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Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production

Abstract

The ever-increasing incidence of antibiotic-resistant infections combined with a weak pipeline of new antibiotics has created a global public health crisis1. Accordingly, novel strategies for enhancing our antibiotic arsenal are needed. As antibiotics kill bacteria in part by inducing reactive oxygen species (ROS)2,3,4, we reasoned that targeting microbial ROS production might potentiate antibiotic activity. Here we show that ROS production can be predictably enhanced in Escherichia coli, increasing the bacteria's susceptibility to oxidative attack. We developed an ensemble approach of genome-scale, metabolic models capable of predicting ROS production in E. coli. The metabolic network was systematically perturbed and its flux distribution analyzed to identify targets predicted to increase ROS production. Targets that were predicted in silico were experimentally validated and further shown to confer increased susceptibility to oxidants. Validated targets also increased susceptibility to killing by antibiotics. This work establishes a systems-based method to tune ROS production in bacteria and demonstrates that increased microbial ROS production can potentiate killing by oxidants and antibiotics.

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Figure 1: Systems approach to enhance microbial ROS production.
Figure 2: In silico predictions and experimental measures of H2O2 and O2 levels.
Figure 3: Evaluation of susceptibility to killing by oxidants.
Figure 4: Evaluation of susceptibility to killing by bactericidal antibiotics and combination treatments with a chemical inhibitor.

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Acknowledgements

This work was supported by the National Institutes of Health Director′s Pioneer Award Program and the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

M.P.B., J.A.W. and J.J.C. designed the study, analyzed the results and wrote the manuscript. Experiments were done by M.P.B., J.A.W., C.S.S. and I.C.M.

Corresponding author

Correspondence to James J Collins.

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Competing interests

The authors have filed US Patent Application no. 61/583,662 covering the method described in the paper.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Figures 1–4 (PDF 596 kb)

Supplementary Data File 1

Constants used to couple ROS generating reactions to their intended reactions within iAF1260 (XLS 9555 kb)

Supplementary Table 1

Potential ROS-generation reactions within iAF1260 (XLSX 24 kb)

Supplementary Table 2

ROS generation within iAF1260 (XLSX 9 kb)

Supplementary Table 3

Enzymes and regulators turned off due to transcriptional regulation in aerobic minimal glucose media (XLSX 31 kb)

Supplementary Table 4

ROS/BM for genetic selections that altered ROS flux (XLSX 11 kb)

Supplementary Table 5

GFP/BM measurements for deletion strains with reporter plasmids (XLSX 10 kb)

Supplementary Table 6

Relative (500nm/420nm) fluorescence ratio of mutant strains (XLSX 9 kb)

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Brynildsen, M., Winkler, J., Spina, C. et al. Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production. Nat Biotechnol 31, 160–165 (2013). https://doi.org/10.1038/nbt.2458

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