MSH2 e o reparo de erros de pareamento em tripanossomatídeos: análises in vitro e in vivo e envolvimento na resposta a estresse oxidativo

Abstract

Recent studies carried out by our group have demonstrated that the species T. cruzi can be divided into three distinct haplogroups, named A, B and C. This division was based upon extensive analyses carried out in several T.cruzi strains of microssatellites and polymorphisms found among various genes, including the MSH2 gene, which encodes a key component of the mismatch repair pathway (MMR). Treatment of parasite cultures with genotoxic agents also suggested that strains belonging to haplogroups B and C present a less efficient MMR when compared to the haplogroup A strains. These results led us to propose that the lower MMR efficiency found in haplogroups B and C could imply in a greater genetic variability in these strains, as indicated by the studies of multigene families present in the T. cruzi genome. It should be noted that strains belonging to haplogroup C are more frequently associated with chronic cases of Chagas disease in Brazil. Thus, it is possible that this higher genetic variability has contributed to a greater adaptation of these strains to the domestic cycle of Chagas disease. Since each haplogroup is characterized by a distinct MSH2 isoform, we decided to further characterize the T. cruzi MSH2 and investigate its role in generating the differences in MMR found between the haplogroups. The MSH2 gene was amplified from the genome of two T. cruzi strains, Colombiana (haplogroup A) and CL Brener (a hybrid strain that contains MSH2 alleles belonging to haplogroups B and C) and their whole coding region was sequenced. We have found 29 aminoacid substitutions between the MSH2 from these strains, some of them in regions described as important for the proteins structural or functional maintenance. In silico analysis indicated that the differences found between the MSH2 of Colombiana and CL Brener do not lead to alterations in protein structure. ATPase assays performed with recombinant proteins purified from E. coli indicated that Colombianas MSH2 presents a higher activity in vitro compared to CL Breners MSH2, suggesting that the SNPs in these strains MSH2 could account for the differences in the response to treatment with genotoxic agents. To compare the activity of CL Brener and Colombianas MSH2 in vivo, a second approach involving the expression of these proteins in Trypanosoma brucei MSH2 knockout cells was attempted. However, this apporach was not successful because microsatellite instability and MNNG resistance assays indicated that the complementation of the MMR pathway in T. brucei through the expression of T. cruzi MSH2 is not possible. On the other hand, this model allowed us to evaluate the role of MSH2 in the response to oxidative stress in these parasites. Similar to T. cruzi MSH2 -/+ single knockout cells, MSH2 -/- T. brucei cells are more sensitive to treatment with hydrogen peroxide than wild type cells. MLH1, the MSH2 counterpart in MMR, is not involved in this process, as shown by the analyses with T. brucei MLH1-/- knockout cells. Since MSH2 from T. cruzi was unable to complement microsatellite instability and MNNG resistance in T. brucei MSH2 -/- cells, and MLH1 is not involved in the response to hydrogen peroxide treatment, we propose that, in trypanosomatids, MSH2 has an additional role in the response to oxidative stress, which is independent from MMR.