Publications by authors named "Miguel A Chiurillo"

22 Publications

  • Page 1 of 1

Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi.

mBio 2021 04 6;12(2). Epub 2021 Apr 6.

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, USA

Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of , the etiologic agent of Chagas disease. Endogenous tagging of () and with 3× showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of and genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized -KO and -KO epimastigotes showed reduced O consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. -KO and -KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in -KOs. Overall, MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes. 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. Using the CRISPR/Cas9 technique, we generated individual () and knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.
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http://dx.doi.org/10.1128/mBio.00540-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092248PMC
April 2021

Signaling pathways involved in environmental sensing in Trypanosoma cruzi.

Mol Microbiol 2020 Oct 9. Epub 2020 Oct 9.

Center for Tropical and Emerging Global Diseases, and Department of Cellular Biology, University of Georgia, Athens, GA, USA.

Trypanosoma cruzi is a unicellular parasite and the etiologic agent of Chagas disease. The parasite has a digenetic life cycle alternating between mammalian and insect hosts, where it faces a variety of environmental conditions to which it must adapt in order to survive. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Major environmental changes include temperature, nutrient availability, ionic composition, pH, osmolarity, oxidative stress, contact with host cells and tissues, host immune response, and intracellular life. Some of the signaling pathways and second messengers potentially involved in the response to these changes have been elucidated in recent years and will be the subject of this review.
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http://dx.doi.org/10.1111/mmi.14621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032824PMC
October 2020

IP receptor-mediated Ca release from acidocalcisomes regulates mitochondrial bioenergetics and prevents autophagy in Trypanosoma cruzi.

Cell Calcium 2020 12 2;92:102284. Epub 2020 Sep 2.

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA, 30602, USA. Electronic address:

In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIPR) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIPR is a Ca release channel gated by IP when expressed in DT40 cells knockout for all vertebrate IP receptors, and is required for Ca uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O consumption, and preventing autophagy. Localization studies revealed its co-localization with an acidocalcisome marker in all life cycle stages of the parasite. Ablation of TcIPR by CRISPR/Cas9 genome editing caused: a) a reduction in O consumption rate and citrate synthase activity; b) decreased mitochondrial Ca transport without affecting the membrane potential; c) increased ammonia production and AMP/ATP ratio; d) stimulation of autophagosome formation, and e) marked defects in growth of culture forms (epimastigotes) and invasion of host cells by infective stages (trypomastigotes). Moreover, TcIPR overexpressing parasites showed decreased metacyclogenesis, trypomastigote host cell invasion and intracellular amastigote replication. In conclusion, the results suggest a modulatory activity of TcIPR-mediated acidocalcisome Ca release on cell bioenergetics in T. cruzi.
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http://dx.doi.org/10.1016/j.ceca.2020.102284DOI Listing
December 2020

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Nat Methods 2020 05 6;17(5):481-494. Epub 2020 Apr 6.

Department of Biochemistry, University of Cambridge, Cambridge, UK.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.
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http://dx.doi.org/10.1038/s41592-020-0796-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200600PMC
May 2020

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

Methods Mol Biol 2020 ;2116:177-197

Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA.

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 Ca transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca 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 (TcIPR), 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.
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http://dx.doi.org/10.1007/978-1-0716-0294-2_13DOI Listing
February 2021

State-of-the-art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids.

J Eukaryot Microbiol 2019 11 7;66(6):981-991. Epub 2019 Jul 7.

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, 30602.

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state-of-the-art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.
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http://dx.doi.org/10.1111/jeu.12747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842398PMC
November 2019

Functional analysis and importance for host cell infection of the Ca-conducting subunits of the mitochondrial calcium uniporter of .

Mol Biol Cell 2019 07 15;30(14):1676-1690. Epub 2019 May 15.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo 13083, Brazil.

