PROLINE TRANS PORT 4. Hampton JR. 1970. Lysine uptake in cultured Trypanosoma cruzi: interactions of competitive inhibitors. J. Protorool. 17, 597-600. 5. Hirato H, Asano A, Brodie AF. 1971. Respiration dependent transport of proline by electron transport particles of Mycobacterium phlei. Biochem. Biophys. Res. Commun. a, 368-74. 6. Isseroff H, Read CP. 1969. Studies on membrane transport. VI. Absorption of amino acids by fasciolid trematodes. Comp. Biochem. Physiol. 30, 1153-9. 7. Kay WW, Gronlund AF. 1969. Amino acid transport in Pseudomonas aeruginosa. J. Bacteriol. 97, 273-81. 8. Krassner SM. 1967. The effect of L-proline on respiration in Leishmania. J . Protorool. 14 (Suppl.), 20. 9. 1969. Proline metabolism in Leishmania tarentolae. Exp. Parasitol. 24, 348-63. 10. ___ , Flory B. 1971. Essential amino acids in the culture of Leishmania tarentolae. J. Parasitol. 57, 917-20. 11. -, - 1972. Proline metabolism in Leishmania donouani promastigotes. J . Protozool. 19,682-5. 12. Law SS, Mukkada AJ. 1975. Transport of L-proline by Leishmania tropica. Abstracts, 50th Annual Meeting, A m . SOG. Parasitologists, p. 82. 13. Lepley PL, Mukkada AJ. Characteristics of a-aminoisobutyric acid transport in Leishmania tropica promastigotes. Abstracts, 50th Annual Meeting, A m . Soc. Parasitol., p. 96. 14. Mukkada AJ. 1977. Tricarboxylic acid and glyoxylate cycles in the Leishmaniae. Acta T r o p . 34, 167-75. 15. ___ , Simon MW. 1977, Leishmania tropica: uptake of methionine by promastigotes. Exp. Paraitol. 42,87-96. 16. _ _ , Schaefer FW 111, Simon MW, Neu C. 1974. Delayed in uitro utilization of glucose by Leishmania tropica promastigotes. J . Protozool. 21, 393-7. 17. Oxender DL, Christensen HN. 1963. Distinct mediating ~
IN
L. tropica
30 1
systems for the transport of neutral amino acids by the Ehrlich cell.
J. Biol. Chem. 238, 3686-99.
18. Ruff MD, Read CP. 1974. Specificity of amino acid transport in Trypanosoma equiperdum. J. Protorool. 21, 368-73. 19. Schaefer FW 111, Mukkada AJ. 1976. Specificity of the glucose transport system in Leishmania tropica promastigotes. J . Protozool. 23, 446-9. 20. ___ , Bell EJ, Etges FJ. 1970. Leishmania tropica: chemostatic cultivation. Exp. Parasitol. 28, 465-72. 21. , Martin E, Mukkada AJ. 1974. The glucose transport system in Leishmania tropica promastigotes. J . Protozool. 21, 592-6. 22. Schwencke NM, Schwencke J. 1969. A proline transport system in Saccharomyces chevalieri. Biochim. Biophys. Acta 173, 3 13-23. 23. Sherman IW, Tanigoshi L. 1974. Incorporation of r4C]amino acids by malarial Plasmodium. 6. Changes in the kinetic constants of amino acid transport during infection. Exp. Parasitol. 35, 369-73. 24. Simon MW, Mukkada AJ. 1977. Leishmania tropica: regulation and specificity of the methionine transport system in promastigotes. Exp. Parasitol. 42, 97-105. 25. Sorouri P. 1955. The nuclear cytology of Leishmania tropica. J. Morphol. 97, 393-405. 26. Sylvester D, Krassner SM. 1976. Proline metabolism in Trypanosoma cruzi epimastigotes. Comp. Biochem. Physiol. 55B, 443-7. 27. Thomas EL, Christensen HN. 1971. Nature of the cosubstrate action of Na+ and neutral amino acids in a transport system. J. Biol. Chem. 246, 1682-8. 28. Tristam H, Neale S. 1968. The activity and specificity of the proline permease in wild-type and analogue-resistant strains of Escherichia coli. J. Gen. Microbiol. 50, 121-37.
