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Altered intracellular polyamines in bloodstream form Trypanosoma brucei brucei: transformation to procyclic trypomastigotes
Acta Tropica, 55(1993)181-190
181
© 1993 Elsevier Science Publishers B.V. All rights reserved 0001-706X/93/$06.00 ACTROP 00330
Altered intracellular polyamines in bloodstream form Trypanosoma brucei brucei: transformation to procyclic trypomastigotes Bruce F. Giffin a'* and Peter P. M c C a n n b aDepartment of Anatomy and Cell Biology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0521, USA and bMarion Merrell Dow, Inc., 9300 Ward Parkway, Kansas City, MO 64114-0480, USA
Received 18 January 1993; revised version received 28 June 1993; accepted 21 July 1993) A monomorphic strain of Trypanosoma brucei brucei (EATRO 1I0) was cultured as long slender bloodstream forms in vitro for 24 h with 100 ~tM Eflornithine HCI. This resulted in depletion of intracellular putrescine, a greater than 50% decrease in spermidine, and a cessation of cell division. These Eflornithine treated trypanosomes were stimulated to synchronously transform to procyclic trypomastigotes by transfer into SDM-79 medium containing the citric acid cycle intermediates citrate and cis-aconitate (CCA). Within 48 h morphological transformation was complete and occurred without any increase in cell numbers. The coadministration of 100 laM putrescine with Eflornithine prevented the depletion of putrescine and abrogated the cytostatic effect of Eflornithine alone, but did not prevent the transformation to procyclic trypomastigotes. Eflornithine-induced short stumpy form trypanosomes resulting from 4 days of culture with 100 ~tM Eflornithine did not transform to procyclic trypomastigotes in SDM-79 medium unless it contained CCA. We conclude that the high concentration of CCA can trigger transformation in all morphological types of bloodstream form trypanosomes, regardless of their threshold of sensitivity for the stimulus. Furthermore, the CCA-stimulated transformation is not dependent on putrescine and can occur independently of cell division. Key words: Eflornithine;
Difluoromethylornithine;
Procyclic
trypomastigote;
Transformation;
Trypanosoma brucei brucei; Citric acid cycle intermediate
Introduction B o t h the i n t e r m e d i a r y m e t a b o l i s m ( E v a n s a n d Brown, 1972; K l e i n et al., 1975; W a n g , 1982; O b g u n d e a n d I k e d i o b i , 1982; Bienen et al., 1983, 1991; R u b e n et al., 1990) a n d the u l t r a s t r u c t u r e (Vickerman, 1965, 1971; B o h r i n g e r a n d Hecker, 1975) o f the A f r i c a n t r y p a n o s o m e s c h a n g e d r a m a t i c a l l y d u r i n g t r a n s f o r m a t i o n f r o m long slender b l o o d s t r e a m f o r m to procyclic t r y p o m a s t i g o t e . T h e m o r p h o l o g i c a l a n d biochemical a l t e r a t i o n s include the a p p e a r a n c e o f a w e l l - d e v e l o p e d m i t o c h o n d r i o n a n d synthesis o f t r i c a r b o x y l i c acid cycle enzymes a n d c y t o c h r o m e s , all o f which are a b s e n t in the b l o o d s t r e a m f o r m ( K r o n i c k a n d Hill, 1974; Hill, 1976; Bienen et al., 1983).
Corresponding author. Fax: + 1 (513) 5584454.
182 Polyamines (putrescine, spermidine and spermine) are involved in cellular growth processes and differentiation (Pegg and McCann, 1982; Kapyako and Janne, 1983; Schindler et al., 1985). Recent studies (Bacchi et al., 1983; deGee et al., 1984; Giffin et al., 1986; Giffin and McCann, 1989) have shown that perturbations in intraceUular polyamines following exposure of long slender bloodstream trypanosomes to Eflornithine HC1 resulted in morphological alteration to the short stumpy form. Eflornithine HC1 (difluoromethylorninthine), the irreversible inhibitor of ornithine decarboxylase (the first enzyme in the polyamine biosynthetic pathway), rapidly depleted the intracellular content of polyamines. This resulted in decreased rates of DNA and RNA synthesis and a subsequent block to cell division (Bacchi and McCann, 1987). Both the morphological alterations and cytostatic effect can be abrogated by coadministration of putrescine, the first polyamine in the biosynthetic pathway. Brun and Schonenberger (1981) have shown that transformation of the long slender bloodstream form can be initiated by citric acid cycle intermediates. The use of this technique and the application of recent methods of in vitro culture of bloodstream (Brun et al., 1981) and procyclic trypomastigotes (Brun and Schonenberger 1979, 1981) enabled the synchronous transformation of monomorphic strains of trypanosomes. In this study we attempted to determine whether polyamines have a role in transformation of monomorphic bloodstream forms to procyclic trypomastigotes; whether cell division is requisite for transformation; and if the Eflornithine-induced short stumpy form trypanosomes have the capacity to transform when stimulated with citric acid cycle intermediates.
