RESUMEN
The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.
Asunto(s)
Daño del ADN , Guanina/análogos & derivados , Estrés Oxidativo , Trypanosoma cruzi/fisiología , Animales , Supervivencia Celular , Células Cultivadas , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/patología , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Femenino , Fibroblastos/parasitología , Expresión Génica , Guanina/metabolismo , Peróxido de Hidrógeno/toxicidad , Macrófagos/parasitología , Ratones , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Parasitemia/parasitología , Parasitemia/patología , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
During acute infection with the parasite Trypanosoma cruzi, the causal agent of Chagas' disease, tissue damage is related to intense tissue parasitism. Here we discuss histological approaches for an optimal visualization and quantification of T. cruzi nests in the heart, the main target organ of the parasite. These analyses are important to evaluate the course of the infection in different experimental models and also can be used to investigate parasite colonization and inflammatory processes in other infected tissues and biopsies.