I . Protozool., 26(2)! 1979, p p . 301-303
0 1979 by the Society of ProtozoologBts
Acetate Oxidation by Bloodstream Forms of Trypanosoma cruzi* ROBERTO DOCAMP0,t FERNANDO S. CRUZ,S WILSON LEON# and GABRIEL A. SCHMUNISt t Znstituto de Quimica Bioldgica, Facultad de Medicina, Universidad de Buenos Aires, Argentina and $ Znstituto de Microbiologia, Universidade Federal do Rio de Janeiro, 20.000 Rio de Janeiro, B r a d
SYNOPSIS. Bloodstream forms of Trypanosoma cruri had a substantial increase in respiration in the presence of acetate. Oxidation of acetate took place via the tricarboxylic acid cycle and involved an antimycin A-sensitive respiratory pathway. Oxygen uptake in the presence of acetate was as sensitive to antimycin A inhibition as was COB production. There was a 6-7% residual O2uptake which was not inhibited by high antimycin concentrations. Human anti-T. czuzi sera had no effect on oxygen uptake.
Index Key Words: Trypanosoma cruri bloodstream forms; acztate oxidation; tricarboxylic acid cycle; antimycin inhibition ; immune sera
THE
aerobic oxidation of acetate has been extensively studied in culture forms of Trypanosoma cruzi ( 2 ) . I n these forms, utilization of acetate does not involve a substantial increase in respiratory rate as would be expected assuming total oxidation. Acetate is oxidized via the tricarboxylic acid cycle and an antimycin A-sensitive respiratory pathway. We now report on the 0, consumption of bloodstream forms of T. cruzi in the presence of acetate.
* This investigation was supported by research grants from the UNPD/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, The Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq), B r a d , and the Financiadora de Projetos de Pesquisa (FINEP convinio no. 362). 5 T o whom all correspondence should be addressed.
MATERIALS AND METHODS Organisms.-Trypanosoma cruzi strain Y bloodstream forms were obtained from batches of 15-20 infected mice (15-20 g) which were bled 7 days postinoculation from the retroorbital sinus using heparinized capillaries. Blood was diluted 1 :4 with tissue culture medium 199 (10) and 1% (w/v) albumin. The blood was centrifuged a t 80 g for 10 min a t room temperature and allowed to stand a t 37 C for 20 min. Finally, the supernatant fluid was centrifuged a t 10,000 g for 30 min, a t 4 C . The final preparation was free from blood cells. I t was resuspended in the saline solution described previously ( 6 ) to give a final concentration of 1-5 X lo7 cells/ml. Reagents.-Analytical grade reagents were used throughout the
ACETATEOxmATIoN
302
TABLE 1. Effect of antimycin A on the oxidation of [I-"C]acetate by bloodstream forms of T. cruzi.* Conc. antimycin A
0, consumption
(SM)
(alO,/h/l08 cells)
0
1.9 5.4 18.2
210.0 110.0 ( 5 2 ) t 13.8 (93) 11.0 (94)
BY
Trypanosoma cruzi
TABLE 2. Effect of several concentrations of immune sera on 0 s uptake and "CO, production from [l-"c] acetate, by bloodstream forms of Trypanosoma cruzi.*
Total "C in remiratom CO , (dpm/h/ Ib cells
x
10")
35.5 17.8 (50) 2.5 (93) 2.2 (941
Exp. 1
1.2 X 10' cells incubated for 1 h. For other experimental conditions, see Materials and Methods. t Percentage inhibition.
RESULTS T o study the aerobic oxidation of acetate by bloodstream forms of Trypanosoma cruzi, the following metabolic parameters were examined: ( a ) endogenous 0,uptake and that resulting from acetate oxidation; ( b ) 14C0, production, total I4C incorporation, and I4C distribution in cells, after oxidation of [1-l4C]acetate.
