THE HISTORY OF THE ACIDOCALCISOME
The first years
As soon as I obtained an MD from the University of Buenos Aires, in Argentina, I started working at the Institute of Biological Chemistry with Julia F. de Boiso on the metabolism of Trypanosoma cruzi. The Director of the Institute and also my PhD Thesis advisor was Andrés O. M. Stoppani. Stoppani was well known in Argentina, in part because he was a disciple of Bernardo Houssay, who won the Nobel Prize in Medicine or Physiology in 1947 for the discovery of the diabetogenic effect of the hypophysis. Stoppani had also been the graduate advisor of another future Nobel Prize winner, Cesar Milstein, who discovered the monoclonal antibodies in 1976.
My first paper in an international journal, almost 30 years ago (1), was on the presence of microbodies (a year later called glycosomes) and an ascorbate peroxidase in T. cruzi. The ascorbate peroxidase is an enzyme usually found in plants, and there were not many precedents for this “plant connection” of trypanosomes at that time. However, the main objective of my Thesis work was to investigate the function of the kinetoplast DNA of T. cruzi and I was mainly working on the metabolic changes of dyskinetoplastic trypanosomes generated by ethidium bromide treatment. The political situation in Argentina was so unstable that the Medical School was almost closed and it was not possible for me to defend my Thesis when I finished it.
A Brazilian experience
Since there was not much to do in Argentina, I got interested in doing postdoctoral work with some of the most active laboratories in the K-DNA area at the time and I applied for several fellowships. I finally got two, one to work with Guy Riou in Paris, and the other, from WHO, to work with Maurice Steinert in Brussels. Unfortunately, family problems prevented me to go to either place and I ended up requesting a change in my WHO fellowship to work in Rio de Janeiro, Brazil. The people at WHO was very nice and they even gave me the opportunity to visit Europe at the end of my fellowship. It was then that I knew Fred Opperdoes and Piet Borst, who had just published their paper on glycosomes, and also Bruce Newton, Guy Riou and Maurice Steinert. I even had some time to do experiments with amastigotes of T.cruzi, in Win Gutteridge’s laboratory in Canterbury. My work in Brazil, with Fernando S. Cruz, was on the trypanocial activity of naphthoquinones in T. cruzi, using b-lapachone as a model compound. We demonstrated redox cycling of this compound with generation of a semiquinone and reactive oxygen species. Our work included the collaboration of Wanderley de Souza, with whom we published the first images of apoptosis in a trypanosome (2) and started a long and fruitful collaboration that has continued over the years.
Back in Argentina
After a couple of years in Rio, where I received a PhD degree in Microbiology (for “merito notorio”) for my work on b-lapachone, I returned to the same Institute in Buenos Aires. I worked more independently after I had the opportunity to present my PhD Thesis in Medicine. I continued the work on free radicals and T. cruzi, this time studying the mode of action of nifurtimox and benznidazole together with my first graduate student, Silvia N.J. Moreno. We also found that antifungic azoles (such as miconazole, and econazole) were effective against T. cruzi in vitro by inhibiting the synthesis of ergosterol (3), a subject later developed by Julio Urbina and his collaborators in Venezuela. With Alberto Boveris, Helmut Sies, Elba Martino and Julio F. Turrens, we demonstrated the absence of catalase and selenium-dependent peroxidases in T. cruzi and proposed this deficiency as a target for chemotherapy (4). Our interest on free radicals led us to bring Ronald P. Mason to Rio de Janeiro to work on crystal violet, a drug used to prevent transmission of Chagas’ disease by blood transfusion. Ronald is a very well known investigator in free radical biology and in the use of electron spin resonance spectroscopy (ESR) and I had worked with him on the detection of the nitro anion radical of nifurtimox during a previous short visit to his laboratory in Research Triangle Park, North Carolina. Since we did not have an ESR spectrometer available in Buenos Aires the only possibility for doing this work using the infective stages of T. cruzi was Rio. We were able to detect the generation of a carbon-centered radical of crystal violet by T. cruzi and demonstrate its role in its cytotoxicity. This led to my first paper in Science (5).
