EXPERIMENTALPARASITOLOGY 71, 350-352 (1990)
RESEARCH BRIEF Stage-Specific Adenylate Cyclase Activity in Trypanosoma brucei SYLVIE RoLIN,**’ STEPHANE HALLEUX,” JACQUELINE VAN SANDE,? JACQUES DUMONT,? ETIENNE PAYS,* AND MAURICE STEINERT* *Department of Molecular Biology, University of Brussels, 67, Rue des Chevaux, 81640 Rhode Saint GenPse, Belgium and tlnstitute of Interdisciplinary Research, School of Medicine, Campus Erasme, B-1070, Belgium
ROLIN, S., HALLEUX, S., VAN SANDE, J., DUMONT, J., PAYS, E., AND STEINERT, M. 1990. Stage-specific adenylate cyclase activity in Trypanosoma brucei. Experimental Parasitology
71, 350-352.
o IWO Academic
press, IIIC.
There have been only a few published studies concerning the role of cyclic nucleotides in Trypanosoma brucei and available information concerns exclusively the bloodstream form of the parasite. Adenylate cyclase and cyclic 3’,5’-adenosine monophosphate phosphodiesterase (CAMP phosphodiesterase) activity have been demonstrated by Martin et al. (1978) and Walter and Opperdoes (1982). Adenylate cyclase is present in a plasma membrane fraction, perhaps the flagellar pocket, whereas CAMP phosphodiesterase appears cytosohc. Changes in CAMP level seem associated with events triggering differentiation from slender to stumpy forms during parasitemia (Mancini and Patton 1981; Reed et al. 1985). In this study we report that the adenylate cyclase activity of bloodstream forms of T. brucei differs from that of insect-specific (procyclic) forms with respect to its response to calcium. Bloodstream forms of T. brucei (stock EATRO 1125, clones AnTat l.lD or 1.3A) were isolated from infected rat blood by the method of Lanham (1968). Procyclic forms, established from bloodstream forms by cultivation at 28°C (Cunningham 1977), were maintained routinely at 28°C by subpassage every 34 days. Assays for adenylate cyclase activity were performed on exponentially growing procyclic and bloodstream forms, permeabilized by “swell dialysis” (Voorheis and Martin 1980). Trypanosomes were harvested by centrifugation and resuspended in a medium of low osmotic strength (55 mM KCI, 1 mM glucose, 5 mM MgCI,, 13.3 m&f Tes, 20 &ml leupeptin, pH 7.5) at 4°C. The cells were preincubated in this medium for variable periods, then samples (20 pl) were added to 80 ~1 of the assay cocktail containing 25 mM Tes (pH
7.4), 0.5 mM CAMP, 10r&f phosphocreatine,50units ’ To whom correspondence should be addressed.
creatine kinase/ml, 10 mM MgCI,, 20 n-&f KCl, 20 &ml leupeptin, 1 &i (#P) ATP (lw Ci/mmol) and 0.5 mM ATP, and incubated for 5 to 20 min. The incubations were performed at 28°C for procyclics and 37°C for bloodstream forms. Reactions were stopped by adding 100 (~1of 2% SDS, 40 mM ATP, 1.4 mM CAMP at pH 7.5. Approximately 20,000 cpm of [3H]cyclic AMP (30 Ci/mmol) was then added to each assay. Blanks were preparedby addingtrypanosomes after the “stop solution.” The CAMP was isolated by two-step chromatography (Salomon 1974). The last eluate was collected directly into 12 ml Insta-Gel II (Packard) and counted by liquid scintillation. It appears that the swell dialysis method can be applied to measure adenylate cyclase activity in procyclic forms, as well as in bloodstream forms (Voorheis 1980). Figure 1 shows that the time course of CAMP production in whole permeabilized procyclic and bloodstream cells is linear during the 20 min of the assay. In order to study the effect of calcium on adenylate cyclase, several experiments were performed under various conditions of preincubation and assay. The range of values found in these experiments was 1.0-6.6 and 2.2-6.5 pmoles of CAMP formed per minute for 10’ bloodstream and procyclic cells, respectively. In agreement with previous studies (Voorheis and Martin 1980 1981), we found that calcium increased the enzyme activity two- to eightfold in bloodstream forms and that Ca’+-dependent activation of bloodstream adenylate cyclase is transient and
decreasesto near Ca*+-independentlevels at 2 m&f Ca*+. However calcium had no effect on adenylate cyclase activity in procyclic forms. Typical doseresponse curves for bloodstream and procyclic forms under identical experimental conditions are plotted in Fig. 2. The mechanism by which calcium activates the bloodstream adenylate cyclase is still unclear, al-
