Molecular and Structural Discrimination of Proline Racemase and Hydroxyproline-2-Epimerase from Nosocomial and Bacterial Pathogens

The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for ‘proline racemase’ virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not ‘one way’ directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on contraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host

La Trypanosoma cruzi proline racemase, un marqueur diagnostique et de suivi pour la Maladie de Chagas ?

Chagas disease caused by Trypanosoma cruzi occurs in Latin and Central America. The parasite is difficult to detect outside the acute phase of infection. No diagnostic means is 100% reliable and does not allow detection of the parasite in the tissues and no vaccine is available. The laboratory characterized the first eukaryotic proline racemase which is also a mitogen for B cells: Trypanosoma cruzi proline racemase (TcPRAC). It is a virulence factor for the parasite, essential for its survival. TcPRAC genes are present in all genotypes of the parasite and are absent in other infectious trypanosomatids. TcPRAC could be a specific marker for the disease. We have demonstrated, using the experimental model, that TcPRAC is indeed a specific diagnostic marker of the infection but especially a marker that would make it possible to assert the effectiveness of a treatment. We were able to follow the infectious process in real time by in vivo imaging. We have produced anti-TcPRAC VHHs capable of binding to intracellular TcPRAC expressed by live parasites in vitro. We have initiated the development of a prototype for the detection of intracellular parasites in vivo. We have been able to optimize new trypanocidal compounds in order to develop new chemotherapies against the disease.La Maladie de Chagas provoquée par Trypanosoma cruzi sévit en Amérique Latine et Centrale. Cette maladie est difficilement décelable en dehors de la phase aiguë de l’infection. Aucun moyen diagnostique n’est fiable à 100% et ne permet la détection du parasite dans les tissus et aucun vaccin n’est disponible. Le laboratoire a caractérisé la première proline racémase eucaryote qui est également un mitogène pour les cellules B : la Trypanosoma cruzi proline racémase (TcPRAC). C'est un facteur de virulence pour le parasite, indispensable à sa survie. Les gènes TcPRAC sont présents dans tous les génotypes du parasite et sont absents chez les autres trypanosomatidés infectieux. La TcPRAC pourrait être un marqueur spécifique de la Maladie. Nous avons mis en évidence, en utilisant le modèle expérimental, que la TcPRAC est effectivement un marqueur diagnostique spécifique de l’infection mais surtout un marqueur qui permettrait d’affirmer l’efficacité d’un traitement. Nous avons été capables de suivre le processus infectieux en temps réel par imagerie in vivo. Nous avons produits des VHH anti-TcPRAC capables de se fixer sur la TcPRAC intracellulaire exprimée par des parasites vivants in vitro. Nous avons entrepris la mise au point d’un prototype pour la détection des parasites intracellulaires in vivo. Nous avons pu optimiser de nouveaux composés trypanocides dans le but de développer de nouvelles chimiothérapies contre la Maladie

The Trypanosoma cruzi proline racemase, a marker for diagnosis and monitoring for Chagas disease ?

La Maladie de Chagas provoquée par Trypanosoma cruzi sévit en Amérique Latine et Centrale. Cette maladie est difficilement décelable en dehors de la phase aiguë de l’infection. Aucun moyen diagnostique n’est fiable à 100% et ne permet la détection du parasite dans les tissus et aucun vaccin n’est disponible. Le laboratoire a caractérisé la première proline racémase eucaryote qui est également un mitogène pour les cellules B : la Trypanosoma cruzi proline racémase (TcPRAC). C'est un facteur de virulence pour le parasite, indispensable à sa survie. Les gènes TcPRAC sont présents dans tous les génotypes du parasite et sont absents chez les autres trypanosomatidés infectieux. La TcPRAC pourrait être un marqueur spécifique de la Maladie. Nous avons mis en évidence, en utilisant le modèle expérimental, que la TcPRAC est effectivement un marqueur diagnostique spécifique de l’infection mais surtout un marqueur qui permettrait d’affirmer l’efficacité d’un traitement. Nous avons été capables de suivre le processus infectieux en temps réel par imagerie in vivo. Nous avons produits des VHH anti-TcPRAC capables de se fixer sur la TcPRAC intracellulaire exprimée par des parasites vivants in vitro. Nous avons entrepris la mise au point d’un prototype pour la détection des parasites intracellulaires in vivo. Nous avons pu optimiser de nouveaux composés trypanocides dans le but de développer de nouvelles chimiothérapies contre la Maladie.Chagas disease caused by Trypanosoma cruzi occurs in Latin and Central America. The parasite is difficult to detect outside the acute phase of infection. No diagnostic means is 100% reliable and does not allow detection of the parasite in the tissues and no vaccine is available. The laboratory characterized the first eukaryotic proline racemase which is also a mitogen for B cells: Trypanosoma cruzi proline racemase (TcPRAC). It is a virulence factor for the parasite, essential for its survival. TcPRAC genes are present in all genotypes of the parasite and are absent in other infectious trypanosomatids. TcPRAC could be a specific marker for the disease. We have demonstrated, using the experimental model, that TcPRAC is indeed a specific diagnostic marker of the infection but especially a marker that would make it possible to assert the effectiveness of a treatment. We were able to follow the infectious process in real time by in vivo imaging. We have produced anti-TcPRAC VHHs capable of binding to intracellular TcPRAC expressed by live parasites in vitro. We have initiated the development of a prototype for the detection of intracellular parasites in vivo. We have been able to optimize new trypanocidal compounds in order to develop new chemotherapies against the disease

