1. Studies on the chemotherapy of parasitic diseases.

We were the first to identify the involvement of free radical metabolites in the mode of action and/or toxicity of a number of antiparasitic drugs, like gentian violet (a), nifurtimox, benznidazole, and metronidazole (2). In the case of gentian violet, a dye that was used to sterilize blood and prevent transmission of Chagas disease by blood transfusion, we demonstrated its conversion to a carbon-centered radical by trypanosomes. This reduction and its trypanocidal action were enhanced by light (a). This led to the exposure to light, before being used, of blood bags needed for transfusion in an area of Brazil, reducing the time needed to sterilize blood.

Our work on the mechanism of free radical generation by metronidazole in trichomonads led to the demonstration that the reaction catalyzed by the pyruvate: ferredoxin oxidoreductase, the enzyme that reduces metronidazole, involves the generation of two free radicals, a thyil radical derived from coenzyme A and a carbon-centered radical derived from pyruvate, to form a high-energy compound: acetyl-CoA (b). This unique reaction was confirmed years later by structural studies (Chabriere et al., Science 249, 2259, 2001).

 We were also the first to find that Trypanosoma cruzi is susceptible to antifungal azole compounds able to inhibit the biosynthesis of sterols (c). Azole compounds are now under clinical trials for Chagas disease. We also found that bisphosphonates, which are used clinically for the treatment of osteoporosis and other bone diseases, were active against multiple parasites and able to produce radical cures in experimental cutaneous leishmaniasis infections (d). We have done extensive studies on the biochemistry and molecular biology of parasites applying a variety of techniques from electron spin resonance spectroscopy to proteomics, transcriptomics, and the study of the immune response to them.

a. 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.

b. 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.

c.  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.

d.   Rodriguez, N., Bailey, B.N., Martin, M.B., Oldfield, E., Urbina, J.A., and Docampo, R. (2002) Radical cure of experimental cutaneous leishmaniasis by the bisphosphonate pamidronate. J. Infect. Dis. 186, 138-140.

 

2. Discovery of the trypanosome mitochondrial calcium uniporter

Form 1961 to 1989 It was though that the mitochondrial calcium uniporter (MCU), which is involved in mitochondrial Ca2+ uptake driven by the mitochondrial membrane potential (m) generated by the respiratory chain, or by ATP hydrolysis, was only present in vertebrates. Our work demonstrated the presence of MCU in trypanosomes (a), and was the basis, together with the absence of MCU in yeasts, for the identification of the molecular nature of MCU in mammals in 2011 (De Stefani et al., Nature 476, 336-340, 2011; Baughman et al., Nature 476, 341-35, 2011). We described the history of this discovery in (b). Recent results from our group (c) indicate that the T. brucei MCU, in contrast to the mouse MCU, is essential for growth and pathogenesis of this parasite and therefore a good target for chemotherapy. The MCU complex is also important for infectivity of Trypanosoma cruzi (d).

a. Docampo, R., and Vercesi, A.E. (1989) Ca2+ transport by coupled Trypanosoma cruzi mitochondria in situ. J. Biol. Chem. 264, 108-111.

b. Docampo and Lukes, J. (2012) Trypanosomes and the solution of a 50-year mitochondrial calcium mystery. Trends Parasitol. 28, 31-37.

c. Huang, G., Vercesi, A.E., and Docampo, R. (2013) Essential regulation of cell bioenergetics in Trypanosoma brucei by the mitochondrial calcium uniporter. Nat. Commun. 4, 2865.

d. Chiurillo, M.A., Lander, N., Bertolini, M.S., Storey, M., Vercesi, A.E., and Docampo, R. (2017) Different roles of mitochondrial calcium uniporter complex in growth and infectivity of Trypanosoma cruzi. mBio 8: e00574-17.

 

3. Discovery of the acidocalcisome

We discovered in trypanosomes a unique organelle, the acidocalcisome (a), an acidic calcium store rich in pyrophosphate and polyphosphate. We later found that this is the only organelle present from bacteria to human cells (b). The organelle was the first membrane-bounded organelle discovered in bacteria (c) that is present in eukaryotes (c) and has a vacuolar proton pyrophosphatase for its acidification in both bacteria and protists. This proton pump that we found in trypanosomes before the description of their genome (d) was previously known to be present only in plants and bacteria. We recently discovered that the inositol 1,4,5-trisphosphate receptor is localized in acidocalcisomes of T. brucei (e), suggesting its involvement in calcium signaling. This is the long sought channel involved in Ca2+ release from this organelle.

a. 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.

b. 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.

c. 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.

Commentary in Science (Editor’s Choice):  How different are we? Science 300, 2005, 2003

Commentary in Nature: Discovery changes view of bacteria, Nature, 423, 909, 2003

Commentary in New Scientist: Rewrite the textbooks. New Scientist, 28 June, 22, 2003

Rated as One of the Discoveries of the Year in Discover magazine: Veterinarian finds evolutionary link between humans and bacteria. Discovery, January 2004, p.45.

 http://discovermagazine.com/2004/jan/biology).

d. 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.

e. Huang, G., Bartlett, P.J., Thomas, A.P., Moreno, S.N.J., and Docampo, R. (2013) Acidocalcisomes of Trypanosoma brucei have an inositol 1,4,5-trisphosphate receptor that is required for growth and infectivity. Proc. Natl. Acad. Sci. USA 110, 1887-1892.

