Papers by Roberto Docampo
mBio
Trypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycol... more Trypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits.
Microbiology Spectrum
Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited ... more Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.
International Journal of Molecular Sciences
The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of ... more The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca2+ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+...
Frontiers in Cellular and Infection Microbiology
Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of ... more Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis, which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in Trypanosoma brucei. In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 (TcGP72) and vacuolar proton pyrophosphatase (TcVP1) with the active (glmS) or inactive (M9) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro. Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in T. cruzi and to validate potential drug targets in this parasite.
Genes
Calcium ion (Ca 2+) serves as a second messenger for a variety of cell functions in trypanosomes.... more Calcium ion (Ca 2+) serves as a second messenger for a variety of cell functions in trypanosomes. Several proteins in the plasma membrane, acidocalcisomes, endoplasmic reticulum, and mitochondria are involved in its homeostasis and in cell signaling roles. The plasma membrane has a Ca 2+ channel for its uptake and a plasma membrane-type Ca 2+-ATPase (PMCA) for its efflux. A similar PMCA is also located in acidocalcisomes, acidic organelles that are the primary Ca 2+ store and that possess an inositol 1,4,5-trisphosphate receptor (IP 3 R) for Ca 2+ efflux. Their mitochondria possess a mitochondrial calcium uniporter complex (MCUC) for Ca 2+ uptake and a Ca 2+ /H + exchanger for Ca 2+ release. The endoplasmic reticulum has a sarcoplasmic-endoplasmic reticulum-type Ca 2+-ATPase (SERCA) for Ca 2+ uptake but no Ca 2+ release mechanism has been identified. Additionally, the trypanosomatid genomes contain other membrane proteins that could potentially bind calcium and await further characterization.
The Journal of eukaryotic microbiology, Jan 15, 2018
Membrane proteins in trypanosomatids are, in general, weakly expressed and confirmation of their ... more Membrane proteins in trypanosomatids are, in general, weakly expressed and confirmation of their subcellular localization frequently requires their overexpression with epitope tags. However, overexpression can lead to mislocalization of the probes. Viswanathan et al. (Nat. Methods, 2015, 12:568) described high performance tags for localization of weakly expressed proteins. We report here the use of these protein tags, named "spaghetti monster," for CRISPR/Cas9-mediated C-terminal endogenous tagging of Trypanosoma cruzi to localize two weakly expressed transient receptor potential channels to acidic compartments. The results indicate that this method will improve the detection of membrane proteins in T. cruzi.
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 2018
To face the high costs of developing new drugs, researchers in both industry and academy are look... more To face the high costs of developing new drugs, researchers in both industry and academy are looking for ways to repurpose old drugs for new uses. In this sense, bisphosphonates that are clinically used for bone diseases have been studied as agents against Trypanosoma cruzi, causative parasite of Chagas disease. In this work, the development of first row transition metal complexes (M = Co, Mn, Ni) with the bisphosphonate ibandronate (iba, Hiba representing the neutral form) is presented. The in-solution behavior of the systems containing iba and the selected 3d metal ions was studied by potentiometry. Mononuclear complexes [M(Hiba)] (x = 0-3) and [M(Hiba)] together with the formation of the neutral polynuclear species [Miba] and [M(Hiba)] were detected for all studied systems. In the solid state, complexes of the formula [M(Hiba)(HO)]·6HO were obtained and characterized. All obtained complexes, forming [M(Hiba)] species under the conditions of the biological studies, were more activ...
Bioorganic & medicinal chemistry, Dec 16, 2017
The obligate intracellular parasite, Trypanosoma cruzi is the etiologic agent of Chagas disease o... more The obligate intracellular parasite, Trypanosoma cruzi is the etiologic agent of Chagas disease or American trypanosomiasis, which is the most prevalent parasitic disease in the Americas. The present chemotherapy to control this illness is still deficient particularly in the chronic stage of the disease. The ergosterol biosynthesis pathway has received much attention as a molecular target for the development of new drugs for Chagas disease. Especially, inhibitors of the enzymatic activity of squalene synthase were shown to be effective compounds on T. cruzi proliferation in in vitro assays. In the present study we designed, synthesized and evaluated the effect of a number of isosteric analogues of WC-9 (4-phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, on T. cruzi growth in tissue culture cells. The selenium-containing derivatives turned out to be extremely potent inhibitors of T. cruzi growth. Certainly, 3-phenoxyphenoxyethyl, 4-phenoxyphenoxyethyl, 4-(3-fluo...
mBio, Jan 9, 2017
Trypanosoma cruzi is the agent of Chagas disease, and the finding that this parasite possesses a ... more Trypanosoma cruzi is the agent of Chagas disease, and the finding that this parasite possesses a mitochondrial calcium uniporter (TcMCU) with characteristics similar to that of mammalian mitochondria was fundamental for the discovery of the molecular nature of MCU in eukaryotes. We report here that ablation of TcMCU, or its paralog TcMCUb, by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca(2+) uptake without affecting the membrane potential of these cells, whereas overexpression of each gene caused a significant increase in the ability of mitochondria to accumulate Ca(2+) While TcMCU-knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of TcMCUb resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced ...
