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Host-Pathogen Interactions

MEMBERS

  • Carlos Robello (PhD, Head)
  • Adriana Parodi-Talice (PhD – Associated Researcher, Facultad de Ciencias)
  • Dolores Piñeyro (PhD – Associated Researcher, Facultad de Medicina)
  • Ma. Laura Chiribao (PhD, Facultad de Medicina)
  • Paula Faral (PhD Student)
  • Gabriela Libisch (PhD Student)
  • Gonzalo Greif (PhD)
  • Cecilia Portela (Tecnician, Facultad de Ciencias)
  • Florencia Díaz (Master student)
  • Fernanda Matto (Master student)
  • Moira Lasserre (Master student)
  • Luisa Berná (Postdoctoral Researcher- INNOVA II)
  • Lucía López (Estudiante Maestría)

MAIN EQUIPMENT

  • DNA Sequencer/analyzer
  • Real Time PCR
  • Microarray reader
  • Microarray hybridizer
  • BioAnalyzer
  • General Molecular Biology Equipment

SERVICES

  1. DNA sequencing (Sanger methodology)
  2. Real Time PCR
  3. Microarrays
  4. Bioanalyzer
  5. Illumina Genome Analyzer
  6. Illumina MiSeq

Formularios

RESEARCH

The Unit of Molecular Biology research is focused on human and animal pathogens, in particular the protozoan parasites T. cruzi, T. vivax and Leishmania, and the prokariote Mycobacterium, with emphasis in genomics and functional genomics of those pathogens, and host-pathogen interactions.

Research lines

Functional Genomics of Host-Parasite Interaction

Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of profilaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion, are important for understanding of disease pathogenesis. In Chagas disease most of the focus was on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions respectively. We studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response), a great number of transcription factors (including the majority of NFκB family members) and host metabolism (cholesterol, fatty acids and phospholipids). These results raise the possibility that early host cell reprogramming is exploited by the parasite to establishment of the initial infection and posterior systemic dissemination.

Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics

The first line treatment for Chagas disease involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. By using a non-targeted MS-based metabolomics approach we studied the metabolic response of T. cruzi to Bzn. Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected. Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi.

Tuberculosis: Genomics and molecular typing

The incidence of tuberculosis (TB) is increasing in high-risk populations in Uruguay, possibly owing to emerging resistance. Mycobacterial interspersed repetitive units (MIRU) genotyping and katG sequence analysis of isoniazid (INH) resistance-associated mutations were performed in 45 INH-resistant Mycobacterium tuberculosis isolates in Uruguayan patients. The genotype distribution among INH-resistant isolates shares features of that of neighbouring countries, with a predominance of Latin American and Mediterranean, T and Haarlem genotypes, although the S genotype was particularly frequent among our isolates. Forty-four per cent of INH-resistant strains harboured the S315T mutation in katG; we found novel katG mutations (W321X, G269T, P232R and G221Wfs1) that could explain INH resistance. More recently, we reported an unusual tuberculosis (TB) outbreak centered on a professional basketball team in Montevideo. The strain, named MtURU-001, was fully sequenced: MtURU-001 has a circular chromosome of 4,378,296 bp, with an average G+C content of 65%, including 4,314 protein-encoding genes, 1 rRNA operon, and 45 tRNA genes. In comparison with M. tuberculosis H37Rv, 4,096 orthologous groups were defined with OrthoMCL and 1,016 polymorphisms were identified using the Burrows-Wheeler Aligner (BWA) and GATK. A subset of 849 polymorphisms (802 single-nucleotide polymorphisms [SNPs] and 47 indels) were inside coding sequences, and 480 affect protein sequences, especially 24 that introduced stop codons disrupting several hypothetical proteins, one transcriptional regulator, 2 genes for the haloacid dehalogenase (HAD) superfamily, and 3 involved in lipid metabolism. Further comparative genomics across this genome may provide genotype-phenotype associations that might explain the rapid progression of this unusual outbreak.

