ARTÍCULO ORIGINAL

Microtubule cytoskeleton distribution using fluorescent
taxoid in Tetratrichomonas didelphidis

SHEILA B LECKE*, TIANA TASCA*/**, ANDRÉ A SOUTO***, FERNANDA P. BORGES*,
RENATA C. M. WILTUSCHING* and GERALDO A DE CARLI*/⁺

ABSTRACT

Tetratrichomonas didelphidis is a flagellated protozoan found in the intestine of opossums. The specimens were stained by the Giemsa method and by FLUTAX-2, an active fluorescent derivative of Taxol which binds to the ab-tubulin polimerized of microtubules of cells. Giemsa stain revealed the morphological features of trichomonads such as four anterior flagella, undulating membrane, axostyle and posterior flagellum. An intense fluorescence was observed in living trophozoites of T. didelphidis and Trichomonas vaginalis (used as control), incubated with FLUTAX-2. An analysis of the composition of the cytoskeleton of T. didelphidis will contribute to understanding the cellular morphology of the parasites.
Key words: Tetratrichomonas didelphidis, microtubule cytoskeleton, fluorescent taxoid.

INTRODUCTION

Few trichomonad species have a proven pathogenic potential for mammals and birds. In general, the species inhabiting trichomonad primary sites, i. e., the large intestine, appear to be non pathogenic. The species with proven pathogenic potential have evolved to inhabit areas other than the large intestine of their hosts¹. Tetratrichomonas didelphidis is a flagellate protozoan found in the intestine, cecum and colon, of the opossum Didelphis marsupialis², D. albiventris³, and Lutreolina crassicaudata⁴. The taxonomy of trichomonads has had a complex history. This species was first described with the name Trichomonas didelphidis⁵. Subsequently, others authors described the species again using the correct genus, Tetratrichomonas⁵.

Microtubules are essential for the maintenance of cell shape and organization; for cytoplasmic transport, motility and division in eukariotic cells. In addition, they are targets of antimitotic drugs, including colchicines, vinblastine, and Taxol (paclitaxel)⁶. Taxol blocks the cell division and caused its death^7-9, being widely used as a microtubule stabilizing agent. It is employed in the treatment of ovarian, breast, lung, head and neck, cervix carcinomas and lymphomas¹⁰. Recently, Taxol is used in experiments of transient invagination of flagella by Tritrichomonas foetus to stabilizing microtubules¹¹ and in the study of resistance to arsenite in Leshmania donovani¹². Fluorescent Taxol derivatives can be extremely useful in the study of cellular microtubules, as well as the mechanisms of microtubule assembly and stabilization by Taxol^13,14. Flutax-2¹⁵ (Figure 1), an active fluorescent derivative of Taxol, binds to ab-tubulin dimer polymerized. It interacts specifically with the Taxol binding site, probably at the outer microtubule surface¹⁶, making them both directly and rapidly observable under fluorescence microscope. Cytoskeleton of Ptk2 epithelial cells, U937 leukemia cells, neuro2A neuroblastoma cells, and Trypanosoma cruzi was stained with FLUTAX¹⁴.

There are few reports regarding the biological aspects of T. didelphidis^2-4,17,18. Take into account that the cytoskeleton of T. didelphidis is completely unknown, in this study we present the analysis of microtubule distribution in living trophozoites of T. didelphidis using the fluorescent taxoid Flutax-2. This probe, 7-O-[N-(4´-fluoresceincarbonyl)-L-alanyl]Taxol (FLUTAX-2), was obtained by the reaction of 7-O-(L-alanyl) Taxol with the corresponding amine-reactive fluorescent dye, as described¹⁵. FLUTAX-2 had ultraviolet (UV)-visible absorbance and fluorescence emission spectra in accordance with the respective chromophores. Analysis by high-performance liquid chromato-graphy (HPLC) FLUTAX-2 showed a purity (228 nm) of 91% (0.3% Taxol). The concentration of FLUTAX-2 was determined spectrophoto-metrically in 0.5% sodium dodecyl sulphate (SDS), 50 mM sodium phosphate buffer, pH 7.0, employing the practical extinction coefficient Î_458nm = 23,100 ± 600 M^-1 cm^-1, obtained by weighing and by SDS solubilization of the taxoid.