An American experience
With Silvia, now my wife, we went to Ronald Mason’s lab in North Carolina (NIEHS/NIH) to continue our work on free radicals. Since trypanosomes were not available there, and we were still interested in working with parasites, we did some work on Tritrichomonas foetus, demonstrating the reduction of metronidazole to a nitro anion radical mediated by the pyruvate ferredoxin oxidoreductase (PFO) or NAD(P)H (6). We also demonstrated, using spin trapping techniques, that the catalytic cycle of PFO involved the generation of two free radicals, a carbon-centered free radical derived from pyruvate and a thyil radical derived from CoA, and that the combination of these two radicals resulted in the formation of acetyl-CoA (7). This was a very novel an unusual reaction and it was with great satisfaction that we saw the confirmation of these studies by structural work done almost 20 years later! (8).
Back in Brazil
After almost 4 years at NIH we returned to Argentina for only a couple of months since the political and economic situation continued to be very unstable, and we went back to Rio. There, we continued our work on crystal violet and found that light and ascorbate increased its trypanocidal activity allowing the use of lower drug concentrations and less time to sterilize blood intended for transfusion (9). This method was later adapted to field conditions and used in some parts of Brazil and was perhaps one of the few practical outcomes of my research. With Anibal Vercesi, in Campinas, we started our work on calcium homeostasis in trypanosomes identifying the mechanism of mitochondrial calcium transport in cells permeabilized with digitonin (10).
The Rockefeller University
After almost 3 years in Brazil, Silvia got a Research Associate position in George A. M. Cross laboratory at the Rockefeller University in New York City and both of us moved to his lab. George was very generous in giving me a lab where I could continue my work on calcium in trypanosomes with support from WHO and NIH grants that I could transfer from Brazil. My main work in George’s lab was on the finding, with Omar Pignataro, of a functional inositol phosphate/diacylglycerol pathway in T. cruzi (11). A year later I moved to the Department of Pathobiology at the University of Illinois at Urbana-Champaign. Silvia came a few months later, after finishing her work at Rockefeller.
The University of Illinois at Urbana-Champaign (UIUC)
For the first few years at UIUC we received periodic visits of Anibal Vercesi and Gustavo Benaim, with whom we described the basic mechanisms that control calcium homeostasis in trypanosomes. The use of digitonin permeabilized cells and intact cells labeled with fura-2 were adapted to the study of these parasites with great success. After a few more papers on free radicals in parasites we definitively abandoned this area and dedicated our full efforts to the molecular characterization of the mechanisms responsible for calcium homeostasis.
In 1992 a paper on calcium homeostasis in Dictyostelium discoideum (12) reported the presence of a Ca2+-ATPase in organelles called the acidosomes, which were thought to be part of the spongiome (an array of tubules and vesicles) of the contractile vacuole apparatus of this slime mold. The name acidosome was given because they were acidic as indicated by their sensitivity to nigericin (a K+/H+ ionophore). Dan Zilberstein and collaborators (13) as well as Larry Ruben and collaborators (14) had also described nigericin-sensitive calcium compartments in L. donovani and T. brucei, respectively. With Anibal Vercesi and Silvia Moreno, we tested whether it was possible to detect a Ca2+-ATPase activity in this acidic compartment of T. brucei. In addition to demonstrate the presence of proton uptake sensitive to vacuolar ATPase (V-H+-ATPase) inhibitors and Ca2+ uptake sensitive to vanadate (Ca2+-ATPase) in permeabilized cells, we found, in fact, that there were organelles in these permeabilized parasites that stained with acridine orange and responded to the inhibitors and ionophores. When I prepared the first version of the paper I named these organelles the acidocalcisomes to indicate that they were acidic and contained calcium (15). Further work in T. cruzi, now using intact cells loaded with fura 2, allowed the physiological characterization of these organelles (16). I remember very vividly working on Christmas Eve and New Year Eve of 1993 finishing the experiments with the infective stages of T. cruzi!
An important aspect of this work was the identification of these organelles at the ultrastructural level. Our best candidates were the polyphosphate granules. These had been described very early in trypanosomes (1908) when they were known as volutin granules (17). Ormerod and collaborators in the 1950’s have characterized them very well from a morphological point of view. Although they were known as polyphosphate granules nobody had ever purified them from trypanosomes or demonstrated that they actually contained polyphosphate. Work by Vickerman and Tetley (18), and later by LeFurgey et al. (19), and Dvorak et al. (20), using X-ray microanalysis had described the presence of large amounts of calcium in these granules. We tried to repeat these experiments at UIUC but, although we got very nice pictures of the granules in the infective forms of T. cruzi, the X-ray microanalysis detector was not working appropriately and we could not do much. In 1995 I was appointed member of the NIH Tropical Medicine and Parasitology Study Section and I had the opportunity to visit Washington DC several times per year. In one of these visits I met at NIH with Jim Dvorak and Richard Leapman, who had done previous work on these granules in T. cruzi, and we planned experiments to demonstrate that acidocalcisomes and polyphosphate granules were the same entity. David Scott, who had joined my lab recently, went to Jim’s lab and succeeded in doing incubations of intact cells with and without nigericin that resolved the issue: the granules that contained calcium increased their K+ concentration after nigericin treatment indicating that they were acidic (21).
