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. 2012 Apr 20;336(6079):315-9.
doi: 10.1126/science.1219192.

Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics

James J Foti  1 , Babho Devadoss, Jonathan A Winkler, James J Collins, Graham C Walker
Affiliations

Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics

James J Foti et al. Science. .

Abstract

A detailed understanding of the mechanisms that underlie antibiotic killing is important for the derivation of new classes of antibiotics and clinically useful adjuvants for current antimicrobial therapies. Our efforts to understand why DinB (DNA polymerase IV) overproduction is cytotoxic to Escherichia coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell death caused by both DinB overproduction and bactericidal antibiotics. We propose a model in which the cytotoxicity of beta-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine lesions, whereas the cytotoxicity of aminoglycosides might additionally result from mistranslation due to the incorporation of 8-oxo-guanine into newly synthesized RNAs.

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Figures

Fig. 1
Fig. 1
Evidence indicating that overproduction of DinB results in DSBs due to closely spaced 8-oxo-dG lesions. A. Lethality of DinB overproduction (black), measured by colony forming units (CFU) per milliliter (ml) relative to time zero, is reduced by thiourea (red), 2,2′-dipyridyl (green), and co-overproduction of MutT (blue). Overproduction of DinB F13V (yellow), which is incapable of incorporating 8-oxo-dG, is not lethal. B. Under anaerobic conditions, DinB overproduction is not cytotoxic; cell viability was assayed by ten-fold serial dilutions of cells overproducing DinB (pDinB + IPTG) and compared to non-induced (pDinB) and vector controls (Vector and Vector + IPTG). C. DinB and DinB F13V (F13V) primer extension analysis using dGTP and 8-oxo-dGTP as the incoming nucleotide and the four different templating bases. The starting primer (P) and extended product (E) are indicated. Lanes 1, 6, 11, and, 16 are unextended primer controls. D. Overproduction of DinB for 3 hours (blue) results in cell filamentation, but does not result in a substantial increase in intracellular OH• levels when compared to an uninduced control (red). The forward-scatter histogram (Cell Size arbitrary units; left) of a DinB overproducing strain suggests cell elongatation. Conversely, a modest increase in the HPF fluorescence signal (1.6 fold) compared to the control is observed in a DinB overproducing strain suggesting that intracellular OH• levels do not substantially increase (OH Signal arbitrary units; right). E. The lethality of DinB overproduction (black) is minimized in a ΔmutM ΔmutY background (purple), but enhanced in a ΔrecA background (blue).
Fig. 2
Fig. 2
The sensitivity of wild-type E. coli cells to killing by ampicillin, norfloxacin, and kanamycin is reduced when incorporation of 8-oxo-dG is minimized. A. Overproduction of the 8-oxo-dGTP sanitizer MutT (blue) in wild-type MG1655 cells was sufficient to significantly reduce the sensitivity of cells to the bactericidal effects of all three classes of drugs compared to the vector control (black). B. Overproduction of the alternative 8-oxo-dGTP sanitizer RibA (yellow) in MG1655 cells is also sufficient to reduce the sensitivity of cells to ampicillin and norfloxacin, but not kanamycin, probably due to its instability (Fig. S5). C. Overproduction of the 8-OH-dATP sanitizer NudB (green) does not reduce the antibiotic sensitivity. D. A mutant strain which lacks the two Y-family DNA polymerases, and expresses an anti-mutator replicative polymerase (dnaE911 ΔdinB ΔumuDC) strain (red) is more resistant to killing by bactericidal antibiotics than wild-type cells (black).
Fig. 3
Fig. 3
Evidence that bactericidal antibiotics cause lethal DSBs. A. A ΔmutM ΔmutY strain is less sensitive than wild-type cells to bactericidal antibiotic killing. B. Deletion of ΔrecA and ΔrecB sensitizes cells to killing by bactericidal antibiotics. Relative CFU/ml of wild-type (black), ΔrecA (red), and ΔrecB (green) cells treated with 2 μg/ml ampicillin, 25 ng/ml norfloxacin, and 3 μg/ml kanamycin. C. Neither ampicillin nor kanamycin treatments for 30 minutes significantly reduce the number of viable wild-type or ΔmutM ΔmutY cells. Norfloxacin treatment for 30 minutes results in a reduction in viable cell number for both wild-type (29% survival) and ΔmutM ΔmutY cells (51% survival). D. Representative fields of wild-type cells after 30 minutes of treatment. Cells containing a DSB (TUNEL positive; green) were overlaid on the propidium iodine staining of all cells (red). Bar = 5 μM. E. Pseudocolor plot of cell size versus TUNEL fluorescence for untreated and treated wild-type and ΔmutM ΔmutY cells at 30 minutes. The vertical line at 10 fluorescence units is the cutoff of TUNEL positive cells which results in 30% of untreated wild-type cells being TUNEL positive consistent with our microscopy results (ca. 25% TUNEL positive: 81 green/292 cells). The number of TUNEL positive wild-type cells increases after antibiotic treatment (top), and ΔmutM ΔmutY cells (bottom) have fewer TUNEL positive cells compared to wild-type. Both cell size (forward scatter) and fluorescence signal (TUNEL signal) are in arbitrary units (A.U.). F. Cell histograms of wild-type (red) and ΔmutM ΔmutY (blue) TUNEL stained cells suggest that ΔmutM ΔmutY cells have fewer dsDNA breaks. G. The percent positive TUNEL labeled cells (≥10 A.U.) determined in panel E is plotted for both wild-type (red) and ΔmutM ΔmutY (blue). H. Plot of the median TUNEL signal for the wild-type and ΔmutM ΔmutY populations shown in panel F.
Fig. 4
Fig. 4
Relative protective effect against killing due to MutT overproduction, mutation of three DNA polymerases (dnaE911 ΔdinB ΔumuDC), and ΔmutM ΔmutY for β-lactams, norfloxacin, and kanamycin suggests oxidation of guanine mediates bactericidal antibiotic-induced cell death. For β-lactams (ampicillin and penicillin), a similar fold rescue is observed for all three conditions. For norfloxacin, a similar degree of protection is afforded by MutT overproduction and mutation of the DNA polymerases, but a lesser degree of protection is observed for ΔmutM ΔmutY. For kanamycin, the greater fold rescue associated with MutT overproduction compared to dnaE911 ΔdinB ΔumuDC and ΔmutM ΔmutY suggests oxidation of the guanine to 8-oxo-rGTP is additionally contributing to cell death.
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