Mechanosensitive Channels Mediate Hypoionic Shock-Induced Aminoglycoside Potentiation against Bacterial Persisters by Enhancing Antibiotic Uptake

Abstract

Improving the efficacy of existing antibiotics is a promising strategy for combating antibiotic-resistant/tolerant bacterial pathogens that have become a severe threat to human health. We previously reported that aminoglycoside antibiotics could be dramatically potentiated against stationary-phase Escherichia coli cells under hypoionic shock conditions (i.e., treatment with ion-free solutions), but the underlying molecular mechanism remains unknown. Here we show that mechanosensitive (MS) channels, a ubiquitous protein family sensing mechanical forces of cell membrane, mediate such hypoionic shock-induced aminoglycoside potentiation. Two-minute treatment under conditions of hypoionic shock (e.g., in pure water) greatly enhances the bactericidal effects of aminoglycosides against both spontaneous and triggered E. coli persisters, numerous strains of Gram-negative pathogens in vitro , and Pseudomonas aeruginosa in mice. Such potentiation is achieved by hypoionic shock-enhanced bacterial uptake of aminoglycosides and is linked to hypoionic shock-induced destabilization of the cytoplasmic membrane in E. coli . Genetic and biochemical analyses reveal that MscS-family channels directly and redundantly mediate aminoglycoside uptake upon hypoionic shock and thus potentiation, with MscL channel showing reduced effect. Molecular docking and site-directed mutagenesis analyses reveal a putative streptomycin-binding pocket in MscS, critical for streptomycin uptake and potentiation. These results suggest that hypoionic shock treatment destabilizes the cytoplasmic membrane and thus changes the membrane tension, which immediately activates MS channels that are able to effectively transport aminoglycosides into the cytoplasm for downstream killing. Our findings reveal the biological effects of hypoionic shock on bacteria and can help to develop novel adjuvants for aminoglycoside potentiation to combat bacterial pathogens via activating MS channels.

CCCP Facilitates Aminoglycoside to Kill Late Stationary-Phase Escherichia coli by Elevating Hydroxyl Radical

“…In particular, the concentration of tobramycin in CCCP-treated cells was significantly lower than that without CCCP treatment during the full window of treatment except for the 0.5 h time point. Further, we applied the cell-growth inhibition assay as we recently reported , to measure the level of tobramycin accumulated in E. coli cells. Consistently, we found that tobramycin, as extracted from 3 h or 5 h CCCP treatment E. coli stationary-phase cells, suppressed cell growth in a weaker strength than that without CCCP treatment (blue frame in Figure E,F), similarly to what occurred in exponential-phase cells (red frame in Figure E).…”

“…36,37 We discovered several chemical adjuvants including 5-methylindole and n-butanol, with which 1 min co-treatment enables aminoglycosides to eradicate bacterial persisters and multidrug-resistant pathogens. 15,69 Besides, we also developed physical stresses for aminoglycoside potentiation, including hypoionic shock 27,70,71 and rapid freezing, 26 both of which activate membrane-localized mechanosensitive channels for enhancing antibiotic uptake.…”

“…22,23 What membrane-localized channels are responsible for their transportation across the membrane? Recent studies from the Blount group 24,25 and us 26,27 more efforts are needed for verification and mechanistic dissection. Aminoglycosides are believed to enter into bacteria via two energy-dependent processes, which strictly rely on the proton motive force (PMF) and particularly on the electric potential (refer to the review in ref 21 and references therein).…”

“…Electric potential or ATP? , What membrane-localized channels are responsible for their transportation across the membrane? Recent studies from the Blount group , and us , suggest that mechanosensitive channels MscL and MscS are potential candidates for aminoglycoside transportation, but more efforts are needed for verification and mechanistic dissection.…”

5-Methylindole kills various bacterial pathogens and potentiates aminoglycoside against methicillin-resistant Staphylococcus aureus

“…The discovery and development of new antibiotics, including structurally modified existing antibiotics, has played a dominant role in this war ( Brown & Wright, 2016 ; Lewis, 2020 ). In addition, improving the efficacy of existing antibiotics has also attracted attentions in the recent decade ( Allison, Brynildsen & Collins, 2011 ; Peng et al, 2015 ; Liu et al, 2019 ; Wright, 2016 ; Zhao et al, 2020 ; Lv et al, 2022a ; Lv et al, 2022b ), given that these existing antibiotics have been well documented in their toxicity, pharmacokinetics, administration and mechanism of actions.…”