Análise de híbridos de diferentes DTUs de Trypanosoma cruzi

Abstract

Trypanosoma cruzi causes Chagas disease, which affects about 8 million people worldwide. As a species, this parasite presents a high genetic diversity, with hybrid and non-hybrid strains. Currently, this parasite is classified into six discrete typing units (DTU): TcI through TcVI. In the literature, there are reports of genetic exchange and hybridization events between different strains, which can impact the diversification and evolution of this organism, and influence characteristics such as its pathogenicity and transmission. Despite its importance, the mechanisms underlying the hybridization phenomenon has not been fully characterized. Previous studies have shown that genes involved in the recombination process, such as RAD51, are more expressed in hybrid strains. Additionally, genes involved in meiosis, such as DMC1 and HAP2, are present in the T. cruzi genome. In this work, we evaluated the formation of hybrids between epimastigotes of different DTUs. Our results revealed that different DTUs have the ability to hybridize. By using FACS, we were able to select possible hybrid clones of the DTUs TcI and TcVI. These clones were genotyped and tested for the ability to grow in culture and to resist gamma radiation exposure. These experiments demonstrated that the selected clones have distinct characteristics from each parental strain. It was also tested whether different culturing conditions (autoclaved versus filtered LIT medium) or the induction of double DNA breakages could increase the frequency of the hybridization events. Among the tested conditions, it was observed that a higher number of hybrid parasites were formed in cultures maintained in filtered medium. This may be related to the higher nutrition quality retained by this sterilizing method. The same FACS protocol was performed to track the hybridization between parasites resistant to two different antibiotics. Hybrid clones could be isolated and selected in the presence of both antibiotics. Once again, parasites were genotyped. However, the PCR results revealed that only one of the antibiotic markers were present. All these results suggest that there is a sexual reproduction pathway in T. cruzi but this event is rare and hard to document.