We report here that the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex subunit that we named and . The predicted structure of the proteins indicates that, as predicted for the and paralogues, 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 Ca uptake, while knockout (KO) of either or led to a loss of mitochondrial Ca uptake, without affecting the mitochondrial membrane potential. -KO and -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 CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues on mitochondrial Ca uptake. In conclusion, the results predict a hetero-oligomeric structure for the MCU complex, with structural and functional differences, as compared with those in the mammalian complex.
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http://dx.doi.org/10.1091/mbc.E19-03-0152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727756PMC
July 2019

MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi.

mBio 2019 05 7;10(3). Epub 2019 May 7.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil

The mitochondrial Ca uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca uptake. MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated and knockout (-KO) cell lines. Ablation of either or showed a significantly reduced mitochondrial Ca uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca concentrations ([Ca]), as occurs with their mammalian orthologs. -KO and -KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles. is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca sensors and gatekeepers of the MCU, preventing Ca uptake under resting conditions and favoring it at high cytosolic Ca concentrations ([Ca]). Using the CRISPR/Cas9 technique, we generated and knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca] but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.
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http://dx.doi.org/10.1128/mBio.00348-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509184PMC
May 2019

Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi.

Methods Mol Biol 2019 ;1955:61-76

Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA.

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.
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http://dx.doi.org/10.1007/978-1-4939-9148-8_5DOI Listing
July 2019

Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of .

J Biol Chem 2018 11 19;293(45):17402-17417. Epub 2018 Sep 19.

From the Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil and

In vertebrate cells, mitochondrial Ca uptake by the mitochondrial calcium uniporter (MCU) leads to Ca-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1α subunit of PDH from nonvertebrate organisms has been described, the Ca-mediated PDP activation has not been studied. In this work, we investigated the Ca sensitivity of two recombinant PDPs from the protozoan human parasites (TcPDP) and (TbPDP) and generated a -KO cell line to establish TcPDP's role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca-sensitive phosphatases. Of note, -KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP/ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, -KO trypomastigotes were impaired in infecting cultured host cells. We conclude that TcPDP is a Ca-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity, and energy metabolism in Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.
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http://dx.doi.org/10.1074/jbc.RA118.004498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231137PMC
November 2018

The mitochondrial calcium uniporter complex in trypanosomes.

Cell Biol Int 2018 Jun 25;42(6):656-663. Epub 2018 Jan 25.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil.

The presence of a conserved mechanism for mitochondrial calcium uptake in trypanosomatids was crucial for the molecular identification of the mitochondrial calcium uniporter (MCU), a long-sought channel present in most eukaryotic organisms. Since then, research efforts to elucidate the role of MCU and its regulatory elements in different biological models have multiplied. MCU is the pore-forming subunit of a multimeric complex (the MCU complex or MCUC) and its predicted structure in trypanosomes is simpler than in mammalian cells, lacking two of its subunits and probably possessing other unidentified components. MCU protein has been characterized in Trypanosoma brucei and Trypanosoma cruzi, the causative agents of African and American trypanosomiasis, respectively. Contrary to its mammalian homolog, TbMCU was found to be essential for cell growth and survival, while its paralog MCUb is an essential protein in T. cruzi. These findings could be further exploited for chemotherapeutic purposes. The emergence of new molecular tools for the genetic manipulation of trypanosomatids has been determinant for the functional characterization of the MCUC components in these organisms. However, further research has to be done to determine the role of each component in intracellular calcium signaling and cell bioenergetics. In this mini-review we summarize the original results on mitochondrial calcium uptake in trypanosomes, how did they contribute to the molecular identification of the MCU, and the functional characterization of the MCUC subunits that has so far been studied in these peculiar eukaryotes.
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http://dx.doi.org/10.1002/cbin.10928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980684PMC
June 2018

Endogenous C-terminal Tagging by CRISPR/Cas9 in .

Bio Protoc 2017 May;7(10)

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil.

