Acta Tropica, 50(1992)79 85
79
© 1991 Elsevier Science Publishers B.V. All rights reserved. 0001-706X/91/$03.50 ACTROP 00169
Phloretin and citrate promote the differentiation rate from epimastigote to metacyclic forms of Trypanosoma cruzi F.J. Adroher 1, A. Osuna*'l and J.A. Lupififiez 2 1Department of Parasitology, Faculty of Pharmacy, University of Granada, Granada, and 2Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Granada, Spain (Received 19 November 1990; accepted 31 May 1991)
We have investigated the effects of the metabolic inhibitors, phloretin, 2-deoxy-o-glucose, maleate and trans-aconitate, as well as two intermediates of the tricarboxylic-acid cycle, acetate and citrate, on the growth and metacyclogenesis of Trypanosoma cruzi, 0.1 mM phloretin increased the percentage of metacyclic forms about 2.7-fold without affecting growth rate, whereas the other inhibitors had no apparent effect on either growth or differentiation rates. The addition of 5 mM citrate stimulated differentiation by about 2.6-fold. When either 10 mM citrate or 10 mM acetate were added, on the other hand, both the growth and differentiation rates were severely inhibited. Key words: Trypanosoma cruzi; Differentiation; Growth; Metabolic inhibitors; Metabolic intermediates
Introduction During recent years it has been observed that during differentiation from one stage to another in the biological cycle of some kinetoplastids their carbohydrate metabolism undergoes a marked change. Thus, Trypanosoma brucei bloodstream forms are highly dependent on glycolysis while the metabolism of the procyclic forms is very oxidative (Bowman et al., 1972; Brown et al., 1973). Bearing in mind the activation of the tricarboxylic-acid (TCA) cycle during the bloodstream-procyclic differentiation of T. brucei, some authors have introduced varying concentrations of several tricarboxylic-acid-cycle intermediates into the composition of the differentiation culture media, thus enhancing the differentiation rates from the bloodstream to procyclic forms of this parasite (Brun and Sch6nenberger, 1981; Simpson et al., 1985; Czichos et al., 1986; Overath et al., 1986). In the same way, the carbohydrate metabolism of T. cruzi changes during the differentiation process from the epimastigote to metacyclic form, the activity of the key glycosomal enzymes responsible for glycolysis being reduced at the same time Correspondence address: Dr. F.J. Adroher, Departamento de Parasitologia, Facultad de Farmacia, Campus Universitario de Cartuja, Universidad de Granada, E-18071 Granada, Spain. *Present address: Department of Parasitology, Faculty of Sciences, University of Granada, E-18001 Granada, Spain.
80 as the activity of the key enzymes for the TCA cycle increases (Adroher et al., 1988b, 1990). The aim of this work has, therefore, been to study the effects of certain glycolysis and TCA-cycle inhibitors and also two intermediates of carbohydrate metabolism on the growth and metacyclogenesis of T. cruzi,
Materials and Methods Parasites and culture conditions The T. cruzi strain used came originally from a human clinical case in Maracay,
Venezuela. It is habitually cultured in our laboratory at 28°C in NNN medium, with a liquid phase made up of Eagle's minimal essential medium with Earle's salts (MEM) plus 20% (v/v) foetal calf serum (FCS), inactivated at 56°C for 30 rain (IFCS). The flagellates were harvested at the end of the exponential growth phase by centrifugation at 3000 × g for 10 rain, washed three times under the same conditions in saline phosphate buffer (PBS), pH 7.2, and finally introduced into the assay medium. The epimastigote forms were inoculated into 17 ml Leighton flasks (Bellco, Vineland, N J) containing 2 ml of Grace's medium supplemented with 10% (v/v) IFCS to a final concentration of approximately 1 × 1 0 6 organisms/ml. The initial pH was always 6.2 and the incubation temperature was 28°C. Parasite counting
Parasite multiplication was ascertained by counting in a Neubauer haemocytometric chamber. The percentage of metacyclic forms was calculated by staining a drop of the medium with 10% (v/v) Giemsa in PBS, and evaluating at least 400 parasites. The terms maximum cell density (MCD) and maximum differentiation (MD) are defined respectively as the mean of the maximum number of organisms, expressed as millions/ml, and as the mean of the maximum percentage of metacyclic forms, for each experiment at different times of culture. Statistical method
Significant values were considered as being P < 0.05 according to Student's t-test. Products
The Grace's medium, MEM and FCS were from Gibco Europe (Gent, Belgium). Chemicals used were from Riedel de Hafin (Seelze, Hannover, F.R.G.) and from Sigma Chemical (St. Louis, MO).