350 OOW4894/W$3.00 Copyright ‘0 1990 by Academic Press. Inc. Au rights of reproduction in any form reserved.
351
T. brucei: ADENYLATE CYCLASE ACTIVITY pmdCAMP, 10’calls
25 _
0
4
0
12
16
M
TlME(min)
FIG. 1. Time course of CAMP production in swollen procyclic and bloodstream forms. Cells (5 x lOs/ml) were preincubated for 3 min at room temperature, as described under Materials and Methods. Samples (20 ~1-10 cells) were added to separate SO-p1portions of assay cocktail for the periods indicated. Each value represents the mean 2 SE of several determinations. Vertical bars represent the standard error of the mean (SE); the absence of bars indicates values fall within the symbol. 0, Procyclic forms; A, bloodstream forms. though similar activation in mammalian cells has been reported to involve calmodulin (or a calmodulin-like protein) that serves as receptor for Ca” (Brostrom et al. 1975; Cheung 1980). It does not seem to depend on a calcium-activated protease, since (i) it occurs in the presence of leupeptin, a protease inhibitor and (ii) it is reversed following chelation of calcium by EGTA (data not shown). A simple interpretation of these results is that an additional adenylate cyclase, stimulable by calcium, is at work in bloodstream forms. This conclusion agrees with observations made at the gene level. During the process of characterization of the variant surface glycoprotein (VSG) gene expression site from bloodstream forms, we discovered a gene (ESAG 4, for Expression Site Associated Gene 4) (Pays et al. 1989) showing significant sequence homology with the adenylate cyclase gene of Saccharomyces cerevisiae (Katakoa et al. 1985).This gene is expressed in bloodstream forms only by a RNA polymerase resistant to a-amanitin. Related sequences are transcribed in both procyclic and bloodstream forms, but by a polymerase sensitive to the drug (Pays et al. 1989).It is tempting to speculate that ESAG4 encodes the calcium-activated bloodstream adenylate cyclase, although its nucleotide sequence does not reveal an obvious consensus for calcium-binding motives. Through the effects of calcium, a relationship can be envisaged between a specific adenylate cyclase activ-
ca++(mM)
FIG. 2. Concentration-effect of calcium on adenylate cyclase activity in procyclic and bloodstream forms. Cells (5 x lO*/ml) were preincubated in swell dialysis medium and assayed for adenylate cyclase activity (3-min preincubation-20-min assay) in the presence of different concentrations of CaCl, (from 0.01 to 2 mikf). Results are expressed as relative amounts of CAMP formed (means ? SE; control = 1). The amount of CAMP produced in the absence of CaClz is 6.5 and 2.2 pmole/min per 10’ procyclic and bloodstream forms, respectively. Cl, Procylic forms; A, bloodstream forms.
ity and processing of the antigenic surface coat. Indeed, in addition to stimulating adenylate cyclase activity, calcium has been reported to induce surface coat release from bloodstream forms (Bowles and Voorheis 1982;Voorheis et al. 1982).We are currently testing the possibility that these two observations may be linked. We thank H. P. Voorheis (Dublin) for helpful advice and aid, and D. Jefferies (Brussels) for comments on the manuscript. This work was supported by the Commission of the European Communities (contract TS2/ 0022-B)B, by the “Communaute Francaise de Belgique” (Convention ARC N” 89/94-134) and by Solvay & Cie (Brussels).
REFERENCES BOWLES,D. J., AND VOORHEIS,H. P. 1982. Release of the surface coat from the plasma membrane of intact bloodstream forms of Trypnnosoma brucei requires Ca2+. FEBS Lett. 139, 17-21. BROSTROM,C. O., HUANG, Y. C., BRECKENIUDGE, B. MC, AND WOLFF, D. J. 1975. Identification of a calcium-binding protein as a calcium dependent regulator of brain adenylate cyclase. Proc. Nat/. Acad. Sci. USA 72, 64-68.
352
ROLIN ET AL.