None Normal
Immune
2
experiments. [1-14C]Acetate (Na salt), purchased from International Chemical & Nuclear Corporation, Irvine, California, was diluted with "cold'' carrier to give a specific activity of 16.65 pCi/mmol. Antimycin A was purchased from Sigma Chemical Co., St. Louis, Missouri. Assays for Metabolic Actiuity.--O, uptake was measured in a Warburg respirometer. All measurements were made in duplicate. Cells were suspended in 3.1 ml of the saline ( 6 ) . Each Warburg flask contained a total volume of 3.2 ml. Incubations were at 37 C. After incubation with 1%-labeled substrate, cells were transferred to an ice bath, then centrifuged at 4 C, and washed twice with 0.5 ml of 0.154 M KCl. The pellets were extracted with 5 ml of 80% (v/v) methanol-H,O. To measure the total radioactivity incorporated by the cells, 100 pl of cell suspension in methanol were transferred to vials containing 10 ml of scintillation solution ( 4 ) . Cell suspensions were centrifuged and the preciDitate discarded. Supernatant fluids (containing the soluble cell fraction) were evaporated in uucuo to 0.4-0.8 ml volumes. The residues were chromatographed with the phenol-H,O and butanol-propionic acid-H,O solvent systems described by Benson et al. ( 1). Radioactive compounds separated by chromatography were identified as described previously ( 7 ) . To measure the formation of respiratory 14CO,, 0.1 mi of 10% (w/v) KOH (carbonate-free) was placed in the center well of the Warburg flask with a filter paper. After incubation, the p H value of the suspension fluid was 7.2. The filter papers were carefully removed from the center wells and transferred directly to counting vials containing scintillation fluid ( 6 ) . Radioactivity was determined in a Beckman LS 250 scintillation spectrometer. Immune Sera.-Human anti-T. cruzi sera were obtained from 2 patients with chronic infections. Both had positive xenodiagnosis and positive antibody titers, as determined by the indirect fluorescent antibody test ( I F T ) ( 1 : 128 and 1 :256), indirect hemagglutination (IH) ( 1 :64 and 1 : 128) and complement fixation ( C F ) (1 :32 and 1 :32). Serum samples from 2 persons with negative serologic findings ( I I F : 1 :2 and 1 :4; IH: 1 :2 and 1 :2; and CF: 1 :2) were used as controls. All tests were carried out as previously described ( 8 ) . Sera were inactivated at 56C for 30 min before use.
Serum
None Normal Immune
Total "C in respiratory (20% ( dpm/h/ 10' cells x 10")
Conc. sefum in reaction mixture
O2consumption
0 1:8 1:64 1:256 1:8 1 :64 1:256
205 215 256 194
260 202
36.5 35.5 37.3 34.4 37.8 34.3 36.3
0 1:16 1:16
207 300 292
37.0 38.1 37.8
(plO,/h/ 108 cells)
205
* 1.2 x 10' cells (Experiment 1) and 2.1 x 10' cells (Experiment 2) incubated for 1 h. For other experimental conditions, see Materials and Methods.
In contrast to the results obtained with culture forms (2, 7 ) ) the addition of acetate to bloodstream forms stimulated the respiratory rate markedly. Bloodstream forms incubated for 1 h with or without 0.01 M acetate had an oxygen consumption of 202 k 81.3 and 9.0 & 1.6 pl O,/h/lOs cells, respectively (mean & SD of 4 experiments). Acetate was oxidized by the bloodsteam forms since radioactivity appeared in respiratory CO, when [l-"%]acetate was used as substrate (Table 1 ) . Although this value does not constitute true total CO, production, since the pH value of the buffer was 7.2 and some amount of CO, might be present in the suspension fluid as HCO,, it is useful for comparative purposes. Total 14C incorporated and I4C in the methanol-water soluble fraction of bloodstream forms (3.3 X 107 cells) were 6.4 and 5.7 dpm/h/l08 cells x 10-3, respectively. The reaction products resulting from the oxidation of [ l-14C]acetate by bloodstream forms were mainly glutamic acid (25%), alanine (8%), succinic acid (3%), aspartic acid (9%), and tricarboxylic acids (10%). It should be noted that: ( a ) the combined radioactivity of the amino acids amounted to more than 40% of the total in the soluble extract; ( b ) radioactivity in glutamic acid proved the labeling of 2-oxoglutaric acid; ( c ) radioactivity in aspartic acid proved the labeling of oxalacetic acid; and ( d ) radioactivity in alanine involved the labeling and decarboxylation of L-malic acid (the "malic" enzyme reaction) ( 3 ) . The rest of the total radioactivity was distributed in succinic and tricarboxylic acids as well as other compounds, difficult to identify because of the low level of radioactivity associated with each. In Table 1, the effects of antimycin A on W O , production from [l-14C]acetate and 0, uptake by bloodstream forms are shown. Antimycin A, a specific inhibitor of mitochondria1 respiration and electron transport ( 11) inhibited the oxidation of [l-14C]acetate by these forms. Production of I4CO, and 0, uptake were affected to about the same degree. The results of experiments with immune sera are shown in Table 2. Several dilutions of these sera had no effect on 0, uptake and on the production of 14C02 from [l-I4C]acetate by bloodstream forms of T . cruzi when compared with cells incubated with normal sera.
