Centre de Recherche en Infectiologie du Centre de Recherche du CHU de Québec and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Quebec City, Québec, Canada.
PLoS Negl Trop Dis. 2023 Jun 29;17(6):e0011458. doi: 10.1371/journal.pntd.0011458. eCollection 2023 Jun.
Most of our understanding of folate metabolism in the parasite Leishmania is derived from studies of resistance to the antifolate methotrexate (MTX). A chemical mutagenesis screen of L. major Friedlin and selection for resistance to MTX led to twenty mutants with a 2- to 400-fold decrease in MTX susceptibility in comparison to wild-type cells. The genome sequence of the twenty mutants highlighted recurrent mutations (SNPs, gene deletion) in genes known to be involved in folate metabolism but also in novel genes. The most frequent events occurred at the level of the locus coding for the folate transporter FT1 and included gene deletion and gene conversion events, as well as single nucleotide changes. The role of some of these FT1 point mutations in MTX resistance was validated by gene editing. The gene DHFR-TS coding for the dihydrofolate reductase-thymidylate synthase was the second locus with the most mutations and gene editing confirmed a role in resistance for some of these. The pteridine reductase gene PTR1 was mutated in two mutants. The episomal overexpression of the mutated versions of this gene, but also of DHFR-TS, led to parasites several fold more resistant to MTX than those overexpressing the wild-type versions. Genes with no known link with folate metabolism and coding for a L-galactolactone oxidase or for a methyltransferase were mutated in specific mutants. Overexpression of the wild-type versions of these genes in the appropriate mutants reverted their resistance. Our Mut-seq approach provided a holistic view and a long list of candidate genes potentially involved in folate and antifolate metabolism in Leishmania.
我们对寄生虫利什曼原虫中叶酸代谢的大部分理解都来源于对叶酸类似物氨甲蝶呤（MTX）耐药性的研究。对 L. major Friedlin 进行化学诱变筛选并选择对 MTX 的耐药性，导致 20 个突变体与野生型细胞相比，MTX 敏感性降低了 2 到 400 倍。这 20 个突变体的基因组序列突出了反复出现的突变（SNP、基因缺失），这些突变发生在已知参与叶酸代谢的基因中，但也发生在新基因中。最常见的事件发生在编码叶酸转运蛋白 FT1 的基因座水平上，包括基因缺失和基因转换事件，以及单核苷酸变化。通过基因编辑验证了其中一些 FT1 点突变在 MTX 耐药性中的作用。编码二氢叶酸还原酶-胸苷酸合酶的基因 DHFR-TS 是第二个突变最多的基因座，基因编辑证实了其中一些基因在耐药性中的作用。在两个突变体中，蝶啶还原酶基因 PTR1 发生了突变。该基因的突变体的质体过量表达，以及 DHFR-TS 的突变体的质体过量表达，导致寄生虫对 MTX 的耐药性比野生型版本高几倍。在特定突变体中，与叶酸代谢没有已知联系、编码 L-半乳糖内酯氧化酶或甲基转移酶的基因发生了突变。在适当的突变体中过量表达这些基因的野生型版本可使它们恢复耐药性。我们的 Mut-seq 方法提供了一个整体的观点和一长串候选基因，这些基因可能参与利什曼原虫中的叶酸和抗叶酸代谢。