Results
We have observed the effect of adding inhibitors glucose metabolism (phloretin and 2-deoxy-o-glucose) and the tricarboxylic-acid cycle (maleate and trans-aconitate) as well as metabolic intermediates (acetate and citrate) to Grace's medium on the growth and differentiation of T. cruzi.
81
Phloretin, an inhibitor of the plasma-membrane transport of glucose, had no effect on the growth of T. cruzi (Fig. IA), but it did provoke a noticeable and significant increase (P < 0.005, Table I) in the differentiation into metacyclic forms when added at a concentration of 0.1 mM (Fig. 1B). Nevertheless, when this inhibitor was added at 1 mM it ceased to have any effect on differentiation. 2-deoxy-D-glucose, an inhibitor of glycolysis, had no effect on either growth or differentiation at any of the concentrations tested (5 mM and 10 mM) (Fig. 1 and Table 1). As for the effects of the two TCA-cycle inhibitors, when maleate was added to the culture medium no change in the growth process was apparent, although there was a very slight increase in the differentiation rate, while the addition of trans-aconitate (1 mM) led also to a slight, although non-significant, increase in differentiation percentages. At higher concentrations this latter inhibitor had no evident effect on either growth or differentiation rates (Fig. 2 and Table 1). Neither acetate nor citrate had any influence on cell density at concentrations of 1 mM and 5 mM, but at 10 mM growth rate was severely inhibited (Figs. 3 and 4, and Table 1). 5 mM citrate produced a significant increase in differentiation rates ( P < 0.005), while a slight increase was observed with 1 mM citrate or 5 mM acetate. When the concentration of both citrate and acetate reached 10 mM, however, the percentage of metacyclic forms diminished drastically along with the growth rates (Figs. 3 and 4, and Table 1).
Discussion We have studied the influence of substances capable of modifying certain specific pathways in the intermediate metabolism of eukaryotic cells. In previous papers (Adroher et al., 1988b, 1990) we have reported important and significant glycolytic and mitochondrial metabolic alternations during the metacyclogenesis of T. cruzi. The activities of glycolytic enzymes (hexokinase and phospho100-
25" B
20" A
~; (J
15
~o
i lo 5
1
2
4 6 8 10 DAYS IN CULTURE
12
O - -
6
,8 10 12 DAYS IN CULTURE
Fig. 1. Growth (A) and differentiation (B) o f Trypanosoma cruzi in Grace's medium ( × ) added with phloretin at 0.1 mM (O) and I mM (O), and 2-deoxy-o-glucose at 5 mM (IS]) and 10mM (11).
82 TABLE 1 Effect of several metabolic inhibitors and intermediate metabolites on maximum cell density (MCD) and maximum differentiation (MD) values of T. cruzi in Grace's medium Substances
MCD a
Day b
MD a
Day c
None Phloretin (0.1 mM) Phloretin (1 mM) 2-deoxy-o-glucose (5 raM) 2-deoxy-D-glucose (10 mM)
28.64_+2.88 30.61 + 1.08 29.96_+ 1.41 28.80_+ 2.83 27.92 _+2. I 1
10.0+0.6 9.5+0.9 9.5+ 1.0 11.5 _+0.5 9.5 4- 0.5
8.1 _+ 1.6 22.0+ 1.2"* 10.2+ 1.3 11.8 4_ 2.3 4.3 _+0.9
11.3 +0.7 11.0+0.6 10.54_0.5 10.0 + 0.5 12.0 4_0.0
None Maleate (1 mM) Maleate (10 mM) Trans-aconitate (l mM) Trans-aconitate ( I 0 mM )
24.60 4-1.16 24.72 4- 2.48 25.76_+ 1.44 24.16_+ 1.13 21.36 _+0.56
10.0___ 1.2 10.0 ___1.2 10.7+0.7 11.5_+0.5 12.0 _+0.0
8.7 + 1.3 11.0 + 2. I 15.3 4-2.9 12.7_+2.3 6.3 + 0.8
11.0___0.6 11.3 4- 0.7 12.0_+0.0 12.0_+0.0 10.7 -+ 0.7
None Acetate (1 mM) Acetate (5 mM) Acetate (10 mM)
23.924-2.08 25.68 ± 3.12 24.16_+2.17 5.92_+0.75*
10.0_+ 1.2 10.5 _+0.5 11.0_+0.6 8.0_+0.0
8.5_+ 1.2 9.3 4- 1.9 12.2_+2.5 1.8_+0.3"
12.0-+_0.0 10.7 -+0.7 12.0+0.0 11.3 ___0.7
None Citrate (1 mM) Citrate (5 mM) Citrate (10 mM)
25.764-2.51 23.20_+2.08 19.84+0.80 4.80_+0.32*
10.0_+0.8 8.7_+0.7 10.0_+ 1.2 11.3 _+0.7
9.3 4_ 1.5 12.1 _+2.2 23.8_+ 1.5"* 1.0+0.2"
12.0_+0.0 11.3+0.7 12.0+ 0.0 10.0_+ 1.2
