Christian M Probst

Fundação Oswaldo Cruz, Rio de Janeiro, Rio de Janeiro, Brazil

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Publications (65)109.06 Total impact

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    ABSTRACT: Trypanosoma cruzi, the etiological agent of Chagas disease, is ingested by triatomines during their bloodmeal on an infected mammal. Aiming to investigate the development and differentiation of T. cruzi inside the intestinal tract of Rhodnius prolixus at the beginning of infection we fed insects with cultured epimastigotes and blood trypomastigotes from infected mice to determine the amount of recovered parasites after ingestion. Approximately 20% of the ingested parasites was found in the insect anterior midgut (AM) 3 h after feeding. Interestingly, a significant reduction (80%) in the numbers of trypomastigotes was observed after 24 h of infection suggesting that parasites were killed in the AM. Moreover, few parasites were found in that intestinal portion after 96 h of infection. The evaluation of the numbers of parasites in the posterior midgut (PM) at the same periods showed a reduced parasite load, indicating that parasites were not moving from the AM. Additionally, incubation of blood trypomastigotes with extracts from R. prolixus AMs revealed that components of this tissue could induce significant death of T. cruzi. Finally, we observed that differentiation from trypomastigotes to epimastigotes is not completed in the AM; instead we suggest that trypomastigotes change to intermediary forms before their migration to the PM, where differentiation to epimastigotes takes place. The present work clarifies controversial points concerning T. cruzi development in insect vector, showing that parasite suffers a drastic decrease in population size before epimastigonesis accomplishment in PM.
    No preview · Article · Jan 2016 · Parasitology
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    ABSTRACT: Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome. The functional characterization of the TcCAT1.1 isoform was performed in two heterologous expression systems. TcCAT subfamily expression was evaluated by real-time PCR in polysomal RNA fractions, and the cellular localization of TcCAT1.1 fused to EGFP was performed by confocal and immunoelectron microscopy. The functional characterization of the TcCAT1.1 isoform was performed in heterologous expression systems. In the S. cerevisiae expression system, TcCAT1.1 showed high affinity for arginine (K m = 0.085 ± 0.04 mM) and low affinity for ornithine (K m = 1.7 ± 0.2 mM). Xenopus laevis oocytes expressing TcCAT1.1 showed a 7-fold increase in arginine uptake when they were pre-loaded with arginine, indicating that transport is enhanced by substrates on the trans side of the membrane (trans-stimulation). Oocytes that were pre-loaded with [(3)H]-arginine displayed a 16-fold higher efflux of [(3)H]-arginine compared with that of the control. Analysis of polysomal RNA fractions demonstrated that the expression of members of the arginine transporter TcCAT subfamily is upregulated under nutritional stress and that this upregulation precedes metacyclogenesis. To investigate the cellular localization of the transporter, EGFP was fused to TcCAT1.1, and fluorescence microscopy and immunocytochemistry revealed the intracellular labeling of vesicles in the anterior region, in a network of tubules and vesicles. TcCAT1.1 is a novel arginine/ornithine transporter, an exchanger expressed in intracellular compartments that is physiologically involved in arginine homeostasis throughout the T. cruzi life cycle. The properties and estimated kinetic parameters of TcCAT1.1 can be extended to other members of the TcCAT subfamily.
    Full-text · Article · Jun 2015 · Parasites & Vectors
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    Full-text · Dataset · Sep 2014
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    ABSTRACT: Background Trypanosoma cruzi, the etiologic agent of Chagas disease, alternates between distinct morphological and functional forms during its life cycle. Axenic multiplication and differentiation processes of this protozoan parasite can be reproduced in vitro, enabling the isolation and study of the different evolutionary forms. Although there are several publications attempting the cultivation of T. cruzi under chemically defined conditions, in our experience none of the published media are capable of maintaining T. cruzi in continuous growth.ResultsIn this work we modified a known chemically defined medium for Trypanosoma brucei growth. The resulting LM14 and LM14B defined media enabled cultivation of five different strains of T. cruzi for more than forty passages until now. The parasite¿s biological characteristics such as morphology and differentiation to metacyclic trypomastigotes were maintained when defined media is used.Conclusions The establishment of a defined medium for T. cruzi cultivation is an important tool for basic biological research allowing several different approaches, providing new perspectives for further studies related to cell biology of this parasite.