Trypanosoma vivax transcriptome

Trypanosoma vivax is the earliest branching African trypanosome. This crucial phylogenetic position makes T. vivax a fascinating model to tackle fundamental questions concerning the origin and evolution of several features that characterize African trypanosomes, such as the Variant Surface Glycoproteins (VSGs) upon which antibody clearing and antigenic variation are based. Other features like gene content and trans-splicing patterns are worth analyzing in this species for comparative purposes. We present a RNA-seq analysis of the bloodstream stage of T. vivax from data obtained using two complementary sequencing technologies (454 Titanium and Illumina). Assembly of 454 reads yielded 13385 contigs corresponding to proteins coding genes (7800 of which were identified). These sequences, their annotation and other features are available through an online database presented herein. Among these sequences, about 1000 were found to be species specific and 50 exclusive of the T. vivax strain analyzed here. Expression patterns and levels were determined for VSGs and the remaining genes. Interestingly, VSG expression level, although being high, is considerably lower than in Trypanosoma brucei. Indeed, the comparison of surface protein composition between both African trypanosomes (as inferred from RNA-seq data), shows that they are substantially different, being VSG absolutely predominant in T. brucei, while in T. vivax it represents only about 55%. This raises the question concerning the protective role of VSGs in T. vivax, hence their ancestral role in immune evasion.It was also found that around 600 genes have their unique (or main) trans-splice site very close (sometimes immediately before) the start codon. Gene Ontology analysis shows that this group is enriched in proteins related to the translation machinery (e.g. ribosomal proteins, elongation factors). This is the first RNA-seq data study in trypanosomes outside the model species T. brucei, hence it provides the possibility to conduct comparisons that allow drawing evolutionary and functional inferences. This analysis also provides several insights on the expression patterns and levels of protein coding sequences (such as VSG gene expression), trans-splicing, codon patterns and regulatory mechanisms. An online T. vivax RNA-seq database described herein could be a useful tool for parasitologists working with trypanosomes.

EDUCATION-COURSES

  1. “Functional Genomics and its applications in biomedicine: Host-Pathogen interaction”. RIIP/UNU-Biolac Course. November 2014.

GRANTS

 

