Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi
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...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2022-10-01
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| Series: | PLoS Neglected Tropical Diseases |
| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9581433/?tool=EBI |
| _version_ | 1797990718245961728 |
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| author | Ana Maria Murta Santi Juliana Martins Ribeiro João Luís Reis-Cunha Gabriela de Assis Burle-Caldas Isabella Fernandes Martins Santos Paula Alves Silva Daniela de Melo Resende Daniella Castanheira Bartholomeu Santuza Maria Ribeiro Teixeira Silvane Maria Fonseca Murta |
| author_facet | Ana Maria Murta Santi Juliana Martins Ribeiro João Luís Reis-Cunha Gabriela de Assis Burle-Caldas Isabella Fernandes Martins Santos Paula Alves Silva Daniela de Melo Resende Daniella Castanheira Bartholomeu Santuza Maria Ribeiro Teixeira Silvane Maria Fonseca Murta |
| author_sort | Ana Maria Murta Santi |
| collection | DOAJ |
| description | 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. Author summary Prostaglandin F2α synthase (PGFS) has been associated with T. cruzi resistance to benznidazole (BZ), but the real involvement of this enzyme in the resistance phenotype is still uncertain since different studies in the literature point in different directions. Here we demonstrated that the deletion of all copies of the PGFS gene in T. cruzi does not affect the parasite’s resistance to BZ or nifurtimox (NFX), but results in reduced tolerance to oxidative stress caused by menadione. The PGFS knockout mutants are less infective, and, at the stationary phase, the parasites have fewer lipid bodies than the control parasites. Thus, our results suggest that this enzyme appears to have a regulatory role in defence against oxidative stress and parasite infectivity. |
| first_indexed | 2024-04-11T08:41:11Z |
| format | Article |
| id | doaj.art-8959fab4bb0846448762a5eaf6da1057 |
| institution | Directory Open Access Journal |
| issn | 1935-2727 1935-2735 |
| language | English |
| last_indexed | 2024-04-11T08:41:11Z |
| publishDate | 2022-10-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Neglected Tropical Diseases |
| spelling | doaj.art-8959fab4bb0846448762a5eaf6da10572022-12-22T04:34:12ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352022-10-011610Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruziAna Maria Murta SantiJuliana Martins RibeiroJoão Luís Reis-CunhaGabriela de Assis Burle-CaldasIsabella Fernandes Martins SantosPaula Alves SilvaDaniela de Melo ResendeDaniella Castanheira BartholomeuSantuza Maria Ribeiro TeixeiraSilvane Maria Fonseca MurtaChagas 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. Author summary Prostaglandin F2α synthase (PGFS) has been associated with T. cruzi resistance to benznidazole (BZ), but the real involvement of this enzyme in the resistance phenotype is still uncertain since different studies in the literature point in different directions. Here we demonstrated that the deletion of all copies of the PGFS gene in T. cruzi does not affect the parasite’s resistance to BZ or nifurtimox (NFX), but results in reduced tolerance to oxidative stress caused by menadione. The PGFS knockout mutants are less infective, and, at the stationary phase, the parasites have fewer lipid bodies than the control parasites. Thus, our results suggest that this enzyme appears to have a regulatory role in defence against oxidative stress and parasite infectivity.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9581433/?tool=EBI |
| spellingShingle | Ana Maria Murta Santi Juliana Martins Ribeiro João Luís Reis-Cunha Gabriela de Assis Burle-Caldas Isabella Fernandes Martins Santos Paula Alves Silva Daniela de Melo Resende Daniella Castanheira Bartholomeu Santuza Maria Ribeiro Teixeira Silvane Maria Fonseca Murta Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi PLoS Neglected Tropical Diseases |
| title | Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi |
| title_full | Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi |
| title_fullStr | Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi |
| title_full_unstemmed | Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi |
| title_short | Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi |
| title_sort | disruption of multiple copies of the prostaglandin f2alpha synthase gene affects oxidative stress response and infectivity in trypanosoma cruzi |
| url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9581433/?tool=EBI |
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