The results are the mean-+_ SEM of three or more experiments. Statistical significance (Student's t-test) compared to control: *P < 0.05; **P < 0.005. aMCD (106 cells/ml) and M D (% metacyclic forms) terms are defined in the Materials and Methods section. bCulture day on which the cell density was maximum in each experiment. ~Culture day on which the differentiation was maximum in each experiment.
100-
25 B
20
to 15 o
lO, o 10
5
2
4 6 8 10 DAYS IN CULTURE
12
0
6
8 10 12 DAYS IN CULTURE
Fig. 2. Growth (A) and differentiation (B) of Trypanosoma cruzi in Grace's medium ( × ) added with maleate at 1 m M (©) and 10mM (O), and trans-aconitate at 1 mM (O) and 10mM (111).
83 100
25 ¸ 8
20
15. 10' 10
5
2
4 6 8 10 DAYS IN CULTURE
12
04
6
8 10 12 DAYS IN CULTURE
Fig. 3. Growth (A) and differentiation (B) of Trypanosoma cruzi in Grace's medium (©) added with acetate at 1 mM ( 0 ) , 5 mM (El), and 10raM (B). 100
25. B
20.
15, 10¸ lO
s
2
4 6 8 lO DAYS IN CULTURE
12
o
,._----; S
8
10
12
OArS ~N CULTURE
Fig. 4. Growth (A) and differentiation (B) of Trypanosoma cruzi in Grace's medium (©) added with citrate at 1 mM (O), 5 m M (Tq), and 10mM (11).
fructokinase) in the glycosomes of the epimastigote forms are higher than in the metacyclic forms. Nevertheless, the activities of mitochondrial enzymes, citrate synthase, NADP-linked isocitrate dehydrogenase and succinate dehydrogenase were higher in the metacyclic forms than in the epimastigotes, which preferably use the carbon skeleton of carbohydrate as their energy source (Cficeres and Fern~indes, 1976). We have also shown that there is a considerable and significant increase in the differentiation rates in parasites grown in a glucose- and fructose-free medium (Adroher et al., 1988a; Osuna et al., 1990). Thus, the flagellates cultured in the presence of 0.1 mM phloretin, an inhibitor of glucose transport in eukaryotic cells, differentiated in higher numbers than the controls (Fig. 1B and Table 1). 2-deoxy-oglucose, however, had no apparent effect on the differentiation of T. cruzi, although
84 it did stimulate the differentiation process of the insect trypanosomatid Herpetomonas samuelpessoai when added at concentrations as high as 30.5 mM (Angluster et al., 1977). Several authors have shown the effect of the TCA-cycle intermediates on the differentiation of bloodstream forms to procyclic forms of T. brucei. Brun and Sch6nenberger (1981) have shown that citrate and/or cis-aconitate stimulate this differentiation and Overath et al. (1986) have reported that when these metabolites are added at 37°C to bloodstream forms an increase in the synthesis of aconitase and proline oxidase takes place. The former enzyme transforms citrate and cis-aconitate into isocitrate and the latter transforms proline into pyrroline-5-carboxylate. In this way, it has been demonstrated that some amino acids, such as proline, glutamate, glutamine, aspartate or asparagine, are capable of inducing metacyclogenesis in T. cruzi (Goldenberg et al., 1984; Contreras et al., 1985; Homsy et al., 1989; Krassner et al., 1990). Likewise, they can be metabolized by eukaryotic cells into pyrroline-5-carboxylate, as mentioned by Homsy et al. (I 989) and Krassner et al. (1990). Furthermore, Overath et al. (1983) have shown that the enzyme pyrroline-5-carboxylate reductase, not expressed in bloodstream forms of T. brucei, appears early in its differentiation process into procyclic forms, which permits it to use proline as a main carbon source. We have tested the effect of acetate and citrate on the growth and metacyclogenesis of T. cruzi. When the concentrations of these substrates were increased from 1 mM to 5 mM the growth rates stayed the same but the percentage of metacyclic forms increased significantly in the media containing 5 mM citrate. At concentrations of 10 mM the two substrates had an inhibitory effect both on growth and differentiation