Active Efflux Leads to Heterogeneous Dissipation of Proton Motive Force by Protonophores in Bacteria

Dai, Le; Krasnopeeva, Ekaterina; Sinjab, Faris; Pilizota, Teuta; Kim, Minsu

doi:10.1128/mbio.00676-21

Cited by 41 publications

(33 citation statements)

References 85 publications

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“…Notably, this time coincided with the onset in roxithromycin-NBD accumulation ( t 0 , indicated by circles and arrows in Figure 2A ), suggesting a link between lag in drug uptake and lag in cell growth. However, other mechanisms might contribute to lag in drug uptake including a positive feedback loop in drug binding or positive feedback between efflux and drug accumulation ( Le et al, 2021 ). Phenotypic variants that continued growing instead did not accumulate roxithromycin-NBD for the entire duration of the treatment ( Figure 2B ).…”

Section: Resultsmentioning

confidence: 99%

Fast bacterial growth reduces antibiotic accumulation and efficacy

Łapińska

Voliotis

Lee

et al. 2022

eLife

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Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here, we hypothesise that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast-growing variants, including efflux that can reduce macrolide accumulation. We used this new knowledge to eradicate variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures.

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Section: Resultsmentioning

confidence: 99%

Fast bacterial growth reduces antibiotic accumulation and efficacy

Łapińska

Voliotis

Lee

et al. 2022

eLife

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“…S3C). The disruption of PMF can interfere with the levels of cellular ATP ( 33 , 34 ). Correspondingly, IBG significantly increased the intracellular ATP levels in a dose-dependent manner ( P < 0.01) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Mechanism of Action of Isopropoxy Benzene Guanidine against Multidrug-Resistant Pathogens

Zhang

Han

et al. 2023

Microbiol Spectr

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“…With mounting evidence of the role of efflux-based resistance in clinically relevant pathogens, , quantifying efflux activity and the resultant tolerance to drugs has implications for strategies aimed at minimizing AMR. Recent studies have measured the accumulation and efflux dynamics , of crucial antibiotics at the single cell ,− and population levels − to capture bacterial export activity. In this study, we measured the population fluorescence intensity of EtBr, known to significantly up-regulate LfrA levels in M. smegmatis , to infer the efflux dynamics in the wild-type and laboratory generated strains with increased basal levels of LfrA.…”

Section: Discussionmentioning

confidence: 99%

The Extent of Antimicrobial Resistance Due to Efflux Pump Regulation

et al. 2022

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A key mechanism driving antimicrobial resistance (AMR) stems from the ability of bacteria to up-regulate efflux pumps upon exposure to drugs. The resistance gained by this up-regulation is pliable because of the tight regulation of efflux pump levels. This leads to temporary enhancement in survivability of bacteria due to higher efflux pump levels in the presence of antibiotics, which can be reversed when the cells are no longer exposed to the drug. Knowledge of the extent of resistance thus gained would inform intervention strategies aimed at mitigating AMR. Here, we combine mathematical modeling and experiments to quantify the maximum extent of resistance that efflux pump up-regulation can confer via phenotypic induction in the presence of drugs and genotypic abrogation of regulation. Our model describes the dynamics of drug transport in and out of cells coupled with the associated regulation of efflux pump levels and predicts the increase in the minimum inhibitory concentration (MIC) of drugs due to such regulation. To test the model, we measured the uptake and efflux as well as the MIC of the compound ethidium bromide (EtBr), a substrate of the efflux pump LfrA, in wild-type Mycobacterium smegmatis mc 2 155, as well as in two laboratory-generated strains. Our model captured the observed EtBr levels and MIC fold-changes quantitatively. Further, the model identified key parameters associated with the resulting resistance, variations in which could underlie the extent to which such resistance arises across different drug−bacteria combinations, potentially offering tunable handles to optimize interventions aimed at minimizing AMR.

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Active Efflux Leads to Heterogeneous Dissipation of Proton Motive Force by Protonophores in Bacteria

Abstract: Various toxic compounds disrupt bacterial physiology. While bacteria harbor defense mechanisms to mitigate the toxicity, these mechanisms are often coupled to the physiological state of the cells and become ineffective when the physiology is severely disrupted.

Cited by 41 publications

References 85 publications

Fast bacterial growth reduces antibiotic accumulation and efficacy

Fast bacterial growth reduces antibiotic accumulation and efficacy

Mechanism of Action of Isopropoxy Benzene Guanidine against Multidrug-Resistant Pathogens

The Extent of Antimicrobial Resistance Due to Efflux Pump Regulation

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