|Immunobiology of Trypanosoma Infections|
|HEAD||Dr. MINOPRIO Paola / firstname.lastname@example.org|
|MEMBERS||Dr. BERNEMAN Armand / BOUTOUT Laurence / Dr. CHAMOND Nathalie / COATNOAN Nicolas / COSSON Alain / D’ARCHIVIO Simon / LOPES COUTINHO Leticia / THOURON Françoise
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 reverse genetic tools in trypanosomes to validate targets for the development of rational therapies.
A novel PRAC orthologue encodes for a functional enzyme in one African Trypanosome.
TcPRAC, the first eukaryotic proline racemase and a B-cell mitogen, is essential to T. cruzi viability, virulence and fate, and is a therapeutic target against Chagas’ disease. New proline racemases and related hydroxyproline epimerases were recently identified and characterized from bacterial nosocomial pathogens, notably Clostridium difficile. However, regardless of the significant degree of gene synteny presented by genomes of the Trypanosomatidae family members and the high conservation ofthe PRAC locus environment, only one TcPRAC orthologue was identified among trypanosomatids : the Trypanosoma vivax proline racemase gene. This major livestock trypanosome is cyclically transmitted between domestic and wild ruminants by tsetse flies, tabanids and other varieties of biting flies. Moreover, the gene encodes for a functional enzyme with molecular and biochemical characteristics equivalent to TcPRAC. Similarly to T. cruzi and other bacterial PRACs, TvPRAC although not possessing a signal for secretion, is also a T-cell independent B-cell mitogen. Studies are in progress to evaluate the biological relevance of TvPRAC in T. vivax trypanosomiasis.
Developing experimental models to study Trypanosoma vivax-based sleeping sickness (Nagana).
The identification of a PRAC in a major parasite of Nagana trypanosomiasis stimulated the development of experimental models to study T. vivax-basedtrypanosomiasis. Detailed analysis of parasitological, haematological, immunological and histopathological parametres of the infection were conducted in several strains of mice infected with T. vivax (coll. G. Jouvion & M. Huerre). 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.
Molecular tools to study gene function in T. cruzi and T. vivax.
Further attempts to develop an inducible RNAi system in T. cruzi (Tc) by importing available components of RNAi machinery from T. brucei (Tb) genome were up to now unfruitful. Therefore, initial viable Tc chimeras possessing Argonaute 1 constructs (TbAGO) inserted in pTREX integrative vector were produced. However, despite the integration of TbAGO plasmid in Tc genome, AGO was not expressed, possibly as a result from the lack of Dicer and siRNA load in the Risk complex. Presently, to circunvent the difficulties of this “RNAi-based” approach, extra efforts are directed to exploit a system that allows the stable expression of transgenes in a controlled/repressible manner. Conditional knockouts for TcPRAC are been produced using a tetracycline-regulatable integrative expression vector, pTcINDEX, kindly provided by M. Taylor and J. Kelly (London School, UK). Moreover, parallel studies are carried out to verify the functionality of T. vivax RNAi machinery and to design specific T. vivax vectors (pKS-GFP and pKS-Luc) for further genetic manipulation of this parasite. The establishment of transfection conditions for T. vivax are in progress.
Further insights in TcPRAC localization during T. cruzi developement.
Transmission electron microscopy studies of non-infective and infective forms of T. cruzi revealed that TcPRAC localizes in the parasite flagellar pocket and within vesicles nearby the cytostome - sites were exocytic/endocytic activities take place in trypanosomes. Thus, as an alternative to active secretion, these sub-cellular localizations may contribute to explain how TcPRAC (and particularly TvPRAC) reaches bloodstream and/or extravascular spaces further triggering non-specific polyclonal B-cell activation. The protein co-localizes as well with transferrin, a marker of parasite reservosomes. Studies of intracellular parasites by confocal laser scanning microscopy and differential contrast interference confirmed that TcPRAC is spread out intracellularly but also concentrated near the kinetoplast, compatible to a flagellar pocket localization. As expected, in vitro treatement of infected cells with pyrole-carboxylic acid (PYC), a competitive inhibitor of PRAC, induced an important dose dependent decrease in both parasite invasion and fate, without affecting host cell viability (coll. M. Alves & W. Degrave, Fiocruz, Brazil). These data provide additional evidences that TcPRAC contributes significantly to the development of T. cruzi in host tissue cells.
Combined approaches toward the identication of new drugs using TcPRAC as a lead.
Rational strategies of medicinal chemistry and innovative molecular dynamics approaches were undertaken to improve PYC affinity and solubility and to search for new TcPRAC inhibitors (coll. Y. Janin & A. Blondel). Making use of the resulting knowledge on the enzyme specificities issued from of a) the chemical synthesis of inhibitor analogues, b) the PRAC crystal structure and c) the low energy transition molecular states of PRAC from its active (wide open) to its liganded (closed) structures, 4 new soluble inhibitors of TcPRAC were identified. Two molecules present higher solubility and increased affinity for the enzyme than PYC. Ongoing studies with in vitro models of infection indicate that these inhibitors are not toxic for host cells but induced a significant reduction of intracellular parasite load and outcome. Complementary studies were performed to derive pharmacophoric/docking models adapted to in silico screening of chemical compound libraries (coll. M. Afshar, Ariana Pharmaceuticals). The analysis of putative inhibitors of the enzyme obtained from virtual screening are underway.
Keywords: Chagas’ disease, Trypanosoma cruzi, African trypanosomiasis, Clostridium difficile, B-cell mitogens, proline racemase, hydroxyproline epimerase
- Chamond N, Cosson A, Coatnoan N, Minoprio P. Proline racemases are conserved mitogens. Characterization of a Trypanosoma vivax proline racemase. Mol. Biochem. Parasitol. 2008, in press.
- 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.
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