Materials and Methods
Trypanosome strain and cultivation in vitro Trypanosoma brucei brucei (EATRO 110) was used in all experiments in this study. This monomorphic strain was obtained from Cyrus J. Bacchi of the Haskins Laboratories at Pace University (New York City, New York). Frozen stabilates were prepared and stored under liquid N2. The growth and isolation of trypanosomes and their cultivation as bloodstream forms in vitro have been describe elsewhere (Giffin et al., 1986; Giffin and McCann, 1989). Transformation protocol Long slender bloodstream form trypanosomes were cultured for 24 h in 75 cm 2 flasks containing 20 ml of medium B (Eagle's Minimal Essential Medium with Earle's salts, 30 mM Hepes, 0.1 mM MEM non-essential amino acid solution, 2 mM glutamine, 20% (v/v) fetal calf serum, 0.2% glucose, 150 units of penicillin and 150 p.g/ml streptomycin (pH 7.4)). At t = - 2 4 h trypanosomes were removed from culture medium by centrifugation at 600 x g for 10 min and resuspended in medium B containing 55 mg sodium pyruvate and 7 mg hypoxanthine per 500 ml (SDM-79). Each flask was supplemented with 600 Ixl of a solution containing 100 mM citric acid, 100 mM cis-aconitate, and 200 mM magnesium acetate as described by Brun
183 and Schonenberger (1979). Putrescine and/or Eflornithine HC1 were added to the medium from 10 × concentrated stock solutions to give a final concentration of 100 p.M. Flasks were incubated at 37°C for 24 h in 5% CO2/95% air. At t = 0 trypanosomes were removed from the medium by centrifugation and resuspended in 20 ml of SDM-79 medium containing citric acid cycle intermediates, Eflornithine HC1, and/or putrescine, and incubated for 48 h at 28°C in flasks containing embryonic bovine trachea fibroblast (EBTr) monolayers. Each day of culture as bloodstream forms and during the transformation to procyclic trypomastigotes, cell counts were made using a Neubauer hemacytometer, air-dried smears prepared for Giemsa staining, and trypanosomes were harvested for polyamine analysis.
Polyamine analysis Polyamines were analyzed in (1-3)x 10 7 trypanosomes after washing two times in 5 ml of phosphate saline and extracting overnight at 4°C in 300 jal of 0.4 M perchloric acid. The extract was filtered by passing through a 0.22 jam Millipore GS filter and 40 jal samples were analyzed using a Dionex D-300 amino acid analyzer (Dionex Corporation, Sunnyvale, CA) as described previously (Giffin et al., 1986).
Chemicals All tissue culture media were obtained from Gibco Laboratories, Grand Island, New York. Eflornithine HC1 was synthesized at the Marion Merrell Dow Research Institute, Cincinnati, OH. All other chemicals were from the Sigma Chemical Co., St. Louis, MO.
Results
When long slender bloodstream form trypanosomes were stimulated to transform by citric acid cycle intermediates without Eflornithine HC1 there was an approximately 2-fold increase in intracellular levels of putrescine as compared to levels found in trypanosomes maintained in culture as bloodstream forms (Fig. 1). Depletion of putrescine (< 0.05 nmol/107 cells) in long slender bloodstream forms by pretreatment with 100 jaM Eflornithine HC1 for 24 h prior to the initiation of transformation did not prevent the morphological alteration associated with this process. However, transformation occurred in the absence of any cell division. The coadministration of 100 jaM putrescine with the Eflornithine HC1 maintained intracellular polyamines at control levels (0.62 nmol/107 cells) and abrogated the cytostatic effect of Eflornithine HCI alone (Fig. 2). Treated and control cells were morphologically identical at 48 h (Fig. 3). Short stumpy form trypanosomes resulting from 4 days of culture in medium B with 100 jaM Eflornithine HC1 were unable to adapt to SDM-79 medium. If, however, citric acid cycle intermediates were present, these forms did transform to procyclic trypomastigotes (Fig. 4).
184
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Fig. 1. Polyaminelevelsduring the transformation of Trypanosorna brucei brucei stimulated by citric acid cycle intermediates (CCA). Long slender bloodstream form trypanosomeswere harvested from rodents with a 3 day parasitemia and subjected to the transformation protocol as described in Materials and Methods. Intracellular levels of putrescine in control (D) and CCA treated (R) trypanosomes. Intracellular levels of spermidine in control (O) and CCA-treated (0) trypanosomes. Discussion
The transformation of Trypanosoma brucei brucei (EATRO 110) to procyclic trypomastigotes has in the past been unsuccessful using standard procedures (Bienen et al., 1980). More recently, Bass and Wang (1991, 1992) were not successful in transforming this EATRO 110 strain to procyclic trypomastigotes using Cunningham's medium (Cunningham, 1977) with and without TCA cycle intermediates. Here we report the successful transformation of this monomorphic strain when the bloodstream forms were cultured in medium B supplemented with citric acid cycle intermediates (Brun and Schonenberger, 1981) above a feeder layer of EBTr fibroblasts for 24 h (Overath et al., 1983) and transferred to SDM-79 medium supplemented with citric acid cycle intermediates. These trypanosomes transformed to procyclic trypomastigotes within 48 h. The results reported in this study indicate that the process of transformation does not have a requirement for putrescine, since trypanosomes completely depleted of their intracellular content of this diamine can undergo successful transformation. Bass and Wang (1991) also demonstrated that the in vitro transformation of long slender, intermediate, and short stumpy bloodstream forms of the pleomorphic strain of Trypanosoma brucei (TREU 667) to procyclic trypomastigotes in Cunningham's medium was not prevented by 1 mM Eflornithine HC1. However, the inhibition of the polyamine biosynthetic pathway did not result in any significant decrease in the
