Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
Evolugate, Gainesville, FL, USA.
Microb Genom. 2021 May;7(5). doi: 10.1099/mgen.0.000553.
Understanding the dynamics and mechanisms of acquired drug resistance across major classes of antibiotics and bacterial pathogens is of critical importance for the optimization of current anti-infective therapies and the development of novel ones. To systematically address this challenge, we developed a workflow combining experimental evolution in a morbidostat continuous culturing device with deep genomic sequencing of population samples collected in time series. This approach was applied to the experimental evolution of six populations of BW25113 towards acquiring resistance to triclosan (TCS), an antibacterial agent in various consumer products. This study revealed the rapid emergence and expansion (up to 100% in each culture within 4 days) of missense mutations in the gene, encoding enoyl-acyl carrier protein reductase, the known TCS molecular target. A follow-up analysis of isolated clones showed that distinct amino acid substitutions increased the drug IC in a 3-16-fold range, reflecting their proximity to the TCS-binding site. In contrast to other antibiotics, efflux-upregulating mutations occurred only rarely and with low abundance. Mutations in several other genes were detected at an earlier stage of evolution. Most notably, three distinct amino acid substitutions were mapped in the C-terminal periplasmic domain of CadC protein, an acid stress-responsive transcriptional regulator. While these mutations do not confer robust TCS resistance, they appear to play a certain, yet unknown, role in adaptation to relatively low drug pressure. Overall, the observed evolutionary trajectories suggest that the FabI enzyme is the sole target of TCS (at least up to the ~50 µm level), and amino acid substitutions in the TCS-binding site represent the main mechanism of robust TCS resistance in . This model study illustrates the potential utility of the established morbidostat-based approach for uncovering resistance mechanisms and target identification for novel drug candidates with yet unknown mechanisms of action.
了解主要类别的抗生素和细菌病原体获得性耐药的动态和机制对于优化当前抗感染治疗和开发新的治疗方法至关重要。为了系统地解决这一挑战，我们开发了一种结合病态生物连续培养装置中的实验进化和种群样本的深度基因组测序的工作流程，该方法应用于 6 个 BW25113 种群向三氯生（TCS）的耐药性实验进化，TCS 是各种消费品中的一种抗菌剂。这项研究揭示了基因中的错义突变的快速出现和扩张（在每个培养物中，在 4 天内高达 100%），该基因编码烯酰基辅酶 A 还原酶，这是 TCS 的已知分子靶标。对分离克隆的后续分析表明，不同的氨基酸取代将药物 IC 提高了 3-16 倍，反映了它们与 TCS 结合位点的接近程度。与其他抗生素不同，外排上调突变很少发生，丰度也很低。在进化的早期阶段还检测到其他几个基因的突变。值得注意的是，CadC 蛋白的 C 端周质结构域中映射了三个不同的氨基酸取代，CadC 蛋白是一种酸应激反应转录调节剂。虽然这些突变不能赋予 TCS 强耐药性，但它们似乎在适应相对低的药物压力方面发挥了一定但未知的作用。总的来说，观察到的进化轨迹表明，FabI 酶是 TCS 的唯一靶标（至少在~50µm 水平），TCS 结合位点的氨基酸取代代表了 在 BW25113 中获得强 TCS 耐药性的主要机制。这项模型研究说明了基于病态生物连续培养装置的既定方法在揭示耐药机制和鉴定作用机制未知的新型候选药物的靶标方面的潜在用途。
