Roberto Docampo Archives - Page 4 of 5 - Center for Tropical and Emerging Global Diseases

CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi

Donna Huber on March 28, 2020

Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite’s life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca²⁺ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca²⁺ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP₃R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.

Noelia Lander, Miguel A. Chiurillo, Roberto Docampo. Methods Mol Biol. 2020;2116:177-197. doi: 10.1007/978-1-0716-0294-2_13.

Isolation and Characterization of Acidocalcisomes from Trypanosomatids

Donna Huber on March 28, 2020

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PP_i) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.

Guozhong Huang, Silvia N. J. Moreno, Roberto Docampo. Methods Mol Biol. 2020;2116:673-688. doi: 10.1007/978-1-0716-0294-2_40.

The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans

Donna Huber on March 17, 2020

Mitochondrial Ca²⁺ transport mediated by the uniporter complex (MCUC) plays a key role in the regulation of cell bioenergetics in both trypanosomes and mammals. Here we report that Trypanosoma brucei MCU (TbMCU) subunits interact with subunit c of the mitochondrial ATP synthase (ATPc), as determined by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Mutagenesis analysis in combination with MYTH assays suggested that transmembrane helices (TMHs) are determinants of this specific interaction. In situ tagging, followed by immunoprecipitation and immunofluorescence microscopy, revealed that T. brucei ATPc (TbATPc) coimmunoprecipitates with TbMCUC subunits and colocalizes with them to the mitochondria. Blue native PAGE and immunodetection analyses indicated that the TbMCUC is present together with the ATP synthase in a large protein complex with a molecular weight of approximately 900 kDa. Ablation of the TbMCUC subunits by RNA interference (RNAi) significantly increased the AMP/ATP ratio, revealing the downregulation of ATP production in the cells. Interestingly, the direct physical MCU-ATPc interaction is conserved in Trypanosoma cruzi and human cells. Specific interaction between human MCU (HsMCU) and human ATPc (HsATPc) was confirmed in vitro by mutagenesis and MYTH assays and in vivo by coimmunoprecipitation. In summary, our study has identified that MCU complex physically interacts with mitochondrial ATP synthase, possibly forming an MCUC-ATP megacomplex that couples ADP and P_i transport with ATP synthesis, a process that is stimulated by Ca²⁺ in trypanosomes and human cells.

IMPORTANCE The mitochondrial calcium uniporter (MCU) is essential for the regulation of oxidative phosphorylation in mammalian cells, and we have shown that in Trypanosoma brucei, the etiologic agent of sleeping sickness, this channel is essential for its survival and infectivity. Here we reveal that that Trypanosoma brucei MCU subunits interact with subunit c of the mitochondrial ATP synthase (ATPc). Interestingly, the direct physical MCU-ATPc interaction is conserved in T. cruzi and human cells.

Guozhong Huang, Roberto Docampo. mBio. 2020 Mar 17;11(2). pii: e00268-20. doi: 10.1128/mBio.00268-20.

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi

Donna Huber on February 27, 2020

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.

Noelia Lander, Teresa Cruz-Bustos, and Roberto Docampo. Front Cell Infect Microbiol. 2020 Feb 27;10:68. doi: 10.3389/fcimb.2020.00068. eCollection 2020.

Synthesis and in vitro evaluation of new 5-substituted 6-nitroimidazooxazoles as antikinetoplastid agents

Donna Huber on February 14, 2020

In continuation of our pharmacomodulation work on the nitroimidazooxazole series, we report the synthesis of new 5-substituted 6-nitroimidazooxazole derivatives. Our aim was to evaluate how functionalization of the 5-position of the 6-nitroimidazooxazole scaffold affects antileishmanial and antitrypanosomal in vitro activities. Twenty-one original compounds were synthesized and evaluated for their in vitro antileishmanial (L. donovani) and antitrypanosomal (T. cruzi) properties. Pallado-catalyzed cross-coupling reactions were used to introduce an aryl or ethynyl aryl substituent in 5-position from a 5-brominated-6-nitroimidazooxazole starting product. Unfortunately, the first series of compounds bearing an aryl group in 5-position presented limited in vitro activities against L. donovani and T. cruzi, with IC₅₀ > 10 μM (vs 0.18 μM and 2.31 μM for the reference drugs amphotericin B and benznidazole respectively). Interestingly, the second series of compounds bearing an ethynyl aryl substituent in 5-position showed more promising, particularly against T. cruzi. Compounds 6a, 6b, 6c, 6g and 6h had better activity than the reference drug benznidazole (0.92 μM ≤ IC₅₀ ≤ 2.18 μM vs IC₅₀ = 2.31 μM), whereas the non-functionalized 2-methyl-6-nitro-2,3-dihydroimidazo [2,1-b]oxazole 2 was not active against T. cruzi (IC₅₀ > 10 μM).