To achieve the C-terminal tagging of endogenous proteins in we use the Cas9/pTREX-n vector (Lander ., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3' end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3' end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer ., 2006) or pMOHX1Tag4H (Lander ., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.
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http://dx.doi.org/10.21769/BioProtoc.2299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531189PMC
May 2017

Different Roles of Mitochondrial Calcium Uniporter Complex Subunits in Growth and Infectivity of .

mBio 2017 05 9;8(3). Epub 2017 May 9.

Departamento de Patología Clínica, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil

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 , or its paralog , by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca 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 While knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced infectivity. Overexpression of , in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU. The finding of a mitochondrial calcium uniporter (MCU) in was essential for the discovery of the molecular nature of this transporter in mammals. In this work, we used the CRISPR/Cas9 technique that we recently developed for to knock out two components of the uniporter: MCU, the pore subunit, and MCUb, which was proposed as a negative regulator of MCU in human cells. In contrast to what occurs in human cells, MCU is not essential, while MCUb is essential for growth, differentiation, and infectivity; has a bioenergetic role; and does not act as a dominant negative subunit of MCU.
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http://dx.doi.org/10.1128/mBio.00574-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424207PMC
May 2017

Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in .

Front Microbiol 2016 22;7:2041. Epub 2016 Dec 22.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo São Paulo, Brazil.

chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in multigene families.
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http://dx.doi.org/10.3389/fmicb.2016.02041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5177640PMC
December 2016

CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor.

J Biol Chem 2016 Dec 28;291(49):25505-25515. Epub 2016 Oct 28.

From the Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo 13083, Brazil and

Methods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIPR (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was co-localized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIPR, endogenously tagged TcIPR showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.
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http://dx.doi.org/10.1074/jbc.M116.749655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207250PMC
December 2016

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists.

J Eukaryot Microbiol 2016 09 15;63(5):679-90. Epub 2016 Jul 15.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil.

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 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.
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http://dx.doi.org/10.1111/jeu.12338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012946PMC
September 2016

Anatomy and evolution of telomeric and subtelomeric regions in the human protozoan parasite Trypanosoma cruzi.

BMC Genomics 2012 Jun 8;13:229. Epub 2012 Jun 8.

Departamento de Microbiologia, Imunologia e Parasitologia Escola Paulista de Medicina, UNIFESP, São Paulo, SP, Brazil.

Background: The subtelomeres of many protozoa are highly enriched in genes with roles in niche adaptation. T. cruzi trypomastigotes express surface proteins from Trans-Sialidase (TS) and Dispersed Gene Family-1 (DGF-1) superfamilies which are implicated in host cell invasion. Single populations of T. cruzi may express different antigenic forms of TSs. Analysis of TS genes located at the telomeres suggests that chromosome ends could have been the sites where new TS variants were generated. The aim of this study is to characterize telomeric and subtelomeric regions of T. cruzi available in TriTrypDB and connect the sequences of telomeres to T. cruzi working draft sequence.

Results: We first identified contigs carrying the telomeric repeat (TTAGGG). Of 49 contigs identified, 45 have telomeric repeats at one end, whereas in four contigs the repeats are located internally. All contigs display a conserved telomeric junction sequence adjacent to the hexamer repeats which represents a signature of T. cruzi chromosome ends. We found that 40 telomeric contigs are located on T. cruzi chromosome-sized scaffolds. In addition, we were able to map several telomeric ends to the chromosomal bands separated by pulsed-field gel electrophoresis.The subtelomeric sequence structure varies widely, mainly as a result of large differences in the relative abundance and organization of genes encoding surface proteins (TS and DGF-1), retrotransposon hot spot genes (RHS), retrotransposon elements, RNA-helicase and N-acetyltransferase genes. While the subtelomeric regions are enriched in pseudogenes, they also contain complete gene sequences matching both known and unknown expressed genes, indicating that these regions do not consist of nonfunctional DNA but are instead functional parts of the expressed genome. The size of the subtelomeric regions varies from 5 to 182 kb; the smaller of these regions could have been generated by a recent chromosome breakage and telomere healing event.

