Tobramycin uptake in Escherichia coli membrane vesicles

Abstract

The uptake of tobramycin was measured in Escherichia coli membrane vesicles prepared in KMES [K ؉ -2-(N-morpholino)ethanesulfonic acid] buffer at pH 6.6. Uptake occurred in vesicles energized with ascorbic acid and phenazine methosulfate, in which the electrical potential (⌬) was ؊120 mV, but not in vesicles energized with D-lactate (⌬ ‫؍‬ ؊95 mV). The addition of nigericin to vesicles energized with D-lactate did not induce tobramycin uptake despite an increase in ⌬ to ؊110 mV. However, when ⌬ was increased or decreased by the addition of nigericin or valinomycin, respectively, uptake in vesicles energized with ascorbic acid and phenazine methosulfate was stimulated or inhibited, respectively, confirming studies with whole cells showing that uptake of aminoglycosides is gated by ⌬ rather than by proton motive force (⌬ H ؉) or ⌬pH. Nethylmaleimide prevented uptake, suggesting that the aminoglycoside transporter is a cytoplasmic membrane protein with accessible sulfhydryl groups. The observation that uptake is gated in vesicles as well as in whole cells suggested that diffusion occurs through a voltage-gated channel. In vesicles preloaded with tobramycin, no efflux occurred after the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone. In susceptible cells, aminoglycosides themselves decreased the magnitude of ⌬. We propose a mechanism of aminoglycoside-induced killing in which aminoglycosides themselves close the voltage-gated channel by decreasing the magnitude of ⌬. Channel closure causes aminoglycosides accumulated prior to the fall in ⌬ to be trapped, which in turn causes irreversible uptake and subsequent bactericidal effects.We have previously shown that the early phase of uptake of aminoglycoside antibiotics by bacteria is regulated by electrical potential (⌬), the electrical component of the protonmotive force (⌬ H ϩ) (19,24,39,40). A critical threshold of ⌬ must be reached for the initiation of aminoglycoside transport. Once this threshold is exceeded, the initial rates of both uptake and killing are proportional to the magnitude of ⌬ (7,9,11,17,20,24,27,32,39,40,53). These observations lent themselves to a chemiosmotic interpretation that active transport of cationic aminoglycosides is driven by ⌬ by a uniport mechanism via a cytoplasmic membrane carrier protein (9, 39). However, uptake which is gated by ⌬ demonstrates properties of a voltageregulated channel.The observations that the early transport of aminoglycosides in whole cells is both energy dependent and gated are difficult to reconcile with most current models of aminoglycoside mechanism of action. One widely accepted unifying hypothesis to explain how the pleiotropic effects of aminoglycosides cause cell death has recently been proposed (18). This hypothesis suggests that aminoglycoside-initiated misreading of mRNA results in altered proteins that fuse to the cytoplasmic membrane, forming many nonspecific channels. These channels are thought to facilitate the uptake or efflux of a variety of substrates, including aminoglycos...

Membrane voltage dysregulation driven by metabolic dysfunction underlies bactericidal activity of aminoglycosides

“…Aminoglycosides are well established to bind and exert pleiotropic effects on ribosomes 5,50,51 , and numerous reports highlighted the importance of maintaining a membrane potential in aminoglycoside activity. This evidence included voltage dependent aminoglycoside uptake 10 and cell death correlated with the citric acid cycle and carbon source 35,52,53 . Metabolic changes can likewise induce changes in membrane voltage and the overall proton motive force.…”

“…The next step is known as the energy dependent phase I (EDP-I) and occurs almost instantaneously upon aminoglycoside treatment 9 . This portion is noted as energy dependent because both respiration inhibitors 10 and differential carbon sources 11 reduced uptake. EDP-I is thought to be the step at which the aminoglycoside enters the cytoplasm 5,11 , is concentration dependent 8 , and occurs in cells that are resistant to or tolerant of aminoglycosides 2,12 .…”

“…Proposed bactericidal mechanisms all stem from this consensus theory of aminoglycoside uptake 2,4,10,13,14 . Once aminoglycosides are inside the cell, several competing theories exist to explain bactericidal activity including membrane breakdown from mistranslated protein 14,15 , reactive oxygen species 4 (ROS), and a positive feedback of drug uptake 2,10 , though there is debate around each 2,13,16 .…”

Rapid Freezing Enables Aminoglycosides To Eradicate Bacterial Persisters via Enhancing Mechanosensitive Channel MscL-Mediated Antibiotic Uptake

“…2C), become relatively tolerant to tobramycin (line 7) after pretreatment with the widely used protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (35,36). This reflects the fact that the aminoglycoside uptake by bacteria under conventional treatment conditions depends on the PMF, which can be dissipated by CCCP (18,37,38). Such persister-like cells, however, were effectively killed by a combined treatment consisting of addition of tobramycin and freezing (lines 6 and 8 in Fig.…”

Cystic fibrosis infections: treatment strategies and prospects

“…Tobramycin uptake in E. coli is regulated by the electrical potential component of the proton motive force and is probably mediated by a voltage‐gated channel in the cytoplasmic membrane that is inhibited by compounds that depolarize the membrane potential (Leviton et al , 1995). Na + , K + , and Cl − are ubiquitous in nature, so much so that elaborate pumping mechanisms for maintaining homeostasis are present in all bacteria.…”