Engineering bacterial traps to understand and inspire next-generation antibiotics

抗菌素耐药性正在上升，而新抗生素的开发却停滞不前：过去常规治愈的感染现在正变得危及生命。这一新出现的重大健康威胁要求对下一代抗生素提出全新的想法和灵感。通过这个项目，我们的目标是提供这样的新想法和灵感，基于对膜靶向抗生素如何杀死细菌以及细菌如何对这种抗生素产生抗药性的更深入理解。这种了解仍然令人惊讶地稀少，主要是由于各种技术限制和细菌细胞膜的复杂性质。我们将克服其中的一些限制，并通过利用我们最近开发的工具以分子尺度的分辨率探测活的细菌表面，使用原子力显微镜(Benn等人，PNAS 2021)。为了使这种显微镜方法能够在活细胞、生长细胞和分裂细胞受到抗生素攻击时实时进行，我们将设计微图案表面，将细菌捕获在适当功能的微流体陷阱中，在其中细菌被充分固定以便于纳米级显微镜，但又不能固定到阻止它们生长和分红。接下来，不同的细菌菌株(敏感和耐药)将在这种陷阱中以纳米分辨率成像，因为它们暴露在不同剂量的抗生素下，基于机器学习的图像分析将用于定量评估细菌在整个细胞周期中的损害，并将其与荧光显微镜测量的细菌细胞死亡相关联。

Antimicrobial resistance is rising whereas the development of new antibiotics has stalled: infections that were routinely cured in the past are now becoming life-endangering. This emerging and major health threat calls for radically new ideas and inspiration for next-generation antibiotics. With this project, we aim to provide such new ideas and inspiration based on a deeper understanding of how membrane-targeting antibiotics can kill bacteria and how bacteria can develop resistance against such antibiotics.Such understanding is still surprisingly scarce, largely due to various technical limitations and due to the complex nature of bacterial cell envelopes. We will overcome some of these limitations and tackle this complexity by making use of our recently developed tools to probe live bacterial surfaces at molecular-scale resolution, using atomic force microscopy (Benn et al., PNAS 2021). To enable such microscopy approaches on live, growing and dividing cells in real time as they are under attack by antibiotics, we will engineer micropatterned surfaces that capture bacteria in suitably functionalised, microfluidic traps within which they are sufficiently immobilised to facilitate nanoscale microscopy, yet not so immobilised that it prevents them from growing and dividing.Different bacterial strains (sensitive and resistant) will next be imaged at nanometre resolution in such traps as they are exposed to varying doses of antibiotics, and machine-learning based image analyses will used to quantitatively assess bacterial damage throughout the cell cycle and to correlate this with bacterial cell death as measured by fluorescence microscopy.