Immunotherapy of Trypanosoma Cruzi Infections

International audienceThe protozoan parasite Trypanosoma cruzi, causative agent of Chagas' disease, is transmitted to man and other mammals by triatominae insects, or 'kissing bugs'. Since its discovery in 1909, by Carlos Chagas, this parasite has been the object of several publications in the domains of immunology, cellular biology and of control gene organization, regulation and expression. Although much progress has been made concerning prophylaxis of Chagas' disease, particularly vector eradication, additional cases of infection and disease development still occur every day throughout the world. Whilst infection was largely limited in the past to vector transmission in endemic areas of Latin America, its impact has increased in terms of congenital and blood transmission, transplants and recrudescence following immunosuppressive states. Reports on new insect vectors adapted to the parasite and domestic animals infected in more developed countries, emphasize the continuing worldwide public health issue. Therapy against this parasite is limited and cure is subjected to several criteria, such as susceptibility of the parasite strain, age of the host and stage of the disease. The ability of Trypanosoma cruzi to induce important and various host immune system dysfunctions makes the development of effective vaccines a laborious and complex task. These considerations strengthen the latent significance of Chagas' disease and encourage the search for new preventive procedures and the research on rational vaccines

Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies

Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi , is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy

Proline racemases are conserved mitogens: characterization of a Trypanosoma vivax proline racemase

International audienceTrypanosoma cruzi proline racemases (TcPRAC) are the only eukaryotic proline racemases described so far. Except their role in the interconversion of free L- and D-proline enantiomers, parasite TcPRACs are involved in major T. cruzi biological pathways. These essential enzymes are implicated in the process of parasite differentiation and the acquisition of virulence during metacyclogenesis and are currently considered as key targets for drug development against Chagas' disease. In this study, we searched for the presence of TcPRAC gene homologues among other trypanosomatid genomes. Despite the high degree of gene synteny observed in Kinetoplastidae genomes, PRAC genes are missing in Trypanosoma brucei, Trypanosoma congolense and Leishmania spp. genomes. Interestingly, we identified a hypothetical PRAC gene in Trypanosoma vivax that is the major hemoparasite responsible for livestock trypanosomiasis, a serious economical impact for most of African and South American countries. We report here that the product of this T. vivax gene is bona fide a proline racemase with an activity comparable to the one we described previously for TcPRAC. Inhibition studies using the pyrrole-2-carboxylic acid confirmed that this compound is a competitive inhibitor for both TcPRAC and TvPRAC enzymes. Similarly to TcPRAC and all members of the racemase family studied so far in other pathogenic and nosocomial bacteria, our results show that TvPRAC is a T-cell-independent B-cell mitogen. Therefore the product of the novel TvPRAC gene identified in T. vivax and reported herein has the potential to be used as a drug target for this parasite-based trypanosomiasis

Proline racemases: insights into Trypanosoma cruzi peptides containing D-proline

Trypanosoma cruzi proline racemases (TcPRAC) are homodimeric enzymes that interconvert the L and D-enantiomers of proline. At least two paralogous copies of proline racemase (PR) genes are present per parasite haploid genome and they are differentially expressed during T. cruzi development. Non-infective epimastigote forms that overexpress PR genes differentiate more readily into metacyclic infective forms that are more invasive to host cells, indicating that PR participates in mechanisms of virulence acquisition. Using a combination of biochemical and enzymatic methods, we show here that, in addition to free D-amino acids, non-infective epimastigote and infective metacyclic parasite extracts possess peptides composed notably of D-proline. The relative contribution of TcPRAC to D-proline availability and its further assembly into peptides was estimated through the use of wild-type parasites and parasites over-expressing TcPRAC genes. Our data suggest that D-proline-bearing peptides, similarly to the mucopeptide layer of bacterial cell walls, may be of benefit to T. cruzi by providing resistance against host proteolytic mechanisms

Prodrugs as new therapies against Chagas disease: In vivo synergy between Trypanosoma cruzi proline racemase inhibitors and benznidazole

International audienceObjective: Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects approximately 6-7 million people worldwide. There are limited available therapies, and they exhibit low efficacy, often high toxicity in chronic cases and some drug resistance. In this study, our objective was to develop ester prodrugs that inhibit proline racemase (TcPRAC), a parasitic enzyme that we have previously identified and characterized as a promising target because of its essential role in the parasite's life cycle and virulence, and to test their activity against T. cruzi.Method: Using structural bioinformatics, we modelled several functional intermediates of the catalytic site between the opened and closed conformations of TcPRAC based on its crystal structures in complex with its competitive inhibitor, pyrrole-2-carboxylic acid. Guided by these intermediates, which were later validated in cocrystals, we designed and evaluated numerous compounds and tested them enzymatically on live parasites and in mice with our quick and straightforward drug screening method, which is based on state-of-the-art bioluminescent T. cruzi parasites injected subcutaneously.Results: Some of our novel compounds specifically inhibited racemase activity, as determined through biochemical assays, and covalently bound to TcPRAC. Furthermore, the corresponding ester prodrugs were effective in killing parasites in vitro. Bioluminescent T. cruzi assays in mice showed that JR1531, a TcPRAC inhibitor prodrug, can kill parasites in living animals, with boosted action when combined with low doses of benznidazole.Conclusions: This approach, based on TcPRAC inhibitor prodrugs in association with low doses of benznidazole, may lead to a more effective, specific and nontoxic therapy against Chagas disease