Nanoscale visualisation of bacteriocins in action on bacterial membranes

抗菌素耐药“超级细菌”的增加促使我们扩大抗菌素策略的曲目。特别令人感兴趣的是针对细菌膜的抗生素，因为对这些抗生素的耐药性很难在不损害细菌整体生存能力的情况下产生。细菌素是很有前途的候选者：例如，乳酸链球菌素已经被广泛用作针对革兰氏阳性细菌的食品防腐剂，但（尚未）对革兰氏阴性细菌有效，例如表皮霉素在其膜破坏模式中显示出广泛的可调性。然而，对真正的细菌膜的作用机制仍然知之甚少，并且需要大量的生物工程来达到下一代抗生素所需的特异性和广谱有效性。这个项目是基于这样一个假设，即这种生物工程和未来更广泛的应用将通过对作用机制的纳米级理解来促进，并且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、荧光标记的细菌素构建物（基于乳酸链球菌素、表皮菌素），并通过AFM观察它们在支持的脂质双分子层上的作用模式。建立并应用单分子全内反射荧光（TIRF）显微镜观察细菌素在模型膜上图像结合的实验方案。通过原子力显微镜观察这些细菌素及其对活细菌的作用方式。开发和应用实验方案，对活菌进行细菌素成像，以及荧光显微镜下的膜破坏（使用脂质和核酸染色），以及超分辨率实验（PALM/STORM）的停流实验。使用这些方法将分子设计与膜破坏和细菌杀死模式联系起来。

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.