PUBLICATIONS

  1. Coitinho C, Greif G, van Ingen J, Laserra P, Robello C, Rivas C. First case of Mycobacterium heckeshornense cavitary lung disease in the Latin America and Caribbean region. New Microbes New Infect. 2015 Dec 18;9:63-5.
  2. Pizzo C, Faral-Tello P, Yaluff G, Serna E,Torres S, Vera N, Saiz C, Robello C, Mahler G. New approach towards the synthesis of selenosemicarbazones, usefulcompounds for Chagas’ disease. Eur J Med Chem. 2016 Feb 15;109:107-13.
  3. Dusfour I, Zorrilla P, Guidez A, Issaly J, Girod R, Guillaumot L, Robello C, Strode C. Deltamethrin Resistance Mechanisms in Aedes aegypti Populations fromThree French Overseas Territories Worldwide. PLoS Negl Trop Dis. 2015 Nov20;9(11).
  4. Lasserre M, Berná L, Greif G, Díaz-Viraqué F, Iraola G, Naya H, Castro-Ramos M, Juambeltz A, Robello C. Whole-Genome Sequences of Mycobacterium bovis StrainMbURU-001, Isolated from Fresh Bovine Infected Samples. Genome Announc. 2015 Nov 5;3(6).
  5. Ubillos L, Freire T, Berriel E, Chiribao ML, Chiale C, Festari MF, Medeiros A,Mazal D, Rondán M, Bollati-Fogolín M, Rabinovich GA, Robello C, Osinaga E. Trypanosoma cruzi extracts elicit protective immune response against chemicallyinduced colon and mammary cancers. Int J Cancer. 2016 Apr 1;138(7):1719-31
  6. Greif G, Rodriguez M, Reyna-Bello A, Robello C, Alvarez-Valin F. Kinetoplast adaptations in American strains from Trypanosoma vivax. Mutat Res. 2015 Mar;773:69-82.
  7. Fernandez-Calero T, Garcia-Silva R, Pena A, Robello C, Persson H, Rovira C, Naya H, Cayota A. Profiling of small RNA cargo of extracellular vesicles shed by Trypanosoma cruzi reveals a specific extracellular signature. Mol Biochem Parasitol. 2015 Jan-Feb;199(1-2):19-28.
  8. Arias DG, Piñeyro MD, Iglesias AA, Guerrero SA, Robello C. Molecular characterization and interactome analysis of Trypanosoma cruzi tryparedoxin II. JProteomics. 2015 Apr 29;120:95-104.
  9. Trochine A, Creek DJ, Faral-Tello P, Barrett MP, Robello C. Bestatin induces specific changes in Trypanosoma cruzi dipeptide pool. Antimicrob Agents Chemother. 2015 May;59(5):2921-5.
  10. Querido JF, Echeverría MG, Marti GA, Costa RM, Susevich ML, Rabinovich JE, Copa A, Montaño NA, Garcia L, Cordova M, Torrico F, Sánchez-Eugenia R, Sánchez-Magraner L, Muñiz-Trabudua X, López-Marijuan I, Rozas-Dennis GS, Diosque P, de Castro AM, Robello C, Rodríguez JS, Altcheh J, Salazar-Schettino PM, Bucio MI, Espinoza B, Guérin DM, Silva MS. Seroprevalence of Triatoma virus (Dicistroviridae: Cripaviridae) antibodies in Chagas disease patients. Parasit Vectors. 2015 Jan 17;8:29.
  11. Márquez VE, Arias DG, Chiribao ML, Faral-Tello P, Robello C, Iglesias AA, Guerrero SA. Redox metabolism in Trypanosoma cruzi. Biochemical characterization of dithiol glutaredoxin dependent cellular pathways. Biochimie. 2014Nov;106:56-67.
  12. Berná L, Iraola G, Greif G, Coitinho C, Rivas CM, Naya H, Robello C. Whole-Genome Sequencing of an Isoniazid-Resistant Clinical Isolate ofMycobacterium tuberculosis Strain MtURU-002 from Uruguay. Genome Announc. 2014Jul 17;2(4). pii: e00655-14.
  13. Giorello FM, Berná L, Greif G, Camesasca L, Salzman V, Medina K, Robello C, Gaggero C, Aguilar PS, Carrau F. Genome Sequence of the Native Apiculate Wine Yeast Hanseniaspora vineae T02/19AF. Genome Announc. 2014 May 29;2(3). pii:e00530-14.
