A new Trypanosoma cruzi genotyping method enables high resolution evolutionary analyses.

BACKGROUND: Trypanosoma cruzi is an important human pathogen in Latin America with nearly seven million people infected. It has a large degree of genetic diversity, classified into six discrete typing units (DTUs), which probably influences its physiological behavior and clinical manifestations. Several genotyping methods are available, with distinct performance on easiness, cost, resolution and applicability; no method excels in all parameters. OBJECTIVES AND METHODS: To devise a molecular method for T. cruzi genotyping, based on polymerase chain reaction (PCR) amplification of a single target with multiple copies in the nuclear genome by large scale sequencing. We have applied this method to 29 T. cruzi isolates, comprising all described DTUs. FINDINGS: We were able to classify all samples into sub DTU level with high robustness. Evolutionary relationship between DTUs were ascertained, suggesting that TcIII and TcIV DTUs are non-hybrid, and DTU IV is more similar to the common ancestral. CONCLUSION: As the TS-LSS method is based on a single PCR reaction, comprising several copies of the target, it is probably useful for clinical samples, when the amount of DNA is a limiting factor. As large scale sequencing systems become more common, the TS-LSS method can be increasingly applied for T. cruzi genotyping.

Decentralising scientific publishing: can the blockchain improve science communication?

We present a decentralised solution for managing scientific communication, based on distributed ledger technologies, also called blockchains. The proposed system aims to solve incentive problems displayed by traditional systems in scientific communication and publication. A minimal working model is presented, defining roles, processes, and expected results from the novel system. The proposed solution is viable, given the current status of blockchain technology, and should lead to a rethinking of current practices and their consequences for scientific communication.

Minimum free energy predicted base pairing in the 39 nt spliced leader and 5' UTR of calmodulin mRNA from Trypanosoma cruzi: influence of the multiple trans-splicing sites.

We analyzed the compositional changes and the stable base pairs in the predicted secondary structure of the 5' UTR calmodulin mRNA in T. cruzi. The three copies of calmodulin in T. cruzi genome display variable position of the trans splicing sites and give rise to several mRNA that differs slightly on 5' UTR composition in the epimastigote stage. We show that the pattern of high probability base pairs in the minimum free energy predicted secondary structures of the calmodulin 5' UTR remains unchanged despite the nucleotide composition variation. However, the 39 nt spliced leader (mini-exon, the 5' exon sequence transferred to trypanosome mRNAs by the mechanism of trans splicing) shows a variable pattern of high and low probability base pairing as consequence of the altered composition of the 5' UTR.

A novel ABCG-like transporter of Trypanosoma cruzi is involved in natural resistance to benznidazole.

Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzi strains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison of TcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1 transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1 transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1 genes of BZ-susceptible and resistant strains were investigated by computational tools.

Cell disruption using a different methodology for proteomics analysis of Trypanosoma cruzi strains.

We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30min of incubation in lysis buffer at 4°C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS(E) approach. ProteinLynx Global Server (version 2.5) with Expression(E) analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract.

Transcription of long hypothetical orfs in Trypanosoma cruzi: the epimastigote stage uses trans-splicing sites that generate short 5' UTRs.

We mapped the 5' UTR for five long hypothetical orfs from Trypanosoma cruzi; each one having a length of more than 10,000 bp. Our aim was to verify the constraints to the length of the 5' UTR and to identify the sites of alternative trans-splicing in the epimastigote stage of three T. cruzi strains. We used reverse transcription PCR to amplify the 5' UTR and demonstrated the transcription of all selected genes as well as additional trans-splicing sites in two of these genes. We observed that the length of the 5' UTR in these genes has a limit, in contrast to previous reports that indicated a trend for longer genes to display a proportionally long 5' UTR. The maximum length of the 5' UTR for the long genes analyzed in the present work is approximately 3% of the orf and, on average, is 1% of the orf length. The poly-pyrimidine tracts used as trans-splicing signal are in the range of 17-53 bases within a distance of 6-59 nt to first spliced-leader acceptor site. T. cruzi populations may use both signals differentially. We conclude that the limit for the 5' UTR length in long genes is determined primarily by the distance to neighboring genes.

The composition of upstream open reading frames (uORF) in four genes from Trypanosoma cruzi typical strains.

Upstream open reading frames (uORF) are small open reading frames located in the 5' untranslated region (5' utr) of a mature mRNA. We analysed in four strains representing the Trypanosoma cruzi groups Tc I, Tc II, Tc IV and Tc VI the uORF present in 5' utr sequences of four genes: P-type H+-ATPase 1, DEAD/H RNA helicase, casein kinase 1.1 and ferredoxin-NADP+ reductase. A segment in the 5' utr at each of these genes encompassing one or more uORF was PCR amplified and sequenced. An analysis of these sequences reveals that the uORF in T. cruzi show minor variations; however, these nucleotide substitutions mirror the divergence of T. cruzi strains into major groups.

Molecular characterization of Trypanosoma cruzi samples derived from Triatoma vitticeps and Panstrongylus geniculatus of the Atlantic rainforest, southeast Brazil.

