Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
Cell Rep. 2024 Nov 26;43(11):114967. doi: 10.1016/j.celrep.2024.114967. Epub 2024 Nov 15.
Bacterial mutant libraries with downregulated antibiotic targets are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi). By modeling depletion levels and adjusting knockdown mutant inocula, we rationally designed and optimized a CRISPR interference-mediated pooled library of essential genes (CIMPLE) approaching coverage of the bacterial essential genome with mutant sensitization. We exposed CIMPLE to an uncharacterized bacterial growth inhibitor structurally different from antibiotics and discovered that it inhibits the essential peptidyl-tRNA hydrolase. Overall, CIMPLE leverages the advantages of arrayed and pooled CRISPRi libraries to uncover unexplored targets for antibiotic action.
具有下调抗生素靶标的细菌突变文库是阐明抗菌化合物作用机制的有用工具，这是抗生素发现的关键步骤。然而，由于在 pooled 生长过程中敲低突变体的不均匀增殖，导致重要靶标意外丢失，因此实现抗菌靶标的基因组覆盖仍然是一个挑战。为了克服这个问题，我们使用 CRISPR 干扰 (CRISPRi) 在抗生素抗性细菌洋葱伯克霍尔德菌中构建了一个基因敲除突变体文库（EGML）。通过模拟耗竭水平并调整敲低突变体接种物，我们合理设计和优化了接近细菌必需基因组覆盖率的 CRISPR 干扰介导的必需基因敲除突变体文库（CIMPLE），从而实现了突变体的敏化。我们将 CIMPLE 暴露于一种与抗生素结构不同的未被表征的细菌生长抑制剂中，并发现它抑制必需的肽基-tRNA 水解酶。总的来说，CIMPLE 利用了基因敲除突变体文库的优势，为探索抗生素作用的未知靶点提供了新的思路。
