Possible role of cAMP in the differentiation of Trypanosoma cruzi

Molecular and Biochemical Parasitology, 22 (1987) 39-43

39

Elsevier MBP 00733

P o s s i b l e role of c A M P in the differentiation of Trypanosoma cruzi Rafael Rangel-Aldao 1, Oscar Allende 1, Francisco Triana 1, Romano Piras 2, Diana Henriquez 2 and Marta Piras 2 ICentro de Investigaciones BiomOdicas (BIOMED), Universidad de Carabobo, Nf~cleo Aragua, Carretera a Turmero, La Morita, Maracay, Venezuela and 2Centro Mddico-Docente La Trinidad, Urb. La Trinidad, Caracas, Venezuela

(Received 27 May 1986; accepted 28 July 1986)

To assess the possible action of cAMP on the cell differentiation of Trypanosoma cruzi, we determined both cAMP levels and cAMP-binding activities of epimastigotes and trypomastigotes of this parasite. Trypomastigotes showed a 4-fold higher cAMP content and a 2.5-fold increase in the specific activity of a cAMP-binding protein with identical properties to that of epimastigotes. The high levels of cAMP present in trypomastigotes strongly suggest a role of this cyclic nucleotide on the differentiation of T. cruzi.

Key words: cAMP; cAMP-binding proteins; Receptors; Epimastigotes; Trypomastigotes; Trypanosoma cruzi; Cell differentiation

Introduction

To study the mechanisms that mediate cell division and differentiation of T r y p a n o s o m a cruzi, it is necessary to identify specific molecules that may be responsible for the intracellular signaling mechanisms that trigger cell differentiation in response to changes in the parasite environment. The knowledge of such mechanisms may allow the design of new drugs capable of interfering with the ability of T. c r u z i to adapt and survive in its vertebrate hosts [1]. In lower eukaryotes, c A M P is an important signal for the control of cell division and differentiation of m a n y organisms [2,3], including Tryp a n o s o m a lewisi [4], T. brucei [5], and Leishmania [6]. In T. cruzi, we have extensively characterized the cAMP-binding activity of epimastigotes [1,7,8], and found that such activity resides in a unique-type of protein with very differCorrespondence address: Dr. R. Rangel-Aldao, Apartado Postal 2296, Maracay, 2101A, Venezuela. Abbreviations: PMSF, phenylmethylsulfonyl fluoride; Buffer

A; Tris-HC1 buffer containing iodoacetic acid and PMSF; Buffer B, Tris-HCl buffer containing iodoacetic acid, PMSF, and NaCI; SDS, sodium dodecyl sulfate.

ent properties from the well conserved cAMPbinding proteins of lower and higher eukaryotes [8]. In order to explore the possible role of c A M P and its cAMP-binding activity in cell differentiation of this parasite, we have determined the intracellular c A M P levels and the cAMP-binding activities of two developmental forms of T. cruzi, epimastigotes and trypomastigotes. Materials and Methods Cell culture and h o m o g e n i z a t i o n . Epimastigotes from the stock E.P. of T. cruzi were grown at 28°C in L I T medium, and harvested at late log phase as described before [7]. Tissue culture-derived trypomastigotes from the same stock of T. cruzi, recently liberated from infected monolayers of Vero cells, were isolated and filtered as reported in a previous publication [9]. c A M P - b i n d i n g assays. W e r e p e r f o r m e d as described by Ueland and Doskeland [10] with [3H]cAMP (26 Ci mmo1-1, The Radiochemical Centre, A m e r s h a m ) at a concentration of 1 IxM of the radioactive nucleotide in 100 Ixl of reaction mixture with 50 m M Tris-HC1 buffer, p H 7.5, 1 m M adenosine, 1 mg m1-1 bovine serum albu-

0166-6851/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

40

min, and sample volumes of 10-40 txl. After incubation for 30 min at 4°C, the reaction is terminated by adding 1 ml of 80% saturated solution of ammonium sulfate, and subsequent filtration on Millipore filters and two 3 ml washings with 60% saturated ammonium sulfate. The wet filters are immersed in 1 ml of 1% sodium dodecyl sulfate (SDS), and counted by liquid scintillation in 7 ml of Aquasol.

