Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli

Abstract

For almost half of a century, we have known that aminoglycoside antibiotics corrupt ribosomes, causing translational misreading, yet it remains unclear whether or not misreading triggers protein misfolding, and possible effects of chaperone action on drug susceptibilities are poorly understood. Here, we show that aminoglycosides cause cytosolic protein misfolding and that chaperonin GroEL/GroES overexpression counters this defect. During aminoglycoside exposure to exponential cultures, chaperonin overexpression protected the bacterial membrane potential, rescued cell growth, and facilitated survival, whereas inhibition of chaperonin expression sensitized bacteria. Overexpression of the DnaK/DnaJ/GrpE chaperone system similarly facilitated survival but did not promote growth of aminoglycoside-treated bacteria. Inhibition of chaperonin expression sensitized bacteria to aminoglycosides as measured by reduced minimum inhibitory concentrations, whereas GroEL/GroES overexpression did not increase minimum inhibitory concentrations. Our observations establish misfolding of cytosolic proteins as an effect of aminoglycoside action and reveal that chaperones, chaperonins in particular, help bacteria cope during early exposure to these drugs.

FIGURE 1.

Aminoglycoside exposure causes bacterial growth inhibition and protein misfolding. A and B, growth and GFP fluorescence, respectively, of MG1655 cells expressing GFP following titration with gentamicin as indicated (n = 2). RFU, relative fluorescence units. C, Western blot of GFP solubility after aminoglycoside treatment for 4 h. The following gentamicin (Gen), streptomycin (Str), kanamycin (Kan), and spectinomycin (Spc) concentrations were used: without (w/o) antibiotic (lanes 1 and 2); 1.5 μg/ml gentamicin, 2 μg/ml streptomycin, 2.5 μg/ml kanamycin, and 10 μg/ml spectinomycin (lanes 3 and 4); 2 μg/ml gentamicin, 4 μg/ml streptomycin, 5 μg/ml kanamycin, and 25 μg/ml spectinomycin (lanes 5 and 6); 2.5 μg/ml gentamicin, 5 μg/ml streptomycin, 7.5 μg/ml kanamycin, and 50 μg/ml spectinomycin (lanes 7 and 8); and 3 μg/ml gentamicin, 10 μg/ml streptomycin, 10 μg/ml kanamycin, and 400 μg/ml spectinomycin (lanes 9 and 10). s and p signify soluble and insoluble cell lysate fractions, respectively. D, Western blot of β-galactosidase following aminoglycoside treatment for 4 h. Lanes 1 and 2, without antibiotic; lanes 3 and 4, 3 μg/ml gentamicin, 5 μg/ml streptomycin, and 50 μg/ml spectinomycin; lanes 5 and 6, 5 μg/ml gentamicin, 10 μg/ml streptomycin, and 100 μg/ml spectinomycin.

FIGURE 2.

Chaperonin overexpression mediates bacterial growth and protein folding upon aminoglycoside exposure. Shown is the growth of MG1655 coexpressing GFP and GroEL/GroES or DnaK/DnaJ/GrpE for 4 h after addition of 20 μg/ml streptomycin (Str; A), 5 μg/ml gentamicin (Gen; B), or 15 μg/ml kanamycin (Kan; C) (n = 2). D, GFP solubility as detected by Western blotting. s and p signify soluble and insoluble cell lysate fractions, respectively. w/o, without.

FIGURE 3.

Chaperone overexpression rescues gentamicin-challenged bacteria. A, cell survival of MG1655 overexpressing GroEL/GroES or DnaK/DnaJ/GrpE during 4 h of treatment with 5 μg/ml gentamicin. B, highlight of survival differences in aminoglycoside-treated GroEL/GroES-overexpressing versus DnaK/DnaJ/GrpE-overexpressing strains (n = 2 (GroEL/GroES), n = 2 (DnaK/DnaJ/GrpE), and n = 4 (empty control)). *, p = 0.0188 for the 4-h data point; **, p = 0.0027; ***, p = 0.0002.

FIGURE 4.

Inhibition of GroEL/GroES expression sensitizes bacteria to aminoglycosides. A, membrane potential following a 4-h treatment with gentamicin. A.U., arbitrary units. B, cell survival time course following treatment with 3 μg/ml gentamicin. ****, p < 0.0001 (n = 2) for the 4-h data point. C, membrane potential following a 4-h treatment with spectinomycin. D, cell survival time course following treatment with 200 μg/ml spectinomycin.

FIGURE 5.

Model of chaperone effects on bacterial killing and survival during early aminoglycoside exposure. An initial limited uptake of aminoglycoside (pink stars) across the intact bacterial envelope and binding to ribosomes promote translational misreading (orange asterisks). Misread polypeptides are encountered co- or post-translationally by several chaperones acting in succession. Limiting chaperone capacity results in protein misfolding, promoting cell killing via membrane permeabilization and possibly depletion of essential cytosolic activities. In contrast, elevated chaperone capacity (GroEL/GroES in particular) shifts the folding trajectory of (essential) misread proteins toward productive folding, hence increasing bacterial survival. om, outer membrane; im, inner membrane; J and K, DnaK/DnaJ/GrpE chaperone system.