Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis.
Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis.
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DOI：
10.1371/journal.ppat.1009063
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发表时间：
2020-11
期刊：
影响因子：
6.7
通讯作者：
Fortune SM
中科院分区：
文献类型：
--
作者：
Hicks ND;Giffen SR;Culviner PH;Chao MC;Dulberger CL;Liu Q;Stanley S;Brown J;Sixsmith J;Wolf ID;Fortune SM
细菌病原体中抗生素抗性的基因组解剖主要集中在赋予超过单一临界药物浓度的生长的遗传变化上。然而，降低对抗生素的敏感性--甚至低于这个临界点--与临床治疗效果不佳有关，包括结核病。结核分枝杆菌的临床菌株在抗生素敏感性方面表现出广泛的数量变化，但这种药物敏感性谱背后的遗传基础仍然不明确。通过全基因组关联研究，我们发现，非同义突变的dnaA，它编码一个重要的和高度保守的DNA复制调节，与临床耐药M。结核菌株我们证明，这些dnaA突变特异性增强M。结核病生存在异烟肼治疗期间通过减少katG的表达，异烟肼的激活剂。为了确定与这种表型相关的DnaA相互作用，我们进行了分枝杆菌中DnaA结合位点的第一个全基因组生物化学作图，揭示了DnaA相互作用位点是临床菌株中复发突变的靶点。在这个DnaA相互作用位点重建临床流行的突变再现了dnaA突变体的表型，这表明M.结核病已经在以前未表征的DnaA途径中进化出突变，该途径定量地增加了对关键的一线抗生素异烟肼的耐药性。发现降低药物敏感性和支持高水平耐药性演变的遗传机制将指导生物标志物的开发，这些生物标志物能够前瞻性地识别临床治疗失败风险的患者。结核病是用针对细菌病原体结核分枝杆菌（Mtb）的抗生素组合治疗的。为了应对抗生素的广泛使用，结核分枝杆菌已经进化出耐药性突变，增加了抑制其生长所需的抗生素数量，并破坏了有效的治疗。导致高水平耐药性的细菌突变已在很大程度上被确定，从而可以开发快速诊断方法。最近的研究表明，中等水平的耐药也会影响患者的预后，然而，我们还不知道可能导致中等耐药的突变范围。在这里，我们利用全基因组关联研究的方法，并确定在细菌DNA复制起始因子dnaA的突变与临床分离株的耐药性。通过产生精确的dnaA突变株，我们确定这些突变赋予一线药物异烟肼的中间水平的耐药性。我们还发现，在基因组中与dnaA物理结合的第二个位点的突变可以对异烟肼产生相同的作用，可能通过相同的途径起作用。这项研究提供了对以前未识别的临床流行变异的深入了解，这些变异可能有助于解释患者结局并指导治疗，以减少治疗失败和随后的高水平耐药演变。
Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced susceptibility to antibiotics—even below this breakpoint—is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug susceptibility remains ill-defined. Through a genome wide association study, we show that non-synonymous mutations in dnaA, which encodes an essential and highly conserved regulator of DNA replication, are associated with drug resistance in clinical M. tuberculosis strains. We demonstrate that these dnaA mutations specifically enhance M. tuberculosis survival during isoniazid treatment via reduced expression of katG, the activator of isoniazid. To identify DnaA interactors relevant to this phenotype, we perform the first genome-wide biochemical mapping of DnaA binding sites in mycobacteria which reveals a DnaA interaction site that is the target of recurrent mutation in clinical strains. Reconstructing clinically prevalent mutations in this DnaA interaction site reproduces the phenotypes of dnaA mutants, suggesting that clinical strains of M. tuberculosis have evolved mutations in a previously uncharacterized DnaA pathway that quantitatively increases resistance to the key first-line antibiotic isoniazid. Discovering genetic mechanisms that reduce drug susceptibility and support the evolution of high-level drug resistance will guide development of biomarkers capable of prospectively identifying patients at risk of treatment failure in the clinic. Tuberculosis disease is treated with a combination of antibiotics targeting the bacterial pathogen Mycobacterium tuberculosis (Mtb). In response to widespread use of antibiotics, Mtb has evolved resistance mutations that increase the amount of antibiotic required to inhibit its growth and undermine effective treatment. The bacterial mutations that cause high-level drug resistance have largely been identified allowing for the development of rapid diagnostics. Recent studies have shown that intermediate levels of resistance can also affect patient outcomes, however, we do not yet know the range of mutations that can cause intermediate resistance. Here we utilize a genome-wide association study approach and identify that mutations in the bacterial DNA replication initiation factor dnaA are associated with drug resistance in clinical isolates. By generating precision dnaA mutant strains we identify that these mutations confer intermediate levels of resistance to the first-line drug isoniazid. We also find that mutations at a second site in the genome physically bound by dnaA can confer the same effect on isoniazid, likely acting through the same pathway. This study provides insight into previously unidentified clinically prevalent variants that may help explain patient outcome and guide therapy to reduce treatment failure and the subsequent evolution of high-level resistance.