BACKGROUND: In rural areas of Espírito Santo state, southeast Brazil, triatomine species attracted by light frequently invade residences. The aim of this study was to investigate the Trypanosoma cruzi discrete typing units (DTUs) harbored by these triatomines. METHODS: Triatomine's intestinal contents were examined, inoculated in mice, and the positive samples were cultivated. Flagellates obtained from infected mice hemoculture were submitted to DNA extraction using a salting-out method and to TcSC5D gene amplification. The amplified samples were sequenced, and polymorphism was analyzed for DTU identification. RESULTS: Three hundred and ninety-four triatomines were identified: Triatoma vitticeps (90.03%), Panstrongylus geniculatus (8.89%), Panstrongylus megistus (0.54%), Panstrongylus diasi (0.27%), and Triatoma tibiamaculata (0.27%). Among the specimens, 251/394 (67.65%) presented flagellated forms similar to T. cruzi. After triatomine intestinal content inoculation into mice, 134 mice presented T. cruzi-like trypomastigotes from Tr. vitticeps and P. geniculatus and 89 samples were positive in hemoculture. Sixty-two samples were analyzed for the TcSC5D gene and TcI, TcII, TcIII, and TcIV DTUs were identified. CONCLUSIONS: We observed T. cruzi DTU diversity in Tr. vitticeps and P. geniculatus, which showed the predominance of TcII and occurrence of TcI, TcIII and TcIV. Triatomines presented high T. cruzi infection rates. Since little is known regarding the possible mammalian hosts that maintain the T. cruzi cycle, further studies are necessary to obtain a better understanding of the parasite transmission cycle in this region.

Detection of mixed infections with Mycobacterium lentiflavum and Mycobacterium avium by molecular genotyping methods.

Three mycobacterial isolates, one from the blood of an HIV-infected patient and two consecutive isolates from a woman with unknown HIV status, had been identified as belonging to the Mycobacterium avium complex by conventional procedures. In both patients, using genetic analysis procedures such as PCR-restriction enzyme analysis (PRA) of the hsp65 gene, a commercially available reverse hybridization-based assay (INNO-LiPA mycobacteria) and/or sequencing analysis of the 16S-23S internal transcribed spacer (ITS), the presence of Mycobacterium lentiflavum was also demonstrated. At the time of detection, both cases were also infected with M. avium, suggesting an underestimation of infection with M. lentiflavum and co-infection with different Mycobacterium species.

Calmodulin Polymerase Chain Reaction-Restriction Fragment Length Polymorphism for Leishmania Identification and Typing.

A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy.

Minicircle organization and diversity in Trypanosoma cruzi populations.

Trypanosoma cruzi strains and isolates can be divided into at least two groups using biochemical and molecular markers such as isoenzymes, ribosomal DNA, mini-exon gene spacers and some maxicircle genes. Despite the accumulating evidence that these major groups are phylogenetically distinct, their kinetoplast minicircle overall organization (i.e. number of conserved regions per length of minicircle molecule) remains conserved in all T. cruzi isolates studied so far, including the two T. cruzi major lineages -T. cruzi I and T. cruzi II - and a third group of uncertain taxonomic status, T. cruzi ZIII. Thus far, despite the extensive intra- and inter-minicircle sequence polymorphism, no group clustering has been observed.

A refined molecular karyotype for the reference strain of the Trypanosoma cruzi genome project (clone CL Brener) by assignment of chromosome markers.

We present a useful refinement of the molecular karyotype of clone CL Brener, the reference clone of the Trypanosoma cruzi Genome Project. The assignment of 210 genetic markers (142 expressed sequence tags (ESTs), seven cDNAs, 32 protein-coding genes, eight sequence tagged sites (STSs), 21 repetitive sequences) to the chromosomal bands separated by pulsed field gel electrophoresis (PFGE) identified 61 chromosome-specific markers, two size-polymorphic chromosomes and seven linkage groups. Fourteen new repetitive elements were isolated in this work and mapped to the chromosomal bands. We found that at least ten repetitive elements can be mapped to each chromosomal band, which may render the whole genome sequence assembly a difficult task. To construct the integrated map of chromosomal band XX, we used yeast artificial chromosome (YAC) overlapping clones and a variety of probes (i.e. known gene sequences, ESTs, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 1.3 Mb, covering 37% of the entire chromosome. We found some degree of polymorphism among YACs derived from band XX. These results are in agreement with data from phylogenetic analysis of T. cruzi which suggest that clone CL Brener is a hybrid genotype [Mol. Biochem. Parasitol. 92 (1998) 253; Proc. Natl. Acad. Sci. USA 98 (2001) 7396]. The physical map of the chromosomal bands, together with the isolation of specific chromosomal markers, will contribute in the global effort to sequence the nuclear genome of this parasite.

Lack of technical specificity in the molecular diagnosis of toxoplasmosis.

The polymerase chain reaction amplification of a fragment of the B1 gene of Toxoplasma gondii coupled to hybridization was performed in 42 patients from Rio de Janeiro, Brazil. The results showed 50% of positivity in the IgM positive toxoplasmosis group, and 12.5% in the positive IgG and negative IgM individuals. The data presented here revealed a lack of specificity of the molecular approach, clearly indicating that the primers used may co-amplify human sequences.