ACETATEOXIDATION BY Trypanosoma cruzi
303
DISCUSSION
LITERATURE C I T E D
In the present investigation we demonstrated a substantial increase in respiratory rate with acetate in bloodstream forms of T . cruzi. The distribution of I4C after oxidation of [l-'*C]acetate by these forms was very similar to that noted in culture forms (2, 7 ) . Among the Krebs-cycle intermediates, the tricarboxylic acids retained most of the label. Labeling of glutamic and aspartic acids indicated the intermediate formation of 2oxoglutaric and oxalacetic acids in the process of acetate oxidation. The foregoing results and the inhibitory effect of antimycin A constitute strong evidence of an operational Krebs cycle and a respiratory chain in bloodstream forms. Although the glyoxylate cycle cannot be excluded, its contribution to acetate oxidation seems to be less important. Although it has been suggested that immune factors can play a significant role in host-induced variations in respiratory exchanges of trypanosomes (13) , the incubation of bloodstream forms of T . cruzi with immune sera did not alter their respiratory rate or the rate of acetate oxidation. Similar results were obtained with T . lewisi ( 1 3 ) . In contrast, immune sera decreased the rate of oxygen consumption of T . vivax (5))T . brucei ( 1 2 ) , and T . gambiense ( 9 ) . This effect may be due to lysis of a certain percentage of the flagellates initially present; this problem had been avoided in our experiments by inactivation of the sera.
1. Benson AA, Bassham JA, Calvin M, Goodale TC, Hass VA, Stepka W. 1950. The path of carbon in photosynthesis. V. Paper chromatography and radioautography of the products. 1. A m . Chem. Soc. 72, 1710-8. 2. Boiso JF, Stoppani AOM. 1973. The mechanism of acetate and pyruvate oxidation by Trypanosoma cruti. J . Prototool. 20, 673-8. 3. Cazzulo JJ, Juan SM, Segura EL. 1977. The malic enzyme from Trypanosoma cruti. J . Gen. Microbiol. 99, 237-41. 4 . Cruz FS, Krassner S. 1973. Assimilatory sulfate reduction by the hemoflagellate Leishmania tarentolae. I . Prototool. 18, 718-22. 5. Desowitz RS. 1956. Effect of antibody on the respiratory rate of Trypanosoma vivax. Nature (London) 177, 132-3. 6. Docampo R, Lopes JN, Cruz FS, De Souza W. 1977. Trypanosoma cruti: ultrastructural and metabolic alterations of epimastigotes by j3-Lapachone. Ex@. Parasitol. 42, 142-9. 7. , Boiso JF, Stoppani AOM. 1978. Tricarboxylic acid cycle operation at the kinetoplast-mitochondrion complex of Trypanosoma cruti. Biochim. Biophys. Acta 502, 466-76. 8. Gonzalez Cappa S, Vattuone NH, Menes S , Schumunis GA. 1973. Humoral antibody response and Ig characterization of the specific agglutinins in rabbit during experimental American trypanosomiases. Exp, Parasitol. 34, 32-9. 9. Masseyeff R, Gombert J. 1963. Inhibition de la respiration de trypanosomes par le serum de malades atteints de Trypanosomiase africaine A Trypanosoma gambiense. Ann. Znst. Pasteur, Paris 104, 115-22. 10. Paul J. 1972. Cell and Tissue Culture, 4th ed., Churchill Levingstone, Edinburgh & London. 11. Rieske JS. 1967. Antimycin A, in Gottlieb D, Shaw PD, eds., Antibiotics Z . Mechanism of Action, Springer Verlag, New York, pp. 542-8. 12. Thurston JP. 1958. The effect of immune sera on the respiration of Trypanosoma brucei in vitro. Parasitology 4.8,463-7. 13. Von Brand T. 1975. Biochemistry of Parasites, 2nd ed., Academic Press, New York.
