Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi

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Abstract
Chagas disease, caused by the protozoan Trypanosoma cruzi, is a serious chronic parasitic disease, currently treated with Nifurtimox (NFX) and Benznidazole (BZ). In addition to high toxicity, these drugs have low healing efficacy, especially in the chronic phase of the disease. The existence of drug-resistant T. cruzi strains and the occurrence of cross-resistance between BZ and NFX have also been described. In this context, it is urgent to study the metabolism of these drugs in T. cruzi, to better understand the mechanisms of resistance. Prostaglandin F2α synthase (PGFS) is an enzyme that has been correlated with parasite resistance to BZ, but the mechanism by which resistance occurs is still unclear. Our results show that the genome of the CL Brener clone of T. cruzi, contains five PGFS sequences and three potential pseudogenes. Using CRISPR/Cas9 we generated knockout cell lines in which all PGFS sequences were disrupted, as shown by PCR and western blotting analyses. The PGFS deletion did not alter the growth of the parasites or their susceptibility to BZ and NFX when compared to wild-type (WT) parasites. Interestingly, NTR-1 transcripts were shown to be upregulated in ΔPGFS mutants. Furthermore, the ΔPGFS parasites were 1.6 to 1.7-fold less tolerant to oxidative stress generated by menadione, presented lower levels of lipid bodies than the control parasites during the stationary phase, and were less infective than control parasites.
Funding Information
  • Fundação Oswaldo Cruz (VPPCB-007-FIO-18-2-94)
  • Convênio Fiocruz-Institut Pasteur-USP
  • Fundação de Amparo à Pesquisa do Estado de Minas Gerais (APQ 02816-21)
  • Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Convênio UGA/FAPEMIG (APQ-04382-16 (D)))
  • Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 304158/2019-4)
  • Fundação Oswaldo Cruz (Convênio Fiocruz-Institut Pasteur-USP)
  • Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Finance Code 001)

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