  14. Trochine A, Creek DJ, Faral-Tello P, Barrett MP, Robello C. Benznidazole biotransformation and multiple targets in Trypanosoma cruzi revealed bymetabolomics. PLoS Negl Trop Dis. 2014 May 22;8(5):e2844.
  15. Palacios F, Abreu C, Prieto D, Morande P, Ruiz S, Fernández-Calero T, Naya H, Libisch G, Robello C, Landoni AI, Gabus R, Dighiero G, Oppezzo P. Activation ofthe PI3K/AKT pathway by microRNA-22 results in CLL B-cell proliferation. Leukemia. 2015 Jan;29(1):115-25.
  16. Chiribao ML, Libisch G, Parodi-Talice A, Robello C. Early Trypanosoma cruzi infection reprograms human epithelial cells. Biomed Res Int. 2014;2014:439501.
  17. Trochine A, Alvarez G, Corre S, Faral-Tello P, Durán R, Batthyany CI, Cerecetto H, González M, Robello C. Trypanosoma cruzi chemical proteomics using immobilized benznidazole. Exp Parasitol. 2014 May;140:33-8.
  18. Martinez A, Peluffo G, Petruk AA, Hugo M, Piñeyro D, Demicheli V, Moreno DM, Lima A, Batthyány C, Durán R, Robello C, Martí MA, Larrieux N, Buschiazzo A, Trujillo M, Radi R, Piacenza L. Structural and molecular basis of theperoxynitrite-mediated nitration and inactivation of Trypanosoma cruziiron-superoxide dismutases (Fe-SODs) A and B: disparate susceptibilities due tothe repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electrontransfer. J Biol Chem. 2014 May 2;289(18):12760-78.
  19. Greif G, Iraola G, Berná L, Coitinho C, Rivas CM, Naya H, Robello C. Complete Genome Sequence of Mycobacterium tuberculosis Strain MtURU-001, Isolated from a Rapidly Progressing Outbreak in Uruguay. Genome Announc. 2014 Jan 23;2(1). pii:e01220-13.
  20. Faral-Tello P, Liang M, Mahler G, Wipf P, Robello C. Imidazolium compounds are active against all stages of Trypanosoma cruzi. Int J Antimicrob Agents. 2014 Mar;43(3):262-8.
  21. Garcia-Silva MR, das Neves RF, Cabrera-Cabrera F, Sanguinetti J, Medeiros LC, Robello C, Naya H, Fernandez-Calero T, Souto-Padron T, de Souza W, Cayota A. Extracellular vesicles shed by Trypanosoma cruzi are linked to small RNApathways, life cycle regulation, and susceptibility to infection of mammalian cells. Parasitol Res. 2014 Jan;113(1):285-304.
  22. Coitinho C, Greif G, Robello C, Laserra P, Willery E, Supply P. Rapidly progressing tuberculosis outbreak in a very low risk group. Eur Respir J. 2014 Mar;43(3):903-6.
  23. Libisch MG, Casás M, Chiribao M, Moreno P, Cayota A, Osinaga E, Oppezzo P, Robello C. GALNT11 as a new molecular marker in chronic lymphocytic leukemia. Gene. 2014 Jan 1;533(1):270-9.
  24. Arias DG, Marquez VE, Chiribao ML, Gadelha FR, Robello C, Iglesias AA, Guerrero SA. Redox metabolism in Trypanosoma cruzi: functional characterization of tryparedoxins revisited. Free Radic Biol Med. 2013 Oct;63:65-77.
  25. Greif G, Ponce de Leon M, Lamolle G, Rodriguez M, Piñeyro D, Tavares-Marques LM, Reyna-Bello A, Robello C, Alvarez-Valin F. Transcriptome analysis of the bloodstream stage from the parasite Trypanosoma vivax. BMC Genomics. 2013 Mar5;14:149.
  26. Gadelha FR, Gonçalves CC, Mattos EC, Alves MJ, Piñeyro MD, Robello C, Peloso EF. Release of the cytosolic tryparedoxin peroxidase into the incubation medium and a different profile of cytosolic and mitochondrial peroxiredoxin expression in H2O2-treated Trypanosoma cruzi tissue culture-derived trypomastigotes. ExpParasitol. 2013 Mar;133(3):287-93.
  27. Michelini FM, Zorrilla P, Robello C, Alché LE. Immunomodulatory activity ofan anti-HSV-1 synthetic stigmastane analog. Bioorg Med Chem. 2013 Jan15;21(2):560-8.
