Lima Davi Alvarenga, Costa-Silva Héllida Marina, Albergaria Karen Stephanie Sebe, Ribeiro Juliana Martins, Resende Daniela de Melo, Santarossa Bruno Alves, Liarte Daniel Barbosa, Calderano Simone Guedes, Murta Silvane Maria Fonseca
Grupo Genômica Funcional de Parasitos, Instituto René Rachou, Fiocruz Minas, Belo Horizonte, Minas Gerais, Brazil.
Laboratório de Ciclo Celular, Instituto Butantan, São Paulo, Brazil.
PLoS Negl Trop Dis. 2025 Sep 11;19(9):e0013479. doi: 10.1371/journal.pntd.0013479. eCollection 2025 Sep.
Glycosomes, peroxisome-like organelles in Trypanosoma cruzi, contain enzymes involved in various metabolic processes, including glycolysis. 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 GAT1 and GAT2. GAT3 transcript levels are downregulated in benznidazole-resistant T. cruzi populations; however, its specific functions remain unknown. Therefore, in this study, we generated GAT3 single-knockout and null mutant lines of the T. cruzi Dm28c strain using the CRISPR/Cas9 system to investigate GAT3 roles in parasite biology. RT-qPCR revealed increased GAT2 transcript levels in the GAT3 null mutant line, without any changes in GAT1 levels. Our findings suggest that GAT3 is not essential for T. cruzi survival, as null mutant parasites showed no growth difference compared to the Cas9-expressing controls. Moreover, the GAT3 single-knockout line exhibited increased resistance to benznidazole, whereas the null mutant line exhibited benznidazole susceptibility similar to the control. Furthermore, both GAT3 single-knockout and null mutant lines showed increased tolerance to hydrogen peroxide-induced oxidative stress. In vitro infection assay of L929 murine fibroblasts revealed that the GAT3 null parasites exhibited a significantly lower infection rate and fewer intracellular amastigotes than the controls. Overall, GAT3 is crucial for T. cruzi infectivity and the regulation of oxidative stress responses, playing key roles in the metabolic regulation and pathogenicity of this parasite.
糖体是克氏锥虫中类似过氧化物酶体的细胞器，含有参与包括糖酵解在内的各种代谢过程的酶。糖体ABC转运蛋白（GATs）通过促进糖体与细胞质之间的代谢物交换，在维持代谢稳态中发挥着至关重要的作用。GAT3是GAT家族的成员，该家族还包括GAT1和GAT2。在对苯硝唑耐药的克氏锥虫群体中，GAT3转录水平下调；然而，其具体功能仍然未知。因此，在本研究中，我们使用CRISPR/Cas9系统构建了克氏锥虫Dm28c株的GAT3单敲除和无效突变株系，以研究GAT3在寄生虫生物学中的作用。RT-qPCR显示，GAT3无效突变株系中GAT2转录水平升高，而GAT1水平没有任何变化。我们的研究结果表明，GAT3对克氏锥虫的存活不是必需的，因为无效突变寄生虫与表达Cas9的对照相比没有生长差异。此外，GAT3单敲除株系对苯硝唑的抗性增加，而无效突变株系对苯硝唑的敏感性与对照相似。此外，GAT3单敲除和无效突变株系对过氧化氢诱导的氧化应激均表现出更高的耐受性。对L929小鼠成纤维细胞的体外感染试验表明，与对照相比，GAT3缺失的寄生虫感染率显著降低，细胞内无鞭毛体数量更少。总体而言，GAT3对克氏锥虫的感染性和氧化应激反应的调节至关重要，在该寄生虫的代谢调节和致病性中发挥关键作用。