Fanny Mathias, AnitaCohen, Youssef Kabri, Núria Waddington Negrão, Maxime D.Crozet, RobertoDocampo, Nadine Azas, Patrice Vanelle. Eur J Med Chem. 2020 Feb 14;191:112146. doi: 10.1016/j.ejmech.2020.112146

Functional analysis and importance for host cell infection of the Ca2+-conducting subunits of the mitochondrial calcium uniporter of Trypanosoma cruzi

Donna Huber on May 15, 2019

We report here that Trypanosoma cruzi, the etiologic agent of Chagas disease, possesses two unique paralogs of the mitochondrial calcium uniporter complex TcMCU subunit that we named TcMCUc, and TcMCUd. The predicted structure of the proteins indicates that, as that predicted for the TcMCU and TcMCUb paralogs, they are composed of two helical membrane-spanning domains, and contain a WDXXEPXXY motif. Overexpression of each gene led to a significant increase in mitochondrial Ca²⁺ uptake while knockout (KO) of either TcMCUc or TcMCUd led to a loss of mitochondrial Ca²⁺ uptake, without affecting the mitochondrial membrane potential. TcMCUc-KO and TcMCUd-KO epimastigotes exhibited reduced growth rate in low glucose medium and alterations in their respiratory rate, citrate synthase activity and AMP/ATP ratio, while trypomastigotes had reduced ability to efficiently infect host cells and replicate intracellularly as amastigotes. By gene complementation of KO cell lines or by a newly developed knock-in approach we also studied the importance of critical amino acid residues of the four paralogs on mitochondrial Ca²⁺ uptake. In conclusion, the results predict a hetero-oligomeric structure for the T. cruzi MCU complex, with structural and functional differences, as compared to those in the mammalian complex.

Miguel A. Chiurillo, Noelia Lander, Mayara S. Bertolini, Anibal E. Vercesi, and Roberto Docampo. 2019. Mol Biol Cell.; mbcE19030152. doi: 10.1091/mbc.E19-03-0152

Pyrophosphate Stimulates the Phosphate-Sodium Symporter of Trypanosoma brucei Acidocalcisomes and Saccharomyces cerevisiae Vacuoles

Donna Huber on April 3, 2019

Inorganic pyrophosphate (PP_i) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PP_i and other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and P_i conductance in Xenopus oocytes expressing a Saccharomyces cerevisiae or Trypanosoma brucei Na⁺/P_i symporter. PP_i is not taken up by Xenopus oocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PP_i generated outward currents in Na⁺/P_i-loaded giant vacuoles prepared from wild-type or pho91Δ yeast strains expressing TbPHO91 but not from the pho91Δ strains. Our results suggest that PP_i, at physiological concentrations, can function as a signaling molecule releasing P_ifrom S. cerevisiae vacuoles and T. brucei acidocalcisomes.

IMPORTANCE Acidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities with S. cerevisiae vacuoles in their structure and composition. Both organelles share a Na⁺/P_isymporter involved in P_i release to the cytosol, where it is needed for biosynthetic reactions. Here we show that PP_i, at physiological cytosolic concentrations, stimulates the symporter expressed in either Xenopus oocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.

Evgeniy Potapenko, Ciro D. Cordeiro, Guozhong Huang, Roberto Docampo. 2019. mSphere; 4(2). pii: e00045-19. doi: 10.1128/mSphere.00045-19.

Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi

Donna Huber on March 15, 2019

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite’s life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have generated homogeneous populations of genetically modified parasites in 4–5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the gene. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines generated by CRISPR/Cas9, and C-terminal tagging of endogenous genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.

Noelia Lander, Miguel A. Chiurillo, Roberto Docampo. 2019. Methods Mol Biol. 2019;1955:61-76. doi: 10.1007/978-1-4939-9148-8_5

Further insights of selenium-containing analogues of WC-9 against Trypanosoma cruzi

Donna Huber on February 28, 2019

As a continuation of our project aimed at searching for new chemotherapeutic agents against American trypanosomiasis (Chagas disease), new selenocyanate derivatives were designed, synthesized and biologically evaluated against the clinically more relevant dividing form of Trypanosoma cruzi, the etiologic agent of this illness. In addition, in order to establish the role of each part of the selenocyanate moiety, different derivatives, in which the selenium atom or the cyano group were absent, were conceived, synthesized and biologically evaluated. In addition, in order to study the optimal position of the terminal phenoxy group, new regioisomers of WC-9 were synthesized and evaluated against T. cruzi. Finally, the resolution of a racemic mixture of a very potent conformationally rigid analogue of WC-9 was accomplished and further tested as growth inhibitors of T. cruzi proliferation. The results provide further insight into the role of the selenocyanate group in its antiparasitic activity.

María N. Chao, María V. Lorenzo-Ocampo, Sergio H. Szajnman, Roberto Docampo, Juan B. Rodriguez. 2019. Bioorganic & Medicinal Chemistry. https://doi.org/10.1016/j.bmc.2019.02.039

5-Diphosphoinositol Pentakisphosphate (5-IP7) Regulates Phosphate Release from Acidocalcisomes and Yeast Vacuoles

Donna Huber on October 12, 2018

Abstract

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate sodium symporter (TbPho91, and Pho91p, in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation, and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi -loaded giant vacuoles prepared from wild type or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ strains, or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Evgeniy Potapenko, Ciro D Cordeiro, Guozhong Huang, Melissa Storey, Christopher Wittwer, Amit K Dutta, Henning J. Jessen, Vincent J. Starai and Roberto Docampo. 2018. Journal of Biological Chemistry; 293:19101-19112.
doi: 10.1074/jbc.RA118.005884

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