Antimicrobial Agents and Chemotherapy, 1997
European Journal of Medicinal Chemistry, Feb 28, 2013
The Journal of eukaryotic microbiology, Jan 17, 2016
Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR... more Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 sy...
The Journal of Eukaryotic Microbiology, Sep 1, 2002
The plasma membrane potential (AT) of procyclic and bloodstream trypomastigotes of Trypanosoma br... more The plasma membrane potential (AT) of procyclic and bloodstream trypomastigotes of Trypanosoma brucei was studied using the potentiometric fluorescent dye bisoxonol. Our results suggest that a proton pump plays a significant role in the regulation of A T in procyclic and bloodstream forms, as evidenced by depolarization of the plasma membrane by H+-ATPase inhibitors (e.g. dicyclohexylcarbo-diimide, N-ethylmaleimide, diethylstilbestrol, and bafilomycin A,). In bloodstream stages the plasma membrane was significantly depolarized by ouabain only when the cells were incubated in sodium-rich buffers indicating that a sodium pump was being inhibited. In contrast, ouabain had no effect on the A T of the procyclic stages in a sodium-rich buffer. However, it induced an additional significant depolarization in these stages when their plasma membrane was already partially depolarized by the H+-ATPase inhibitor dicyclohexylcarbo-diimide, indicating the presence of an ouabain-sensitive sodium pump whose activity is masked by the H+-ATPase. Unlike procyclic forms, the A T of bloodstream-stage trypomastigotes was markedly sensitive to extracellular Na+ and K+ concentrations. Thus, there are significant differences between procyclic and blooodstream forms in the maintenance of-the A* and in their permeability to cations.
Pnas, 2006
Inorganic polyphosphate is an abundant component of acidocalcisomes of bacteria and unicellular e... more Inorganic polyphosphate is an abundant component of acidocalcisomes of bacteria and unicellular eukaryotes. Human platelet dense granules strongly resemble acidocalcisomes, and we recently showed that they contain substantial amounts of polyphosphate, which is secreted upon platelet activation. We now report that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V, which in turn results in abrogation of the function of the natural anticoagulant protein, tissue factor pathway inhibitor. Polyphosphate was also found to delay clot lysis by enhancing a natural antifibrinolytic agent, thrombin-activatable fibrinolysis inhibitor. Polyphosphate is unstable in blood or plasma, owing to the presence of phosphatases. We propose that polyphosphate released from platelets or microorganisms initially promotes clot formation and stability; subsequent degradation of polyphosphate by blood phosphatases fosters inhibition of clotting and activation of fibrinolysis during wound healing. factor V | platelets | tissue factor pathway inhibitor | acidocalcisomes | dense granules
Protist, 2005
The elemental composition and stoichiometric profile of elements present in acidocalcisomes of di... more The elemental composition and stoichiometric profile of elements present in acidocalcisomes of different genera of the Trypanosomatidae family (insect, plant, and mammalian parasites) submitted to parallel cultivation conditions were studied. X-ray microanalysis using transmission electron microscopy in conjunction with a morphometric approach was used to investigate the elemental content, number, distribution, and volumetric density of acidocalcisomes of different species. Microanalytical data showed that the different parasites possess the same elemental composition (oxygen, sodium, magnesium, phosphorus, calcium, iron, and zinc) in their acidocalcisomes. However, the relative concentrations of the elements varied among species, but not within acidocalcisomes of individual species. Iron was detected in acidocalcisomes of all species analyzed, characterizing this element as a constituent of these organelles. Taken together, the results strongly indicate a speciesspecific composition of acidocalcisomes in trypanosomatid parasites.
ACS chemical biology, May 23, 2016
Trypanosomatid parasites are the causative agents of many neglected tropical diseases including t... more Trypanosomatid parasites are the causative agents of many neglected tropical diseases including the leishmaniases, Chagas disease, and human African trypanosomiasis. They exploit unusual vacuolar soluble pyrophosphatases (VSPs), absent in humans, for cell growth and virulence and as such, are drug targets. Here, we report the crystal structures of VSP1s from Trypanosoma cruzi and T. brucei, together with that of the T. cruzi protein bound to a bisphosphonate inhibitor. Both VSP1s form a hybrid structure containing an (N-terminal) EF-hand domain fused to a (C-terminal) pyrophosphatase domain. The two domains are connected via an extended loop of about 17 residues. Crystallographic analysis and size exclusion chromatography indicate that the VSP1s form tetramers containing head-to-tail dimers. Phosphate and diphosphate ligands bind in the PPase substrate-binding pocket and interact with several conserved residues, and a bisphosphonate inhibitor (BPH-1260) binds to the same site. Based...
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Papers by Roberto Docampo