rates (Figs. 3 and 4, and Table 1). The use of inhibitors of Krebs' cycle, such as trans-aconitate and maleate, had no effect on either growth or differentiation rates, at least at the concentrations assayed (Fig. 2 and Table I). The factors involved in the metacyclogenetic mechanism are multiple. Elsewhere (Adroher et al., 1988a; Osuna et al., 1990) we have shown the existence of several apparently independent factors: the presence of NaC1 and the absence of monosaccharides, and the sum of their combined effects. Many other authors have described numerous such factors involved in this differentiation process. In this paper we have noted the positive effect of exogenous citrate at certain concentrations on the metacyclogenesis of T. cruzi. It would appear, however, that the basal levels of metacyclogenesis that occur under control conditions do not depend upon endogenous citrate, since trans-aconitate did not reduce the metacyclic percentage to below that of the control medium, despite the fact that this compound inhibits aconitase, the enzyme which transforms citrate into cis-aconitate. Moreover, Overath et al. (1986), working with T. brucei, have reported that the addition of citrate and/or cis-aconitate to the differentiation media increases the synthesis of aconitase and proline oxidase. At the moment we are furthering our research to discover exactly which metabolite in the citrate metabolism is that responsible for triggering differentiation in T. cruzi.
Acknowledgments We thank Dr. Rocio Benitez-Rodriguez for her advice and collaboration, and Dr. John Trout of the Scientific Translation Service of Granada University for revising the English text.
85
References Adroher, F.J., Lupi~ifiez, J.A. and Osuna, A. (1988a) Influence of saccharides and sodium chloride on growth and differentiation of Trypanosoma cruzi. Cell Differ. 22, 165-170. Adroher, F.J., Osuna, A. and Lupififiez, J.A. (1988b) Differential energetic metabolism during Trvpanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase, and succinate dehydrogenase. Arch. Biochem. Biophys. 267, 252-261. Adroher, F.J., Osuna, A. and Lupififiez, J.A. (1990) Differential energetic metabolism during Trypanosoma cruzi differentiation. I1. Hexokinase, phosphofructokinase, and pyruvate kinase. Mol. Cell. Biochem. 94, 71-82. Angluster, J., Bunn, M.M. and De Souza, W. (1977) Effect of 2-deoxy-o-glucose on differentiation of Herpetomonas samuelpessoai. J. Parasitol. 63, 922-924. Bowman, I.B.R., Srivastava, H.K. and Flynn, I.W. (1972) Adaptations in oxidative metabolism during the transformation of Trypanosoma rhodesiense from bloodstream into culture form. In: H. Van den Bossche (Ed.), Comparative biochemistry of parasites. Academic Press, New York, London, pp. 329-342. Brown, R.C., Evans, D.A. and Vickerman, K. (1973) Changes in oxidative metabolism and ultrastructure accompanying differentiation of the mitochondrion in Trypanosoma brucei. Int. J. Parasitol. 3,691-704. Brun, R. and Sch6nenberger, M. (1981) Stimulating effect of citrate and cis-aconitate on the transformation of Trypanosoma brucei bloodstream forms to procyclic forms in vitro. Z. Parasitenkd. 66, 17-24. Cficeres, O. and Fernfindes, J.F. (1976) Glucose metabolism, growth and differentiation of Trypanosoma cruzi. Rev. Bras. Biol. 36, 397-410. Contreras, V.T., Salles, J.M., Thomas, N., Morel, C.M. and Goldenberg, S. (1985) In vitro differentiation of Trypanosoma cruzi under chemically defined conditions. Mol. Biochem. Parasitol. 16, 315-327. Czichos, J., Nonnengaesser, C, and Overath, P. (1986) Trypanosoma brucei: cis-aconitate and temperature reduction as triggers of synchronous transformation of bloodstream to procyclic trypomastigotes in vitro. Exp. Parasitol. 62, 283-291. Goldenberg, S., Contreras, V.T., Salles, J.M., Bonaldo, M.C., Lima Franco, M.P.A. de, Linss, J., Lafaille, J., Valle, D. and Morel, C.M. (1984) Facts and hypothesis on Trvpanosoma cruzi differentiation. Mem. Inst. Oswaldo Cruz 79 (Suppl.) 