185
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Fig. 2. Cell proliferation and morphologicaltransformation in long slender bloodstream trypanosomes pretreated with either 100 laM EflornithineHC1 (©) or 100 laM Eflornithineand 100/aM putrescine(O) prior to initiation of transformation by citric acid cycleintermediates. Pretreatment was in medium B at 37°C for 24 h. The transformation protocol is described in Materials and Methods. rate of cellular proliferation. In contrast, the monomorphic strain used in our studies did not undergo cellular proliferation during transformation to procyclic trypomastigotes when depleted of putrescine by exposure to 100 IxM Eflornithine. In addition to transformation occurring in the absence of putrescine, the transformation of bloodstream forms to procyclic trypomastigotes does not have a requisite for cellular proliferation. That the transforming cells did not proliferate was not unexpected in light of the established role of the polyamines in cellular growth processes, particularly in cells undergoing rapid proliferation (Pegg and McCann, 1982). Blahushova et al. (1984) also report transformation in the absence of cell division. Their pleomorphic strain of Trypanosoma brucei brucei (STIB 247) successfully completes transformation when D N A replication and cell division are blocked by hydroxyurea. The cessation of cell division following exposure of trypanosomes to Eflornithine in our studies, but not in the studies of Bass and Wang, may result from the different culture media used or differences each strain has for polyamines during cellular proliferation. However, other investigators have demonstrated a specific role for polyamines in the regulation of differentiation using Eflornithine HC1. Polyamines induced differentiation in some cell systems, while in others differentiation was inhibited (Heby et al., 1987). We have shown that long slender bloodstream trypanosomes transform to short stumpy forms when intracellular putrescine is depleted following exposure to Eflornithine HC1 (Giffin et al., 1986; Giffin and McCann, 1989). A form morphologically intermediate between the long slender form and the
187
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Fig. 4. The capacity of Eflornithine-HCl-induced short stumpy form trypanosomes to transform in SDM79 medium. Long slender bloodstream forms were cultured in medium B with 100 laM Eflornithine HC1 for 4 days, after which they were removed from the medium by centrifugation, introduced into 75 cm2 tissue culture flasks without EBTr fibroblast monolayers containing 20 ml of SDM-79 medium with (@) and without (A) citric acid cycle intermediates and incubated at 28°C. s h o r t s t u m p y f o r m h a d the c a p a c i t y to t r a n s f o r m to procyclic t r y p o m a s t i g o t e s when i n t r o d u c e d into S D M - 7 9 . M o r e o v e r , this t r a n s f o r m a t i o n was a c c o m p a n i e d by r a p i d cell division. D a t a f r o m this c u r r e n t s t u d y s u p p o r t the o b s e r v a t i o n t h a t the E f l o r n i t h i n e - i n d u c e d i n t e r m e d i a t e forms can t r a n s f o r m . H o w e v e r , the proliferative c o m p o n e n t o f the t r a n s f o r m a t i o n process is d e p e n d e n t on the t r y p a n o s o m e m a i n t a i n ing its i n t r a c e l l u l a r c o n t e n t o f putrescine. W h e n l o n g slender b l o o d s t r e a m forms o f either p l e o m o r p h i c ( G h i o t t o et al., 1979) or m o n o m o r p h i c (Giffin et al., 1986) strains o f Trypanosoma brucei are i n t r o d u c e d into semi-defined m e d i a (Brun a n d Jenni, 1977; Brun a n d S c h o n e n b e r g e r , 1979) w i t h o u t citric acid cycle intermediates, they die within 24 h. O n l y when i n t e r m e d i a t e a n d s h o r t s t u m p y forms o f the p l e o m o r p h i c strain were present o r the long slender forms were e x p o s e d to T C A cycle i n t e r m e d i a t e s was there successful t r a n s f o r m a t i o n to procyclic t r y p o m a s t i g o t e s . In contrast, the m o n o m o r p h i c E A T R O 110 strain o f Trypanosoma brucei was f o u n d i n c a p a b l e o f t r a n s f o r m a t i o n to procyclic t r y p o m a s t i gotes when i n t r o d u c e d into C u n n i n g h a m ' s m e d i u m with or w i t h o u t citric acid cycle i n t e r m e d i a t e s (Bass a n d W a n g , 1991). These investigators also f o u n d c o n s i d e r a b l e
Fig. 3. The morphology of trypanosomes after 48 h of citric acid cycle intermediate-stimulated transformation (x 900). (A) Control; (B) transformation in the absence of 100 laM Eflornithine HC1; (C) transformation in the presence of 100 ~tM Eflornithine HC1 and 100 laM putrescine. The pretreatment of long slender bloodstream form trypanosomes in medium B and the transformation protocol are described in Materials and Methods.