  28. Coitinho C, Greif G, Robello C, van Ingen J, Rivas C. Identification of Mycobacterium tuberculosis complex by polymerase chain reaction of Exact TandemRepeat-D fragment from mycobacterial cultures. Int J Mycobacteriol. 2012Sep;1(3):146-8.
  29. Greif G, Coitinho C, Rivas C, van Ingen J, Robello C. Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates in Uruguay. Int J Tuberc Lung Dis. 2012 Jul;16(7):947-9.
  30. García G, Libisch G, Trujillo-Cenóz O, Robello C, Russo RE. Modulation of gene expression during early stages of reconnection of the turtle spinal cord. J Neurochem. 2012 Jun;121(6):996-1006.
  31. Peloso EF, Gonçalves CC, Silva TM, Ribeiro LH, Piñeyro MD, Robello C, GadelhaFR. Tryparedoxin peroxidases and superoxide dismutases expression as well as ROS release are related to Trypanosoma cruzi epimastigotes growth phases. ArchBiochem Biophys. 2012 Apr 15;520(2):117-22.
  32. Chiribao ML, Libisch MG, Osinaga E, Parodi-Talice A, Robello C. Cloning, localization and differential expression of the Trypanosoma cruzi TcOGNT-2 glycosyl transferase. Gene. 2012 May 1;498(2):147-54.
  33. Gascue C, Tan PL, Cardenas-Rodriguez M, Libisch G, Fernandez-Calero T, LiuYP, Astrada S, Robello C, Naya H, Katsanis N, Badano JL. Direct role of Bardet-Biedl syndrome proteins in transcriptional regulation. J Cell Sci. 2012Jan 15;125(Pt 2):362-75.
  34. Peñagaricano F, Zorrilla P, Naya H, Robello C, Urioste JI. Gene expression analysis identifies new candidate genes associated with the development of black skin spots in Corriedale sheep. J Appl Genet. 2012 Feb;53(1):99-106.
  35. Peloso Ede F, Vitor SC, Ribeiro LH, Piñeyro MD, Robello C, Gadelha FR. Role of Trypanosoma cruzi peroxiredoxins in mitochondrial bioenergetics. J BioenergBiomembr. 2011 Aug;43(4):419-24.
  36. Piñeyro MD, Parodi-Talice A, Portela M, Arias DG, Guerrero SA, Robello C. Molecular characterization and interactome analysis of Trypanosoma cruzi tryparedoxin 1. J Proteomics. 2011 Aug 24;74(9):1683-92.
  37. Schijman AG, Bisio M, Orellana L, Sued M, Duffy T, Mejia Jaramillo AM, CuraC, Auter F, Veron V, Qvarnstrom Y, Deborggraeve S, Hijar G, Zulantay I, LuceroRH, Velazquez E, Tellez T, Sanchez Leon Z, Galvão L, Nolder D, Monje Rumi M, LeviJE, Ramirez JD, Zorrilla P, Flores M, Jercic MI, Crisante G, Añez N, De Castro AM, Gonzalez CI, Acosta Viana K, Yachelini P, Torrico F, Robello C, Diosque P,Triana Chavez O, Aznar C, Russomando G, Büscher P, Assal A, Guhl F, Sosa EstaniS, DaSilva A, Britto C, Luquetti A, Ladzins J. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Negl Trop Dis. 2011 Jan 11;5(1):e931.
  38. Piñeyro MD, Arcari T, Robello C, Radi R, Trujillo M. Tryparedoxin peroxidases from Trypanosoma cruzi: high efficiency in the catalytic elimination of hydrogen peroxide and peroxynitrite. Arch Biochem Biophys. 2011 Mar 15;507(2):287-95.
  39. Dujardin JC, Herrera S, do Rosario V, Arevalo J, Boelaert M, Carrasco HJ, Correa-Oliveira R, Garcia L, Gotuzzo E, Gyorkos TW, Kalergis AM, Kouri G, LarragaV, Lutumba P, Macias Garcia MA, Manrique-Saide PC, Modabber F, Nieto A, Pluschke G, Robello C, Rojas de Arias A, Rumbo M, Santos Preciado JI, Sundar S, Torres J, Torrico F, Van der Stuyft P, Victoir K, Olesen OF. Research priorities for neglected infectious diseases in Latin America and the Caribbean region. PLoSNegl Trop Dis. 2010 Oct 26;4(10):e780.

CONTACT

robello@pasteur.edu.uy