39-44. Homsy, J.J., Granger, B. and Krassner, S.M. (1989) Some factors inducing formation of metacyclic stages of Trypanosoma cruzi. J. Protozool. 36, 150 153. Krassner, S.M., Granger, B., Phermsangngnarn, P., Le, T. and Linden, V. (1990) Further studies on substrates inducing metacyclogenesis in Trypanosoma cruzi. J. Protozool. 37, 128-132. Osuna, A., Adroher, F.J. and Lupi/tfiez, J.A. (1990) Influence of electrolytes and non-electrolytes on growth and differentiation of Trypanosoma cruzi. Cell Differ. Develop. 30, 89-95. Overath, P., Czichos, J., Stock, U. and Nonnengaesser, C. (1983) Repression of glycoprotein synthesis and release of surface coat during transformation of Trypanosoma brucei. EMBO J. 2, 1721-1728. Overath, P., Czichos, J. and Haas, C. (1986) The effect of citrate/cis-aconitate on oxidative metabolism during transformation of Trypanosoma brucei. Eur. J. Biochem. 160, 175-182. Simpson, A.M., Hughes, D. and Simpson, L. (1985) Trypanosoma brucei: Differentiation of in vitrogrown bloodstream trypomastigotes into procyclic forms. J. Protozool. 32, 672-677.
79
© 1991 Elsevier Science Publishers B.V. All rights reserved. 0001-706X/91/$03.50 ACTROP 00169
Phloretin and citrate promote the differentiation rate from epimastigote to metacyclic forms of Trypanosoma cruzi F.J. Adroher 1, A. Osuna*'l and J.A. Lupififiez 2 1Department of Parasitology, Faculty of Pharmacy, University of Granada, Granada, and 2Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Granada, Spain (Received 19 November 1990; accepted 31 May 1991)
We have investigated the effects of the metabolic inhibitors, phloretin, 2-deoxy-o-glucose, maleate and trans-aconitate, as well as two intermediates of the tricarboxylic-acid cycle, acetate and citrate, on the growth and metacyclogenesis of Trypanosoma cruzi, 0.1 mM phloretin increased the percentage of metacyclic forms about 2.7-fold without affecting growth rate, whereas the other inhibitors had no apparent effect on either growth or differentiation rates. The addition of 5 mM citrate stimulated differentiation by about 2.6-fold. When either 10 mM citrate or 10 mM acetate were added, on the other hand, both the growth and differentiation rates were severely inhibited. Key words: Trypanosoma cruzi; Differentiation; Growth; Metabolic inhibitors; Metabolic intermediates
Introduction During recent years it has been observed that during differentiation from one stage to another in the biological cycle of some kinetoplastids their carbohydrate metabolism undergoes a marked change. Thus, Trypanosoma brucei bloodstream forms are highly dependent on glycolysis while the metabolism of the procyclic forms is very oxidative (Bowman et al., 1972; Brown et al., 1973). Bearing in mind the activation of the tricarboxylic-acid (TCA) cycle during the bloodstream-procyclic differentiation of T. brucei, some authors have introduced varying concentrations of several tricarboxylic-acid-cycle intermediates into the composition of the differentiation culture media, thus enhancing the differentiation rates from the bloodstream to procyclic forms of this parasite (Brun and Sch6nenberger, 1981; Simpson et al., 1985; Czichos et al., 1986; Overath et al., 1986). In the same way, the carbohydrate metabolism of T. cruzi changes during the differentiation process from the epimastigote to metacyclic form, the activity of the key glycosomal enzymes responsible for glycolysis being reduced at the same time Correspondence address: Dr. F.J. Adroher, Departamento de Parasitologia, Facultad de Farmacia, Campus Universitario de Cartuja, Universidad de Granada, E-18071 Granada, Spain. *Present address: Department of Parasitology, Faculty of Sciences, University of Granada, E-18001 Granada, Spain.
