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Activated ClpP kills persisters and eradicates a chronic biofilm infection

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Abstract

Chronic infections are difficult to treat with antibiotics but are caused primarily by drug-sensitive pathogens. Dormant persister cells that are tolerant to killing by antibiotics are responsible for this apparent paradox. Persisters are phenotypic variants of normal cells and pathways leading to dormancy are redundant, making it challenging to develop anti-persister compounds. Biofilms shield persisters from the immune system, suggesting that an antibiotic for treating a chronic infection should be able to eradicate the infection on its own. We reasoned that a compound capable of corrupting a target in dormant cells will kill persisters. The acyldepsipeptide antibiotic (ADEP4) has been shown to activate the ClpP protease, resulting in death of growing cells. Here we show that ADEP4-activated ClpP becomes a fairly nonspecific protease and kills persisters by degrading over 400 proteins, forcing cells to self-digest. Null mutants of clpP arise with high probability, but combining ADEP4 with rifampicin produced complete eradication of Staphylococcus aureus biofilms in vitro and in a mouse model of a chronic infection. Our findings indicate a general principle for killing dormant cells—activation and corruption of a target, rather than conventional inhibition. Eradication of a biofilm in an animal model by activating a protease suggests a realistic path towards developing therapies to treat chronic infections.

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Figure 1
Figure 2: Quantitative proteomic analysis of S. aureus cells treated with ADEP4 reveals extensive protein degradation.
Figure 3: ADEP4 kills persisters.
Figure 4: ADEP4 with rifampicin eradicates a variety of S. aureus strains.
Figure 5: ADEP4 kills a S. aureus biofilm and in combination with rifampicin eradicates the population.
Figure 6: ADEP4 in combination with rifampicin eradicates a deep-seated mouse biofilm infection.

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Acknowledgements

We thank B. Wright and C. Blinn of AstraZeneca for assisting with the establishment of the mouse infection model, R. E. Lee, M. Pollastri and Z. Maglika for critical discussions and advice, I. Keren and S. Rowe for reading of the manuscript, H. Brewer, V. Petyuk and D. Camp II for assistance with proteomics, and Z. Zheng for assistance with ChemDraw. This work was supported by NIH award T-RO1 AI085585 to K.L., by Arietis Corporation to M.D.L and K.C., by the NIH-NIAID IAA Y1-AI-8401 to J.N.A. and P41 GM103493-11 to R.D.S. Proteomic analysis was performed in the EMSL, a DOE-BER national scientific user facility at Pacific Northwest National Laboratory (PNNL). PNNL is a multi-program national laboratory operated by Battelle Memorial Institute for the DOE under contract DE-AC05-76RLO 1830.

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Contributions

B.P.C., M.D.L., K.C. and K.L. designed the study, analysed results and wrote the manuscript. B.P.C. performed in vitro antibiotic susceptibility assays, collected samples for proteomics, and performed biofilm susceptibility studies and mouse infection models. V.M.I. assisted with in vitro susceptibility assays. E.S.N. and J.N.A. performed i-TRAQ proteomics and analysed results. L.E.F. participated in mouse infection model experiments. S.N.L performed hollow-fibre experiments. M.D.L. was responsible for histopathology. R.D.S. provided the proteomics measurement capabilities.

Corresponding author

Correspondence to K. Lewis.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Combinations of conventional antibiotics against stationary phase S. aureus.

Data are representative of 3 independent experiments. Error bars represent s.d.

Extended Data Figure 2 ADEP4 resistant strains are heat sensitive.

Wild-type S. aureus ATCC 33591 and 9 ADEP4 resistant isolates with mutations in clpP were grown for 20 h in MHB at 44 °C in 96-well polystyrene plates. Data are representative of 3 independent experiments. Error bars represent s.d.

Extended Data Figure 3 ADEP4 with rifampicin eradicates S. aureus in a hollow-fibre infection model.

Antibiotics were delivered at concentrations mimicking human dosing, while the concentration of ADEP was varied over time to match the pharmacokinetics in the mouse model. Data are representative of 3 independent experiments. Error bars represent s.d.

Extended Data Figure 4 Conventional bactericidal antibiotics target active processes in bacterial cells (green) resulting in death.

In a dormant persister (blue), the antibiotic binds an inactive target, producing no effect. ADEP4 activates and dysregulates ClpP in growing cells and in dormant persisters, resulting in eradication of the bacterial population.

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41586_2013_BFnature12790_MOESM98_ESM.mp3

Researcher Kim Lewis discusses how a compound could help treat the chronic infections that antibiotics fail to fight off.

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Conlon, B., Nakayasu, E., Fleck, L. et al. Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503, 365–370 (2013). https://doi.org/10.1038/nature12790

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