Conclusions: The lack of synteny in the subtelomeric regions suggests that genes located in these regions are subject to recombination, which increases their variability, even among homologous chromosomes. The presence of typical subtelomeric genes can increase the chance of homologous recombination mechanisms or microhomology-mediated end joining, which may use these regions for the pairing and recombination of free ends.
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http://dx.doi.org/10.1186/1471-2164-13-229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418195PMC
June 2012

CYP2D6 gene variants in urban/admixed and Amerindian populations of Venezuela: pharmacogenetics and anthropological implications.

Ann Hum Biol 2012 Mar;39(2):137-42

Laboratorio de Genética Molecular Dr Jorge Yunis-Turbay, Decanato de Ciencias de la Salud, Universidad Centroccidental Lisandro Alvarado, Barquisimeto 3001, Venezuela.

Background: Differences in genes encoding enzymes involved in the biotransformation of a large number of compounds, such as CYP2D6, are related to inter-individual and inter-ethnic variability in the metabolism of many drugs, which have also been linked to susceptibility to cancer and other health outcomes. Therefore, populations are likely to benefit from inclusion in pharmacogenetic research studies.

Aim: To determine the frequency of functionally important allele variants of CYP2D6 gene in a sample of an Urban/admixed and five Amerindian Venezuelan populations.

Subjects And Methods: DNA of 328 unrelated volunteers was analysed for the presence of CYP2D6 *2, *3, *4, *5, *6 and *10 variants.

Results: The frequency in the Urban/admixed population for *2, *3, *4, *5, *6 and *10 alleles was 37.9%, 0%, 13.4%, 2.0%, 1.2% and 4.0%, respectively. In the Bari population, the prevalence of *4 allele associated with decreased enzyme activity was observed in 42.5%, whereas the poor metabolizer genotype *4/*4 was found in 25%. In the Panare, Pemon, Warao and Wayuu populations the *4 allele was found in 5.4%, 2.5%, 1.7% and 4.2%, respectively. The *10 allele frequency found in Amerindians (0.0-6.3%) was lower than reported for Asians.

Conclusion: The results are consistent with the known genetic admixture origin of most Venezuela populations. Nevertheless, the observed significant differences among Amerindians highlight the need for pharmacogenetic studies taking into account biogeographical and anthropological considerations.
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http://dx.doi.org/10.3109/03014460.2012.656703DOI Listing
March 2012

Usefulness of 12 Y-STRs for forensic genetics evaluation in two populations from Venezuela.

Leg Med (Tokyo) 2008 Mar 5;10(2):107-12. Epub 2007 Nov 5.

Unidad de Genética Médica, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela.

The distribution of allele frequencies and haplotypes for 12 STRs loci, (DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438 and DYS439) on the Y-chromosome from two Venezuelan populations were determined in 173 DNA samples of unrelated males living in Caracas (62) and Maracaibo (111). Some parameters of forensic importance were calculated. AMOVA and genetic distances between these populations were estimated. The results confirmed Y-STR genotypes as useful markers for forensic genetics analysis.
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http://dx.doi.org/10.1016/j.legalmed.2007.08.005DOI Listing
March 2008

Trypanosoma rangeli expresses a gene of the group II trans-sialidase superfamily.

Mol Biochem Parasitol 2005 Jul 9;142(1):133-6. Epub 2005 Apr 9.

Decanato de Medicina, Universidad Centroccidental Lisandro Alvarado, Barquisimeto, Estado Lara, Venezuela.

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http://dx.doi.org/10.1016/j.molbiopara.2005.03.012DOI Listing
July 2005

Comparative study of Trypanosoma rangeli and Trypanosoma cruzi telomeres.

Mol Biochem Parasitol 2002 Apr;120(2):305-8

Instituto de Biologia Experimental, Universidad Central de Venezuela, Apartado 47525, 1041-A Caracas, Venezuela.

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http://dx.doi.org/10.1016/s0166-6851(02)00005-1DOI Listing
April 2002