Abstract
The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.
Highlights
Due to its central cellular role and the relatively easy accessibility for extracellular agents, the bacterial cytoplasmic membrane is a major target for antimicrobial compounds such as membranetargeting peptides (Yeaman and Yount, 2003; Brogden, 2005; Wimley and Hristova, 2011)
We provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo
We have shown that the membrane potential is crucial for correct cellular localization of certain membrane proteins in B. subtilis and E. coli (Strahl and Hamoen, 2010)
Summary
Due to its central cellular role and the relatively easy accessibility for extracellular agents, the bacterial cytoplasmic membrane is a major target for antimicrobial compounds such as membranetargeting peptides (Yeaman and Yount, 2003; Brogden, 2005; Wimley and Hristova, 2011). Disrupting the membrane function by an antimicrobial compound can simultaneously inhibit several essential processes and amounts to a serious assault on a bacterial cell. Dissipation of the membrane potential is either the sole mechanism of action, or contributes to the potency of the compound This can either be caused by formation of ionconducting membrane pores, by otherwise increasing membrane ion-permeability, or by acting as an ion carrier (Yeaman and Yount, 2003; Brogden, 2005; Wimley and Hristova, 2011).
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