    Full-text · Article · Sep 2014 · BMC Microbiology
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    ABSTRACT: The STINGRAY system has been conceived to ease the tasks of integrating, analyzing, annotating and presenting genomic and expression data from Sanger and Next Generation Sequencing (NGS) platforms. STINGRAY includes: (a) a complete and integrated workflow (more than 20 bioinformatics tools) ranging from functional annotation to phylogeny; (b) a MySQL database schema, suitable for data integration and user access control; and (c) a user-friendly graphical web-based interface that makes the system intuitive, facilitating the tasks of data analysis and annotation. STINGRAY showed to be an easy to use and complete system for analyzing sequencing data. While both Sanger and NGS platforms are supported, the system could be faster using Sanger data, since the large NGS datasets could potentially slow down the MySQL database usage. STINGRAY is available at http://stingray.biowebdb.org and the open source code at http://sourceforge.net/projects/stingray-biowebdb/.
    Full-text · Article · Mar 2014 · BMC Research Notes
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    ABSTRACT: Leishmaniasis is an infectious disease caused by Leishmania species. Leishmania amazonensis is a New World Leishmania species belonging to the Mexicana complex, which is able to cause all types of leishmaniasis infections. The L. amazonensis reference strain MHOM/BR/1973/M2269 was sequenced identifying 8,802 codifying sequences (CDS), most of them of hypothetical function. Comparative analysis using six Leishmania species showed a core set of 7,016 orthologs. L. amazonensis and Leishmania mexicana share the largest number of distinct orthologs, while Leishmania braziliensis presented the largest number of inparalogs. Additionally, phylogenomic analysis confirmed the taxonomic position for L. amazonensis within the “Mexicana com-plex”, reinforcing understanding of the split of New and Old World Leishmania. Potential non-homologous isofunctional enzymes (NISE) were identified between L. amazonensis and Homo sapiens that could provide new drug targets for development.
    Full-text · Article · Feb 2014 · Evolutionary bioinformatics online
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    ABSTRACT: Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.
    Full-text · Article · Jun 2013 · PLoS ONE
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    ABSTRACT: BACKGROUND: Simian malaria is still an open question concerning the species of Plasmodium parasites and species of New World monkeys susceptible to the parasites. In addition, the lingering question as to whether these animals are reservoirs for human malaria might become important especially in a scenario of eradication of the disease. To aid in the answers to these questions, monkeys were surveyed for malaria parasite natural infection in the Amazonian state of Rondônia, Brazil, a state with intense environmental alterations due to human activities, which facilitated sampling of the animals. METHODS: Parasites were detected and identified in DNA from blood of monkeys, by PCR with primers for the 18S rRNA, CSP and MSP1 genes and sequencing of the amplified fragments. Multiplex PCR primers for the 18S rRNA genes were designed for the parasite species Plasmodium falciparum and Plasmodium vivax, Plasmodium malariae/Plasmodium brasilianum and Plasmodium simium. RESULTS: An overall infection rate of 10.9% was observed or 20 out 184 monkey specimens surveyed, mostly by P. brasilianum. However, four specimens of monkeys were found infected with P. falciparum, two of them doubly infected with P. brasilianum and P. falciparum. In addition, a species of monkey of the family Aotidae, Aotus nigriceps, is firstly reported here naturally infected with P. brasilianum. None of the monkeys surveyed was found infected with P. simium/P. vivax. CONCLUSION: The rate of natural Plasmodium infection in monkeys in the Brazilian state of Rondônia is in line with previous surveys of simian malaria in the Amazon region. The fact that a monkey species was found that had not previously been described to harbour malaria parasites indicates that the list of monkey species susceptible to Plasmodium infection is yet to be completed. Furthermore, finding monkeys in the region infected with P. falciparum clearly indicates parasite transfer from humans to the animals. Whether this parasite can be transferred back to humans and how persistent the parasite is in monkeys in the wild so to be efficient reservoirs of the disease, is yet to be evaluated. Finding different species of monkeys infected with this parasite species suggests indeed that these animals can act as reservoirs of human malaria.
