National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Pathumthani, Thailand.
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Pathumthani, Thailand.
Acta Trop. 2024 Oct;258:107360. doi: 10.1016/j.actatropica.2024.107360. Epub 2024 Aug 12.
A new superior bacteria complementation model was achieved for testing antifolate compounds and investigating antifolate resistance in the dihydrofolate reductase (DHFR) enzyme of the malaria parasite. Earlier models depended on the addition of trimethoprim (TMP) to chemically suppress the host Escherichia coli (Ec) DHFR function. However, incomplete suppression of EcDHFR and potential interference of antibiotics needed to maintain plasmids for complementary gene expression can complicate the interpretations. To overcome such limitations, the folA (F) and thyA (T) genes were genetically knocked out (Δ) in E. coli BL21(DE3). The resulting EcΔFΔT cells were thymidine auxotroph where thymidine supplementation or functional complementation with heterologous DHFR-thymidylate synthase (TS) is needed to restore the loss of gene functions. When tested against pyrimethamine (PYR) and its analogs designed to target Plasmodium falciparum (Pf) DHFR-TS, the 50 % inhibitory concentration values obtained from EcΔFΔT surrogates expressing wildtype (PfTM4) or double mutant (PfK1) DHFR-TS showed strong correlations to the results obtained from the standard in vitro P. falciparum growth inhibition assay. Interestingly, while TMP had little effect on the susceptibility to PYR and analogs in EcΔFΔT expressing PfDHFR-TS, it hypersensitized the chemically knockdown E. coli BL21(DE3) expressing PfTM4 DHFR-TS but desensitized the one carrying PfK1 DHFR-TS. The low intrinsic expression level of PfTM4 in E. coli BL21(DE3) by western blot analysis may explain the hypersensitive to antifolates of chemical knockdown bacteria surrogate. These results demonstrated the usefulness of EcΔFΔT surrogate as a new tool for antifolate antimalarial screening with potential application for investigation of antifolate resistance mechanism.
建立了一种新的用于测试抗叶酸化合物和研究疟原虫二氢叶酸还原酶（DHFR）酶中抗叶酸耐药性的新型超细菌互补模型。早期的模型依赖于添加三甲氧苄氨嘧啶（TMP）来化学抑制宿主大肠杆菌（Ec）DHFR 功能。然而，不完全抑制 EcDHFR 和维持互补基因表达所需的抗生素的潜在干扰可能会使解释复杂化。为了克服这些限制，将 folA（F）和 thyA（T）基因在大肠杆菌 BL21（DE3）中遗传敲除（Δ）。由此产生的 EcΔFΔT 细胞是胸苷营养缺陷型，需要胸苷补充或异源 DHFR-胸苷酸合酶（TS）的功能互补才能恢复基因功能的丧失。当用嘧啶并嘧啶（PYR）及其设计用于靶向恶性疟原虫 DHFR-TS 的类似物进行测试时，用野生型（PfTM4）或双突变体（PfK1）DHFR-TS 表达的 EcΔFΔT 替代物获得的 50%抑制浓度值与从标准体外恶性疟原虫生长抑制测定获得的结果显示出很强的相关性。有趣的是，虽然 TMP 对 EcΔFΔT 中表达 PfDHFR-TS 的 PYR 和类似物的敏感性影响不大，但它使化学敲低的 EcBL21（DE3）表达 PfTM4 DHFR-TS 的敏感性增加，但使携带 PfK1 DHFR-TS 的敏感性降低。Western blot 分析表明，PfTM4 在大肠杆菌 BL21（DE3）中的内在表达水平较低，这可能解释了化学敲低细菌替代物对抗叶酸药物敏感的原因。这些结果表明 EcΔFΔT 替代物作为一种新的抗叶酸抗疟筛选工具的有用性，具有研究抗叶酸耐药机制的潜在应用。
