion zone (curved arrow in Fig. 4). Ultrathin sections stained only with lead citrate (2 min). Fig. 3, X20,000; Fig. 4, ~15,000. Bar = 1 pm. 139

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Pyridine alone did not suppress the staining. After application of the postformalin ammoniacal silver technique, the cell suspension has a brown to black color which can also be seen by light microscopy, mainly when the amplitude contrast method associated with Nomarski optics is used. The reaction product is seen mainly in spherical structures with a mean diameter of 0.7 ,um. Treatment of the cells with the ammoniacal silver and formalin solutions interferes with the preservation of cell structure. Some empty cytoplasmic areas were occasionally seen. In the electron microscope the reaction product appears in the form of electron-opaque particles with diameters of about 20 nm. They are found either isolated or in contact with each other. The silver particles are found mainly in the hydrogenosomes (Fig. 6). Not all hydrogenosomes, however, showed silver particles. Other large dense membranebounded structures also do not react. After exposure of T. foetus cells to 10% acetic anhydride in pyridine for 90 min before AS treatment, no silver particles are seen in the hydrogenosomes. Pyridine alone does not suppress the reaction. DISCUSSION

Protozoa of the family Trichomonadidae, which includes agents of important human and veterinary diseases, have a large number of filamentous and tubular structures which form the mastigont, a group of structures which has been studied by electron microscopy by using either thin sections or negative staining technique. In addition they have a spherical structure which has been designated as paracostal granules, paraxostylar granules, chromatic granules, or hydrogenosomes (Honigberg et al. 1971; Nielsen and Diemer 1976; Lindmark and Muller 1973). The results obtained by us confirm previous morphological studies on Tritrichomonas foetus (Inoki et al. 1961; Simpson and White 1964; Honigberg et al. 1971). We

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will discuss here only aspects related to the structures which result in reaction product when the periodic acid - thiosemicarbazidesilver proteinate, the ammoniacal silver or the ethanolic phosphotungstic acid techniques are used. The postformalin ammoniacal silver technique was fust introduced in light microscopy by Black and Ansley (1964), who used it for investigations related to the biological functions of histones (Black and Ansley 1964, 1966). Experimental studies using isolated histones suggest that when a yellow color appears after interaction of the histone with AS a lysin-rich histone is indicated, whereas the black staining is associated with arginine-rich histones (Black and Ansley 1966). However, the authors pointed out that “differences in the AS staining may reflect differences in reactivity of amino and guanidino groups rather than differences in the relative content of lysine and arginine residues in the histone.” MacRae and Meetz (1970) introduced the AS technique to electron microscopy applying it to localize histones in the erythropoietic cells of the chick. They show that stem cells and early erythroblasts exhibit little reaction while more differentiated cells such as polychromatophilic erythrocytes and reticulocytes exhibit more silver granules. More recently this technique has been applied to localize basic proteins in erythroid precursors from patients with anemia (Kass and Gray 1975), in polymorphonuclear leukocytes (MacRae and Spitznagel 1975; Brown and Wood 1978), in eosinophils (Pimenta et al. 1979), and in two developmental stages of Trypanosoma cruzi (Souto-Padron and de Souza 1978). In all these studies the reaction product appears as electron-dense particles. In T. foetus, silver particles are observed only in hydrogenosomes. A few granules are seen in the cytoplasm. However, we could not associate them with any particular structure of the cell. T. foetus submitted to the AS method after acetyla-

FIG. 5. Tritrichomonas foetus submitted to the ethanolic phosphotungstic acid technique after previous acetylation under conditions which block most free amino groups. A weak reaction is seen in the microtubules which form the axostyle (M). No reaction product is seen in the hydrogenosomes (H). ~30,000. Bar = 0.5 pm. FIG. 6. Tritrichomonas foetus submitted to the postformal in ammoniacal silver method. Silver particles are seen only in the hydrogenosomes (H). ~22,000. Bar = 0.5 pm.

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tion did not show silver particles, thus confirming its specificity. Phosphotungstic acid is an anionic stain introduced to electron microscopy by Hall et al. (1945) and widely used in negative staining (Haschmeyer and Myers 1972). In cytochemistry PTA has been used to detect polysaccharides (Pease 1970) and proteins. Bloom and Aghajanian (1968) suggest that E-PTA binds principally to proteins rich in lysine, arginine, and histidine which are localized at synapses. Sheridan and Barrnett (1969) use it to locate nuclear proteins, presumably histones, in the meiotic chromosomes of the prophase nuclei of lily microsporocytes. Reaction product is observed in the nuclear chromatin, in the nucleoli, in material found in the nuclear membrane pore, in the annulate lamellae, and in the lateral element of the synaptinemal complex. Silverman and Glick (1969), on the basis of histochemical experiments, suggest that PTA selectively stains proteins. Pease (1966) has used PTA to detect polysaccharides associated with the exterior surface (glycocalix) of epithelial cells. However, the results we have recently obtained show that E-PTA, under the conditions described under Materials and Methods, does not detect carbohydrates. In Trypanosoma cruzi, E-PTA reaction product is seen in the kinetoplast, in the nucleus, and in some vesicles; all of them are structures which do not show any reaction product when the PA-TSC-SP technique is used (SoutoPadron and de Souza 1978). A similar result is obtained when the same technique is applied to other trypanosomatids where reaction product is seen also at the region of adhesion between the cell body and the flagellum (Souto-Padron and de Souza 1979). In addition, reaction product is associated with the conoid, rhoptries, and micronemes of the sporozoa Toxoplasma go&ii, structures which do not have carbohydrates (de Souza and Souto-Padron 1978). Biochemical studies suggest that the rhoptries of the sporozoa Plasmodium