    Full-text · Dataset · Jun 2013
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    ABSTRACT: Background Simian malaria is still an open question concerning the species of Plasmodium parasites and species of New World monkeys susceptible to the parasites. In addition, the lingering question as to whether these animals are reservoirs for human malaria might become important especially in a scenario of eradication of the disease. To aid in the answers to these questions, monkeys were surveyed for malaria parasite natural infection in the Amazonian state of Rondônia, Brazil, a state with intense environmental alterations due to human activities, which facilitated sampling of the animals. Methods Parasites were detected and identified in DNA from blood of monkeys, by PCR with primers for the 18S rRNA, CSP and MSP1 genes and sequencing of the amplified fragments. Multiplex PCR primers for the 18S rRNA genes were designed for the parasite species Plasmodium falciparum and Plasmodium vivax, Plasmodium malariae/Plasmodium brasilianum and Plasmodium simium. Results An overall infection rate of 10.9% was observed or 20 out 184 monkey specimens surveyed, mostly by P. brasilianum. However, four specimens of monkeys were found infected with P. falciparum, two of them doubly infected with P. brasilianum and P. falciparum. In addition, a species of monkey of the family Aotidae, Aotus nigriceps, is firstly reported here naturally infected with P. brasilianum. None of the monkeys surveyed was found infected with P. simium/P. vivax. Conclusion The rate of natural Plasmodium infection in monkeys in the Brazilian state of Rondônia is in line with previous surveys of simian malaria in the Amazon region. The fact that a monkey species was found that had not previously been described to harbour malaria parasites indicates that the list of monkey species susceptible to Plasmodium infection is yet to be completed. Furthermore, finding monkeys in the region infected with P. falciparum clearly indicates parasite transfer from humans to the animals. Whether this parasite can be transferred back to humans and how persistent the parasite is in monkeys in the wild so to be efficient reservoirs of the disease, is yet to be evaluated. Finding different species of monkeys infected with this parasite species suggests indeed that these animals can act as reservoirs of human malaria.
    Full-text · Article · Jun 2013 · Malaria Journal
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    ABSTRACT: CBA macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. Employing a transcriptomic approach, we previously showed the up-regulation of the genes involved in the classical pathway of macrophage activation in resistant mice. However, microarray analyses do not evaluate changes in gene expression that occur after translation. To circumvent this analytical limitation, we employed a proteomics approach to increase our understanding of the modulations that occur during infection and identify novel targets for the control of Leishmania infection. To identify proteins whose expression changes in CBA macrophages infected with L. major or L. amazonensis, protein extracts were obtained and digested and the peptides were characterized using multi-dimensional liquid chromatography coupled with tandem mass spectrometry analyses. A total of 162 proteins were selected as potentially modulated. Using biological network analyses, these proteins were classified as primarily involved in cellular metabolism and grouped into cellular development biological networks. This study is the first to use a proteomics approach to describe the protein modulations involved in cellular metabolism during the initial events of Leishmania-macrophage interaction. Based on these findings, we hypothesize that these differentially expressed proteins likely play a pivotal role in determining the course of infection.
    Full-text · Article · Apr 2013 · Microbes and Infection
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    ABSTRACT: Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.
    Full-text · Article · Apr 2013 · PLoS ONE
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    Dataset: Figure S3
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    ABSTRACT: Absence of apoptotic markers in the EC50/72 h response. (A) Analysis of phosphatidylserine exposure, based on double-staining with annexin-V-FITC and PI. As an example, data are plotted for 120 hours of exposure to 32 µM ketoconazole (ii) or 50 µM lovastatin (iii), and the control cell pattern is shown (i). (B) DNA laddering assay; total DNA was isolated from control cultures (0) and from drug-treated cells after 24 to 120 hours of drug exposure (indicated at the top). We separated 5 µg of DNA by electrophoresis in a 1.5% agarose gel andstained with ethidium bromide; M lanes contain the 1 kb Plus DNA ladder. Similar results were indicated for the two SBIs and the name of the drug used is therefore not indicated. (TIF)
