Estabilidade de microssatélite do DNA nuclear e mitocondrial de Trypanosoma cruzi

Abstract

The Trypanosoma cruzi (T. cruzi) is the etiologic agent of Chagas Disease. This sickness occurs predominantly in Latin America and is transmitted mainly by faeces of triatomines insects. About 10 million peoples are infected around the world, mostly in Central and South America, and more than 25 million are in risk of infection. The etiologic agent is a hemoflagellate of the Trypanosomatidae family, characterized by the presence of a flagellum, and only one mitochondria. The mitochondrial DNA is presented as a structure known as Kinetoplast. The DNA repair system is responsible for preserving the genomic stability, correcting lesions in DNA caused by ambiental and metabolic sources. In the meantime, some lesions remain, leading to mutation or disrupting the replication. Among several ways to repair the DNA, the Mismatch Repair System (MMR) is one between many pathways that the cell possesses to deal with chemical and physical genome damages. The main MMR protein is the MSH2-MSH6 heterodimer, whose functions and properties are still being clarified. Besides the MMR participation, is becoming clearer its involvement in the oxidative stress resistance, mainly against the 8oxoG lesion. The TcMSH2 and TcMSH6 genes are codified in the nucleus and they are responsible to repair the nuclear genetic content. To guarantee the cell survival, in the face of oxidative stress in physiologic and/or induced stressing situation during the evolutive life cycle in metabolically aggressive environment, the repair of the only one mitochondria has to be adequate, being so, its important to study the parasite efforts to protect its genetic content. This present work aimed to describe the microsatellite stability of the T. cruzi mitochondrial and nuclear DNA. The evaluation of the microsatellites stability where developed in CL Brener T. cruzi wild type strain, hemi and double knockout MSH2 gene and heminockout MSH6 gene, and also the Silvio wild type strain. The CL Brener microsatellite stability was assessed after 50 generations of the cell, and it was separated in 2 groups: control or treated with 50M of hydrogen peroxide. The Silvio microsatellite stability was assessed after 30 generations of the cell, separated in 2 groups just like CL Brener. Two approaches were applied: denaturating gel electrophoresis in ALF sequencer and the sequencing of a repetitive DNA previously identified. Besides that, also was realized the sequencing of a mitochondrial intergenic region of CL Brener to look for any mutation. The analysis showed that even without the MSH2 gene or with MSH2 or MSH6 hemiknockout in CL Brener cells, no mutations in the mitochondrial DNA were found, also in induced oxidative stress situation, suggesting that the mitochondria MMR pathway posses others proteins involved or another pathways are responsible to take care of this kind of lesion. Meanwhile in nucleus none of 9 microsatellite loci developed instability, however, the locus SCLE11 in the CL Brener hemi knockout strain showed a new microsatellite allele in the zero generation, suggesting that the instability occurred among the strain generation until the beginning of this work. The absence of instability in two wild type strains and the lack of the MSH2/MSH6 heterodimer in CL Brener suggest that there may be others proteins involved in the recognition or another repair pathway involved in the repair of this kind of lesion.