Pushing the envelope: atomic force microscopy imaging of the bacterial outer membrane during growth and division

挑战极限：生长和分裂过程中细菌外膜的原子力显微镜成像

• 批准号: BB/X00760X/1
• 负责人: Bart Hoogenboom
• 金额: $ 61.35万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX00760X%2F1
• 关键词: Pushing; envelope; atomic; force; microscopy

Gram-negative bacteria are among the microbes with the highest potential to develop resistance against commonly used antibiotics, such that previously harmless infections can become severe and potentially life-threatening diseases. This is in part because these bacteria (including A. baumannii, P. aeruginosa, and enterobacteriaceae such as Salmonella and E. coli) are protected by an outer membrane that limits entry of antibiotics into the cell.Based on light - and particularly fluorescence - microscopy, it has been suggested that this outer membrane organises its building blocks in rather sophisticated ways, including the formation of protein islands that may also play a role in how efficiently bacteria clear antibiotics. Quite generally, light microscopy has been a most powerful tool to discover and understand biological phenomena that define the life, growth, division, and death of cells. Yet its resolution on living cells is mostly insufficient to resolve cells at molecular length scales.Atomic force microscopy (AFM) is an alternative technique that can probe single molecules by gently tracing their contours with a sharp probe. We have recently used AFM to resolve the outer membrane of living bacteria at molecular-scale resolution, thereby revealing how the membrane segregates into different protein-enriched and lipid-enriched domains.Here, noting that bacteria can divide every ~20 minutes under favourable conditions, we propose to develop methods that will enable us to carry out such AFM experiments on growing and dividing cells, with the aim to better understand how the outer membrane facilitates/adapts to bacterial growth and division, including the synthesis and insertion of new membrane components.

革兰氏阴性细菌是最有可能对常用抗生素产生耐药性的微生物之一，因此以前无害的感染可能会变成严重的、可能危及生命的疾病。部分原因是这些细菌（包括鲍曼不动杆菌、铜绿假单胞菌以及沙门氏菌和大肠杆菌等肠杆菌科细菌）受到外膜的保护，限制抗生素进入细胞。基于光，特别是荧光 -在显微镜下观察，有人认为这种外膜以相当复杂的方式组织其构件，包括蛋白质岛的形成，这也可能在细菌清除抗生素的效率中发挥作用。一般来说，光学显微镜一直是发现和理解定义细胞生命、生长、分裂和死亡的生物现象的最强大工具。然而，它对活细胞的分辨率大多不足以在分子长度尺度上解析细胞。原子力显微镜 (AFM) 是一种替代技术，可以通过用锋利的探针轻轻追踪单个分子的轮廓来探测单个分子。我们最近使用 AFM 以分子尺度分辨率解析活细菌的外膜，从而揭示膜如何分离成不同的蛋白质富集和脂质富集区域。在这里，注意到细菌在有利的条件下每约 20 分钟即可分裂一次，我们建议开发方法，使我们能够对生长和分裂的细胞进行此类 AFM 实验，目的是更好地了解外膜如何促进/适应细菌生长和分裂，包括新膜成分的合成和插入。