Pyrophosphate and the vacuolar H+ pyrophosphatase
In 1997 Julio Urbina went to the UIUC to spend a Sabbatical with Eric Oldfield in the Department of Chemistry. I had known Julio for several years and it was very natural for us to collaborate during his stay in Urbana. Since the nature of the abundant phosphorous compounds present in acidocalcisomes was still a mistery and Julio would have easy access to several NMR spectrometers, we decided to collaborate in the identification of these compounds by 31P NMR. With Ben Moreno and Brian Bailey in Eric’s lab, we were able to show that T. cruzi and other trypanosomes possess very large amounts of pyrophosphate and that this is preferentially located in the acidocalcisomes (22).
While this was happening Hong-gang Lu, in my lab, was able to clone the gene of the first pump described in acidocalcisomes, the Ca2+-ATPase, which co-localized with the vacuolar H+-ATPase (23). The presence of these pumps made the acidocalcisomes look very similar to the plant vacuoles and I remember reading an article where it was mentioned that plant vacuoles contain both a V-H+-ATPase and a V-H+-PPase. This, together with the finding of large amount of PPi, led us to believe that perhaps this was also the case with acidocalcisomes. David Scott successfully tested pyrophosphate-driven proton uptake in permeabilized cells (24). A few days later I called Philip Rea, who discovered the V-H+-PPase in plant vacuoles to ask him for antibodies. These were used by Wanderley de Souza and Marlene Benchimol, who were visiting our lab, to show the staining of acidocalcisomes (24). The discovery of this enzyme was also important because it was the marker that we needed to purify the organelle, a process that David developed with great success in T. cruzi (24), and was later used by Claudia Rodrigues to isolate acidocalcisomes from T. brucei (25) and L. donovani (26).
The recent years
The recent years have been very exciting. We improved the isolation method for acidocalcisomes (27), found acidocalcisomes in several trypanosomatid and Apicomplexan parasites (28), algae (29), slime molds (30), bacteria (31) and human platelets (32), identify their chemical composition, cloned, expressed and localized a number of pumps, channels and exchangers in their membranes and started to investigate their functional roles (reviewed in 16).
Our most recent work in human platelets demonstrated that polyphosphate was released upon thrombin stimulation. Since polyphosphate is polyanionic, like heparin, I though that perhaps polyphosphate was an anticoagulant and I contacted Jim Morrisey, an expert in coagulation at the University of Illinois to test this potential effect. With Stephanie Smith, Nicole Mutch, Deepak Baskar and Peter Rohloff we found not only that polyphosphate was not an anticoagulant but that it has a potent procoagulant effect accelerating clotting by activating the contact pathway and accelerating factor V activation, which in turn results in abrogation of a natural anticoagulant (tissue factor pathway inhibitor), and delayed clot lysis by enhancing a natural antifibrinolytic agent (thrombin-activatable fibrinolysis inhibitor) (33). This physiological role of polyphosphate is very novel and another evidence of the power of trypanosome biology in leading to unexpected results of wide significance.