    Preview · Dataset · Jan 2013
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    Dataset: Figure S4
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    ABSTRACT: Flow cytometry analysis of T. cruzi necrotic death in response to treatment with SBIs at the EC100/24 h of SBIs. (A) Overlay histograms of Fluo-4-AM-stained cultures (with or without 1 mM EGTA) exposed to 100 µM lovastatin (i) or 120 µM ketoconazole (ii) from 0.5 to 12 hours. (B) Mitochondrial membrane depolarization; plots in (i) and (ii) show overlay histograms of R123-stained cultures exposed to EC100/24 h of lovastatin and ketoconazole, respectively; time-dependent mitochondrial depolarization with respect to control cells is clearly visible (iii). (C) Cell viability analysis; the percentage dead cells was determined by staining with the vital dye propidium iodide (ii) or from light scatter pattern (i) (data for 12 hours of exposure to lovastatin are plotted as an example). (TIF)
    Preview · Dataset · Jan 2013
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    ABSTRACT: The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs) ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC(50)/72 h) or killing all cells within 24 hours (EC(100)/24 h). Incubation with inhibitors at the EC(50)/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC(50)/72 h. By contrast, treatment with SBIs at the EC(100)/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP), culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the "point of no return" in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite.
    Preview · Article · Jan 2013 · PLoS ONE
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    Dataset: Figure S2
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    ABSTRACT: T. cruzi staining with the acidotophic fluorescent dye LysoTracker® Red (LTR) DND-99 (Invitrogen). The parasites were treated for 72 hours with 32 µM ketoconazole (row 2), 50 µM lovastatin (row 3) or left untreated (row 1), stained with 0.5 µM LTR and fixed for fluorescence microscopy analysis. Column A: DIC; column B: DNA dye Hoechst 33342; column C: acidic vesicles stained with LTR; column D: superimposition of B and C. Note the stronger staining in the posterior region of SBI-treated parasites. (TIF)
    Preview · Dataset · Jan 2013
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    Dataset: Figure S1
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    ABSTRACT: Morphological changes in response to treatment with SBIs at the EC50/72 h, as observed by light microscopy analysis of stained smears. Left column: control cells; middle column: ketoconazole; right column: lovastatin. The numbers on the left side of the figure refer to the number of days of drug exposure. Black arrows indicate cells with two nuclei and/or kinetoplast. The highlighted boxes show details of cells with large numbers of acidic vesicles in their posterior parts. Bars indicate 10 µm, except for highlighted cells (0.5 µm). (TIF)
    Preview · Dataset · Jan 2013
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    Dataset: Figure S5
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    ABSTRACT: Analysis of DNA fragmentation in an in situ TUNEL assay. Parasites were treated for 12 hours with the EC100 dose of ketoconazole or lovastatin (indicated on the left) and fixed for TUNEL experiments. DNase I-treated parasites were used as a positive control for the assay, together with normal parasites (negative control). DNA was stained with Hoechst 33342, and images were artificially colored in green to improve the visualization of overlay images. Note the TUNEL staining mostly in the kinetoplast region of SBI-treated parasites. Bars indicate 10 µm. (TIF)
    Preview · Dataset · Jan 2013
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    Full-text · Dataset · Jan 2013
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    Full-text · Dataset · Jan 2013
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    Full-text · Dataset · Jan 2013

Publication Stats

542 Citations
109.06 Total Impact Points

Institutions

  • 2013
    • Fundação Oswaldo Cruz
      • Laboratório de Biologia Molecular de Tripanossomatídeos (BIOMOLTRIP) (ICC)
      Rio de Janeiro, Rio de Janeiro, Brazil
  • 2008-2013
    • Fundação Carlos Chagas
      San Paulo, São Paulo, Brazil
    • Virginia Commonwealth University
      • Center for the Study of Biological Complexity
      Richmond, VA, United States
  • 2011
    • Roche Institute of Molecular Biology
      Nutley, New Jersey, United States
  • 2004-2009
    • Instituto de Biologia Molecular do Paraná
      Curityba, Paraná, Brazil
  • 2007
    • Pontifícia Universidade Católica do Paraná (PUC-PR)
      Curityba, Paraná, Brazil