The bacterial cytoplasmic membrane (CM) is the first line of defense which protects cells from the harmful influences of exogenous molecules. Given this, a recurring theme in the ongoing development of antimicrobial compounds is the disruption of the phospholipid packing of the CM, which weakens the efficacy of this protective barrier. A novel variation of this approach is to instead forcibly activate one or more of the protein channels spanning the CM, creating an efficient route for molecules to freely cross into the cell. For example, emerging literature has demonstrated the feasibility of chemically activating the mechanosensitive (MS) channels, which protect the cell against acute changes in cellular volume by allowing the membrane to expand as the channels open. We present here an examination of the influence of activated MS channels on the passive uptake of a pair of hydrophobic ions in colloidal suspensions of living E. coli. As a preliminary test, we activated the MS channels by inducing a persistent osmotic shock. Specifically, bacteria were suspended in either a solution of phosphate buffered saline (PBS, channels closed) or distilled deionized water (di-water, channels open), which forced the MS channels to adopt a permanent open geometry. Time-resolved second harmonic light scattering was then used to quantify the rate of transport of the antimicrobial dye, malachite green (MG), across the CM depending upon whether the MS channels were open or closed. Similarly, fluorescence microscopy was used to monitor the propensity for transport of the viability stain, propidium (Pro), under the very same conditions. When the cells were suspended in water (channels open), both MG and Pro freely diffused across the CM. However, when the cells were instead suspended in PBS (channels closed), Pro was completely unable to cross the CM. Further, the effect was observed to be largely reversable. When water cells were washed and resuspended in PBS (i.e., forcing the MS channels to close), transport of Pro across the CM ceased. More importantly, the overall viability of the colony was shown to be completely preserved, suggesting that suspension in di-water did not kill the cells. The potential application of activated MS channels for improving antimicrobial efficacy will be discussed.

Activating mechanosensitive channels to increase passive diffusion across the bacterial cytoplasmic membrane

ACS Spring 2022

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