During our time in Illinois several graduate students (Fernanda Gadelha, Tetsuya Furuya, Nicole VanderHeyden, Julian Wong, Brian Bailey, Peter Rohloff, Mike Okura, Yan Ling, Corinna Kashuba, Jessica Graves, Rosa Martinez, Youhong Wang, Maria L. Salto, Claudia Rodrigues, Kildare Miranda, Mauricio Vieira, Alexis Fernández, Ianina Conte, Ileana Cuevas, Carlos Labriola, Sergio Szajman, and Esteban Ravaschino), postdoctoral fellows (Hong-gang Lu, Li Zhong, David A. Scott, Janet Hill, Norma Marchesini, Shuhong Luo, Felix A. Ruiz, Andrea Montalvetti, Jianmin Fang, and Manfredo Seufferheld) and visiting professors (Omar Pignataro, Mecia de Oliveira, Eva Carnieri, Julio Turrens, Wanderley de Souza, Marlene Benchimol, Sergio Uyemura, Anibal Vercesi, Edu Hoffman, Maria Grijalba, Rosana Catisti, Gustavo Benaim, Julio Urbina, Juan Bautista Rodriguez, and Noris Rodriguez) have been involved in this work or in other subjects that appeared interesting to us or to them. Our philosophy has been to go after interesting areas and give as much freedom as possible to our students in pursuing interesting topics.
Since January 2005 we are at the Center for Tropical and Global Emerging Diseases and Department of Cellular Biology of the University of Georgia.
1. Docampo, R., Boiso, J.F. de, and Stoppani, A.O.M. (1976) Localization of peroxidase activity in Trypanosoma cruzi microbodies. Experientia 32, 365-370.
2. Docampo, R., Lopes, J.N., Cruz, F.S., and De Souza, W. (1977) Trypanosoma cruzi: Ultrastructural and metabolic alterations of epimastigotes by b-lapachone. Exp. Parasitol. 42, 142-149.
3. Docampo, R., Moreno, S.N.J., Turrens, J.F., Katzin, A.M., Gonzalez-Cappa, S.M., and Stoppani, A.O.M. (1981) Biochemical and ultrastructural alterations produced by miconazole and econazole in Trypanosoma cruzi. Mol. Biochem. Parasitol. 3, 169-180.
4. Boveris, A., Sies, H., Martino, E.E., Docampo, R., Turrens, J.F., and Stoppani, A.O.M. (1980) Deficient metabolic utilization of H202 in Trypanosoma cruzi. Biochem. J. 188, 643-648.
5. Docampo, R., Moreno, S.N.J., Muniz, R.P.A., Cruz, F.S., and Mason, R.P. (1983) Light-enhanced free radical formation and trypanocidal action of gentian violet (crystal violet). Science 220, 1292-1295, 1983.
6. Moreno, S.N.J., Mason, R.P., and Docampo, R. (1984) Distinct reduction of nitrofurans and metronidazole to free radical metabolites by Tritrichomonas foetus hydrogenosomal and cytosolic enzymes. J. Biol. Chem.. 259, 8252-8259.
7. Docampo, R., Moreno, S.N.J., and Mason, R.P. (1987) Free radical intermediates in the reaction of pyruvate:ferredoxin oxidoreductase in Tritrichomonas foetus hydrogenosomes. J. Biol. Chem. 262, 12417-12420.
8. Chabriere, E., Vernede, X., Guigliarelli, B., Charon, M.H., Hatchikian, E.C., and Fontecilla-Camps, J.C. (2001) Crystal structure of the free radical intermediate of pyruvate:ferredoxin oxidoreductase. Science 294, 2559-2563
9. Docampo, R., Moreno, S.N.J., and Cruz, F.S. (1988) Enhancement of the cytotoxicity of crystal violet against Trypanosoma cruzi in the blood by ascorbate. Mol. Biochem. Parasitol. 27, 241-248.
10. Docampo, R., and Vercesi, A.E. (1989) Ca2+ transport by coupled Trypanosoma cruzi mitochondria in situ. J. Biol. Chem. 264, 108-111.
11. Docampo, R., and Pignataro, O.P. (1991) The inositol phosphate/diacylglycerol signalling pathway in Trypanosoma cruzi. Biochem. J. 275, 407-411.
12. Rooney,E.K., and Gross,J.D. (1992) ATP-drive Ca2+/H+ antiport in acid vesicles from Dictyostelium discoideum. Proc. Natl. Acad. Sci. USA 89, 8025-8029.
13. Philosoph, H., and Zilberstein, D. (1989) Regulation of intracellular calcium in promastigotes of the human protozoan parasite Leishmania donovani. J. Biol. Chem. 264, 10420-10424.
14. Ruben, L, Hutchinson, A, and Moehlman J. (1991) Calcium homeostasis in Trypanosoma brucei. Identification of a pH sensitive non-mitochondrial calcium pool. J. Biol. Chem. 266, 24351-24358.