MATERIALS AND METHODS

The TDLC01 strain of T. didelphidis used in this study was isolated from the swabbed rectum of L. crassicaudata and cultivated in Diamond's¹⁹ modified trypticase-yeast extract-starch (TYS) medium, without maltose and with starch solution, pH 7.5 (5 mg/ml)¹⁷. The cells were grown in vitro at 28ºC (± 0.5) in TYS medium supplemented with 10 % (v/v) heat inactivated bovine serum, penicillin (1000 IU/ml) and streptomycin sulphate (1 mg/ml). Certain samples were frozen and maintained at -196°C with 5% (v/v) of dimethyl sulfoxide (DMSO) as previously described²⁰.

One Trichomonas vaginalis strain was used in this study as control for FLUTAX-2 binding²¹. The 30238 strain was obtained from the American Type Culture Collection (ATCC). The strain was cultivated axenically in vitro at 37°C, in trypticase-yeast extract-maltose (TYM), Diamond medium¹⁹ without agar, pH 6.0, supplemented with 10% heat inactivated bovine serum, penicillin (1,000 IU/ml) and streptomycin sulphate (1 mg/ml). The trichomonads in the logarithmic phase of growth exhibited more than 95% mobility and normal morphology.

Trophozoites of T. didelphidis and T. vaginalis strains were counted in a hemocytometer to obtain 10⁶ cells/ml. Intact cells were incubated with FLUTAX-2 (1 mM, final concentration) at 37°C, during 40 min, and observed in a Zeiss fluorescence microscope.

RESULTS AND DISCUSSION

Figures 2 and 3 are from T. didelphidis TDLC01 strain, stained with Giemsa as previously described³, to show the protozoan structure by light microscopy. There were four anterior flagella unequal in size in the mature individuals, and a well-developed undulating membrane extended almost the entire length of the body (Figures 2, 3). The undulating membrane often exhibits several bold waves and the outer margin of the membrane consists of the accessory filament, which extends into a free posterior flagellum (Figures 2, 3). There was a spheroidal, ellipsoidal, or ovoid nucleus situated in the anterior portion of the body (Figures 2, 3), clearly discernible in most living and stained specimens. Anterior to the nucleus was a blepharoplast where the flagella were inserted (Figures 2, 3). Posterior to the nucleus the capitulum continues as a slender, hyaline, somewhat attenuating axostylar trunk that courses near the anteroposterior axis of the organism, its terminal segment projecting approximately one-third the cell length beyond the posterior surface of the flagellate (Figure 2). Both trichomonad species displayed an intense fluorescence in the axostyle, centrossome and flagella of living cells, strongly suggesting the microtubule composition of these structures (Figures 4 - 6). Figures 4 and 5 show the trophozoites of T. didelphidis after incubation with FLUTAX-2. Structures such as centrossome, axostyle and the anterior flagella jointed, can be observed (Figure 4). Figure 6, 30238 strain, show the same structures as above: the centrossome, the axostylar position through the cell shape and, the anterior flagella, arranged in two groups, as described in some organisms²².

As shown by Lopes et al²³, using specific monoclonal antibodies, T. vaginalis presents a-tubulin at the flagella, basal bodies and the axostyle. ab-tubulin is present at the axostyle and flagella too. Our data are in accordance with them²³, since FLUTAX-2 binds to ab-tubulin dimmers, for both trichomonad species. The axostyle contains acetylated tubulin²³, a characteristic isoform of stable microtubules²⁴, contrary to the findings of others who claim that this structure is not permanent^25-27.

The direct observation of microtubules with FLUTAX-2 in living T. didelphidis and T. vaginalis trophozoites presents an advantage over indirect immunofluorescence and does not require microinjection of individual cells to introduce the probe. As shown by Evangelio et al ¹⁴, it allows diverse morphological applications, including for example, the taxonomy and cell cycle characte-rization of flagellates and ciliates. Moreover, the major advantage of this method is the absence of cell fixation, and consequent death, which can mask the true morphological characteristics of living cells.

Our results showed the binding of FLUTAX-2 in two trichomonad species. An analysis of the composition of the cytoskeleton of T. didelphidis will contribute to understanding the biochemical aspects involved in cellular morphology and behavior as well as the mechanisms related to host-parasite interactions.

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Acknowledgements. The authors thank Dr. Sérgio De Meda Lamb, Pharmacy School Chairman for his support; Dr. Denise C. Machado, IPB, PUCRS, for microscope assistance. S. B. L. is recipient of a Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) fellowship (# 00501323), Brazil, and T. T. of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. This study received grant from CNPq (# 470148/ 01-2)
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* Laboratório de Parasitologia Clínica, Faculdade de Farmácia
** Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
*** Laboratório de Síntese Orgânica, Faculdade de Química, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil.
+ Corresponding author. FAX: + 55 51 33332.2582. E-mail gdecarli@portoweb.com.br