Determination of intracellular cAMP content. The cAMP content of epimastigotes and trypomastigotes of T. cruzi, was determined by radioimmunoassay using a kit from New England Nuclear (NEX-132), which employs acetylation of endogenous cAMP and succinyl cAMP tyrosine methyl ester-lZSI as a tracer. For a typical experiment, 2-5 × 108 cells were pelleted and resuspended with 500 ~xl of 0.1 N HC1 and submitted to three cycles of freeze and thawing using a solid CO2/acetone bath. The lysed cells were then boiled for 5 min and the volume of the suspension carefully measured. At this point, aliquots of 5-20 ~1 were taken to measure proteins by the method of Schaterle and Pollack [11]. The suspension was then centrifuged at 15000 x g for 3 min in a microcentrifuge and the cAMP concentration measured in the resulting supernatant with tripliclate samples of 10, 20, 30 and 40 ~1. Acetylation of the samples and cAMP determination was performed as recommended in the manufacturer's instructions, except for the following modifications: at the end of the incubation with the respective antibodies, the immunoprecipitates were filtered, instead of centrifuged, through G F A filters (25 mm diameter, from Whatman) and washed three times with 5 ml each of 0.05 M acetate buffer, pH 6.2. The filters were dried and immersed in 5 ml of omnifluor/toluene (4 g 1-1, w/v) and the radioactivity measured by liquid scintillation. Values are expressed as pmol of cAMP per mg of total protein. The intracellular concentrations of cAMP (~M) in epimastigotes and trypomastigotes were calculated from the respective cell volumes of these parasite forms, measured by [14C]inulin exclusion as described by Damper and Patton [12]. These values corresponded to 30 p~m3 and 12 ~m 3, respectively, for epimastigotes and trypomastigotes.

Statistical analyses of cAMP content of epimastigotes and trypomastigotes were made using the Student's t test.

DEAE-Sephacel chromatography. Cell pellets of 2 x 10 9 trypomastigotes or 1 x 10 9 epimastigotes, were resuspended in 500 ~1 of 50 mM TrisHC1 buffer, pH 7.5, containing 1 mM iodoacetic acid, 1 mM phenylmethylsulfonyl fluoride (PMSF) (buffer A), and homogenized by three cycles of freeze and thawing using a solid CO2/acetone bath. The respective homogenates were then chromatographed in a DEAE-Sephacel column (bed volume = 2 ml), previously equilibrated in the same buffer at 4°C. After washing with 10 ml of buffer A, containing 50 mM NaC1 (buffer B), each column was eluted with a linear gradient of 15 ml of buffer B vs 15 ml of 0.5 M NaC1 in buffer A. Fractions [20] of 1.5 ml each were collected and assayed (500 ~1) for [3H]cAMP binding activities. The concentration of NaCI in each fraction was measured with the aid of a conductivity meter. Sedimentation in sucrose density gradients. Cell pellets of 6 x 108 trypomastigotes or 2 x 108 epimastigotes, were resuspended in 100 p~l of buffer A and homogenized as described above. The homogenates were then subjected to sedimentation in sucrose density gradients [13] as described in a previous publication [1]. Fractions [25] of 200 p~l each were collected and assayed for [3H]cAMP binding activities. Sedimentation coefficient markers were bovine serum albumin ($20,w=4.31), alcohol dehydrogenase ($20,w=7.61), and catalase (S20,w: 11.3).

Results

Cyclic A M P content of epimastigotes and trypomastigotes of T. cruzi. Table I shows that trypomastigotes contain 4.3 times more intracellular cAMP than epimastigotes (2.62 -+ 0.29 pmol (mg protein) -1 vs 0.6 -+ 0.19 pmol(mg protein)-1). Such differences were also apparent when the respective values of cAMP were expressed as I~M concentrations (Table I), calculated from the measured cell volumes of both forms of T. cruzi (see Methods).

41 TABLE I Cyclic AMP content of epimastigotes and trypomastigotes of T. cruzi Parasite form

cAMP content (pmol (rag protein) -1)

(txM)

Epimastigotes

0.60 -+ 0.19 (9)

0.13 ± 0.04

Trypomastigotes

2.62 -+ 0,29 (6)

0.54 ± 0.06

Pellets containing live epimastigotes or trypomastigotes (5 x 107 cells) of T. cruzi, were analyzed for their cAMP content as described under Methods. The numbers in parenthesis refer to the number of experiments performed. P < 0.001.

cAMP-binding activities of epimastigotes and trypomastigotes of T. cruzi. T h e elevated c A M P levels of t r y p o m a s t i g o t e s o b s e r v e d in Table I, were also paralleled by a 2.6-fold increase in the c A M P binding activity of t r y p o m a s t i g o t e h o m o g e n a t e s , when c o m p a r e d with epimastigotes (see Table II). T h e c A M P - b i n d i n g activities of both forms of T. cruzi gave a 10 fold difference when assayed by two widely used m e t h o d s of c A M P - b i n d i n g det e r m i n a t i o n (Table II). This fact indicated a similarity in the two c A M P - b i n d i n g activities of epimastigotes and trypomastigotes, and a dissimilarity of b o t h proteins with respect to the other well c o n s e r v e d c A M P - b i n d i n g proteins of e u k a r y o t e s [8].

