Epidemiology, biochemistry and evolution of Trypanosoma cruzi lineages based on ribosomal RNA sequences

Universidade de São PauloInstituto Oswaldo CruzUniversidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region

BACKGROUND: The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. RESULTS: We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. CONCLUSION: The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network

Trypanosoma cruzi benznidazole susceptibility in vitro does not predict the therapeutic outcome of human Chagas disease

Therapeutic failure of benznidazole (BZ) is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC50 to BZ between 7.6-32 µM. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC50 of the strains varied from 15.6 ± 3-51.4 ± 1 µM. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.FAPESPCNP

Auxin production by the plant trypanosomatid Phytomonas\ud serpens and auxin homoeostasis in infected tomato fruits

Previously we have characterized the complete gene encoding a pyruvate decarboxylase (PDC)/indolepyruvate\ud decarboxylase (IPDC) of Phytomonas serpens, a trypanosomatid highly abundant in tomato fruits. Phylogenetic analyses\ud indicated that the clade that contains the trypanosomatid protein behaves as a sister group of IPDCs of γ-proteobacteria.\ud Since IPDCs are key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid (IAA), the ability for IAA\ud production by P. serpens was investigated. Similar to many microorganisms, the production of IAA and related indolic\ud compounds, quantified by high performance liquid chromatography, increased inP. serpens media in response to amounts\ud of tryptophan. The auxin functionality was confirmed in the hypocotyl elongation assay. In tomato fruits inoculated with\ud P. serpensthe concentration of free IAA had no significant variation, whereas increased levels of IAA-amide and IAA-ester\ud conjugates were observed. The data suggest that the auxin produced by the flagellate is converted to IAA conjugates,\ud keeping unaltered the concentration of free IAA. Ethanol also accumulated inP. serpens-conditioned media, as the result of\ud a PDC activity. In the article we discuss the hypothesis of the bifunctionality of P. serpens PDC/IPDC and provide a\ud three-dimensional model of the enzyme.FAPESP) (grant number 2010/50957-1)(CNPq) (grant number 304793/2009-4)FAPESP (grant number 2008/50209-5

Drug discovery for Chagas disease should consider Trypanosoma cruzi strain diversity.

This opinion piece presents an approach to standardisation of an important aspect of Chagas disease drug discovery and development: selecting Trypanosoma cruzi strains for in vitro screening. We discuss the rationale for strain selection representing T. cruzi diversity and provide recommendations on the preferred parasite stage for drug discovery, T. cruzi discrete typing units to include in the panel of strains and the number of strains/clones for primary screens and lead compounds. We also consider experimental approaches for in vitro drug assays. The Figure illustrates the current Chagas disease drug-discovery and development landscape

Evidence for T Cell Help in the IgG Response against Tandemly Repetitive Trypanosoma cruzi

The tandemly repetitive Trypanosoma cruzi B13 protein is an immunodominant antigen among Chagas disease patients. Such repetitive domains may behave as T-independent antigens. However, T cells can recognize B13 epitopes in an HLA class II-restricted fashion and could potentially provide cognate T cell help and boost antibody titers. We assessed whether the presence of HLA class II molecules able to present B13 epitopes to T cells could affect anti-B13 IgG levels in a cognate fashion, in both major clinical forms of chronic Chagas disease. We found no difference between anti-B13 IgG antibody levels between patients carrying HLA class II molecules associated to T cell responses or other alleles. The predominant anti-B13 IgG subclass was IgG1, with negligible IgG2, suggesting a T-dependent, noncognate help for antibody production. In addition, the finding of increased anti-B13 IgG levels in sera from CCC patients indicates that clinical presentation is associated with increased anti-B13 antibody levels

Recommendations from a Satellite Meeting (International Symposium to Commemorate the 90th Anniversary of the Discovery of Chagas Disease, April 11-16 1999, Rio de Janeiro, Brazil)

During this symposium the standardization of the nomenclature of Trypanosoma cruzi strains was discussed, in a parallel session, with a view to facilitating the use and understanding of a common nomenclature that would serve not only taxonomists but the general community of researchers working with T. cruzi. The diversity in the behavior and morphology of T. cruzi isolates was soon recognized after the discovery of Chagas disease. Since then a variety of biochemical and molecular techniques have revealed the great genetic diversity present in strains of this parasite. Different investigators have described this diversity by using various terms. Correlation between this diversity and the complex epidemiological and clinical manifestations of the disease has however been hindered by the lack of a common nomenclature. Recent studies have indicated a convergence among investigators regarding the clustering of strains of T. cruzi, into two principal groups. This consensus, together with the report of a meeting on the standardization of methods for T. cruzi classification held in Panama (unpublished document TDR/EPICHA-TCC/85.3 Geneva, World Health Organization, 1985), form the basis of the recommendations outlined in this document

Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences

Traditional molecular and biochemical methods, such as schizodeme analysis, karyotyping, DNA fingerprinting, and enzyme electrophoretic profiles, have shown a large variability among Trypanosoma cruzi isolates. In contrast to those results, polymerase chain reaction (PCR) amplification of sequences from the 24Sα ribosomal RNA gene and from the mini-exon gene nontranscribed spacer indicated a dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 86 T. cruzi field stocks (68 isolated from humans with defined presentations of Chagas' disease and 18 from triatomines) derived from four Brazilian geographic areas were typed by the PCR assay based on the DNA sequences of the mini-exon and 24Sα rRNA genes. These stocks were ordered into the two major T. cruzi lineages. Lineage I was associated mainly with human isolates and lineage 2 with the sylvatic cycle of the parasite