Immunobiology of Trypanosoma Infections  

  MEMBERSBLOM-POTAR Marie Christine / BOUTOUT Laurence / COATNOAN Nicolas / COSSON Alain / D’ARCHIVIO Simon / Dr. GOYARD Sophie / MEDINA Mathieu

  Annual Report

The laboratory’s primary goal is to identify molecules which are both implicated in parasite evasion of the host’s immune system and could ultimately be used as targets in the development of new anti-trypanosomiasis therapies. The teamhas been studying experimental models of Chagas’ disease and Sleeping sickness, two of the “most neglected diseases” that afflict the poor and powerless in developing regions of sub-Saharan Africa, Asia and the Americas. Together they cause an estimated 500,000 to 1 million deaths annually. Chagas’ disease, the third largest disease burden in Latin America, is caused by Trypanosoma cruzi. It affects at least 15 million people and 100 million people are at risk. Human and animal trypanosomiasis (Nagana) are major public health concerns in 35 sub-Saharan countries. The livestock challenge, which is mainly caused by Trypanosoma vivax, causes about 3 million cattle deaths annually, has a severe impact on African’s agriculture, and was recently introduced into South America and Mauritius. No vaccines have yet been developed against these seriously disabling or life-threatening illnesses.

Trypanosoma vivax possesses a functional proline racemase.

Trypanosoma cruzi proline racemase (TcPRAC), the first eukaryotic proline racemase and a B-cell mitogen, is an essential enzyme to T. cruzi viability, virulence and fate, and is a therapeutic target against Chagas’ disease. Previous studies of T. cruzi, Trypanosoma brucei and Leishmania major genomes showed a high degree of gene synteny. Given that TcPRAC appears to be involved in the key developmental processes of T. cruzi, we hypothesized that PRAC may also be present - and share similar biological functions - in other trypanosomatids. Although several megabases of synteny blocks were indeed observed, an analysis of the TcPRAC locus showed that the PRAC gene was present only in T. vivax. We have shown that TvPRAC is functional and the protein it codes for has been fully characterized biochemically (TvPRAC). Our results also showed that 2-pyrrole-carboxylic acid (PYC) is a competitive inhibitor of TvPRAC, as it is for TcPRAC and Clostridium difficile PRACs. Similarly, we found that TvPRAC possesses mitogenic activity for B cells.

Trypanosoma vivax, a new experimental model for the study of Nagana.

Given the importance of animal trypanosomiasis and our recent results on the validation of TcPRAC as a therapeutic target, we decided to check if TvPRAC also plays a key role in the metabolism and virulence of T. vivax. This question would be easier to answer if, similarly to T. brucei, this parasite possesses functional RNAi machinery. Initially, we confirmed that the necessary RNAi elements, i.e. AGO and DICER homologue genes, are indeed present in the T. vivax genome. The products of these hypothetical genes present conserved domains that are necessary for RNAi activity. We then demonstrated that these genes are transcribed into mature mRNAs. Transfection of T. vivax blood forms with tubulin siRNA generated a characteristic “fat” phenotype showing severe cytoskeletal disorganization due to the parasite’s inability to synthesize tubulin. These encouraging results indicate that T. vivax possesses functional RNAi machinery and these studies will be pursued in the laboratory.

Very few laboratories have attempted to develop a reference model for T. vivax. We decided to further develop such a model of infection in mice then use it to study the immunobiology of the infectious process and characterize some of the key players in the immunopathology of animal trypanosomiasis. Sustained and reproducible infections were obtained with C57BL/6, BALB/c and Outbred (Swiss) mice, and this already successfully supported the analysis made of the infection’s parasitological, histological and pathological parameters observed in the field, and the its dynamics (coll. N. Chamond, G. Jouvion & M. Huerre). Hallmarks of cattle trypanosomiasis were also observed, namely severe acute anemia, thrombocytopenia and a reduced number of B lymphocytes. We observed that the splenomegaly that preceeds organ necrosis primarily involves CD4+ T cells and macrophages.These models will be useful to better explore the immunobiology of T. vivax infection and essential to elucidate, for instance, the fonction of TvPRAC in vivo.