188 variability in the ability of three other monomorphic strains of Trypanosoma brucei to transform in vitro in Cunninghams's medium (Bass and Wang, 1992). These differences presumably reflect variations in the transformation protocols and composition of the media. The use of citric acid cycle intermediates should be regarded as an artificial means of inducing transformation in monomorphic strains of Trypanosoma in so far as the process occurs in the absence of pleomorphism. Recently, however, Bass and Wang (1991) demonstrated that all three morphological forms of the pleomorphic strain of Trypanosoma brucei (TRUE 667) could transform with similar time-courses into procyclic trypomastigotes when introduced into Cunningham's medium. The addition of cis-aconitate and L-citrate to the medium decreased the lag time but did not alter the dynamics of the transformation. In our studies, the long slender trypanosomes become procyclic trypomastigotes without passing through the stage of short stumpy forms. They do, however, become intermediate in morphology. It has been widely held that trypanosomes survive in the midgut of the tsetse fly because of morphological changes associated with transformation from long slender to short stumpy form trypanosomes, and the simultaneous physiological activation of the mitochondrion that permits the cell to make the transition from depending exclusively on glycolysis for ATP production to synthesis of ATP by oxidative phosphorylation (Bowman and Flynn, 1976; Bienan et al., 1991). Since the intermediate trypanosomes resulting from treatment of the monomorphic EATRO 110 strain with Eflornithine and all three bloodstream forms of the pleomorphic TRUE 667 strain can transform to procyclic trypomastigotes when introduced into SDM-79 and Cunningham's medium, respectively, it is apparent that, under these in vitro conditions, morphological alterations are not absolutely coupled to those prerequisite biochemical changes permitting successful transition of the bloodstream forms to life in the vector. There is an optimum state of biochemical preadaptation for successful transformation to procyclic trypomastigote associated with the pleomorphism of the three bloodstream forms. Our observations using Eflornithine-induced bloodstream forms of the EATRO 110 strain of Trypanosoma brucei suggest that there is a threshold of sensitivity to the stimulus for transformation associated with the three morphological forms, the intermediate form being the most sensitive, the long slender less, and short stumpy form least sensitive. In our studies described here, the very high concentration of citric acid cycle intermediates (3 mM) may optimally activate the TCA cycle (Brun and Schonenberger, 1981) and thereby bypass any normal gradient of activation. Consequently, all three cell types are stimulated to transform, regardless of their threshold of sensitivity and the presence of absence of the natural trigger(s) for transformation. This hypothesis needs to be confirmed. Since transformation of the bloodstream forms to procyclic trypomastigotes is a critical event in the life cycle of the trypanosome, further studies are needed to elucidate the molecular events involved in the regulation of this process. Only when the details have been worked out will the possibility exist for the design of rational chemotherapeutic interventions directed at controlling trypanosomal transformation. References
Bacchi, C.J., Garafalo, J., Mochenhaupt, D., McCann, P.P., Diekema, K.A., Pegg, A.E., Nathan, H.C., Mullaney, E.A., Chunosoff, L., Sjoerdsma, A. and Hunter, S.H. (1983) In vivo effects of OL-~-
189 difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei. Mol. Biochem. Parasitol. 7,209-225. Bass, K.E. and Wang, C.C. (1991) The in vitro differentiation of pleomorphic Trypanosoma brucei from bloodstream into procyclic form requires neither intermediary nor short-stumpy stage. Mol. Biochem. Parasitol. 44, 261-270. Bass, K.E. and Wang, C.C. (1992) Transient inhibition of protein synthesis accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms. Mol. Biochem. Parasitol. 56, 129-140. Bienen, E.J., Hammadi, E. and Hill, G.C. (1980) Initiation of trypanosome transformation from bloodstream trypomastigotes to procyclic trypomastigotes. J. Parasitol. 66, 680-682. Bienen, E.J., Hill, G.C. and Shin, K-O. (1983) Elaboration of mitochondrial function during Trypanosoma brucei differentiation. Mol. Biochem. Parasitol. 7, 75-86. Bienen, E.J., Saric, M., Pollakis, G., Grady, R.W. and Clarkson, A.B. (1991) Mitochondrial development in Trypanosoma brucei brucei transitional bloodstream forms. Mol. Biochem. Parasitol. 45, 185-192. Blahushova, A., Markus, A. and Nonynkova, E. (1984) Trypanosoma brucei brucei: in vitro transformation of bloodstream to procyclic forms. J. Protozool. 31, 49A. Bohringer, S. and Hecker, H. (1975) Quantitative ultrastructural investigations of the life cycle of Trypanosoma brucei: a morphometric analysis. J. Protozool. 22, 463-467 (1975). Bowman, I.B.R. and Flynn, I.W. (1976) Oxidative metabolism of trypanosomes. In: Biology of the Kinetoplastida, Vol. I, ed. by W.H.R. Lumsden and D.A. Evans, pp. 436-467. Academic Press, New York. ./ Brun, R. and Jenni, L. (1977) A new semi-defined medium for Trypanosoma brucei sspp. Acta Trop. 34, 21-22. Brun, R., Jenni, L., Schonenberger, M. and Schell, K.F. (1981) In vitro cultivation of bloodstream forms Trypanosoma brucei, 7". rhodesiense and T. gambiense. J. Protozool. 28, 470-479. Brun, R. and Schonenberger, M. (1979) Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Acta Trop. 36,289-292. Brun, R. and Schonenberger, M. (1981) Stimulating effect of citrate and cis-aconitate on the transformation of T. brucei bloodstream forms to procyclic forms in vitro. Parasitenbunde 66, 17-24. Cunningham, I. (1977) New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J. Protozool. 24, 325-329. deGee, A.L.W., Carstens, P.H.B., McCann, P.P. and Mansfield, J.M. (1984) Role of antibody in the elimination of trypanosomes after DL-ct-difluoromethylornithine chemotherapy. J. Parasitol. 69, 818-822 (1984). Evans, D.A. and Brown, R.C. (1972) The utilization of glucose and proline by culture forms of T. brucei. J. Protozool. 