80 as the activity of the key enzymes for the TCA cycle increases (Adroher et al., 1988b, 1990). The aim of this work has, therefore, been to study the effects of certain glycolysis and TCA-cycle inhibitors and also two intermediates of carbohydrate metabolism on the growth and metacyclogenesis of T. cruzi,
Materials and Methods Parasites and culture conditions The T. cruzi strain used came originally from a human clinical case in Maracay,
Venezuela. It is habitually cultured in our laboratory at 28°C in NNN medium, with a liquid phase made up of Eagle's minimal essential medium with Earle's salts (MEM) plus 20% (v/v) foetal calf serum (FCS), inactivated at 56°C for 30 rain (IFCS). The flagellates were harvested at the end of the exponential growth phase by centrifugation at 3000 × g for 10 rain, washed three times under the same conditions in saline phosphate buffer (PBS), pH 7.2, and finally introduced into the assay medium. The epimastigote forms were inoculated into 17 ml Leighton flasks (Bellco, Vineland, N J) containing 2 ml of Grace's medium supplemented with 10% (v/v) IFCS to a final concentration of approximately 1 × 1 0 6 organisms/ml. The initial pH was always 6.2 and the incubation temperature was 28°C. Parasite counting
Parasite multiplication was ascertained by counting in a Neubauer haemocytometric chamber. The percentage of metacyclic forms was calculated by staining a drop of the medium with 10% (v/v) Giemsa in PBS, and evaluating at least 400 parasites. The terms maximum cell density (MCD) and maximum differentiation (MD) are defined respectively as the mean of the maximum number of organisms, expressed as millions/ml, and as the mean of the maximum percentage of metacyclic forms, for each experiment at different times of culture. Statistical method
Significant values were considered as being P < 0.05 according to Student's t-test. Products
The Grace's medium, MEM and FCS were from Gibco Europe (Gent, Belgium). Chemicals used were from Riedel de Hafin (Seelze, Hannover, F.R.G.) and from Sigma Chemical (St. Louis, MO).
Results
We have observed the effect of adding inhibitors glucose metabolism (phloretin and 2-deoxy-o-glucose) and the tricarboxylic-acid cycle (maleate and trans-aconitate) as well as metabolic intermediates (acetate and citrate) to Grace's medium on the growth and differentiation of T. cruzi.
81
Phloretin, an inhibitor of the plasma-membrane transport of glucose, had no effect on the growth of T. cruzi (Fig. IA), but it did provoke a noticeable and significant increase (P < 0.005, Table I) in the differentiation into metacyclic forms when added at a concentration of 0.1 mM (Fig. 1B). Nevertheless, when this inhibitor was added at 1 mM it ceased to have any effect on differentiation. 2-deoxy-D-glucose, an inhibitor of glycolysis, had no effect on either growth or differentiation at any of the concentrations tested (5 mM and 10 mM) (Fig. 1 and Table 1). As for the effects of the two TCA-cycle inhibitors, when maleate was added to the culture medium no change in the growth process was apparent, although there was a very slight increase in the differentiation rate, while the addition of trans-aconitate (1 mM) led also to a slight, although non-significant, increase in differentiation percentages. At higher concentrations this latter inhibitor had no evident effect on either growth or differentiation rates (Fig. 2 and Table 1). Neither acetate nor citrate had any influence on cell density at concentrations of 1 mM and 5 mM, but at 10 mM growth rate was severely inhibited (Figs. 3 and 4, and Table 1). 5 mM citrate produced a significant increase in differentiation rates ( P < 0.005), while a slight increase was observed with 1 mM citrate or 5 mM acetate. When the concentration of both citrate and acetate reached 10 mM, however, the percentage of metacyclic forms diminished drastically along with the growth rates (Figs. 3 and 4, and Table 1).