CHEUNG, W. Y. 1980. Calmodulin plays a pivotal role
in celhtlar regulation. Science 207, 19-27. CUNNINGHAM, I. 1977. New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. Protozoology 24, 325-329. KATAKOA, J., BROEK, D., AND WIGLER, M. 1985. DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell 43, 493-50s. LANHAM, S. M. 1968. Separation of trypanosomes
from the blood of infected rats and mice by anionexchangers. Nature (London) 218, 1273-1274. MANCINI, P. E., AND PATTON, C. L. 1981. Cyclic 3’:5’ adenosine monophosphate levels during the developmental cycle of Trypanosoma brucei brucei in the rat. Mol. Biochem. Parasitol. 3, 19-31. MARTIN, B. R., VOORHEIS, H. P., AND KENNEDY, E. L. 1978.Adenylate cyclase in bloodstream forms of Ttypanosoma (ttypanozoon) brucei sp. Biochem. J. 175, 207-212. PAYS, E., TEBABI, P., PAYS, A., COQUELET, H., REVELARD, P., SALMON, D., AND STEINERT, M. 1989. The genes and transcripts of an antigen gene expression site from T. brucei. Cell 57, 835-845. REED, S. L., FIERER,A. S., GODDARD,D. R., COLMERAUER,M. E. M., AND DAVIS, C. E. 1985. Effect
of theophylline on differentiation of Trypanosoma brucei. Infect. Immun. 49, 844-847. SALOMON,Y., LONDOS,C., AND RODBELL, M. 1974. A highly sensitive assay for adenylate cyclase. Anal. Biochem. 58, 541-548. VOORHEIS,H. P., AND MARTIN, B. R. 1980. ‘Swell dialysis’ demonstrates that adenylate cyclase in Trypanosoma brucei is regulated by calcium ions. Eur. J. Biochem. 113, 223-227. VOORHEIS,H. P., AND MARTIN, B. R. 1981. Characteristics of the calcium mediated mechanism activating adenylate cyclase in Ttypanosoma brucei. Eur. J. Biochem. 116,471477. VOORHEIS,H. P., BOWLES,D. J., AND SMITH, G. A. 1982. Characteristics of the release of the surface coat proteins from bloodstream forms of Trypanosoma brucei. J. Biol. Chem. 10, 23w2304. WALTER, R. D., AND OPPERDOES, F. R. 1982. Subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinase and phosphoprotein phosphatase in Trypanosoma brucei. Mol. Biothem. Parasitol. 6. 287-295. Received 3 April 1990; accepted with revision 19 June 1990
RESEARCH BRIEF Stage-Specific Adenylate Cyclase Activity in Trypanosoma brucei SYLVIE RoLIN,**’ STEPHANE HALLEUX,” JACQUELINE VAN SANDE,? JACQUES DUMONT,? ETIENNE PAYS,* AND MAURICE STEINERT* *Department of Molecular Biology, University of Brussels, 67, Rue des Chevaux, 81640 Rhode Saint GenPse, Belgium and tlnstitute of Interdisciplinary Research, School of Medicine, Campus Erasme, B-1070, Belgium
ROLIN, S., HALLEUX, S., VAN SANDE, J., DUMONT, J., PAYS, E., AND STEINERT, M. 1990. Stage-specific adenylate cyclase activity in Trypanosoma brucei. Experimental Parasitology
71, 350-352.
o IWO Academic
press, IIIC.
There have been only a few published studies concerning the role of cyclic nucleotides in Trypanosoma brucei and available information concerns exclusively the bloodstream form of the parasite. Adenylate cyclase and cyclic 3’,5’-adenosine monophosphate phosphodiesterase (CAMP phosphodiesterase) activity have been demonstrated by Martin et al. (1978) and Walter and Opperdoes (1982). Adenylate cyclase is present in a plasma membrane fraction, perhaps the flagellar pocket, whereas CAMP phosphodiesterase appears cytosohc. Changes in CAMP level seem associated with events triggering differentiation from slender to stumpy forms during parasitemia (Mancini and Patton 1981; Reed et al. 1985). In this study we report that the adenylate cyclase activity of bloodstream forms of T. brucei differs from that of insect-specific (procyclic) forms with respect to its response to calcium. Bloodstream forms of T. brucei (stock EATRO 1125, clones AnTat l.lD or 1.3A) were isolated from infected rat blood by the method of Lanham (1968). Procyclic forms, established from bloodstream forms by cultivation at 28°C (Cunningham 1977), were maintained routinely at 28°C by subpassage every 34 days. Assays for adenylate cyclase activity were performed on exponentially growing procyclic and bloodstream forms, permeabilized by “swell dialysis” (Voorheis and Martin 1980). Trypanosomes were harvested by centrifugation and resuspended in a medium of low osmotic strength (55 mM KCI, 1 mM glucose, 5 mM MgCI,, 13.3 m&f Tes, 20 &ml leupeptin, pH 7.5) at 4°C. The cells were preincubated in this medium for variable periods, then samples (20 pl) were added to 80 ~1 of the assay cocktail containing 25 mM Tes (pH
7.4), 0.5 mM CAMP, 10r&f phosphocreatine,50units ’ To whom correspondence should be addressed.