19, 686-690. Ghiotto, V., Brun, R., Jenni, L. and Hecker, H. (1979) Trypanosoma brucei morphometric changes and loss of infectivity during transformation of bloodstream forms to procyclic culture forms in vitro. Exp. Parasitol. 48,447-456. Giffin, B.F., McCann, P.P., Bitonti, A.J. and Bacchi, C.J. (1986) Polyamine depletion following exposure to DL-~-difluoromethylornithine both in vivo and in vitro initiates morphological alterations and mitochondrial activation in a monomorphic strain of Trypanosoma brucei brucei. J. Protozool. 33, 239-243. Giffin, B.F. and McCann, P.P. (1989) Physiological activation of the mitochondrion and the transformation capacity of DFMO induced intermediate and short stumpy bloodstream form trypanosomes. Am. J. Trop. Med. Hyg. 40,487-493. Heby, O., Luk, G.D. and Schindler, J. (1987) Polyamine synthesis inhibitors act as both inducers and suppressors of cell differentiation. In: Inhibition of Polyamine Metabolism: Biological Significance and Basis for New Therapies, ed. by P.P. McCann, A.E. Pegg, and A. Sjoerdsma, p. 165-183. Academic Press, New York. Hill, G.C. (1976) Electron transport systems in kinetoplastida. Biochim. Biophys. Acta 456, 149-163. Jennings, F.W., Urguhart, G.M., Murray, P.K. and Miller, B.M. (1980) "Berenil" and nitroimidazole combinations in the treatment of Trypanosoma brucei infection with central nervous system involvement. Int. J. Parasitol. 10, 27-32. Kapyako, K. and Janne, J. (1983) Stimulation of melanotic expression in murine melanoma cells exposed to polyamine antimetabolites. Biochem. Biophys. Res. Commun. 113, 18. Klein, R.A., Linstead, D.J. and Wheeler, M.V. (1975) Carbon dioxide fixation in trypanosomatids. Parasitology 71, 93-107.
190 Kronick, P. and Hill, G.C. (1974) Evidence for the functioning of cytochrome o in kinetoplastida. Biochim. Biophys. Acta 368, 173-180. Obgunude, P.O.J. and Ikediobi, C.O. (1982) Effect of nitrobenzylthioinosinate on the toxicity of tubercidin and ethidium against Trypanosoma garnbiense. Acta Trop. 39, 219-224. 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. Pegg, A.E. and McCann, P.P. (1982) Polyamine metabolism and function: A brief review. Am. J. Physiol. 243, C212-C221. Ruben, L., Haghighat, N. and Campbell, A. (1990) Cyclical differentiation of Trypanosoma brucei involves changes in the cellular complement of calmodulin-binding proteins. Exp. Parasitol. 70, 144-153. Schindler, J., Kelly, M. and McCann, P.P. (1985) The response of several murine embryonal carcinoma cell lines to stimulation of differentiation by a-difluoromethylornithine. J. Cell Physiol. 122, 1-6. Vickerman, K. (1965) The mechanism of cyclical development in trypanosome of the Trypanosoma brucei sub-group: A hypothesis based on ultrastructural observations. Trans. R. Soc. Trop. Med. Hyg. 56, 487-495. Vickerman, K. (1971) Morphological and physiological considerations of extracellular blood protozoa. In: Ecology and Physiology of Parasites, ed. by A.M. Fallis, pp. 48-91. University of Toronto Press, Toronto. Wang, C.C. (1982) Current problems in anti-parasite chemotherapy. Trends Biochem. Sci. 7, 354-356.
181
© 1993 Elsevier Science Publishers B.V. All rights reserved 0001-706X/93/$06.00 ACTROP 00330
Altered intracellular polyamines in bloodstream form Trypanosoma brucei brucei: transformation to procyclic trypomastigotes Bruce F. Giffin a'* and Peter P. M c C a n n b aDepartment of Anatomy and Cell Biology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0521, USA and bMarion Merrell Dow, Inc., 9300 Ward Parkway, Kansas City, MO 64114-0480, USA
Received 18 January 1993; revised version received 28 June 1993; accepted 21 July 1993) A monomorphic strain of Trypanosoma brucei brucei (EATRO 1I0) was cultured as long slender bloodstream forms in vitro for 24 h with 100 ~tM Eflornithine HCI. This resulted in depletion of intracellular putrescine, a greater than 50% decrease in spermidine, and a cessation of cell division. These Eflornithine treated trypanosomes were stimulated to synchronously transform to procyclic trypomastigotes by transfer into SDM-79 medium containing the citric acid cycle intermediates citrate and cis-aconitate (CCA). Within 48 h morphological transformation was complete and occurred without any increase in cell numbers. The coadministration of 100 laM putrescine with Eflornithine prevented the depletion of putrescine and abrogated the cytostatic effect of Eflornithine alone, but did not prevent the transformation to procyclic trypomastigotes. Eflornithine-induced short stumpy form trypanosomes resulting from 4 days of culture with 100 ~tM Eflornithine did not transform to procyclic trypomastigotes in SDM-79 medium unless it contained CCA. We conclude that the high concentration of CCA can trigger transformation in all morphological types of bloodstream form trypanosomes, regardless of their threshold of sensitivity for the stimulus. Furthermore, the CCA-stimulated transformation is not dependent on putrescine and can occur independently of cell division. Key words: Eflornithine;
Difluoromethylornithine;
Procyclic
trypomastigote;
Transformation;
Trypanosoma brucei brucei; Citric acid cycle intermediate
Introduction B o t h the i n t e r m e d i a r y m e t a b o l i s m ( E v a n s a n d Brown, 1972; K l e i n et al., 1975; W a n g , 1982; O b g u n d e a n d I k e d i o b i , 1982; Bienen et al., 1983, 1991; R u b e n et al., 1990) a n d the u l t r a s t r u c t u r e (Vickerman, 1965, 1971; B o h r i n g e r a n d Hecker, 1975) o f the A f r i c a n t r y p a n o s o m e s c h a n g e d r a m a t i c a l l y d u r i n g t r a n s f o r m a t i o n f r o m long slender b l o o d s t r e a m f o r m to procyclic t r y p o m a s t i g o t e . T h e m o r p h o l o g i c a l a n d biochemical a l t e r a t i o n s include the a p p e a r a n c e o f a w e l l - d e v e l o p e d m i t o c h o n d r i o n a n d synthesis o f t r i c a r b o x y l i c acid cycle enzymes a n d c y t o c h r o m e s , all o f which are a b s e n t in the b l o o d s t r e a m f o r m ( K r o n i c k a n d Hill, 1974; Hill, 1976; Bienen et al., 1983).
