The history of the acidocalcisome
In 1992 a paper on calcium homeostasis in Dictyostelium discoideum (1) reported the presence of a Ca2+-ATPase in organelles called the acidosomes, which were thought to be part 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 (2) as well as Larry Ruben and collaborators (3) had also described nigericin-sensitive calcium compartments in L. donovani and T. brucei, respectively. With Anibal E. Vercesi and Silvia N.J. Moreno, we decided to test whether it was possible to detect a Ca2+-ATPase activity in the calcium-containing acidic compartment of T. brucei. We demonstrated the presence of proton uptake sensitive to vacuolar ATPase (V-H+-ATPase) inhibitors, and Ca2+ uptake sensitive to vanadate (Ca2+-ATPase) in permeabilized cells, and we found, that there were organelles in these parasites that stained with Acridine orange and were responsible for these responses to the inhibitors and ionophores. We named these organelles the acidocalcisomes to indicate that they were acidic and contained calcium (4). Further work in T. cruzi, now using intact cells loaded with Fura 2-AM, allowed the physiological characterization of these organelles (5).
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 (6). Ormerod and collaborators in the 1950’s 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 (7), and later by LeFurgey et al. (8), and Dvorak et al. (9), using X-ray microanalysis had described the presence of large amounts of calcium in these granules. We tried to repeat these experiments at the University of Illinois at Urbana-Champaign (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 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 planned experiments to demonstrate that acidocalcisomes and polyphosphate granules were the same entity. David Scott, who had joined our 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 (10).
Pyrophosphate and the vacuolar H+ pyrophosphatase
In 1997 Julio Urbina, from the Instituto Venezolano de Investigaciones Científicas, went to the UIUC to spend a sabbatical with Eric Oldfield in the Department of Chemistry. We 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 phosphorus compounds present in acidocalcisomes was still a mystery and Julio would have easy access to several NMR spectrometers, we decided to collaborate in the identification of these compounds using 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 (11). Felix Ruiz latter found that polyphosphate was also abundant in acidocalcisomes (12), a finding that was confirmed using 31P-NMR (13, 14).
While this was happening Hong-gang Lu, in our 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 (15) in acidocalcisomes. The presence of these pumps made the acidocalcisomes look very similar to the plant vacuoles and it was known 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 (13). A few days later we called Philip Rea, who discovered the V-H+-PPase in plant vacuoles to ask him for antibodies. Wanderley de Souza and Marlene Benchimol, who were visiting our lab, used these antibodies to show the staining of acidocalcisomes (16). 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 (16), and was later used by Claudia Rodrigues to isolate acidocalcisomes from T. brucei (17) and L. donovani (18).
Ubiquitous presence of acidocalcisomes from bacteria to human cells
During the following years we improved the isolation method for acidocalcisomes (19), found acidocalcisomes in several trypanosomatid and Apicomplexan parasites (20), algae (21), slime molds (22), bacteria (23) and human platelets (24), identified 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 25). Our work in human platelets (24) demonstrated that polyphosphate was released upon thrombin stimulation. Since polyphosphate is polyanionic, like heparin, we though that perhaps polyphosphate was an anticoagulant and we contacted Jim Morrissey, an expert in blood coagulation at the UIUC to test this potential effect. 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) (26). This physiological role of polyphosphate was very novel and another evidence of the power of trypanosome biology in leading to unexpected results of wide significance.
Work at the University of Georgia
More recent work was in the characterization of enzymes and transporters present in acidocalcisomes, such as that involved in polyphosphate synthesis and translocation, the vacuolar transporter chaperone complex or VCT (27,28), the Ca2+ release channel, inositol 1,4,5-trisphosphate receptor (29.30), and a number of exchangers and transporters identified by proteomic analyses (31,32). We also investigated the biogenesis of the organelles (33) and their relation with the contractile vacuole complex of T. cruzi (34). We found acidocalcisomes in other species, notably in eggs from insects (35), hens (36), and sea urchins (37), and we identified the mast cell granules and human basophils as organelles related to acidocalcisomes with a potential function of their polyphosphate content in inflammation and blood coagulation (38). Interestingly, the synthesis of acidocalcisome polyphosphate is linked to the inositol polyphosphate pathway in trypanosomes (39).
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18. 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.
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21. 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.
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24. 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.
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27. Fang, J., Rohloff, P., Miranda, K., and Docampo, R. (2007) Ablation of a small transmembrane protein of Trypanosoma brucei (TbVTC1) involved in the synthesis of polyphosphate alters acidocalcisome biogenesis and function, and leads to a cytokinesis defect. Biochem. J. 407, 161-170.
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34. Niyogi, S., Jimenez, V., Girard-Dias, W., de Souza, W., Miranda, K., and Docampo, R. (2015) Rab32 is essential for maintaining functional acidocalcisomes and for growth and infectivity of Trypanosoma cruzi. J. Cell Sci. 128, 2363-2373.
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36. Ramos, I., Miranda, K., Ulrich, P., Ingram, P., LeFurgey, A., Machado, E.A., de Souza, W., and Docampo, R. (2010) Calcium- and polyphosphate-containing acidocalcisomes in chicken egg yolk. Biol. Cell 102, 421-434.
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38. Moreno-Sanchez, D., Hernandez-Ruiz, L., Ruiz, F.A., and Docampo, R. (2012) Polyphosphate is a novel pro-inflammatory regulator of mast cells and is located in acidocalcisomes. J. Biol. Chem. 287, 28435-28444.
39. Cordeiro, C.D., Saiardi, A., and Docampo, R. (2017) The inositol pyrophosphate synthesis pathway in Trypanosoma brucei is linked to polyphosphate synthesis in acidocalcisomes. Mol Microbiol. 106: 319-333.