Chromosomal Resistance to Metronidazole in Clostridioides difficile Can Be Mediated by Epistasis between Iron Homeostasis and Oxidoreductases
Chromosomal Resistance to Metronidazole in Clostridioides difficile Can Be Mediated by Epistasis between Iron Homeostasis and Oxidoreductases
复制标题
DOI：
10.1128/aac.00415-20
复制
发表时间：
2020-08-01
影响因子：
4.9
通讯作者：
Hurdle, Julian G.
中科院分区：
文献类型：
--
作者：
Deshpande, Aditi;Wu, Xiaoqian;Hurdle, Julian G.
临床分离的艰难梭菌对甲硝唑的染色体耐药已出现，但其遗传机制尚不清楚。这进一步受到无法在实验室中产生自发的甲硝唑耐药突变体以解释临床分离株中的遗传变异的阻碍。因此，我们在非致突变ATCC 700057中构建了错配修复突变子，以调查从头耐药机制的突变景观。在单独的实验进化中，增变子采用了确定性的抗性路径，截断亚铁转运蛋白FeoB 1作为低水平抗性的第一步机制。ATCC 700057中feoB 1的缺失降低了细胞内铁含量，似乎使细胞转向黄素氧还蛋白介导的氧化还原酶反应，这对甲硝唑的细胞作用不利。更高水平的耐药性是从连续获得突变到铁氧还蛋白/黄素氧还蛋白氧化还原酶（PFOR;由nifJ编码）的催化结构域，同义密码子改变为推定的xdh（黄嘌呤脱氢酶;由CD 630 31770编码），可能影响mRNA的稳定性，最后是失活铁硫簇调节因子（IscR）的移码和点突变。用催化死亡的Cas9使nifJ、xdh或iscR基因沉默揭示了涉及这些基因的抗性仅在feoBl失活时发生;即，仅在feoB 1缺失突变体中观察到抗性，而在同基因野生型（WT）亲本中未观察到抗性。有趣的是，C.在基因互补后，携带nifJ突变的艰难梭菌感染（CDI）相关菌株减少。这一观察结果支持了PFOR突变是临床菌株甲硝唑耐药机制之一的观点。我们的研究结果表明，甲硝唑耐药的C。艰难梭菌是复杂的，涉及多遗传机制，可能与铁依赖性和氧化还原代谢途径交叉。
Chromosomal resistance to metronidazole has emerged in clinical Clostridioides difficile isolates, but the genetic mechanisms remain unclear. This is further hindered by the inability to generate spontaneous metronidazole-resistant mutants in the lab to interpret genetic variations in clinical isolates. We therefore constructed a mismatch repair mutator in nontoxigenic ATCC 700057 to survey the mutational landscape for de novo resistance mechanisms. In separate experimental evolutions, the mutator adopted a deterministic path to resistance, with truncation of the ferrous iron transporter FeoB1 as a first-step mechanism of low-level resistance. Deletion of feoB1 in ATCC 700057 reduced the intracellular iron content, appearing to shift cells toward flavodoxin-mediated oxidoreductase reactions, which are less favorable for metronidazole's cellular action. Higher-level resistance evolved from sequential acquisition of mutations to catalytic domains of pyruvate-ferredoxin/flavodoxin oxidoreductase (PFOR; encoded by nifJ), a synonymous codon change to putative xdh (xanthine dehydrogenase; encoded by CD630 31770), likely affecting mRNA stability, and last, frameshift and point mutations that inactivated the iron-sulfur cluster regulator (IscR). Gene silencing of nifJ, xdh, or iscR with catalytically dead Cas9 revealed that resistance involving these genes occurred only when feoB1 was inactivated; i.e., resistance was seen only in the feoB1 deletion mutant and not in the isogenic wild-type (WT) parent. Interestingly, metronidazole resistance in C. difficile infection (CDI)-associated strains carrying mutations in nifJ was reduced upon gene complementation. This observation supports the idea that mutation in PFOR is one mechanism of metronidazole resistance in clinical strains. Our findings indicate that metronidazole resistance in C. difficile is complex, involving multigenetic mechanisms that could intersect with iron-dependent and oxidoreductive metabolic pathways.