Abstract

A challenge in the treatment of Staphylococcus aureus infections is the high prevalence of methicillin-resistant S. aureus (MRSA) strains and the formation of non-growing, dormant ‘persister’ subpopulations that exhibit high levels of tolerance to antibiotics1,2,3 and have a role in chronic or recurrent infections4,5. As conventional antibiotics are not effective in the treatment of infections caused by such bacteria, novel antibacterial therapeutics are urgently required. Here we used a Caenorhabditis elegans–MRSA infection screen6 to identify two synthetic retinoids, CD437 and CD1530, which kill both growing and persister MRSA cells by disrupting lipid bilayers. CD437 and CD1530 exhibit high killing rates, synergism with gentamicin, and a low probability of resistance selection. All-atom molecular dynamics simulations demonstrated that the ability of retinoids to penetrate and embed in lipid bilayers correlates with their bactericidal ability. An analogue of CD437 was found to retain anti-persister activity and show an improved cytotoxicity profile. Both CD437 and this analogue, alone or in combination with gentamicin, exhibit considerable efficacy in a mouse model of chronic MRSA infection. With further development and optimization, synthetic retinoids have the potential to become a new class of antimicrobials for the treatment of Gram-positive bacterial infections that are currently difficult to cure.

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Acknowledgements

This study was supported by National Institutes of Health grant P01 AI083214 to M.S.G., F.M.A. and E.M., by National Science Foundation grant CMMI-1562904 to H.G., and by National Institute of General Medical Sciences grant 1R35GM119426 and National Science Foundation grant NSF1755698 to W.M.W. D.V.T. is supported by National Eye Institute grant EY028222. We thank the Institute of Chemistry and Cell Biology-Longwood at Harvard Medical School for providing the chemical libraries used in this study. We thank L. Rice for providing the E. faecium strains, K. Bayles and J. Endres for providing plasmid pBK123, J. Saavedra for assistance with next-generation sequencing library preparation, and S. Khalid for providing the atomic structures and force fields of the phosphatidylglycerol, Lys-PG and DPG lipids. The simulations reported were performed on resources provided by the Extreme Science and Engineering Discovery Environment through grant MSS090046 and the Center for Computation and Visualization at Brown University.

Author information

Affiliations

  1. Division of Infectious Diseases, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island 02903, USA

    • Wooseong Kim
    • , Gabriel Lambert Hendricks
    • , Steven Shen
    • , Wen Pan
    • , Kiho Lee
    • , Rajmohan Rajamuthiah
    • , Beth Burgwyn Fuchs
    •  & Eleftherios Mylonakis
  2. School of Engineering, Brown University, Providence, Rhode Island 02903, USA

    • Wenpeng Zhu
    • , Nico Fricke
    •  & Huajian Gao
  3. Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA

    • Daria Van Tyne
    •  & Michael S. Gilmore
  4. Department of Microbiology and Immunobiology, Harvard Medical School, Massachusetts 02115, USA

    • Daria Van Tyne
    •  & Michael S. Gilmore
  5. Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA

    • Andrew D. Steele
    • , Colleen E. Keohane
    •  & William M. Wuest
  6. Emory Antibiotic Resistance Center, Emory University, Atlanta, Georgia 30322, USA

    • Andrew D. Steele
    • , Colleen E. Keohane
    •  & William M. Wuest
  7. Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    • Annie L. Conery
    •  & Frederick M. Ausubel
  8. Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Annie L. Conery
    •  & Frederick M. Ausubel
  9. Department of Engineering Sciences and Applied Mathematics, Northwestern University, Evanston, Illinois 60208, USA

    • Petia M. Vlahovska

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Contributions

W.K., A.L.C., R.R., B.B.F., F.M.A. and E.M. designed the chemical screen. W.K., B.B.F. and R.R. performed the chemical screen. W.K. designed, performed and analysed MIC assays, dose–response C. elegans infection assays, membrane permeability assays, time-kill assays and transmission electron microscopy experiments. W.K. and D.V.T. designed, performed and analysed the selection of resistant mutants and whole genome sequencing. W.K., N.F. and P.M.V. designed, performed and analysed giant unilamellar vesicle experiments. W.K., W.Z. and H.G. designed, performed and analysed molecular dynamics simulations. A.D.S., C.E.K. and W.M.W. synthesized analogues. W.K. and B.B.F. designed, performed and analysed toxicity tests. W.K., G.L.H., S.S., W.P. and K.L. designed, performed and analysed animal studies. A.L.C., B.B.F., P.M.V., W.M.W., M.S.G., H.G., F.M.A. and E.M. contributed reagents, materials and/or analysis tools. E.M. supervised the project. W.K., W.Z., G.L.H., W.M.W., H.G., F.M.A. and E.M. wrote the manuscript.

Competing interests

F.M.A. and E.M. have interests in Genma Biosciences, Inc. and Octagon Therapeutics, Inc., companies that are engaged in developing antimicrobial compounds. The interests of E.M. and F.M.A. were reviewed and are managed by Rhode Island Hospital (E.M.) and Massachusetts General Hospital and Partners HealthCare (F.M.A.) in accordance with their conflict of interest policies. The remaining authors declare no competing interests.