Trypanosoma cruzi peptides contain D-proline

We further hypothesized that TcPRAC contributes to parasite evasion strategies by making D-proline available for post-translational addition into protein chains. This hypothesis is appealing since the incorporation of D- amino acid residues (D-aa) into peptide sequences would render them less immunogenic. To detect the presence of D-aa (free or bound to peptide chains) we used enzymatic fractionation of parasite extracts, thin layer preparative/chiral chromatography and a D-aa oxidase test developed in the laboratory. We observed that concentrations of free D-aas and in particular D-proline bound to peptides increased during parasite acquisition of virulence. Also, and consistent with our hypothesis, parasite extracts overexpressing TcPRAC genes possessed more D-proline containing-peptides than wild type parasites. Although not yet proven, the presence of D-proline in peptides exposed by T. cruzi may favor parasite persistence and multiplication inside the cytoplasm of host cells through lower susceptibility to lysosome-delivered proteases.

Multidisciplinary approach leads to the identification of new TcPRAC inhibitors

Our results strongly supported the notion that TcPRAC is a putative target for the development of both chemotherapy and/or immunotherapeutics. Our next step was to focus on : i. modifying the known inhibitor of TcPRAC (PYC) to make it water soluble (in coll. with Y. Janin); ii. in silico screening of libraries and pharmacophore design after further pharmacodynamic studies of the enzyme (outsourced to Ariana-Pharma) and iii. virtual screening of chemical libraries and models of the enzyme’s catalytic site deduced by structural bioinformatics (in coll. with A. Blondel). Our previous studies revealed that the catalytic site of the enzyme is small, what could be seen as a challenge for the identification of putative enzyme inhibitors. To widen the chemical space accessible to virtual screening, intermediate conformations between liganded (closed) and free (opened) enzyme structures were calculated to take account of the flexibility of TcPRAC observed upon ligand binding. Most importantly, these transitional conformation models were used to perform docking simulations for several thousand molecules in accessible chemical libraries. Two of these compounds, Oxo-PA and Br-OxoPA, were selected and shown to exert 3-6 times more inhibition than PYC. The Ki of these molecules is lower (close to 1 mM) than that of PYC (6 mM). Preliminary data reveals that Oxo-PA and Br-OxoPA hamper parasite/host cell interaction. These novel inhibitors would not have been selected had the screening been based on crystallographic structure alone.

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


Legende de la Figure :Electronic microscopy of T. cruzi trypomastigotes: figure depicts TcPRAC inside the flagella pocket and reservosomes (A) or nearby vesicles (B). IMF of epimastigotes (C-F) or amastigotes inside Vero cells (G-H) showing intracytoplasmic localization of TcPRAC; controls only with DAPI (C and G).



- Chamond N, Cosson A, Coatnoan N, Minoprio P. (2009) Proline racemases are conserved mitogens. Characterization of a Trypanosoma vivax proline racemase. Mol. Biochem. Parasitol. 165 : 170-9. PMID: 19428664

- Coatnoan, N., Berneman, A., Chamond, N., and Minoprio, P. (2009). Proline racemases: insights into Trypanosoma cruzi peptides containing D-proline. Mem Inst Oswaldo Cruz 104 295-300. PMID: 19753488

- Goytia M, Chamond N, Cosson A, Coatnoan N, Hermant D, Berneman A, Minoprio P. (2007) Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens. PLoS ONE. 2: 885. PMID: 17849014

- Buschiazzo A, Goytia M, Schaeffer F, Degrave W, Shepard W, Grégoire C, Chamond N, Cosson A, Berneman A, Coatnoan N, Alzari PM, Minoprio P. (2006) Crystal structure, catalytic mechanism, and mitogenic properties of Trypanosoma cruzi proline racemase. Proc Natl Acad Sci U S A. 103: 1705-10. PMID: 16446443

- Chamond N, Goytia M, Coatnoan N, Barale JC, Cosson A, Degrave WM, Minoprio P. (2005) Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity. Mol Microbiol. 58:46-60. PMID: 16164548

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