Sucrose density gradients of the cAMP-binding activities of epimastigotes and trypomastigotes of T. cruzi. T o establish w h e t h e r the c A M P - b i n d i n g activities of epimastigotes and trypomastigotes c o r r e s p o n d e d to the same type of r e c e p t o r [7], as suggested by the results indicated in Table II, we

analysed such activities by direct m e t h o d s of molecular separation. Fig. 1 shows that both c A M P binding activities present in h o m o g e n a t e s of the two forms of the parasite, displayed a similar behaviour u p o n centrifugation on sucrose-density gradients. As r e p o r t e d for epimastigotes [1], the trypomastigotes also displayed one single p e a k of c A M P - b i n d i n g activity with a $20.w=8.2, as det e r m i n e d f r o m the experimental data depicted in Fig. 1.

Behaviour on DEAE-Sephacel chromatography of the cAMP-binding activities of epimastigotes and trypomastigotes of T. cruzi. In agreement with the results o b t a i n e d by sedimentation on sucrose density gradients, Fig. 2 shows the similar behaviour of the c A M P - b i n d i n g activities of h o m o g e nates of epimastigotes and trypomastigotes during chromatography on DEAE-Sephacel. H o m o g e n a t e s of both forms of T. cruzi yielded one single p e a k of c A M P - b i n d i n g activity, which eluted at about 0.15-0.18 M NaC1 (Fig. 2).

TABLE II Specific cAMP binding activity of homogenates of epimastigotes and trypomastigotes of T. cruzi Parasite form

[3H]cAMP binding (pmol (mg of protein) 1)

Ratio 2:1

Method 1

Method 2

Epimastigotes

0.24 -+ 0.01

2.5 _+ 0.4 (10)

10.4

Trypomastigotes

0.57 - 0.03

6.4 _+ 0.2 (8)

11.2

Epimastigotes or trypomastigotes (1 × 1 0 9 cells) of T. cruzi, were homogenized and assayed in duplicates, for their cAMP binding activity in the presence of 1 ~M [3H]cAMP and 500 txM adenosine as described under Methods. Method 1 corresponds to Gilman's [14] and Method 2 to Ueland and Doskeland [10]. The numbers in parenthesis refer to the number of experiments performed. The ratio 2:1 represents the value obtained by dividing the results of method 1 by method 2. P<0.05.

42

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Fig. 1. Sucrose density gradient of the cAMP-binding activity of epimastigotes and trypomastigotes of T. cruzi. Homogenates of epimastigotes (3.4 x 10 s cells) (A), or trypomastigotes (6 x 108 cells) (B), were subjected to sedimentation in sucrose density gradients as indicated under Methods.

Discussion

The results presented in Table I showed that trypomastigotes contained 4 times more cAMP than epimastigotes. These latter cellular forms, capable of proliferation, displayed cAMP levels (0.13 ~xM) comparable to those reported by Strickler and Patton [4] for the reproducing forms of Trypanosoma lewisi (0.16 txM), whereas the non reproducing trypomastigotes of T. cruzi showed values somewhat higher (0.54 p~M) than those reported for the non proliferative forms, of T. lewisi (0.32 p~M) [4]. These results also give further support to the hypothesis of cAMP as being a negative regulator of the cell division of

Fig. 2. D E A E - S e p h a c e l chromatography of the cAMP-binding activities of epimastigotes and trypomastigotes of T. cruzi. Live pelleted epimastigotes (2 x 109 cells) (A) or trypomastigotes (1 x 10 9 cells) (B), were resuspended in 0.5 ml of buffer A, homogenized and chromatograpbed on a column of D E A E - S e p h a c e l (bed volume = 2 ml) as described under Methods.

trypanosomes [4,5], including T. cruzi. It was important to test in T. cruzi the validity of the above hypothesis, because we have previously found in epimastigotes of this parasite, a unique type of cAMP-binding protein which could be exploited chemotherapeutically because of its many differences from the evolutionary conserved counterparts of the vertebrate hosts of T. cruzi [1]. The results of Table II, show that this type of cAMP-binding activity is also present at elevated levels (2.5 x) in trypomastigotes, and displayed a similar behaviour to the one described in epimastigotes when assayed by two widely used methods of assay of cAMP-binding activity [7]. In this respect, the trypomastigote