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SOUZA

contain polypeptides rich in histidine which may be involved in the process of penetration of this protozoa inside a host cell (Kilejian 1976). The results obtained with T. foetus also support the idea that E-PTA does not detect polysaccharides. In addition, previous acetylation of the cells under conditions which block most free amino groups (Pearse 1968) greatly reduced the E-PTA staining. When we used the PA-TSC-SP technique, reaction product was associated with structures such as the plasma membrane, the membrane of some intracellular structures such as the Golgi complex, pinocytotic vesicles, hydrogenosomes, and mainly with glycogen particles. These structures do not react with E-PTA. E-PTA reaction product is seen to be associated with the hydrogenosomes, in the costa, in the microtubules which form the basal bodies, flagella, axostyle, and pelta, in the clockwise curving rootlet filaments associated with the basal bodies, in the filaments which radiate from the peripheral microtubules of flagellum number 2, and the region of adherence of the recurrent flagellum to the cell body (undulate membrane). All the microtubules show the same intense reaction, a result which has not been obtained in other cells. Gordon and Bensch (1968) found a strong staining in the peripheral doublet microtubules and a less intense staining in the central pair of microtubules of the flagellum of the guinea pig sperm. In trypanosomatids less intense or no staining is observed in the central microtubules (Souto-Padron and de Souza 1978, 1979) The zone of adherence of the recurrent flagellum to the cell body appears to be specialized as previously observed in trypanosomatids (de Souza et al. 1978, 1979). A plate which shows an intense PTA staining is seen below the inner face of the cell body plasma membrane. The hydrogenosome is an organelle which contains enzymes which participate in the metabolism of pyruvate formed in lophurae

Tritrichomonas

foetus:

CARBOHYDRATES

glycolysis (Lindmark and Muller 1973; Muller 1975). This organelle appears to be rich in basic proteins since it gives intense reaction with either the E-PTA or the AS technique. Its membrane, however, shows reaction product when the PA-TSC-SP method, which detects carbohydrates, is used. The absence of reaction product, indicative of the presence of carbohydrates, in the costa of cells submitted to the PATSC-SP method was not expected in view of the results recently described by Sledge et al. (1978). Analysis of purified costa of Tritrichomonas foetus is shown to be composed of about 95% carbohydrate (mainly glucose) and 5% protein. Our results agree with those recently reported for some trichomonad species (Amos et al. 1979). Using a histochemical method these authors could not detect PAS-stained material in the costa. Biochemical analysis (SDS-gel electrophoresis) showed the presence of three main classes of proteins. As suggested by Amos et al. (1979) it is possible that the preparations of Sledge et al. (1978) contained glycogen which is abundant in the surrounding cytoplasm. Our results suggest that the E-PTA and AS techniques detect different proteins. Reaction product is seen with both techniques only in the hydrogenosomes, suggesting that the AS technique is more specific. Results recently obtained with other cell types support this assertion. Although an intense staining is seen in the rhoptries and micronemes of E-PTA-treated T. gondii cells, no silver particles are seen in these structures (de Souza and Souto-Padr6n 1978; de Souza, unpublished observations). In eosinophils and mast cells similar results have been obtained (Pimenta et al., 1979). However, we cannot at the present time know which basic amino acids have more affinity to either E-PTA or AS. Studies are being carried out in different cell types as well as in isolated polypeptides with a defined composition of amino acids in order to

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clarify the specificity of these two techniques . ACKNOWLEDGMENTS

We thank Mr. A. L. de Oliveira for help with photography and Mrs. Zelia de Freitas for secretarial assistance. The technical assistance of Miss Silvia R. V. Sales was very much appreciated. This work has been supported by Brasil’s Conselho National de Desenvolvimento Cientifico e Tecnoldgico (CNPq), by Conselho de Ensino e Pesquisa da UFRJ, and by Financiadora de Estudos e Projetos (FINEP). REFERENCES AMOS, W. B., GRIMSTONE, A. V., ROTHSCHILD, L. J., AND ALLEN, R. D. 1979. Structure, protein compo-

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