ACKNOWLEDGEMENT The authors thank Prof. L. R. Travassos (Department of General Microbiology, UFRJ) for help with the manuscript.
IN
L. tropica
30 1
systems for the transport of neutral amino acids by the Ehrlich cell.
J. Biol. Chem. 238, 3686-99.
18. Ruff MD, Read CP. 1974. Specificity of amino acid transport in Trypanosoma equiperdum. J. Protorool. 21, 368-73. 19. Schaefer FW 111, Mukkada AJ. 1976. Specificity of the glucose transport system in Leishmania tropica promastigotes. J . Protozool. 23, 446-9. 20. ___ , Bell EJ, Etges FJ. 1970. Leishmania tropica: chemostatic cultivation. Exp. Parasitol. 28, 465-72. 21. , Martin E, Mukkada AJ. 1974. The glucose transport system in Leishmania tropica promastigotes. J . Protozool. 21, 592-6. 22. Schwencke NM, Schwencke J. 1969. A proline transport system in Saccharomyces chevalieri. Biochim. Biophys. Acta 173, 3 13-23. 23. Sherman IW, Tanigoshi L. 1974. Incorporation of r4C]amino acids by malarial Plasmodium. 6. Changes in the kinetic constants of amino acid transport during infection. Exp. Parasitol. 35, 369-73. 24. Simon MW, Mukkada AJ. 1977. Leishmania tropica: regulation and specificity of the methionine transport system in promastigotes. Exp. Parasitol. 42, 97-105. 25. Sorouri P. 1955. The nuclear cytology of Leishmania tropica. J. Morphol. 97, 393-405. 26. Sylvester D, Krassner SM. 1976. Proline metabolism in Trypanosoma cruzi epimastigotes. Comp. Biochem. Physiol. 55B, 443-7. 27. Thomas EL, Christensen HN. 1971. Nature of the cosubstrate action of Na+ and neutral amino acids in a transport system. J. Biol. Chem. 246, 1682-8. 28. Tristam H, Neale S. 1968. The activity and specificity of the proline permease in wild-type and analogue-resistant strains of Escherichia coli. J. Gen. Microbiol. 50, 121-37.
I . Protozool., 26(2)! 1979, p p . 301-303
0 1979 by the Society of ProtozoologBts
Acetate Oxidation by Bloodstream Forms of Trypanosoma cruzi* ROBERTO DOCAMP0,t FERNANDO S. CRUZ,S WILSON LEON# and GABRIEL A. SCHMUNISt t Znstituto de Quimica Bioldgica, Facultad de Medicina, Universidad de Buenos Aires, Argentina and $ Znstituto de Microbiologia, Universidade Federal do Rio de Janeiro, 20.000 Rio de Janeiro, B r a d
SYNOPSIS. Bloodstream forms of Trypanosoma cruri had a substantial increase in respiration in the presence of acetate. Oxidation of acetate took place via the tricarboxylic acid cycle and involved an antimycin A-sensitive respiratory pathway. Oxygen uptake in the presence of acetate was as sensitive to antimycin A inhibition as was COB production. There was a 6-7% residual O2uptake which was not inhibited by high antimycin concentrations. Human anti-T. czuzi sera had no effect on oxygen uptake.
Index Key Words: Trypanosoma cruri bloodstream forms; acztate oxidation; tricarboxylic acid cycle; antimycin inhibition ; immune sera
THE
aerobic oxidation of acetate has been extensively studied in culture forms of Trypanosoma cruzi ( 2 ) . I n these forms, utilization of acetate does not involve a substantial increase in respiratory rate as would be expected assuming total oxidation. Acetate is oxidized via the tricarboxylic acid cycle and an antimycin A-sensitive respiratory pathway. We now report on the 0, consumption of bloodstream forms of T. cruzi in the presence of acetate.
* This investigation was supported by research grants from the UNPD/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, The Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq), B r a d , and the Financiadora de Projetos de Pesquisa (FINEP convinio no. 362). 5 T o whom all correspondence should be addressed.