creatine kinase/ml, 10 mM MgCI,, 20 n-&f KCl, 20 &ml leupeptin, 1 &i (#P) ATP (lw Ci/mmol) and 0.5 mM ATP, and incubated for 5 to 20 min. The incubations were performed at 28°C for procyclics and 37°C for bloodstream forms. Reactions were stopped by adding 100 (~1of 2% SDS, 40 mM ATP, 1.4 mM CAMP at pH 7.5. Approximately 20,000 cpm of [3H]cyclic AMP (30 Ci/mmol) was then added to each assay. Blanks were preparedby addingtrypanosomes after the “stop solution.” The CAMP was isolated by two-step chromatography (Salomon 1974). The last eluate was collected directly into 12 ml Insta-Gel II (Packard) and counted by liquid scintillation. It appears that the swell dialysis method can be applied to measure adenylate cyclase activity in procyclic forms, as well as in bloodstream forms (Voorheis 1980). Figure 1 shows that the time course of CAMP production in whole permeabilized procyclic and bloodstream cells is linear during the 20 min of the assay. In order to study the effect of calcium on adenylate cyclase, several experiments were performed under various conditions of preincubation and assay. The range of values found in these experiments was 1.0-6.6 and 2.2-6.5 pmoles of CAMP formed per minute for 10’ bloodstream and procyclic cells, respectively. In agreement with previous studies (Voorheis and Martin 1980 1981), we found that calcium increased the enzyme activity two- to eightfold in bloodstream forms and that Ca’+-dependent activation of bloodstream adenylate cyclase is transient and
decreasesto near Ca*+-independentlevels at 2 m&f Ca*+. However calcium had no effect on adenylate cyclase activity in procyclic forms. Typical doseresponse curves for bloodstream and procyclic forms under identical experimental conditions are plotted in Fig. 2. The mechanism by which calcium activates the bloodstream adenylate cyclase is still unclear, al-
350 OOW4894/W$3.00 Copyright ‘0 1990 by Academic Press. Inc. Au rights of reproduction in any form reserved.
351
T. brucei: ADENYLATE CYCLASE ACTIVITY pmdCAMP, 10’calls
25 _
0
4
0
12
16
M
TlME(min)
FIG. 1. Time course of CAMP production in swollen procyclic and bloodstream forms. Cells (5 x lOs/ml) were preincubated for 3 min at room temperature, as described under Materials and Methods. Samples (20 ~1-10 cells) were added to separate SO-p1portions of assay cocktail for the periods indicated. Each value represents the mean 2 SE of several determinations. Vertical bars represent the standard error of the mean (SE); the absence of bars indicates values fall within the symbol. 0, Procyclic forms; A, bloodstream forms. though similar activation in mammalian cells has been reported to involve calmodulin (or a calmodulin-like protein) that serves as receptor for Ca” (Brostrom et al. 1975; Cheung 1980). It does not seem to depend on a calcium-activated protease, since (i) it occurs in the presence of leupeptin, a protease inhibitor and (ii) it is reversed following chelation of calcium by EGTA (data not shown). A simple interpretation of these results is that an additional adenylate cyclase, stimulable by calcium, is at work in bloodstream forms. This conclusion agrees with observations made at the gene level. During the process of characterization of the variant surface glycoprotein (VSG) gene expression site from bloodstream forms, we discovered a gene (ESAG 4, for Expression Site Associated Gene 4) (Pays et al. 1989) showing significant sequence homology with the adenylate cyclase gene of Saccharomyces cerevisiae (Katakoa et al. 1985).This gene is expressed in bloodstream forms only by a RNA polymerase resistant to a-amanitin. Related sequences are transcribed in both procyclic and bloodstream forms, but by a polymerase sensitive to the drug (Pays et al. 1989).It is tempting to speculate that ESAG4 encodes the calcium-activated bloodstream adenylate cyclase, although its nucleotide sequence does not reveal an obvious consensus for calcium-binding motives. Through the effects of calcium, a relationship can be envisaged between a specific adenylate cyclase activ-
ca++(mM)
FIG. 2. Concentration-effect of calcium on adenylate cyclase activity in procyclic and bloodstream forms. Cells (5 x lO*/ml) were preincubated in swell dialysis medium and assayed for adenylate cyclase activity (3-min preincubation-20-min assay) in the presence of different concentrations of CaCl, (from 0.01 to 2 mikf). Results are expressed as relative amounts of CAMP formed (means ? SE; control = 1). The amount of CAMP produced in the absence of CaClz is 6.5 and 2.2 pmole/min per 10’ procyclic and bloodstream forms, respectively. Cl, Procylic forms; A, bloodstream forms.