15. Vercesi, A.E., Moreno, S.N.J., and Docampo, R. (1994) Ca2+/H+ exchange in acidic vacuoles of Trypanosoma brucei. Biochem. J. 304, 227-233.
16. Docampo, R., Scott, D.S., Vercesi, A.E., and Moreno, S.N.J. (1995) Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi. Biochem. J. 310, 1005-1012.
17. Docampo, R., de Souza, W., Miranda, K., Rohloff, P., and Moreno, S.N.J. (2005) Acidocalcisomes- conserved from bacteria to man, Nat. Rev. Microbiol. 3, 251-261.
18. Vickerman, K, and Tetley, L. (1977) Recent ultrastructural studies on trypanosomes. Ann. Soc. Belge Med. Trop. 57, 441-455.
19. LeFurgey, A., Ingram, P., and Blum, J. J. (1990) Elemental composition of polyphosphate-containing vacuoles and cytoplasm of Leishmania major. Mol. Biochem. Parasitol. 1990;40:77-86
20. Dvorak, J. A., Engel, J. C., Leapman, R. D., Swyt, C. R., and Pella, P. A. (1988) Trypanosoma cruzi: elemental composition hetereogeneity of cloned stocks. Mol. Biochem. Parasitol. 31, 19-26.
21. Scott, D.A., Docampo, R., Dvorak, J.A., Shi, S., and Leapman, R.D. (1997) In situ compositional analysis of acidocalcisomes in Trypanosoma cruzi. J. Biol. Chem., 272, 28020-28029.
22. Urbina, J.A., Moreno, B., Vierkotter, S., Oldfield, E., Payares, G., Sanoja, C., Bailey, B.N., Yan, W., Scott, D.A., Moreno, S.N.J., and Docampo, R. (1999) Trypanosoma cruzi contains major pyrophosphate stores and its growth in vitro and in vivo is blocked by pyrophosphate analogs. J. Biol. Chem. 274, 33609-33615.
23. Lu, H.-G., Zhong, L., de Souza, W., Benchimol, M., Moreno, S.N.J., and Docampo, R. (1998) Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi. Mol. Cell. Biol. 18, 2309-2323.
24. Scott, D.A., de Souza, W., Benchimol, M., Zhong, L., Lu, H.-g., Moreno, S.N.J., and Docampo, R. (1998) Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi. J. Biol. Chem. 273, 22151-22158.
25. Rodrigues, C.O., Scott, D.A., and Docampo, R. (1999) Characterization of a vacuolar pyrophosphatase in Trypanosoma brucei and its localization to acidocalcisomes. Mol. Cell. Biol. 19, 7712-7723.
26. Rodrigues, C.O., Scott, D.A., and Docampo, R. (1999) Presence of a vacuolar H+-pyrophosphatase in promastigotes of Leishmania donovani and its localization to a different compartment from the vacuolar H+-ATPase. Biochem. J. 340, 759-766.
27. Scott, D.A., and Docampo, R. (2000) Characterization of isolated acidocalcisomes of Trypanosoma cruzi. J. Biol. Chem. 275, 24215-24221.
28. Docampo, R., and Moreno, S.N.J. (2001) The acidocalcisome. Mol. Biochem. Parasitol. 33, 151-159.
29. Ruiz, F.A., Marchesini, N., Seufferheld, M., Govindjee, and Docampo, R. (2001) The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton pumping pyrophosphatase and are similar to acidocalcisomes. J. Biol. Chem. 276, 46196-46203.
30. Marchesini, N., Ruiz, F.A., Vieira, M., and Docampo, R. (2002) Acidocalcisomes are linked to the contractile vacuole of Dictyostelium discoideum. J. Biol. Chem. 277, 8146-8153.
31. Seufferheld, M., Vieira, M.C.F., Ruiz, F.A., Rodrigues, C.O., Moreno, S.N.J., and Docampo, R. (2003) Identification in bacteria of organelles similar to acidocalcisomes of unicellular eukaryotes. J. Biol. Chem. 278, 29971-29978.
32. Ruiz, F. A., Lea, C. R., Oldfield, E., and Docampo, R. (2004) Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J. Biol. Chem. 279, 44250-44257.
33. Smith, S., Mutch, N.J., Baskar, D., Rohloff, P., Docampo, R., and Morrissey, J.M. (2006) Polyphosphate- a new modulator of blood coagulation and fibrinolysis. Proc. Natl Acad. Sci. U.S.A., in press.