Immunobiology of Trypanosoma Infections  


  HEADDr. MINOPRIO Paola / pmm@pasteur.fr
  MEMBERSDr. BERNEMAN Armand / BOUTOUT Laurence / Dr. CHAMOND Nathalie / COATNOAN Nicolas / COSSON Alain / Dr. GOYTIA Maira / HERMANT Daniel


  Annual Report

Chagas’ disease and Sleeping sickness, respectively caused by American and African trypanosomes, are two of the ‘most neglected’ and seriously disabling or life-threatening diseases for which vaccines do not exist and treatments are missing or inadequate. Trypanosomes provoke a broad range of disturbances in the host immunological system to circumvent its defense mechanisms. The main goals of the team are: 1. to identify and study structural and functional components involved in host–pathogen interactions, primarily those implicated in the mechanisms allowing micro-organisms to escape from the host immune responses; 2. to better understand the biological properties of those components as well as their role in micro-organism development and acquisition of virulence and 3. to develop new reverse genetic tools in American trypanosomes to validate targets for drug development.

New proline racemases and hydroxyproline epimerases from trypanosomatides and bacterial pathogens. The first eukaryotic proline racemase (TcPRAC), a T. cruzi B-cell mitogen essential to the parasite viability, virulence and fate, is a therapeutic target against Chagas’ disease. Data mining of released genome databases with minimal essential TcPRAC motifs unveiled two new PRACs, isolated respectively from the African Trypanosoma vivax and the nosocomial bacteria Clostridium difficile, and five novel bacterial Hydroxyproline-2-Epimerases (HyPRE), namely from Pseudomonas aeruginosa, Burkholderia pseudomallei and 3 Brucella species (B. abortus, B. suis and B. mellitensis). Genes and respective functional enzymes were fully characterized. Specific shape and hydrophobicity constraints of the catalytic pockets and precise sequence elements determine substrate accessibility, enzyme specificities and allow in silico discrimination of PRAC and HyPRE enzymes (see Figure). Similarly to PRACs, HyPREs are strong B-cell mitogens.

TcPRAC localization and its involvement in the expression of proteins bearing D-proline. Intracellular localization of PRAC throughout T. cruzi development was further addressed in collaboration with L. Coutinho Lopes, M. Alves, N. Soeiro and W. Degrave (FIOCRUZ-RJ, Brazil). Transmission electron microscopy revealed that PRAC localizes in the parasite flagellar pocket and within vesicles nearby the cytostome, sites of exocytosis/endocytosis in trypanosomes. Confocal fluorescence showed that PRAC co-localize as well with transferrin, a marker of parasite reservosomes. Chemistry, proteomics and mass spectrometry combined approaches confirmed the presence of different isoforms of PRAC all the way through metacyclogenesis, the differential expression of several stage-specific proteins and the presence of D-proline-bearing proteins mainly in infective parasites. The post-translational addition of D-proline as single or blocks of residues to ribosome synthesized protein chains expressed by the parasite may well confer to such ‘D-pro’- containing peptides resistance to host proteolytic enzymes and contribute to parasite persistence in the host. Isolation and characterization of those proteins are in progress.

Developing an inducible RNAi system in T. cruzi. Aiming at studying the biological significance of genes involved in parasite differentiation and acquisition of virulence, experiments were undertaken to develop an inducible RNAi system in T. cruzi (Tc). Chimeric Tc/Tb parasites are been produced by exporting available T. brucei (Tb) RNAi machinery components into T. cruzi genome. At a first glance, T. cruzi transfected with tagged Tb Argonaute 1 (AGO 1) constructs inserted in pTREX integrative vector will be ultimately transfected with appropriate TbDicer (DCR) constructs. Similar studies are being conducted with T. vivax that equally possess RNAi elements in the genome.

Identification, validation and optimization of TcPRAC inhibitors. Saturation of TcPRAC catalytic site with a known inhibitor induces conformational changes of the protein thus precluding its interaction with B-cell ligands. Furthermore, increasing amounts of this inhibitor significantly affect parasite growth inside host cells. In collaboration with Y. Janin, A. Blondel and M. Afshar (Institut Pasteur and Ariana Pharmaceuticals) and using the resulting knowledge on PRAC crystal structure, low energy transition states of PRAC/ligand molecular dynamics and chemical synthesis of inhibitor analogues, optimum pharmacophoric/docking models were derived in silico and are now used for the virtual screening of chemical compound libraries. A simple medium/high throughput in vitro test was developed to test the activity of putative inhibitors of the enzyme.

Keywords: Chagas’ disease, Trypanosoma cruzi, African trypanosomiasis, Clostridium difficile, B-cell mitogens, proline racemase, hydroxyproline epimerase

I2t.jpg

Ribbon model of Trypanosoma cruzi Proline Racemase (green) and Pseudomonas aeruginosa Hydroxyproline Epimerase (purple) subunits. Despite the overall similarities of the 3D-structures (upper panels), closer view of the catalytic pockets reveal polarity and hydrophobicity constraints responsible for ligand specificity (lower panels). Catalytic residues are shown in orange and proline and hydroxyproline were modeled inside the reaction centers.



  Publications

- Goytia M, Chamond N, Cosson A, Coatnoan N, Hermant D, Berneman A, Minoprio P. Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens. PLoS ONE. 2007 2(9): 885.

- Buschiazzo A, Goytia M, Schaeffer F, Degrave W, Shepard W, Grégoire C, Chamond N, Cosson A, Berneman A, Coatnoan N, Alzari PM, Minoprio P. Crystal structure, catalytic mechanism, and mitogenic properties of Trypanosoma cruzi proline racemase. Proc Natl Acad Sci U S A. 2006 103: 1705-10.

- Chamond N, Goytia M, Coatnoan N, Barale JC, Cosson A, Degrave WM, Minoprio P. Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity. Mol Microbiol. 2005 58:46-60.

- Minoprio P, Chamond N, Degrave WM, Berneman A. Identification and characterization of racemases, definition of protein signatures, and a test for detecting D-amino acid and for screening molecules capable of inhibiting the activity of racemase, especially proline racemase, 2004 US Pat. N°7,262,015

- Chamond N, Grégoire C, Coatnoan N, Rougeot C, Freitas-Junior LH, da Silveira JF, Degrave WM, Minoprio P. Biochemical characterization of proline racemases from the human protozoan parasite Trypanosoma cruzi and definition of putative protein signatures. J Biol Chem. 2003 278(18):15484-94.



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Activity Reports 2007 - Institut Pasteur
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