Global Gene Expression Profiling through the Complete Life Cycle of Trypanosoma vivax

The parasitic flagellate Trypanosoma vivax is a cause of animal trypanosomiasis across Africa and South America. The parasite has a digenetic life cycle, passing between mammalian hosts and insect vectors, and a series of developmental forms adapted to each life cycle stage. Each point in the life cycle presents radically different challenges to parasite metabolism and physiology and distinct host interactions requiring remodeling of the parasite cell surface. Transcriptomic and proteomic studies of the related parasites T. brucei and T. congolense have shown how gene expression is regulated during their development. New methods for in vitro culture of the T. vivax insect stages have allowed us to describe global gene expression throughout the complete T. vivax life cycle for the first time. We combined transcriptomic and proteomic analysis of each life stage using RNA-seq and mass spectrometry respectively, to identify genes with patterns of preferential transcription or expression. While T. vivax conforms to a pattern of highly conserved gene expression found in other African trypanosomes, (e.g. developmental regulation of energy metabolism, restricted expression of a dominant variant antigen, and expression of 'Fam50' proteins in the insect mouthparts), we identified significant differences in gene expression affecting metabolism in the fly and a suite of T. vivax-specific genes with predicted cell-surface expression that are preferentially expressed in the mammal ('Fam29, 30, 42') or the vector ('Fam34, 35, 43'). T. vivax differs significantly from other African trypanosomes in the developmentally-regulated proteins likely to be expressed on its cell surface and thus, in the structure of the host-parasite interface. These unique features may yet explain the species differences in life cycle and could, in the form of bloodstream-stage proteins that do not undergo antigenic variation, provide targets for therapy

Phylogenetic and syntenic data support a single horizontal transference to a Trypanosoma ancestor of a prokaryotic proline racemase implicated in parasite evasion from host defences

Abstract\ud \ud Background\ud Proline racemase (PRAC) enzymes of Trypanosoma cruzi (TcPRAC), the agent of Chagas disease, and Trypanosoma vivax (TvPRAC), the agent of livestock trypanosomosis, have been implicated in the B-cells polyclonal activation contributing to immunosuppression and the evasion of host defences. The similarity to prokaryotic PRAC and the absence in Trypanosoma brucei and Trypanosoma congolense have raised many questions about the origin, evolution, and functions of trypanosome PRAC (TryPRAC) enzymes.\ud \ud \ud Findings\ud We identified TryPRAC homologs as single copy genes per haploid genome in 12 of 15 Trypanosoma species, including T. cruzi and T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli, T. conorhini and T. lewisi, all parasites of mammals. Polymorphisms in TcPRAC genes matched T. cruzi genotypes: TcI-TcIV and Tcbat have unique genes, while the hybrids TcV and TcVI contain TcPRACA and TcPRACB from parental TcII and TcIII, respectively. PRAC homologs were identified in trypanosomes from anurans, snakes, crocodiles, lizards, and birds. Most trypanosomes have intact PRAC genes. T. rangeli possesses only pseudogenes, maybe in the process of being lost. T. brucei, T. congolense and their allied species, except the more distantly related T. vivax, have completely lost PRAC genes.\ud \ud \ud \ud Conclusions\ud The genealogy of TryPRAC homologs supports an evolutionary history congruent with the Trypanosoma phylogeny. This finding, together with the synteny of PRAC loci, the relationships with prokaryotic PRAC inferred by taxon-rich phylogenetic analysis, and the absence in trypanosomatids of any other genera or in bodonids or euglenids suggest that a common ancestor of Trypanosoma gained PRAC gene by a single and ancient horizontal gene transfer (HGT) from a Firmicutes bacterium more closely related to Gemella and other species of Bacilli than to Clostridium as previously suggested. Our broad phylogenetic study allowed investigation of TryPRAC evolution over long and short timescales. TryPRAC genes diverged to become species-specific and genotype-specific for T. cruzi and T. rangeli, with resulting genealogies congruent with those obtained using vertically inherited genes. The inventory of TryPRAC genes described here is the first step toward the understanding of the roles of PRAC enzymes in trypanosomes differing in life cycles, virulence, and infection and immune evasion strategies.U.S. National Institutes of Health (NIH)U.S. National Science Foundation (NSF)SENACYT (Secretaría Nacional de Ciencia, Tecnología e Innovación)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP (grant #2013/14622-3, São Paulo Research Foundation)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade de São Paulo (USP

Trypanosoma vivax Infections: Pushing Ahead with Mouse Models for the Study of Nagana. II. Immunobiological Dysfunctions

