Department of Pathogen, Biology, International Cancer Center, Shenzhen University Medical School, Shenzhen, Guangdong, China.
Department of Fundamental Microbiology, University of Lausanne, , Switzerland.
mSystems. 2024 Jul 23;9(7):e0128923. doi: 10.1128/msystems.01289-23. Epub 2024 Jun 5.
Antibiotic resistance and tolerance remain a major problem for the treatment of staphylococcal infections. Identifying genes that influence antibiotic susceptibility could open the door to novel antimicrobial strategies, including targets for new synergistic drug combinations. Here, we developed a genome-wide CRISPR interference library for , demonstrated its use by quantifying gene fitness in different strains through CRISPRi-seq, and used it to identify genes that modulate susceptibility to the lipoglycopeptide dalbavancin. By exposing the library to sublethal concentrations of dalbavancin using both CRISPRi-seq and direct selection methods, we not only found genes previously reported to be involved in antibiotic susceptibility but also identified genes thus far unknown to affect antibiotic tolerance. Importantly, some of these genes could not have been detected by more conventional transposon-based knockout approaches because they are essential for growth, stressing the complementary value of CRISPRi-based methods. Notably, knockdown of a gene encoding the uncharacterized protein KapB specifically sensitizes the cells to dalbavancin, but not to other antibiotics of the same class, whereas knockdown of the Shikimate pathway showed the opposite effect. The results presented here demonstrate the promise of CRISPRi-seq screens to identify genes and pathways involved in antibiotic susceptibility and pave the way to explore alternative antimicrobial treatments through these insights.IMPORTANCEAntibiotic resistance is a challenge for treating staphylococcal infections. Identifying genes that affect how antibiotics work could help create new treatments. In our study, we made a CRISPR interference library for and used this to find which genes are critical for growth and also mapped genes that are important for antibiotic sensitivity, focusing on the lipoglycopeptide antibiotic dalbavancin. With this method, we identified genes that altered the sensitivity to dalbavancin upon knockdown, including genes involved in different cellular functions. CRISPRi-seq offers a means to uncover untapped antibiotic targets, including those that conventional screens would disregard due to their essentiality. This paves the way for the discovery of new ways to fight infections.
抗生素耐药性和耐受性仍然是治疗葡萄球菌感染的主要问题。确定影响抗生素敏感性的基因可能为新型抗菌策略打开大门，包括新的协同药物组合的靶点。在这里，我们为 开发了一个全基因组 CRISPR 干扰文库，通过 CRISPRi-seq 量化不同菌株中的基因适应性来证明其用途，并使用它来鉴定调节糖肽类抗生素 dalbavancin 敏感性的基因。通过使用 CRISPRi-seq 和直接选择方法将文库暴露于亚致死浓度的 dalbavancin 下，我们不仅发现了先前报道的与抗生素敏感性相关的基因，还鉴定了迄今为止未知的影响抗生素耐受性的基因。重要的是，由于这些基因对生长至关重要，一些基因无法通过更传统的转座子敲除方法检测到，这强调了基于 CRISPRi 的方法的互补价值。值得注意的是，下调编码未鉴定蛋白 KapB 的基因特异性使细胞对 dalbavancin 敏感，但对同一类别的其他抗生素不敏感，而下调莽草酸途径则产生相反的效果。这里呈现的结果表明，CRISPRi-seq 筛选可用于鉴定与抗生素敏感性相关的基因和途径，并为通过这些见解探索替代抗菌治疗铺平道路。
抗生素耐药性是治疗葡萄球菌感染的挑战。确定影响抗生素作用的基因可以帮助开发新的治疗方法。在我们的研究中，我们为 构建了一个 CRISPR 干扰文库，并使用该文库来确定哪些基因对生长至关重要，还绘制了对抗生素敏感性很重要的基因图谱，重点是糖肽类抗生素 dalbavancin。通过这种方法，我们鉴定了敲低后改变对 dalbavancin 敏感性的基因，包括涉及不同细胞功能的基因。CRISPRi-seq 提供了一种发现未开发抗生素靶点的方法，包括由于其必需性而被传统筛选忽略的靶点。这为发现新的抗感染方法铺平了道路。