Corresponding author

Correspondence to Eleftherios Mylonakis.

Reviewer Information Nature thanks F. DeLeo and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Information

    This file contains Supplementary Tables 1-9, full legends for Supplementary Videos 1-17, a Supplementary Discussion, Supplementary Methods, Supplementary Figure 1, Supplementary References and NMR Spectra (Supplementary Figures 2-61).

Zip files

  1. 1.

    Supplementary Videos 1-5

    CD437, CD1530, and adarotene disrupt GUVs. GUVs consisting of DOPC/DOPG (7:3) labeled with 18:1 Liss Rhod PE (0.05%) were treated with 10 µg/ml (10X MIC) CD437 (Supplementary Video 1), 10 µg/ml (10X MIC) CD1530 (Supplementary Video 2), 20 µg/ml (10X MIC) adarotene (Supplementary Video 3), 20 µg/ml adapalene (Supplementary Video 4), or 0.1% DMSO (Supplementary Video 5). After adding compounds at t=0 sec, changes in each GUV were recorded using a fluorescent microscope (63x objective, Ex=460 nm, Em=483 nm). Experiments were repeated 3 times with similar results.

  2. 2.

    Supplementary Videos 6-9

    Molecular dynamics of CD437 (Supplementary Video 6), CD1530 (Supplementary Video 7), adarotene (Supplementary Video 8) and adapalene (Supplementary Video 9) interacting with mixed 108PG/72lys PG/10DPG lipid bilayers. In the videos of MD simulations, the retinoids and sodium ions are depicted as large spheres, and phospholipids are represented as chains. The atoms in retinoids, phospholipids and sodium ions are colored as follows: hydrogen, white; oxygen, red; nitrogen, dark blue; carbon, cyan; phosphorus, orange; sodium, lavender. Water molecules are set to be transparent for clarity. The outer blue lines indicate the period boundaries of the simulation boxes. Simulations were repeated 5 times with similar results.

  3. 3.

    Supplementary Videos 10-13

    Molecular dynamics of CD437 (Supplementary Video 10), CD1530 (Supplementary Video 11), adarotene (Supplementary Video 12) and adapalene (Supplementary Video 13) interacting with mixed lipid bilayers at a DOPC:DOPG ratio of 7:3. In the videos of MD simulations, the retinoids and sodium ions are depicted as large spheres, and phospholipids are represented as chains. The atoms in retinoids, phospholipids and sodium ions are colored as follows: hydrogen, white; oxygen, red; nitrogen, dark blue; carbon, cyan; phosphorus, orange; sodium, lavender. Water molecules are set to be transparent for clarity. The outer blue lines indicate the period boundaries of the simulation boxes. Simulations were repeated 5 times with similar results.

  4. 4.

    Supplementary Videos 14-15

    Molecular dynamics of CD437 carboxylic glucuronide (Supplementary Video 14) and phenolic hydroxyl glucuronide (Supplementary Video 15) interacting with mixed lipid bilayers at a DOPC:DOPG ratio of 7:3. In the videos of MD simulations, the CD437-glucuronides and sodium ions are depicted as large spheres, and phospholipids are represented as chains. The atoms in CD437-glucuronides, phospholipids and sodium ions are colored as follows: hydrogen, white; oxygen, red; nitrogen, dark blue; carbon, cyan; phosphorus, orange; sodium, lavender. Water molecules are set to be transparent for clarity. The outer blue lines indicate the period boundaries of the simulation boxes. Simulations were repeated 5 times with similar results.

  5. 5.

    Supplementary Videos 16-17

    Supplementary Video 16 shows the molecular dynamics of Analog 2 interacting with mixed 108PG/72lys PG/10DPG lipid bilayers. In the videos of MD simulations, Analog 2 and sodium ions are depicted as large spheres, and phospholipids are represented as chains. The atoms in Analog 2, phospholipids and sodium ions are colored as follows: hydrogen, white; oxygen, red; nitrogen, dark blue; carbon, cyan; phosphorus, orange; sodium, lavender. Water molecules are set to be transparent for clarity. The outer blue lines indicate the period boundaries of the simulation boxes. Simulations were repeated 5 times with similar results. Supplementary Video 17 shows the molecular dynamics of Analog 3 interacting with mixed lipid bilayers at a DOPC:DOPG ratio of 7:3. In the videos of MD simulations, Analog 3 and sodium ions are depicted as large spheres, and phospholipids are represented as chains. The atoms in Analog 3, phospholipids and sodium ions are colored as follows: hydrogen, white; oxygen, red; nitrogen, dark blue; carbon, cyan; phosphorus, orange; sodium, lavender. Water molecules are set to be transparent for clarity. The outer blue lines indicate the period boundaries of the simulation boxes. Simulations were repeated 5 times with similar results.

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https://doi.org/10.1038/nature26157

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