 

4. Discovery of the role of polyphosphate in blood coagulation and inflammation

The dense granules of human platelets have similar electron-dense characteristics to acidocalcisomes of prokaryotes and early branching eukaryotes and we discovered that they also possess polyphosphate indicating that acidocalcisomes are the only organelles conserved from bacteria to human cells. (a). We found that activation of platelets led to the release of polyphosphate (a) suggesting a role for this polymer in the blood. We found that platelet polyphosphate was a potent modulator of the blood clotting system (b). In particular, we reported that polyphosphate triggered the contact pathway of clotting, accelerated factor V activation, and delayed fibrinolysis (b). We found that polyphosphate is also present in serotonin-containing mast cell granules and in basophils granules, is released when mast cells are activated, and has a pro-inflammatory action (c).

a. 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.

b. Smith, S.A., Mutch, N.J., Baskar, D., Rohloff, P., Docampo, R.*, and Morrissey, J.H.* (2006) Polyphosphate modulates blood coagulation and fibrinolysis (*joint senior authors) Proc. Natl. Acad. Sci. U.S.A. 103, 903-908 [PM1347979].

Highlighted on the cover of PNAS and with a brief commentary in the “In This Issue” section: Polyphosphates promote clot formation and stability. Proc Natl Acad Sci USA 103:827, 2006.

Commentary in Chemical & Engineering News: Clot Plot Tickens, Chemical & Engineering News, January 16, 2006 http://pubs.acs.org/cen/news/84/i03/8403notw9.html

Commentary in ASBMB Today: Polyphosphate speeds blood clotting; helps clots last longer. ASBMB Today, April 2006, page 8-9.

Selected by the Faculty of Medicine 1000: http://f1000.com/10299

c. 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.

 

5. Development of the CRISPR/Cas9 system for gene knockout, tagging and complementation in T. cruzi

Few genetic tools were available to work with T. cruzi until our recent introduction of the CRISPR/Cas9 technique for gene knockout (a), endogenous gene tagging (b), and gene complementation (c). Previous attempts to generate null mutants by homologous recombination frequently yielded parasites bearing the two plasmid replacements but also extra copies of the genes through aneuploidy or gene amplification. The RNA interference (RNAi) technique for gene knockdown was unavailable because the RNAi machinery is absent in T. cruzi. The introduction of this new methodology is revolutionizing the study of this parasite (d).

a. Lander, N., Li, Z.H., Niyogi, S., and Docampo, R. (2015) CRISPR/Cas9-induced disruption of paraflagellar rod proteins 1 and 2 genes in Trypanosoma cruzi reveals their role in flagellar attachment. mBio 6 (4), e01012-15. 

b. Lander, N., Chiurillo, M.A., Storey, M., Vercesi, A.E., and Docampo, R. (2016) CRISPR-Cas9-mediated C-terminal tagging of Trypanosoma cruzi genes reveals the acidocalcisome localization of the inositol-1,4,5-trisphosphate receptor. J. Biol. Chem. 291, 25505-25515.

c. Chiurillo, M.A., Lander, N., Bertolini, M.S., Storey, M., Vercesi, A.E., and Docampo, R. (2017) Different roles of mitochondrial calcium uniporter complex subunits in growth and infectivity of Trypanosoma cruzi. mBio 8 (2): e00574-17.

d. Lander, N., Chiurillo, M.A., and Docampo, R. (2016) Genome editing by CRISPR/Cas9: a game change in the genetic manipulation of protists. J. Eukaryot. Microbiol. 63, 679-690.

 

6. Studies on the role of the contractile vacuole complex in T. cruzi

Although a contractile vacuole complex (CVC), composed by a large vacuole or bladder and a number of tubules known as the spongiome, was described in 1959 in Trypanosoma cruzi, the organelle was almost forgotten until we found that it possesses an aquaporin or water channel (the first described in a CVC) and has an important role in parasite recovery after hyposmotic stress (a) and shrinking after hyperosmotic conditions (b). We recently discovered that, in addition to its role is osmoregulation, the CVC of T. cruzi is a trafficking hub and is involved in the traffic of proteins to the plasma membrane (c) and to acidocalcisomes (d) of this parasite.

a. Rohloff, P., Montalvetti, A., and Docampo, R. (2004) Acidocalcisomes and the contractile vacuole are involved in osmoregulation in Trypanosoma cruzi. J. Biol. Chem. 279, 52270-52281.

b, Li ZH, Alvarez VE, De Gaudenzi JG, Sant'anna C, Frasch AC, Cazzulo JJ, Docampo R. (2011) Hyperosmotic stress induces aquaporin-dependent cell shrinkage, polyphosphate synthesis, amino acid accumulation and global gene expression changes in Trypanosoma cruzi. J Biol Chem. 286, 43959-43971.

c. Niyogi, S., Mucci, J., Campetella, O., and Docampo, R. (2014) Rab11 regulates trafficking of trans- sialidase to the plasma membrane through the contractile vacuole complex of Trypanosoma cruzi. PLoS Path. 10 (6): e1004224.

d. 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.