Molecular analysis of the hypervariable regions in the minicircle of kinetoplastic genome in Trypanosoma cruzi, agent of Chagas'disease
Résumé
Chagas'disease is a parasitosis caused by a flagellate protozoan : Trypanosoma cruzi, which remains one of the most important tropical disease in Latin America, which affect about 12 million people, and 100 million people remaining at risk. Trypanosoma cruzi, possess only one mitochondrion that extends throughout the cell body, called a kinetoplast, which contains a mass of DNA, it consist of a network of maxicercles and minicercles molecules associated with each other. Each minicircle are organized into one to four conserved regions in an equal number of variable regions. In the present work, we investigated the impacts of the sequences (variable region) in the clonal evolution and genetic recombination on the polymorphism kDNA, and the RNA guides coding- role of minicircles variable sequences among phylogeneticaly distant strains. Nineteen stocks representatives of the eco-epidemiological genetic diversity of T. cruzi (lineage I and lineages IIb, IId and IIe) were cultured and collected. After extraction of DNA the hypervariable regions was amplified by PCR and 330pb amplicons were cloned in pCR2.1 plasmids vector. The plasmid inserts were sequenced using Big Dye methods and ABI prism automatic sequencing equipment. We obtained 157 sequences of the hypervariable regions of T. cruzi, and 12 sequences of T. cruzi marinkellei. The comparison shown the unexpected great diversity however, the sequences of each lineages are unique, i.e. not found in other lineages. For the lineages I and IIb, the variability of the domain was so high that the same sequence was repeated only few times. In the contrast, the lineage IId and IIe exhibited a significant homology groups, with the sequences are conserved for each lineage. The high variability of obtained sequences did not allowed an evolution analyses. We also investigate the potential sequences of RNA guides in hypervariable domain of kDNA. We found of several putative gRNA sequences that T. cruzi and the probably edited genes. Our data suggest that the phylogenetic diversity of T. cruzi obtained of the sequences for the variable region is not correlated to nuclear diversity data. The other hand, the interested for gRNA identified is the knowledge the editing mechanism and could be used as a new arm in the fight against the parasite.