43 c A M P - b i n d i n g p r o t e i n was also similar to o t h e r s that we h a v e r e p o r t e d in s e v e r a l T r y p a n o s o m a t i d a e , such as T. brucei, Leishmania tropica, a n d Crithidia luciliae [15]. Figs. 1 a n d 2, d e m o n s t r a t e d t h a t t h e c A M P b i n d i n g activity o f t r y p o m a s t i g o t e s r e s i d e d in a p e a k with p r o p e r t i e s such as s e d i m e n t a t i o n coefficient ($20,w=8.2), and elution from D E A E - S e p h a c e l c h r o m a t o g r a p h y (Fig. 2) that w e r e similar to t h e e p i m a s t i g o t e s c A M P - b i n d i n g p r o t e i n . T h e s e results, t a k e n t o g e t h e r with t h o s e p r e s e n t e d in T a b l e II, strongly suggest that all the c A M P - b i n d i n g activity o f t r y p o m a s t i g o t e s res i d e d in t h e s a m e t y p e o f r e c e p t o r t h a t we h a v e d e s c r i b e d for e p i m a s t i g o t e s ( C A R P T ) [1,7] a n d that c o n t r a r y to t h e case o f o t h e r T r y p a n o s o m a t i d a e , such as T. brucei a n d T. gambiense w h e r e two t y p e s o f c A M P - b i n d i n g p r o t e i n s h a v e b e e n d e s c r i b e d [15,16], t h e a c t i o n of c A M P in t r y p o m a s t i g o t e s o f T. cruzi a p p e a r s to involve o n e single t a r g e t p r o t e i n . T h e e l e v a t e d c A M P levels o f t r y p o m a s t i g o t e s

with r e s p e c t to the e p i m a s t i g o t e s suggest a role for c A M P in t h e i n t r a c e l l u l a r signaling m e c h a nism t h a t triggers the cell d i f f e r e n t i a t i o n of T. cruzi. T h e i n c r e a s e d specific activity o f a c A M P b i n d i n g p r o t e i n in t r y p o m a s t i g o t e s , with p r o p e r ties similar to the o n e d e s c r i b e d in e p i m a s t i g o t e s ( T a b l e II, Figs. 1 a n d 2), suggests that the a c t i o n of c A M P in the d i f f e r e n t i a t i o n o f T. cruzi is p r o b a b l y m e d i a t e d by this t y p e of r e c e p t o r .

Acknowledgements W e t h a n k D r . E. C a y a m a for his critical c o m m e n t s on t h e m a n u s c r i p t . This i n v e s t i g a t i o n received financial support from the U N D P / W O R L D B A N K / W H O S p e c i a l P r o g r a m m e for R e s e a r c h a n d T r a i n i n g in T r o p i c a l D i s e a s e s , a n d f r o m G r a n t s CICS-1 a n d C I C S - 6 f r o m C o n s e j o N a c i o n a l de I n v e s t i g a c i o n e s Cientificas y T e c n o l 6 gicas ( C O N I C I T ) , and from C O D E C I H , a w a r d e d to R . R - A .

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9 Piras, M.M., Piras, R. and Henriquez, D. (1982) Changes in morphology and infectivity of cell culture-derived trypomastigotes of Trypanosoma cruzi. Mol. Biochem. Parasitol. 6, 67-81. 10 Ueland, P.M. and Doskeland, S.O. (1976) Adenosine 3':5'cyclic monophosphate-dependence of protein kinase isoenzymes from mouse liver. Biochem. J. 157, 117-126. 11 Schaterle, G.R. and Pollack, R.L. (1973) A simplified method for the quantitative assay of small amounts of protein in biological materials. Anal. Biochem. 51,654-655. 12 Damper, D. and Patton, C.L. (1976) Pentamidine transport and sensitivity in brucei-group trypanosomes. J. Protozool. 23, 349-356. 13 Martin, R.G. and Ames, B.N. (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. Biol. Chem. 236, 1372-1379. 14 Gilman, A.G. (1970) A protein binding assay for adenosine 3':5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A. 67, 305-312. 15 Rangel-Aldao, R. and Opperdoes, F.R. (1984) Subcellular distribution and partial characterization of the cyclic AMP-binding proteins of Trypanosoma brucei. Mol. Biochem. Parasitol. 10,231-241. 16 Walter, R.D. (1978) Adenosine 3':5'-cyclic monophosphate binding proteins from Trypanosoma gambiense. Hoppe-Seyler's Z. Physiol. Chem. 359, 607-612.