Nanoscale visualisation of bacteriocins in action on bacterial membranes

细菌素在细菌膜上作用的纳米级可视化

• 批准号: 2870122
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2870122
• 关键词: Nanoscale; visualisation; bacteriocins; action; bacterial

The rise in antimicrobial-resistant "superbugs" urges us to expand the repertoire of antimicrobial strategies. Of particular interest are antibiotics that target bacterial membranes, since resistance to these antibiotics is hard to develop without compromising bacterial viability overall. Bacteriocins are promising candidates: e.g. nisin is already widely used as a food preservative that targets Gram-positive bacteria, but is not (yet) effective against Gram-negative bacteria, and e.g. epidermicin shows wide tuneability in its modes of membrane disruption. However, mechanisms of action on real bacterial membranes remain poorly understood, and substantial bioengineering remains necessary to reach the specificity and broad-range effectiveness required for next-generation antibiotics.This project is based on the presumption that such bioengineering and prospective wider application will be facilitated by a nanoscale understanding of mechanisms of action, and on the hypothesis that this understanding can be achieved by an integrative microscopy approach applied to living bacteria.Our objectives are:To prepare functional, fluorescently labelled bacteriocin constructs (based on nisin, epidermicin) and to visualise their modes of action by AFM on supported lipid bilayers.To develop and apply experimental protocols to image binding bacteriocins on model membranes by single-molecule total internal reflection fluorescence (TIRF) microscopy.To visualise these bacteriocins and their modes of action by AFM on live bacteria.To develop and apply experimental protocols to image bacteriocins on live bacteria, as well as the membrane disruption (using lipid and nucleic acid staining) by fluorescence microscopy, as well as stop-flow experiments for super-resolution experiments (PALM/STORM).To use these methods to correlate molecular design with modes of membrane disruption and bacterial killing.

耐药性“超级细菌”的增加促使我们扩大抗菌策略的范围。特别令人感兴趣的是靶向细菌膜的抗生素，因为对这些抗生素的耐药性很难在不损害细菌整体生存力的情况下发展。细菌素是有希望的候选物：例如乳链菌肽已经被广泛用作靶向革兰氏阳性细菌的食品防腐剂，但对革兰氏阴性细菌（尚未）有效，并且例如表皮霉素在其膜破坏模式中显示出广泛的耐受性。然而，对真实的细菌膜的作用机制仍然知之甚少，为了达到下一代抗生素所需的特异性和广泛的有效性，仍然需要大量的生物工程。并假设这种理解可以通过应用于活细菌的综合显微镜方法来实现。我们的目标是：制备功能性荧光标记细菌素构建体（基于乳链菌肽，表皮霉素）并通过原子力显微镜观察它们在支持的脂质双层上的作用模式。分子全内反射荧光（TIRF）显微镜。通过AFM观察这些细菌素及其在活细菌上的作用模式。开发和应用实验方案来成像活细菌上的细菌素，以及膜破裂（使用脂质和核酸染色）通过荧光显微镜，以及用于超分辨实验的停流实验（PALM/STORM）。使用这些方法将分子设计与膜破坏和细菌杀灭模式相关联。