Discussion We have studied the influence of substances capable of modifying certain specific pathways in the intermediate metabolism of eukaryotic cells. In previous papers (Adroher et al., 1988b, 1990) we have reported important and significant glycolytic and mitochondrial metabolic alternations during the metacyclogenesis of T. cruzi. The activities of glycolytic enzymes (hexokinase and phospho100-
25" B
20" A
~; (J
15
~o
i lo 5
1
2
4 6 8 10 DAYS IN CULTURE
12
O - -
6
,8 10 12 DAYS IN CULTURE
Fig. 1. Growth (A) and differentiation (B) o f Trypanosoma cruzi in Grace's medium ( × ) added with phloretin at 0.1 mM (O) and I mM (O), and 2-deoxy-o-glucose at 5 mM (IS]) and 10mM (11).
82 TABLE 1 Effect of several metabolic inhibitors and intermediate metabolites on maximum cell density (MCD) and maximum differentiation (MD) values of T. cruzi in Grace's medium Substances
MCD a
Day b
MD a
Day c
None Phloretin (0.1 mM) Phloretin (1 mM) 2-deoxy-o-glucose (5 raM) 2-deoxy-D-glucose (10 mM)
28.64_+2.88 30.61 + 1.08 29.96_+ 1.41 28.80_+ 2.83 27.92 _+2. I 1
10.0+0.6 9.5+0.9 9.5+ 1.0 11.5 _+0.5 9.5 4- 0.5
8.1 _+ 1.6 22.0+ 1.2"* 10.2+ 1.3 11.8 4_ 2.3 4.3 _+0.9
11.3 +0.7 11.0+0.6 10.54_0.5 10.0 + 0.5 12.0 4_0.0
None Maleate (1 mM) Maleate (10 mM) Trans-aconitate (l mM) Trans-aconitate ( I 0 mM )
24.60 4-1.16 24.72 4- 2.48 25.76_+ 1.44 24.16_+ 1.13 21.36 _+0.56
10.0___ 1.2 10.0 ___1.2 10.7+0.7 11.5_+0.5 12.0 _+0.0
8.7 + 1.3 11.0 + 2. I 15.3 4-2.9 12.7_+2.3 6.3 + 0.8
11.0___0.6 11.3 4- 0.7 12.0_+0.0 12.0_+0.0 10.7 -+ 0.7
None Acetate (1 mM) Acetate (5 mM) Acetate (10 mM)
23.924-2.08 25.68 ± 3.12 24.16_+2.17 5.92_+0.75*
10.0_+ 1.2 10.5 _+0.5 11.0_+0.6 8.0_+0.0
8.5_+ 1.2 9.3 4- 1.9 12.2_+2.5 1.8_+0.3"
12.0-+_0.0 10.7 -+0.7 12.0+0.0 11.3 ___0.7
None Citrate (1 mM) Citrate (5 mM) Citrate (10 mM)
25.764-2.51 23.20_+2.08 19.84+0.80 4.80_+0.32*
10.0_+0.8 8.7_+0.7 10.0_+ 1.2 11.3 _+0.7
9.3 4_ 1.5 12.1 _+2.2 23.8_+ 1.5"* 1.0+0.2"
12.0_+0.0 11.3+0.7 12.0+ 0.0 10.0_+ 1.2
The results are the mean-+_ SEM of three or more experiments. Statistical significance (Student's t-test) compared to control: *P < 0.05; **P < 0.005. aMCD (106 cells/ml) and M D (% metacyclic forms) terms are defined in the Materials and Methods section. bCulture day on which the cell density was maximum in each experiment. ~Culture day on which the differentiation was maximum in each experiment.
100-
25 B
20
to 15 o
lO, o 10
5
2
4 6 8 10 DAYS IN CULTURE
12
0
6
8 10 12 DAYS IN CULTURE
Fig. 2. Growth (A) and differentiation (B) of Trypanosoma cruzi in Grace's medium ( × ) added with maleate at 1 m M (©) and 10mM (O), and trans-aconitate at 1 mM (O) and 10mM (111).
83 100
25 ¸ 8
20
15. 10' 10
5
2
4 6 8 10 DAYS IN CULTURE
12
04
6
8 10 12 DAYS IN CULTURE
Fig. 3. Growth (A) and differentiation (B) of Trypanosoma cruzi in Grace's medium (©) added with acetate at 1 mM ( 0 ) , 5 mM (El), and 10raM (B). 100
25. B
20.
