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

  1. Giancarlo Noe Bruni
  2. Joel M Kralj  Is a corresponding author
  1. BioFrontiers Institute and the Department of Molecular, Cellular, Developmental Biology, University of Colorado Boulder, United States
5 figures, 3 videos, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Voltage is not necessary for aminoglycoside uptake or inner membrane pore formation in E. coli but is required for bactericidal activity.

(A) Colony forming units (CFUs) of untreated cells (blue) over four time points compared to cells treated with 50 µM CCCP (yellow), 100 µg/mL kanamycin (orange), and 50 µM CCCP + 100 µg/mL kanamycin …

Figure 1—figure supplement 1
Aminoglycosides enter cells and induce ribosomal dissociation in the abscence of membrane voltage.

(A) Ribosomal sucrose gradient depth plotted against 254 nm absorbance from E. coli in treatment conditions from Figure 1C. (B) Ratio of the area under the curve for the 30S + 50S to 70S peaks from …

Figure 2 with 4 supplements
Voltage and calcium exhibit altered electrophysiological flux in response to aminoglycosides.

(A) Time traces of GCaMP6 fluorescence from single cells treated with 0 µg/mL (blue shades) and 100 µg/mL (orange shades) kanamycin. Individual cells display non-oscillatory transients. (B) The …

Figure 2—figure supplement 1
Kanamycin induces voltage and calcium transients.

(A) A histogram of the fraction of cells with a given standard deviation of PROPS fluorescence in the absence (blue) and presence (orange) of 100 μg/mL kanamycin. (B,C) Untreated cells have …

Figure 2—figure supplement 2
Calculating moving standard deviation from treated cells.

(A) Top - GCaMP6 intensity traces from single cells marked by different colors under no external antibiotic concentrations. Bottom – Moving standard deviation traces from the same cells in the top, …

Figure 2—figure supplement 3
Only aminoglycosides induce calcium transients.

(A) All aminoglycosides tested (kanamycin, streptomycin, apramycin, gentamicin) exhibit calcium transients in a concentration dependent fashion. For each compound tested, the fraction of cells …

Figure 2—figure supplement 4
High pH is necessary to induce calcium transients.

(A) The average of the moving SD from a population of cells at pH 5.95 and pH 7.99, either treated with 0 or 100 µg/mL kanamycin. Each curve averages three biological replicates. Shading around the …

Figure 3 with 1 supplement
Single-cell calcium flux predicts cellular aminoglycoside response.

(A) Strip chart of cells expressing GCaMP6f. Cells were imaged in PMM alone for 5 hr, then exposed to 10 µg/mL kanamycin for 4 hr. After 4 hr, PMM alone was flowed in for an additional 26 hr. The …

Figure 3—figure supplement 1
Cells that did not experience calcium transients are not genetically resistant.

(A–C) Cells that regrow after 1 treatment of kanamycin are not genetically resistant. (A) Strip chart of E. coli with 0 µg/mL kanamycin (t = 0–2 hr), 10 µg/mL kanamycin (t = 2–6 hr), 0 µg/mL …

Figure 4 with 1 supplement
Voltage toggles between bactericidal and bacteriostatic activity in aminoglycoside-treated cells.

(A) Single-cell traces of GCaMP6f intensity over time upon treatment with kanamycin (blue bar, top), or with kanamycin followed by CCCP (yellow bar, bottom). (B) Mean GCaMP6f moving SD of biological …

Figure 4—figure supplement 1
Catastrophic calcium transients require a membrane potential as indicated by low pH protection.

(A–D) Catastrophic calcium transients require a membrane potential as indicated by low pH protection. (A) Random single-cell traces of GCaMP6f intensity over time upon treatment with kanamycin (pink …

Figure 5 with 3 supplements
ATP dysregulation precedes voltage-induced bactericidal killing.

(A) iATPSnFR ratios from E. coli treated with vehicle (blue) or 10 µg/mL gentamicin (orange). The ratio of iATPSnFR (488 nm) to mRuby (561 nm) indicates ATP concentration. Each trace averages two …

Figure 5—figure supplement 1
ATP and membrane potential measurements are consistent with ATP dysregulation.

(A,B) Low pH and CCCP maintain the rise in the iATPSnFR1.0/mRuby ratio upon gentamicin treatment when compared to controls. (A) ATP measured in PMM pH 7.5 (blue) or low pH (orange) upon treatment …

Figure 5—figure supplement 2
Basal membrane potential measured in strains tested that protect against aminoglycosides does not explain the protective effect.

(A) Violin plots of individual clonal populations of E. coli treated with 200 nM TMRM measured by flow cytometry, with additional strain and treatment indicated on the x-axis. CCCP concentration is …

Figure 5—figure supplement 3
Proposed model of aminoglycoside-induced cell death in E. coli.

(1) Uptake of aminoglycosides has been extensively studied and is not shown in detail here, although our data indicates that aminoglycoside uptake can occur in the absence of membrane potential. (2) …

Videos

Video 1
Video of E. coli expressing GCaMP6f-mScarlet upon treatment with 100 µg/mL kanamycin.