Corresponding author. Fax: + 1 (513) 5584454.
182 Polyamines (putrescine, spermidine and spermine) are involved in cellular growth processes and differentiation (Pegg and McCann, 1982; Kapyako and Janne, 1983; Schindler et al., 1985). Recent studies (Bacchi et al., 1983; deGee et al., 1984; Giffin et al., 1986; Giffin and McCann, 1989) have shown that perturbations in intraceUular polyamines following exposure of long slender bloodstream trypanosomes to Eflornithine HC1 resulted in morphological alteration to the short stumpy form. Eflornithine HC1 (difluoromethylorninthine), the irreversible inhibitor of ornithine decarboxylase (the first enzyme in the polyamine biosynthetic pathway), rapidly depleted the intracellular content of polyamines. This resulted in decreased rates of DNA and RNA synthesis and a subsequent block to cell division (Bacchi and McCann, 1987). Both the morphological alterations and cytostatic effect can be abrogated by coadministration of putrescine, the first polyamine in the biosynthetic pathway. Brun and Schonenberger (1981) have shown that transformation of the long slender bloodstream form can be initiated by citric acid cycle intermediates. The use of this technique and the application of recent methods of in vitro culture of bloodstream (Brun et al., 1981) and procyclic trypomastigotes (Brun and Schonenberger 1979, 1981) enabled the synchronous transformation of monomorphic strains of trypanosomes. In this study we attempted to determine whether polyamines have a role in transformation of monomorphic bloodstream forms to procyclic trypomastigotes; whether cell division is requisite for transformation; and if the Eflornithine-induced short stumpy form trypanosomes have the capacity to transform when stimulated with citric acid cycle intermediates.
Materials and Methods
Trypanosome strain and cultivation in vitro Trypanosoma brucei brucei (EATRO 110) was used in all experiments in this study. This monomorphic strain was obtained from Cyrus J. Bacchi of the Haskins Laboratories at Pace University (New York City, New York). Frozen stabilates were prepared and stored under liquid N2. The growth and isolation of trypanosomes and their cultivation as bloodstream forms in vitro have been describe elsewhere (Giffin et al., 1986; Giffin and McCann, 1989). Transformation protocol Long slender bloodstream form trypanosomes were cultured for 24 h in 75 cm 2 flasks containing 20 ml of medium B (Eagle's Minimal Essential Medium with Earle's salts, 30 mM Hepes, 0.1 mM MEM non-essential amino acid solution, 2 mM glutamine, 20% (v/v) fetal calf serum, 0.2% glucose, 150 units of penicillin and 150 p.g/ml streptomycin (pH 7.4)). At t = - 2 4 h trypanosomes were removed from culture medium by centrifugation at 600 x g for 10 min and resuspended in medium B containing 55 mg sodium pyruvate and 7 mg hypoxanthine per 500 ml (SDM-79). Each flask was supplemented with 600 Ixl of a solution containing 100 mM citric acid, 100 mM cis-aconitate, and 200 mM magnesium acetate as described by Brun
183 and Schonenberger (1979). Putrescine and/or Eflornithine HC1 were added to the medium from 10 × concentrated stock solutions to give a final concentration of 100 p.M. Flasks were incubated at 37°C for 24 h in 5% CO2/95% air. At t = 0 trypanosomes were removed from the medium by centrifugation and resuspended in 20 ml of SDM-79 medium containing citric acid cycle intermediates, Eflornithine HC1, and/or putrescine, and incubated for 48 h at 28°C in flasks containing embryonic bovine trachea fibroblast (EBTr) monolayers. Each day of culture as bloodstream forms and during the transformation to procyclic trypomastigotes, cell counts were made using a Neubauer hemacytometer, air-dried smears prepared for Giemsa staining, and trypanosomes were harvested for polyamine analysis.
Polyamine analysis Polyamines were analyzed in (1-3)x 10 7 trypanosomes after washing two times in 5 ml of phosphate saline and extracting overnight at 4°C in 300 jal of 0.4 M perchloric acid. The extract was filtered by passing through a 0.22 jam Millipore GS filter and 40 jal samples were analyzed using a Dionex D-300 amino acid analyzer (Dionex Corporation, Sunnyvale, CA) as described previously (Giffin et al., 1986).
Chemicals All tissue culture media were obtained from Gibco Laboratories, Grand Island, New York. Eflornithine HC1 was synthesized at the Marion Merrell Dow Research Institute, Cincinnati, OH. All other chemicals were from the Sigma Chemical Co., St. Louis, MO.