15, 10¸ lO
s
2
4 6 8 lO DAYS IN CULTURE
12
o
,._----; S
8
10
12
OArS ~N CULTURE
Fig. 4. Growth (A) and differentiation (B) of Trypanosoma cruzi in Grace's medium (©) added with citrate at 1 mM (O), 5 m M (Tq), and 10mM (11).
fructokinase) in the glycosomes of the epimastigote forms are higher than in the metacyclic forms. Nevertheless, the activities of mitochondrial enzymes, citrate synthase, NADP-linked isocitrate dehydrogenase and succinate dehydrogenase were higher in the metacyclic forms than in the epimastigotes, which preferably use the carbon skeleton of carbohydrate as their energy source (Cficeres and Fern~indes, 1976). We have also shown that there is a considerable and significant increase in the differentiation rates in parasites grown in a glucose- and fructose-free medium (Adroher et al., 1988a; Osuna et al., 1990). Thus, the flagellates cultured in the presence of 0.1 mM phloretin, an inhibitor of glucose transport in eukaryotic cells, differentiated in higher numbers than the controls (Fig. 1B and Table 1). 2-deoxy-oglucose, however, had no apparent effect on the differentiation of T. cruzi, although
84 it did stimulate the differentiation process of the insect trypanosomatid Herpetomonas samuelpessoai when added at concentrations as high as 30.5 mM (Angluster et al., 1977). Several authors have shown the effect of the TCA-cycle intermediates on the differentiation of bloodstream forms to procyclic forms of T. brucei. Brun and Sch6nenberger (1981) have shown that citrate and/or cis-aconitate stimulate this differentiation and Overath et al. (1986) have reported that when these metabolites are added at 37°C to bloodstream forms an increase in the synthesis of aconitase and proline oxidase takes place. The former enzyme transforms citrate and cis-aconitate into isocitrate and the latter transforms proline into pyrroline-5-carboxylate. In this way, it has been demonstrated that some amino acids, such as proline, glutamate, glutamine, aspartate or asparagine, are capable of inducing metacyclogenesis in T. cruzi (Goldenberg et al., 1984; Contreras et al., 1985; Homsy et al., 1989; Krassner et al., 1990). Likewise, they can be metabolized by eukaryotic cells into pyrroline-5-carboxylate, as mentioned by Homsy et al. (I 989) and Krassner et al. (1990). Furthermore, Overath et al. (1983) have shown that the enzyme pyrroline-5-carboxylate reductase, not expressed in bloodstream forms of T. brucei, appears early in its differentiation process into procyclic forms, which permits it to use proline as a main carbon source. We have tested the effect of acetate and citrate on the growth and metacyclogenesis of T. cruzi. When the concentrations of these substrates were increased from 1 mM to 5 mM the growth rates stayed the same but the percentage of metacyclic forms increased significantly in the media containing 5 mM citrate. At concentrations of 10 mM the two substrates had an inhibitory effect both on growth and differentiation rates (Figs. 3 and 4, and Table 1). The use of inhibitors of Krebs' cycle, such as trans-aconitate and maleate, had no effect on either growth or differentiation rates, at least at the concentrations assayed (Fig. 2 and Table I). The factors involved in the metacyclogenetic mechanism are multiple. Elsewhere (Adroher et al., 1988a; Osuna et al., 1990) we have shown the existence of several apparently independent factors: the presence of NaC1 and the absence of monosaccharides, and the sum of their combined effects. Many other authors have described numerous such factors involved in this differentiation process. In this paper we have noted the positive effect of exogenous citrate at certain concentrations on the metacyclogenesis of T. cruzi. It would appear, however, that the basal levels of metacyclogenesis that occur under control conditions do not depend upon endogenous citrate, since trans-aconitate did not reduce the metacyclic percentage to below that of the control medium, despite the fact that this compound inhibits aconitase, the enzyme which transforms citrate into cis-aconitate. Moreover, Overath et al. (1986), working with T. brucei, have reported that the addition of citrate and/or cis-aconitate to the differentiation media increases the synthesis of aconitase and proline oxidase. At the moment we are furthering our research to discover exactly which metabolite in the citrate metabolism is that responsible for triggering differentiation in T. cruzi.
Acknowledgments We thank Dr. Rocio Benitez-Rodriguez for her advice and collaboration, and Dr. John Trout of the Scientific Translation Service of Granada University for revising the English text.
85
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