The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi

Ceres Luciana Alves,Erich Birelli Tahara,Sérgio Danilo Junho Pena,Maria Carolina Elias,Carlos Renato Machado,Selma Da Silva Santos,Luciana De Oliveira Andrade,Isabela Cecília Mendes,Paula Andrea Marin,Glória Regina Franco,Marcelo Santos Da Silva,Bruno Marçal Repolês,Andréa Mara Macedo,Pedro Henrique Nascimento Aguiar,Alessandra Aparecida Guarneri

doi:10.1038/s41598-018-31541-z

Ceres Luciana Alves, Erich Birelli Tahara + Show 13 more

Open Access

https://doi.org/10.1038/s41598-018-31541-z

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Abstract

Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 – a recombinase involved in homologous recombination – in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism.

Highlights

Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity
In order to verify whether the elapsed time taken to resume T. cruzi logarithmic growth upon exposure to ionizing radiation differed between naturally-occurring non-hybrid strains and CL Brener – this may indicate the existence of intra-specific variations with regard to the efficiency of DNA repair processes in this parasite, we treated epimastigotes from naturally-occurring non-hybrid strains – namely Sylvio (TcI) and Esmeraldo (TcII) – in parallel with epimastigotes from CL Brener strain with the dose of 500 Gy of ionizing radiation
In order to experimentally check, under laboratory conditions, the possibility of assessing the rates of genetic exchange in T. cruzi cells, we developed an experimental assay based on cellular incorporation of two distinguishable halogenated thymidine analogues, namely 5′-chloro-2′-deoxyuridine (CldU) and 5′-iodo-2′-deoxyuridine (IdU), whose presence in the genetic material can be detected by immunostaining through the use of specific antibodies that generate distinct signals – red for CldU, and green for IdU – in labeled cells

Summary

Introduction

Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Back in 2003, Gaunt et al were able to identify events of genetic exchange by detecting the presence of T. cruzi fused-cell hybrids isolated from the mammalian host carrying two different drug-resistance markers (neomycin and hygromycin B), each one coming from distinct populations of T. cruzi I12, suggesting that genetic exchange could take place in specific life cycle phases[12]. It is not clear yet if the mechanisms of such genetic exchange in www.nature.com/scientificreports/. T. cruzi are similar to those observed in other parasites such as Leishmania and T. brucei, which present sexual recombination driven by meiosis[15,16]

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