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

Abstract

Aminoglycosides are broad-spectrum antibiotics whose mechanism of bactericidal activity has been under debate. It is widely accepted, however, that membrane voltage potentiates aminoglycoside activity, which is ascribed to voltage dependent drug uptake. In this paper, we measured the single cell response of Escherichia coli treated with aminoglycosides and discovered that the bactericidal action arises not from the downstream effects of voltage dependent drug uptake, but rather directly from dysregulated membrane potential. In the absence of voltage, aminoglycosides are taken into cells and exert bacteriostatic effects by inhibiting translation. However, cell killing was immediate upon re-polarization. The hyperpolarization arose from altered ATP flux, which induced a reversal of the F1Fo-ATPase to hydrolyze ATP and generated the deleterious voltage. Heterologous expression of an ATPase inhibitor from Salmonella completely eliminated bactericidal activity, while loss of the F-ATPase significantly reduced the electrophysiological response to aminoglycosides. Our data support a model of voltage induced death, which could be resolved in real-time at the single cell level, and separates the mechanisms of aminoglycoside bacteriostasis and bactericide in E. coli.

Genome wide functional screen for calcium transients inE. coliidentifies decreased membrane potential adaptation to persistent DNA damage

“…Yet, the cells were metabolically active for 8 hours after treatment. This intermediate time period, between treatment and cell death, is similar to previous results with aminoglycosides 19 , and suggests this could be an important consideration during antibiotic treatment if those dying cells can still signal to neighbors, and influence the population level activity.…”

“…To test the resolving power of the screen, CCCP and apramycin were used as positive controls, respectively. These chemical treatments had Z' factors of 0.32 for CCCP (low transients 6 ), and a Z' factor of 0.28 for apramycin, an aminoglycoside that induces increased transients 19 (Fig 2A, Supp fig 2). Though the Z' score was < 0.5, we considered it sufficient to move forward into a genome wide screen by accepting potential false positives during the primary data collection.…”

“…Considering our current model, these data are also in agreement with the F1Fo-ATPase as a sink of membrane voltage. We expected that inhibiting or eliminating that pathway would result in a higher overall membrane potential and increased calcium transients 15 . Measurements with TMRM confirmed the higher membrane potential as well (Supp Fig 3).…”

“…The constitutive GCaMP-mScarlet plasmid was generated in a previous publication 19 and is available on Addgene (#158979). The plasmid was transfected into E. coli knockouts by using the Transformation and Storage Solution (TSS) buffer in a 96 well plate.…”

“…In bacteria, intracellular calcium concentration changes have been associated with infection 7 , cell division 8 , motility 9,10 , along with other important cellular processes 3 . Bacteria are known to tightly regulate the cytoplasmic calcium levels 11,12 , while changes in the chemical or mechanical environment have been shown to induce calcium transients 6,[13][14][15] . Given the overwhelming importance of calcium in eukaryotes, as well as the many potential bacterial signals, it is critical to understand exactly how and why bacteria regulate calcium levels, as well as the proteins that bind calcium and exert calcium-responsive cellular functions.…”