Choe Donghui, Lee Eunju, Song Yoseb, Kim Sun Chang, Jeong Ki Jun, Palsson Bernhard, Cho Byung-Kwan, Cho Suhyung
Department of Bioengineering, University of California San Diego, La Jolla, California, USA.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea.
mSystems. 2025 Jul 22;10(7):e0035325. doi: 10.1128/msystems.00353-25. Epub 2025 Jun 16.
Bacterial genes serve diverse cellular functions and many affect fitness in response to environmental challenges. We employed CRISPR interference screening to investigate the fitness effect of each gene in exposed to gentamicin, aiming to understand the cellular defense mechanisms. Our findings revealed that ribosomal proteins, ribosome-associated proteins, toxin-antitoxin systems, and outer membrane proteins strongly influence the fitness of in gentamicin. Notably, gentamicin-induced fitness changes resembled those under anaerobic conditions, where resistance to gentamicin was observed. Specifically, genes related to the biosynthesis of cofactors and electron carriers, crucial for the respiratory system, showed reduced essentiality under both gentamicin and anaerobic conditions, suggesting a disruption in membrane potential leading to limited gentamicin uptake. Transcriptomic and genome-wide binding analyses identified the two-component system CpxR as a key regulator of respiratory systems in response to gentamicin. Our study provides insights into cellular defense mechanisms, offering potential strategies for combating antibiotic resistance.IMPORTANCEBacteria can adapt to a variety of stressful environments, including antibiotic exposure. The mechanisms underlying antibiotic resistance remain an active area of investigation. Clustered regularly interspaced short palindromic repeats (CRISPR) interference enables specific silencing of gene expression, allowing researchers to assess the fitness effects of gene knockdowns under given conditions. Using genome-wide CRISPR interference screening on exposed to gentamicin, we identified anaerobic-like fitness effects of genes involved in respiration and the maintenance of membrane potential-key processes that facilitate gentamicin entrance into the cell. Transcriptomic analysis and immunoprecipitation assays further indicated that the two-component system CpxR modulates respiratory adaptations in response to gentamicin challenge. These findings shed light on the development of antibiotic resistance in bacteria and may offer new insight into strategies for treating gentamicin-resistant pathogens.
细菌基因具有多种细胞功能，许多基因在应对环境挑战时会影响适应性。我们采用CRISPR干扰筛选来研究每个基因在暴露于庆大霉素时的适应性效应，旨在了解细胞防御机制。我们的研究结果表明，核糖体蛋白、核糖体相关蛋白、毒素-抗毒素系统和外膜蛋白对在庆大霉素环境中的适应性有强烈影响。值得注意的是，庆大霉素诱导的适应性变化类似于在厌氧条件下观察到的情况，在厌氧条件下对庆大霉素有抗性。具体而言，对于呼吸系统至关重要的辅因子和电子载体生物合成相关基因，在庆大霉素和厌氧条件下的必需性都降低了，这表明膜电位的破坏导致庆大霉素摄取受限。转录组学和全基因组结合分析确定双组分系统CpxR是呼吸系统响应庆大霉素的关键调节因子。我们的研究为细胞防御机制提供了见解，为对抗抗生素耐药性提供了潜在策略。
重要性
细菌能够适应各种应激环境，包括抗生素暴露。抗生素耐药性的潜在机制仍然是一个活跃的研究领域。成簇规律间隔短回文重复序列（CRISPR）干扰能够特异性沉默基因表达，使研究人员能够评估在给定条件下基因敲低的适应性效应。通过对暴露于庆大霉素的细菌进行全基因组CRISPR干扰筛选，我们确定了参与呼吸作用和维持膜电位的基因具有类似厌氧的适应性效应，而这些关键过程促进了庆大霉素进入细胞。转录组分析和免疫沉淀试验进一步表明，双组分系统CpxR调节对庆大霉素挑战的呼吸适应。这些发现揭示了细菌抗生素耐药性的发展，并可能为治疗耐庆大霉素病原体提供新的策略见解。
