Study of important components of the DNA homologous recombination repair in Toxoplasma gondii: RAD51 and BRCA2

CRISTALDI, CONSTANZA; SALDARRIAGA CARTAGENA, ANA M.; VANAGAS L; ANGEL SO

Congreso; Host Pathogen Interaction Meeting 21; 2021

INTRODUCTION: Toxoplasma gondii is an obligate intracellular parasite, is responsible of toxoplasmosis infection. Due to the toxicity of the currents treatments against toxoplasmosis, there is an intensive search for new drugs against the parasite that may be innocuous for the host cell. There are conserved components of the homologous recombination DNA repair (HRR) pathway in T.gondii that could present unique characteristics which make them attractive therapeutic targets. Among them, a putative BRCA2 was identified in silico, this protein interacts with the recombinase RAD51, generating an essential complex for the HRR. Genotoxic drugs like camptothecin (CPT) or analogues with less cell toxicity, have been used before to generate DNA damage, inhibiting T.gondii proliferation, which make them potential candidates as anti-T. gondii therapy. OBJECTIVES: To induce DNA damage in T. gondii through genotoxic drugs and affect proteins involved in the HRR pathway, such as BRCA2 and RAD51, leading to unresolved DNA damage, thus eliminating the parasite with little harm to the host cell. MATERIALS AND METHODS: For our study, BRCA2 and RAD51 genes were cloned and expressed in bacteria to obtain recombinant proteins used to produce specific mouse and rabbit polyclonal antibodies, which were then used to detect their presence by western blot and their cellular localization by indirect immunofluorescence (IFA). We used the analogues of CPT: topotecan and 10-hydroxycamptothecin, in intracellular parasites. Both drugs were assayed first in parasites that express the red fluorescent protein and toxicity, in host cells was also evaluated. RESULTS AND CONCLUSIONS: The antibodies against BRCA2 and RAD51 would allow us to make progress in the understanding of the complex BRCA2-RAD51 in the HRR pathway, which is fundamental to study the DNA repair in presence and absence of genotoxic drugs.