MATERIALS AND METHODS Organisms.-Trypanosoma cruzi strain Y bloodstream forms were obtained from batches of 15-20 infected mice (15-20 g) which were bled 7 days postinoculation from the retroorbital sinus using heparinized capillaries. Blood was diluted 1 :4 with tissue culture medium 199 (10) and 1% (w/v) albumin. The blood was centrifuged a t 80 g for 10 min a t room temperature and allowed to stand a t 37 C for 20 min. Finally, the supernatant fluid was centrifuged a t 10,000 g for 30 min, a t 4 C . The final preparation was free from blood cells. I t was resuspended in the saline solution described previously ( 6 ) to give a final concentration of 1-5 X lo7 cells/ml. Reagents.-Analytical grade reagents were used throughout the
ACETATEOxmATIoN
302
TABLE 1. Effect of antimycin A on the oxidation of [I-"C]acetate by bloodstream forms of T. cruzi.* Conc. antimycin A
0, consumption
(SM)
(alO,/h/l08 cells)
0
1.9 5.4 18.2
210.0 110.0 ( 5 2 ) t 13.8 (93) 11.0 (94)
BY
Trypanosoma cruzi
TABLE 2. Effect of several concentrations of immune sera on 0 s uptake and "CO, production from [l-"c] acetate, by bloodstream forms of Trypanosoma cruzi.*
Total "C in remiratom CO , (dpm/h/ Ib cells
x
10")
35.5 17.8 (50) 2.5 (93) 2.2 (941
Exp. 1
1.2 X 10' cells incubated for 1 h. For other experimental conditions, see Materials and Methods. t Percentage inhibition.
RESULTS T o study the aerobic oxidation of acetate by bloodstream forms of Trypanosoma cruzi, the following metabolic parameters were examined: ( a ) endogenous 0,uptake and that resulting from acetate oxidation; ( b ) 14C0, production, total I4C incorporation, and I4C distribution in cells, after oxidation of [1-l4C]acetate.
None Normal
Immune
2
experiments. [1-14C]Acetate (Na salt), purchased from International Chemical & Nuclear Corporation, Irvine, California, was diluted with "cold'' carrier to give a specific activity of 16.65 pCi/mmol. Antimycin A was purchased from Sigma Chemical Co., St. Louis, Missouri. Assays for Metabolic Actiuity.--O, uptake was measured in a Warburg respirometer. All measurements were made in duplicate. Cells were suspended in 3.1 ml of the saline ( 6 ) . Each Warburg flask contained a total volume of 3.2 ml. Incubations were at 37 C. After incubation with 1%-labeled substrate, cells were transferred to an ice bath, then centrifuged at 4 C, and washed twice with 0.5 ml of 0.154 M KCl. The pellets were extracted with 5 ml of 80% (v/v) methanol-H,O. To measure the total radioactivity incorporated by the cells, 100 pl of cell suspension in methanol were transferred to vials containing 10 ml of scintillation solution ( 4 ) . Cell suspensions were centrifuged and the preciDitate discarded. Supernatant fluids (containing the soluble cell fraction) were evaporated in uucuo to 0.4-0.8 ml volumes. The residues were chromatographed with the phenol-H,O and butanol-propionic acid-H,O solvent systems described by Benson et al. ( 1). Radioactive compounds separated by chromatography were identified as described previously ( 7 ) . To measure the formation of respiratory 14CO,, 0.1 mi of 10% (w/v) KOH (carbonate-free) was placed in the center well of the Warburg flask with a filter paper. After incubation, the p H value of the suspension fluid was 7.2. The filter papers were carefully removed from the center wells and transferred directly to counting vials containing scintillation fluid ( 6 ) . Radioactivity was determined in a Beckman LS 250 scintillation spectrometer. Immune Sera.-Human anti-T. cruzi sera were obtained from 2 patients with chronic infections. Both had positive xenodiagnosis and positive antibody titers, as determined by the indirect fluorescent antibody test ( I F T ) ( 1 : 128 and 1 :256), indirect hemagglutination (IH) ( 1 :64 and 1 : 128) and complement fixation ( C F ) (1 :32 and 1 :32). Serum samples from 2 persons with negative serologic findings ( I I F : 1 :2 and 1 :4; IH: 1 :2 and 1 :2; and CF: 1 :2) were used as controls. All tests were carried out as previously described ( 8 ) . Sera were inactivated at 56C for 30 min before use.