Results
When long slender bloodstream form trypanosomes were stimulated to transform by citric acid cycle intermediates without Eflornithine HC1 there was an approximately 2-fold increase in intracellular levels of putrescine as compared to levels found in trypanosomes maintained in culture as bloodstream forms (Fig. 1). Depletion of putrescine (< 0.05 nmol/107 cells) in long slender bloodstream forms by pretreatment with 100 jaM Eflornithine HC1 for 24 h prior to the initiation of transformation did not prevent the morphological alteration associated with this process. However, transformation occurred in the absence of any cell division. The coadministration of 100 jaM putrescine with the Eflornithine HC1 maintained intracellular polyamines at control levels (0.62 nmol/107 cells) and abrogated the cytostatic effect of Eflornithine HCI alone (Fig. 2). Treated and control cells were morphologically identical at 48 h (Fig. 3). Short stumpy form trypanosomes resulting from 4 days of culture in medium B with 100 jaM Eflornithine HC1 were unable to adapt to SDM-79 medium. If, however, citric acid cycle intermediates were present, these forms did transform to procyclic trypomastigotes (Fig. 4).
184
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Fig. 1. Polyaminelevelsduring the transformation of Trypanosorna brucei brucei stimulated by citric acid cycle intermediates (CCA). Long slender bloodstream form trypanosomeswere harvested from rodents with a 3 day parasitemia and subjected to the transformation protocol as described in Materials and Methods. Intracellular levels of putrescine in control (D) and CCA treated (R) trypanosomes. Intracellular levels of spermidine in control (O) and CCA-treated (0) trypanosomes. Discussion
The transformation of Trypanosoma brucei brucei (EATRO 110) to procyclic trypomastigotes has in the past been unsuccessful using standard procedures (Bienen et al., 1980). More recently, Bass and Wang (1991, 1992) were not successful in transforming this EATRO 110 strain to procyclic trypomastigotes using Cunningham's medium (Cunningham, 1977) with and without TCA cycle intermediates. Here we report the successful transformation of this monomorphic strain when the bloodstream forms were cultured in medium B supplemented with citric acid cycle intermediates (Brun and Schonenberger, 1981) above a feeder layer of EBTr fibroblasts for 24 h (Overath et al., 1983) and transferred to SDM-79 medium supplemented with citric acid cycle intermediates. These trypanosomes transformed to procyclic trypomastigotes within 48 h. The results reported in this study indicate that the process of transformation does not have a requirement for putrescine, since trypanosomes completely depleted of their intracellular content of this diamine can undergo successful transformation. Bass and Wang (1991) also demonstrated that the in vitro transformation of long slender, intermediate, and short stumpy bloodstream forms of the pleomorphic strain of Trypanosoma brucei (TREU 667) to procyclic trypomastigotes in Cunningham's medium was not prevented by 1 mM Eflornithine HC1. However, the inhibition of the polyamine biosynthetic pathway did not result in any significant decrease in the
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Fig. 2. Cell proliferation and morphologicaltransformation in long slender bloodstream trypanosomes pretreated with either 100 laM EflornithineHC1 (©) or 100 laM Eflornithineand 100/aM putrescine(O) prior to initiation of transformation by citric acid cycleintermediates. Pretreatment was in medium B at 37°C for 24 h. The transformation protocol is described in Materials and Methods. rate of cellular proliferation. In contrast, the monomorphic strain used in our studies did not undergo cellular proliferation during transformation to procyclic trypomastigotes when depleted of putrescine by exposure to 100 IxM Eflornithine. In addition to transformation occurring in the absence of putrescine, the transformation of bloodstream forms to procyclic trypomastigotes does not have a requisite for cellular proliferation. That the transforming cells did not proliferate was not unexpected in light of the established role of the polyamines in cellular growth processes, particularly in cells undergoing rapid proliferation (Pegg and McCann, 1982). Blahushova et al. (1984) also report transformation in the absence of cell division. Their pleomorphic strain of Trypanosoma brucei brucei (STIB 247) successfully completes transformation when D N A replication and cell division are blocked by hydroxyurea. The cessation of cell division following exposure of trypanosomes to Eflornithine in our studies, but not in the studies of Bass and Wang, may result from the different culture media used or differences each strain has for polyamines during cellular proliferation. However, other investigators have demonstrated a specific role for polyamines in the regulation of differentiation using Eflornithine HC1. Polyamines induced differentiation in some cell systems, while in others differentiation was inhibited (Heby et al., 1987). We have shown that long slender bloodstream trypanosomes transform to short stumpy forms when intracellular putrescine is depleted following exposure to Eflornithine HC1 (Giffin et al., 1986; Giffin and McCann, 1989). A form morphologically intermediate between the long slender form and the
187