The movie was taken using 488 nm excitation and a 40x air objective imaged onto an sCMOS camera. The movie was taken at a sampling rate of 1 image per minute for 16 hr. This movie has been corrected …

Video 2
Video of E. coli expressing GCaMP6f-mScarlet with no kanamycin addition.

The video was taken using 488 nm excitation and a 40x air objective imaged onto an sCMOS camera. The movie was taken at a sampling rate of 1 image per minute for 16 hr. This movie has been corrected …

Video 3
Video of E. coli expressing GCaMP6f-mScarlet switching the medium from PMM (0–5 hr), PMM + 10 µg/mL kanamycin (5–9 hr), PMM (9–35 hr).

The movie was taken at a sampling rate of 1 image per minute for 29 hr. This movie has been corrected for uneven illumination, XY drift, and background as mentioned in the Materials and methods. The …

Tables

Table 1
Measurements of basal membrane voltage for protective strains
Strain + treatmentFluorescent dyeMean
emission (AU)
SD of
emission (AU)
p-value (T-test)Voltage estimates
(mV)
Voltage estimate
variance
BW+CCCPTMRM399.86104.090.02−133.66−34.79
BW+NoneTMRM716.6747.761−150−10
DK8+NoneTMRM1123.41218.670.08−162.59−31.65
mgtC118+NoneTMRM1062.52202.430.09−161.03−30.68
BW+CCCPTMRM380.7423.37<0.01−130.17−7.99
BW+pH6.0TMRM535.7224.2<0.01−139.73−6.31
BW+pH7.5TMRM773.0418.51−150−3.59
DK8+pH7.5TMRM1674.418.67<0.01−171.64−0.89
BW+CCCPDiOC617.432.550.01−57.69−8.45
BW+NoneDiOC6471.0762.411−150−19.87
DK8+NoneDiOC6724.67162.210.1−162.06−36.27
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)E. coli K-12 BW25113Yale Coli Genetic Stock CenterCGSC#: 7636
Strain, strain background (Escherichia coli)BW25113 ΔnuoADharmacon KeioOEC4987-213603796
Strain, strain background (Escherichia coli)BW25113 ΔnuoBDharmacon KeioOEC4987-213603795
Strain, strain background (Escherichia coli)BW25113 ΔnuoHDharmacon KeioOEC4987-213603791
Strain, strain background (Escherichia coli)BW25113 ΔnuoIDharmacon KeioOEC4987-213603790
Strain, strain background (Escherichia coli)BW25113 ΔatpADharmacon KeioOEC4987-213606163
Strain, strain background (Escherichia coli)BW25113 ΔatpBDharmacon KeioOEC4987-213606167
Strain, strain background (Escherichia coli)BW25113 ΔatpCDharmacon KeioOEC4987-213605824
Strain, strain background (Escherichia coli)BW25113 ΔatpDDharmacon KeioOEC4987-213605825
Strain, strain background (Escherichia coli)BW25113 ΔatpEDharmacon KeioOEC4987-213606166
Strain, strain background (Escherichia coli)BW25113 ΔatpFDharmacon KeioOEC4987-213606165
Strain, strain background (Escherichia coli)BW25113 ΔatpGDharmacon KeioOEC4987-213607977
Strain, strain background (Escherichia coli)BW25113 ΔatpHDharmacon KeioOEC4987-213606164
Strain, strain background (Escherichia coli)E. coli DK8 1100∆(uncB-uncC)ilv::TnlORubinstein lab (created in Guo et al., 2019)DK8
Strain, strain background (Escherichia coli)BL21(DE3)Sigma-AldrichCMC0016Electrocompetent cells
Transfected construct (bacterial)Plasmid: pKL09-GCaMP6f-mScarlet bb118This paperAddgene #pendingGCaMP6-mScarlet
Transfected construct (bacterial)Plasmid: pKL10-mRuby-iATPSnFR1.0 bb118This paperAddgene # pendingmRuby-ATPSnfr
Transfected construct (bacterial)Plasmid: pKL12-GCaMP pHuji bb118This paperAddgene # pendingGCaMP6-pHuji
Transfected construct (bacterial)Plasmid: pKL13-MgtC bb118This paperAddgene # pendingS. typhimurium mgtC expression plasmid
Transfected construct (bacterial)Plasmid: pKL11-GCaMP6f bb100This paperAddgene # pendingGCaAMP6
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Chemical compound, drugHydrochloric acidSigma320331
Chemical compound, drugLow melt agaroseVWR97064–134
Chemical compound, drugSodium hydroxideSigma795429
Chemical compound, drugKanamycin sulfateSigma60615–5G
Chemical compound, drugGentamicin sulfateSigma345814
Chemical compound, drugApramycinSigmaA2024-1G
Chemical compound, drugStreptomycin sulfateSigmaS9137
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