Serum
None Normal Immune
Total "C in respiratory (20% ( dpm/h/ 10' cells x 10")
Conc. sefum in reaction mixture
O2consumption
0 1:8 1:64 1:256 1:8 1 :64 1:256
205 215 256 194
260 202
36.5 35.5 37.3 34.4 37.8 34.3 36.3
0 1:16 1:16
207 300 292
37.0 38.1 37.8
(plO,/h/ 108 cells)
205
* 1.2 x 10' cells (Experiment 1) and 2.1 x 10' cells (Experiment 2) incubated for 1 h. For other experimental conditions, see Materials and Methods.
In contrast to the results obtained with culture forms (2, 7 ) ) the addition of acetate to bloodstream forms stimulated the respiratory rate markedly. Bloodstream forms incubated for 1 h with or without 0.01 M acetate had an oxygen consumption of 202 k 81.3 and 9.0 & 1.6 pl O,/h/lOs cells, respectively (mean & SD of 4 experiments). Acetate was oxidized by the bloodsteam forms since radioactivity appeared in respiratory CO, when [l-"%]acetate was used as substrate (Table 1 ) . Although this value does not constitute true total CO, production, since the pH value of the buffer was 7.2 and some amount of CO, might be present in the suspension fluid as HCO,, it is useful for comparative purposes. Total 14C incorporated and I4C in the methanol-water soluble fraction of bloodstream forms (3.3 X 107 cells) were 6.4 and 5.7 dpm/h/l08 cells x 10-3, respectively. The reaction products resulting from the oxidation of [ l-14C]acetate by bloodstream forms were mainly glutamic acid (25%), alanine (8%), succinic acid (3%), aspartic acid (9%), and tricarboxylic acids (10%). It should be noted that: ( a ) the combined radioactivity of the amino acids amounted to more than 40% of the total in the soluble extract; ( b ) radioactivity in glutamic acid proved the labeling of 2-oxoglutaric acid; ( c ) radioactivity in aspartic acid proved the labeling of oxalacetic acid; and ( d ) radioactivity in alanine involved the labeling and decarboxylation of L-malic acid (the "malic" enzyme reaction) ( 3 ) . The rest of the total radioactivity was distributed in succinic and tricarboxylic acids as well as other compounds, difficult to identify because of the low level of radioactivity associated with each. In Table 1, the effects of antimycin A on W O , production from [l-14C]acetate and 0, uptake by bloodstream forms are shown. Antimycin A, a specific inhibitor of mitochondria1 respiration and electron transport ( 11) inhibited the oxidation of [l-14C]acetate by these forms. Production of I4CO, and 0, uptake were affected to about the same degree. The results of experiments with immune sera are shown in Table 2. Several dilutions of these sera had no effect on 0, uptake and on the production of 14C02 from [l-I4C]acetate by bloodstream forms of T . cruzi when compared with cells incubated with normal sera.
ACETATEOXIDATION BY Trypanosoma cruzi
303
DISCUSSION
LITERATURE C I T E D
In the present investigation we demonstrated a substantial increase in respiratory rate with acetate in bloodstream forms of T . cruzi. The distribution of I4C after oxidation of [l-'*C]acetate by these forms was very similar to that noted in culture forms (2, 7 ) . Among the Krebs-cycle intermediates, the tricarboxylic acids retained most of the label. Labeling of glutamic and aspartic acids indicated the intermediate formation of 2oxoglutaric and oxalacetic acids in the process of acetate oxidation. The foregoing results and the inhibitory effect of antimycin A constitute strong evidence of an operational Krebs cycle and a respiratory chain in bloodstream forms. Although the glyoxylate cycle cannot be excluded, its contribution to acetate oxidation seems to be less important. Although it has been suggested that immune factors can play a significant role in host-induced variations in respiratory exchanges of trypanosomes (13) , the incubation of bloodstream forms of T . cruzi with immune sera did not alter their respiratory rate or the rate of acetate oxidation. Similar results were obtained with T . lewisi ( 1 3 ) . In contrast, immune sera decreased the rate of oxygen consumption of T . vivax (5))T . brucei ( 1 2 ) , and T . gambiense ( 9 ) . This effect may be due to lysis of a certain percentage of the flagellates initially present; this problem had been avoided in our experiments by inactivation of the sera.
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ACKNOWLEDGEMENT The authors thank Prof. L. R. Travassos (Department of General Microbiology, UFRJ) for help with the manuscript.
