Nanoscale visualisation of bacteriocins in action on bacterial membranes

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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.