Abstract
Recent evidence suggests that metabolic shutdown alone does not fully explain how bacteria exhibit phenotypic antibiotic tolerance. In an attempt to investigate the range of starvation-induced physiological responses underlying tolerance development, we found that active maintenance of the transmembrane proton motive force (PMF) is essential for prolonged expression of antibiotic tolerance in bacteria. Eradication of tolerant sub-population could be achieved by disruption of PMF using the ionophore CCCP, or through suppression of PMF maintenance mechanisms by simultaneous inhibition of the phage shock protein (Psp) response and electron transport chain (ETC) complex activities. We consider disruption of bacterial PMF a feasible strategy for treatment of chronic and recurrent bacterial infections.
Highlights
Recent evidence suggests that metabolic shutdown alone does not fully explain how bacteria exhibit phenotypic antibiotic tolerance
In order to unravel the range of active cellular mechanisms required for eliciting and maintaining prolonged phenotypic tolerance to antibiotics, nutrient starvation was chosen as an induction factor in this work because of the following reasons: (i) starvation was previously shown to induce a significantly stronger tolerance phenotype than compounds that inhibit bacterial growth under nutrient-rich conditions[8], suggesting that starvation-induced tolerance probably involves inducible defensive mechanisms and deserves more in-depth and systematic exploration; (ii) nutrient starvation is commonly encountered by bacteria during the infection process[9], starvation-induced tolerance responses should be investigated comprehensively at the transcriptional and physiological level; (iii) it is a readily manipulated test condition, which facilitates investigation into the key mechanisms underlying the formation of antibiotic tolerance[5,10,11]
We found that active maintenance of proton motive force (PMF) is essential for starvation-induced tolerance, and that disruption of PMF resulted in the eradication of the entire antibiotic-tolerant subpopulation
Summary
Recent evidence suggests that metabolic shutdown alone does not fully explain how bacteria exhibit phenotypic antibiotic tolerance. Efflux activities played an active role in conferring the bacterial tolerance phenotype through upregulation of the expression level of a range of multidrug efflux genes, such as tolC, acrA, acrB, acrD, emrA, emrB, macA, and macB7 In this previous study, time-lapse imaging of antibiotic-tolerant cells containing fluorescence-labeled TolC confirmed that high expression of tolC directly led to the formation of antibiotictolerant subpopulation. In order to unravel the range of active cellular mechanisms required for eliciting and maintaining prolonged phenotypic tolerance to antibiotics, nutrient starvation was chosen as an induction factor in this work because of the following reasons: (i) starvation was previously shown to induce a significantly stronger tolerance phenotype than compounds that inhibit bacterial growth under nutrient-rich conditions[8], suggesting that starvation-induced tolerance probably involves inducible defensive mechanisms and deserves more in-depth and systematic exploration; (ii) nutrient starvation is commonly encountered by bacteria during the infection process[9], starvation-induced tolerance responses should be investigated comprehensively at the transcriptional and physiological level; (iii) it is a readily manipulated test condition, which facilitates investigation into the key mechanisms underlying the formation of antibiotic tolerance[5,10,11]. Our findings provide the molecular basis for devising new approaches to eradicate antibiotic-tolerant subpopulation through PMF disruption
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