ity and processing of the antigenic surface coat. Indeed, in addition to stimulating adenylate cyclase activity, calcium has been reported to induce surface coat release from bloodstream forms (Bowles and Voorheis 1982;Voorheis et al. 1982).We are currently testing the possibility that these two observations may be linked. We thank H. P. Voorheis (Dublin) for helpful advice and aid, and D. Jefferies (Brussels) for comments on the manuscript. This work was supported by the Commission of the European Communities (contract TS2/ 0022-B)B, by the “Communaute Francaise de Belgique” (Convention ARC N” 89/94-134) and by Solvay & Cie (Brussels).
REFERENCES BOWLES,D. J., AND VOORHEIS,H. P. 1982. Release of the surface coat from the plasma membrane of intact bloodstream forms of Trypnnosoma brucei requires Ca2+. FEBS Lett. 139, 17-21. BROSTROM,C. O., HUANG, Y. C., BRECKENIUDGE, B. MC, AND WOLFF, D. J. 1975. Identification of a calcium-binding protein as a calcium dependent regulator of brain adenylate cyclase. Proc. Nat/. Acad. Sci. USA 72, 64-68.
352
ROLIN ET AL.
CHEUNG, W. Y. 1980. Calmodulin plays a pivotal role
in celhtlar regulation. Science 207, 19-27. CUNNINGHAM, I. 1977. New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. Protozoology 24, 325-329. KATAKOA, J., BROEK, D., AND WIGLER, M. 1985. DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell 43, 493-50s. LANHAM, S. M. 1968. Separation of trypanosomes
from the blood of infected rats and mice by anionexchangers. Nature (London) 218, 1273-1274. MANCINI, P. E., AND PATTON, C. L. 1981. Cyclic 3’:5’ adenosine monophosphate levels during the developmental cycle of Trypanosoma brucei brucei in the rat. Mol. Biochem. Parasitol. 3, 19-31. MARTIN, B. R., VOORHEIS, H. P., AND KENNEDY, E. L. 1978.Adenylate cyclase in bloodstream forms of Ttypanosoma (ttypanozoon) brucei sp. Biochem. J. 175, 207-212. PAYS, E., TEBABI, P., PAYS, A., COQUELET, H., REVELARD, P., SALMON, D., AND STEINERT, M. 1989. The genes and transcripts of an antigen gene expression site from T. brucei. Cell 57, 835-845. REED, S. L., FIERER,A. S., GODDARD,D. R., COLMERAUER,M. E. M., AND DAVIS, C. E. 1985. Effect
of theophylline on differentiation of Trypanosoma brucei. Infect. Immun. 49, 844-847. SALOMON,Y., LONDOS,C., AND RODBELL, M. 1974. A highly sensitive assay for adenylate cyclase. Anal. Biochem. 58, 541-548. VOORHEIS,H. P., AND MARTIN, B. R. 1980. ‘Swell dialysis’ demonstrates that adenylate cyclase in Trypanosoma brucei is regulated by calcium ions. Eur. J. Biochem. 113, 223-227. VOORHEIS,H. P., AND MARTIN, B. R. 1981. Characteristics of the calcium mediated mechanism activating adenylate cyclase in Ttypanosoma brucei. Eur. J. Biochem. 116,471477. VOORHEIS,H. P., BOWLES,D. J., AND SMITH, G. A. 1982. Characteristics of the release of the surface coat proteins from bloodstream forms of Trypanosoma brucei. J. Biol. Chem. 10, 23w2304. WALTER, R. D., AND OPPERDOES, F. R. 1982. Subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinase and phosphoprotein phosphatase in Trypanosoma brucei. Mol. Biothem. Parasitol. 6. 287-295. Received 3 April 1990; accepted with revision 19 June 1990
