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Glycosomal ABC transporters (GATs) play a vital role in maintaining metabolic homeostasis by facilitating metabolite exchange between glycosomes and the cytoplasm. GAT3 is a member of the GAT family, which also includes <jats:italic>GAT1<\/jats:italic> and <jats:italic>GAT2<\/jats:italic>. <jats:italic>GAT3<\/jats:italic> transcript levels are downregulated in benznidazole-resistant <jats:italic><jats:italic>T. cruzi<\/jats:italic><\/jats:italic> populations; however, its specific functions remain unknown. Therefore, in this study, we generated <jats:italic>GAT3<\/jats:italic> single-knockout and null mutant lines of the <jats:italic><jats:italic>T. cruzi<\/jats:italic><\/jats:italic> Dm28c strain using the CRISPR\/Cas9 system to investigate GAT3 roles in parasite biology. RT-qPCR revealed increased <jats:italic>GAT2<\/jats:italic> transcript levels in the <jats:italic>GAT3<\/jats:italic> null mutant line, without any changes in <jats:italic>GAT1<\/jats:italic> levels. Our findings suggest that <jats:italic>GAT3<\/jats:italic> is not essential for <jats:italic><jats:italic>T. cruzi<\/jats:italic><\/jats:italic> survival, as null mutant parasites showed no growth difference compared to the Cas9-expressing controls. Moreover, the <jats:italic>GAT3<\/jats:italic> single-knockout line exhibited increased resistance to benznidazole, whereas the null mutant line exhibited benznidazole susceptibility similar to the control. Furthermore, both <jats:italic>GAT3<\/jats:italic> single-knockout and null mutant lines showed increased tolerance to hydrogen peroxide-induced oxidative stress. <jats:italic><jats:italic>In vitro<\/jats:italic><\/jats:italic> infection assay of L929 murine fibroblasts revealed that the <jats:italic>GAT3<\/jats:italic> null parasites exhibited a significantly lower infection rate and fewer intracellular amastigotes than the controls. Overall, <jats:italic>GAT3<\/jats:italic> is crucial for <jats:italic><jats:italic>T. cruzi<\/jats:italic><\/jats:italic> infectivity and the regulation of oxidative stress responses, playing key roles in the metabolic regulation and pathogenicity of this parasite.<\/jats:p>","DOI":"10.1371\/journal.pntd.0013479","type":"journal-article","created":{"date-parts":[[2025,9,11]],"date-time":"2025-09-11T17:28:16Z","timestamp":1757611696000},"page":"e0013479","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Glycosomal ABC transporter 3 (GAT3) deletion enhances the oxidative stress responses and reduces the infectivity of Trypanosoma cruzi"],"prefix":"10.1371","volume":"19","author":[{"given":"Davi Alvarenga","family":"Lima","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"H\u00e9llida Marina","family":"Costa-Silva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karen Stephanie Sebe","family":"Albergaria","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juliana Martins","family":"Ribeiro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela de Melo","family":"Resende","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bruno Alves","family":"Santarossa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel Barbosa","family":"Liarte","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simone Guedes","family":"Calderano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8523-2155","authenticated-orcid":true,"given":"Silvane Maria Fonseca","family":"Murta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,9,11]]},"reference":[{"key":"pntd.0013479.ref001","unstructured":"WHO. 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