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Fig. 4. The capacity of Eflornithine-HCl-induced short stumpy form trypanosomes to transform in SDM79 medium. Long slender bloodstream forms were cultured in medium B with 100 laM Eflornithine HC1 for 4 days, after which they were removed from the medium by centrifugation, introduced into 75 cm2 tissue culture flasks without EBTr fibroblast monolayers containing 20 ml of SDM-79 medium with (@) and without (A) citric acid cycle intermediates and incubated at 28°C. s h o r t s t u m p y f o r m h a d the c a p a c i t y to t r a n s f o r m to procyclic t r y p o m a s t i g o t e s when i n t r o d u c e d into S D M - 7 9 . M o r e o v e r , this t r a n s f o r m a t i o n was a c c o m p a n i e d by r a p i d cell division. D a t a f r o m this c u r r e n t s t u d y s u p p o r t the o b s e r v a t i o n t h a t the E f l o r n i t h i n e - i n d u c e d i n t e r m e d i a t e forms can t r a n s f o r m . H o w e v e r , the proliferative c o m p o n e n t o f the t r a n s f o r m a t i o n process is d e p e n d e n t on the t r y p a n o s o m e m a i n t a i n ing its i n t r a c e l l u l a r c o n t e n t o f putrescine. W h e n l o n g slender b l o o d s t r e a m forms o f either p l e o m o r p h i c ( G h i o t t o et al., 1979) or m o n o m o r p h i c (Giffin et al., 1986) strains o f Trypanosoma brucei are i n t r o d u c e d into semi-defined m e d i a (Brun a n d Jenni, 1977; Brun a n d S c h o n e n b e r g e r , 1979) w i t h o u t citric acid cycle intermediates, they die within 24 h. O n l y when i n t e r m e d i a t e a n d s h o r t s t u m p y forms o f the p l e o m o r p h i c strain were present o r the long slender forms were e x p o s e d to T C A cycle i n t e r m e d i a t e s was there successful t r a n s f o r m a t i o n to procyclic t r y p o m a s t i g o t e s . In contrast, the m o n o m o r p h i c E A T R O 110 strain o f Trypanosoma brucei was f o u n d i n c a p a b l e o f t r a n s f o r m a t i o n to procyclic t r y p o m a s t i gotes when i n t r o d u c e d into C u n n i n g h a m ' s m e d i u m with or w i t h o u t citric acid cycle i n t e r m e d i a t e s (Bass a n d W a n g , 1991). These investigators also f o u n d c o n s i d e r a b l e
Fig. 3. The morphology of trypanosomes after 48 h of citric acid cycle intermediate-stimulated transformation (x 900). (A) Control; (B) transformation in the absence of 100 laM Eflornithine HC1; (C) transformation in the presence of 100 ~tM Eflornithine HC1 and 100 laM putrescine. The pretreatment of long slender bloodstream form trypanosomes in medium B and the transformation protocol are described in Materials and Methods.
188 variability in the ability of three other monomorphic strains of Trypanosoma brucei to transform in vitro in Cunninghams's medium (Bass and Wang, 1992). These differences presumably reflect variations in the transformation protocols and composition of the media. The use of citric acid cycle intermediates should be regarded as an artificial means of inducing transformation in monomorphic strains of Trypanosoma in so far as the process occurs in the absence of pleomorphism. Recently, however, Bass and Wang (1991) demonstrated that all three morphological forms of the pleomorphic strain of Trypanosoma brucei (TRUE 667) could transform with similar time-courses into procyclic trypomastigotes when introduced into Cunningham's medium. The addition of cis-aconitate and L-citrate to the medium decreased the lag time but did not alter the dynamics of the transformation. In our studies, the long slender trypanosomes become procyclic trypomastigotes without passing through the stage of short stumpy forms. They do, however, become intermediate in morphology. It has been widely held that trypanosomes survive in the midgut of the tsetse fly because of morphological changes associated with transformation from long slender to short stumpy form trypanosomes, and the simultaneous physiological activation of the mitochondrion that permits the cell to make the transition from depending exclusively on glycolysis for ATP production to synthesis of ATP by oxidative phosphorylation (Bowman and Flynn, 1976; Bienan et al., 1991). Since the intermediate trypanosomes resulting from treatment of the monomorphic EATRO 110 strain with Eflornithine and all three bloodstream forms of the pleomorphic TRUE 667 strain can transform to procyclic trypomastigotes when introduced into SDM-79 and Cunningham's medium, respectively, it is apparent that, under these in vitro conditions, morphological alterations are not absolutely coupled to those prerequisite biochemical changes permitting successful transition of the bloodstream forms to life in the vector. There is an optimum state of biochemical preadaptation for successful transformation to procyclic trypomastigote associated with the pleomorphism of the three bloodstream forms. Our observations using Eflornithine-induced bloodstream forms of the EATRO 110 strain of Trypanosoma brucei suggest that there is a threshold of sensitivity to the stimulus for transformation associated with the three morphological forms, the intermediate form being the most sensitive, the long slender less, and short stumpy form least sensitive. In our studies described here, the very high concentration of citric acid cycle intermediates (3 mM) may optimally activate the TCA cycle (Brun and Schonenberger, 1981) and thereby bypass any normal gradient of activation. Consequently, all three cell types are stimulated to transform, regardless of their threshold of sensitivity and the presence of absence of the natural trigger(s) for transformation. This hypothesis needs to be confirmed. Since transformation of the bloodstream forms to procyclic trypomastigotes is a critical event in the life cycle of the trypanosome, further studies are needed to elucidate the molecular events involved in the regulation of this process. Only when the details have been worked out will the possibility exist for the design of rational chemotherapeutic interventions directed at controlling trypanosomal transformation. References
Bacchi, C.J., Garafalo, J., Mochenhaupt, D., McCann, P.P., Diekema, K.A., Pegg, A.E., Nathan, H.C., Mullaney, E.A., Chunosoff, L., Sjoerdsma, A. and Hunter, S.H. (1983) In vivo effects of OL-~-
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