Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Biophore Building, Lausanne, Switzerland.
Guangdong Key Laboratory for Genome Stability and Human Disease Prevention, Department of Pharmacology, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, China.
Elife. 2022 Jun 24;11:e75607. doi: 10.7554/eLife.75607.
Antibiotic resistance in the important opportunistic human pathogen is on the rise. This is particularly problematic in the case of the β-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (ubsets of ISPR nterference ibraries xtracted by fluorescence activated ell orting coupled to next generation uencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by a reduced availability of cell wall precursors at the site of cell wall synthesis due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier that is responsible for transporting these precursors across the cell membrane. The data suggest that, whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant . These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.
重要的人体机会致病菌的抗生素耐药性正在上升。在β-内酰胺类抗生素阿莫西林的情况下，这尤其成问题，因为阿莫西林是一线治疗药物。因此，发现能够杀死或重新敏化耐阿莫西林肺炎球菌的靶标至关重要。为此，我们使用称为 sCRilecs-seq 的基于 CRISPR 干扰的全基因组单细胞基因沉默筛选（通过荧光激活细胞分选提取的 ISPR 干扰文库亚组与下一代测序相结合）。由于阿莫西林会影响生长和分裂，因此 sCRilecs-seq 用于鉴定负责维持适当细胞大小的靶标。我们的筛选揭示了下调甲羟戊酸途径会导致细胞广泛伸长。对这种表型的进一步研究表明，由于细胞膜上未端磷酸酯（Und-P）的产生受到限制，导致细胞壁合成部位的细胞壁前体可用性降低，从而导致该表型。Und-P 是负责将这些前体穿过细胞膜运输的脂质载体。数据表明，尽管 Und-P 水平降低，但肽聚糖合成仍在继续，而细胞收缩则被特异性阻断。我们成功地利用了这一知识，创建了一种联合治疗策略，将 FDA 批准的药物氯米酚（Und-P 合成抑制剂）与阿莫西林联合使用。我们的结果表明，氯米酚增强了阿莫西林的抗菌活性，并且联合治疗使耐阿莫西林的肺炎球菌重新敏感。这些发现可以为开发针对越来越多的难以治疗的耐阿莫西林肺炎球菌感染的解决方案提供起点。