Trypanosoma vivax is the main species involved in trypanosomosis, but very little is known about the immunobiology of the infective process caused by this parasite. Recently we undertook to further characterize the main parasitological, haematological and pathological characteristics of mouse models of T. vivax infection and noted severe anemia and thrombocytopenia coincident with rising parasitemia. To gain more insight into the organism's immunobiology, we studied lymphocyte populations in central (bone marrow) and peripherical (spleen and blood) tissues following mouse infection with T. vivax and showed that the immune system apparatus is affected both quantitatively and qualitatively. More precisely, after an initial increase that primarily involves CD4+ T cells and macrophages, the number of splenic B cells decreases in a step-wise manner. Our results show that while infection triggers the activation and proliferation of Hematopoietic Stem Cells, Granulocyte-Monocyte, Common Myeloid and Megacaryocyte Erythrocyte progenitors decrease in number in the course of the infection. An in-depth analysis of B-cell progenitors also indicated that maturation of pro-B into pre-B precursors seems to be compromised. This interferes with the mature B cell dynamics and renewal in the periphery. Altogether, our results show that T. vivax induces profound immunological alterations in myeloid and lymphoid progenitors which may prevent adequate control of T. vivax trypanosomosis

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

Genetic Engineering of Trypanosoma (Dutonella) vivax and In Vitro Differentiation under Axenic Conditions

Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite's biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites' biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents

Trypanosoma vivax Infections: Pushing Ahead with Mouse Models for the Study of Nagana. I. Parasitological, Hematological and Pathological Parameters

African trypanosomiasis is a severe parasitic disease that affects both humans and livestock. Several different species may cause animal trypanosomosis and although Trypanosoma vivax (sub-genus Duttonella) is currently responsible for the vast majority of debilitating cases causing great economic hardship in West Africa and South America, little is known about its biology and interaction with its hosts. Relatively speaking, T. vivax has been more than neglected despite an urgent need to develop efficient control strategies. Some pioneering rodent models were developed to circumvent the difficulties of working with livestock, but disappointedly were for the most part discontinued decades ago. To gain more insight into the biology of T. vivax, its interactions with the host and consequently its pathogenesis, we have developed a number of reproducible murine models using a parasite isolate that is infectious for rodents. Firstly, we analyzed the parasitical characteristics of the infection using inbred and outbred mouse strains to compare the impact of host genetic background on the infection and on survival rates. Hematological studies showed that the infection gave rise to severe anemia, and histopathological investigations in various organs showed multifocal inflammatory infiltrates associated with extramedullary hematopoiesis in the liver, and cerebral edema. The models developed are consistent with field observations and pave the way for subsequent in-depth studies into the pathogenesis of T. vivax - trypanosomosis

Les populations lymphocitaires de la souris infectee par le Trypanosoma cruzi

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Les racémases et épimérases d'acides aminés (l'exemple de la TcPRAC comme cible pour le développement d'une thérapie contre l'infection par Trypanosoma cruzi)

Le Trypanosoma cruzi est l agent étiologique de la Maladie de Chagas, pathologie affectant 20 millions de personnes en Amérique latine. Les traitements actuels sont inadéquats, le développement de nouvelles cibles thérapeutiques est essentiel. Nous avons approfondi les recherches sur les Proline Racémases de T. cruzi qui possèdent aussi une activité mitogénique des cellules B de l hôte. Nous avons apporté les évidences pour consolider la TcPRAC comme cible thérapeutique contre la Maladie de Chagas. La TcPRAC est essentielle pour le parasite, la surexpression augmente la différentiation en formes infectantes et l invasion des cellules hôte, c est un facteur de virulence. L étude démontre de manière formelle le mécanisme réactionnel pour les PRACs, et la dissociation de l activité enzymatique dépendante de deux résidus catalytiques par sous-unité et de l activité mitogénique dépendante d épitopes conformationnels de l enzyme non liée à un ligand. Par ailleurs, nous mettons en évidence, chez divers microorganismes pathogènes, une autre PRAC et des hydroxyproline épimérases, dont le gène est publié pour la première fois grâce à nos travaux. L analyse des séquences a permis de discriminer les activités racémase et épimérase.Trypanosoma cruzi is the etiologic agent of Chagas Disease affecting 20 million people in Latin America. Drugs available are inadequate, and the development of new therapeutic targets is essential. In this study, we focus on Proline Racemases of T. cruzi (TcPRAC) that present mitogenic and racemic activities. We present proof of concept for TcPRAC as a drug target against Chagas Disease. TcPRAC is essential for the parasite, and is a virulence factor as its hyperexpression increases differentiation into infective forms and host cell s invasion. The research demonstrates the definitive enzymatic mechanism for PRACs, and the dissociation of enzymatic activity, which relies on two catalytic residues per subunit, and the mitogenic acitivy, relying on conformational epitopes of the free enzyme. Furthermore, we show another PRAC enzyme and 5 new hydroxyproline epimerases, for which the sequence of the gene is published for the first time by our work, in 6 other pathogenic microorganisms. Analysis of the sequences allowed us to discriminate between the racemase and the epimerase activities.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF