An antibiotic from an uncultured bacterium binds to an immutable target

Rhythm Shukla; Aaron J Peoples; Kevin C Ludwig; Sourav Maity; Maik G N Derks; Stefania De Benedetti; Annika M Krueger; Bram J A Vermeulen; Theresa Harbig; Francesca Lavore; Raj Kumar; Rodrigo V Honorato; Fabian Grein; Kay Nieselt; Yangping Liu; Alexandre M J J Bonvin; Marc Baldus; Ulrich Kubitscheck; Eefjan Breukink; Catherine Achorn; Anthony Nitti; Christopher J Schwalen; Amy L Spoering; Losee Lucy Ling; Dallas Hughes; Moreno Lelli; Wouter H Roos; Kim Lewis; Tanja Schneider; Markus Weingarth

doi:10.1016/j.cell.2023.07.038

An antibiotic from an uncultured bacterium binds to an immutable target

Rhythm Shukla, Aaron J Peoples, Kevin C Ludwig, Sourav Maity, Maik G N Derks, Stefania De Benedetti, Annika M Krueger, Bram J A Vermeulen, Theresa Harbig, Francesca Lavore, Raj Kumar, Rodrigo V Honorato, Fabian Grein, Kay Nieselt, Yangping Liu, Alexandre M J J Bonvin, Marc Baldus, Ulrich Kubitscheck, Eefjan Breukink, Catherine AchornAnthony Nitti, Christopher J Schwalen, Amy L Spoering, Losee Lucy Ling, Dallas Hughes, Moreno Lelli, Wouter H Roos, Kim Lewis, Tanja Schneider^*, Markus Weingarth^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55PP, lipid II, and lipid III WTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Original language	English
Pages (from-to)	4059-4073.e27
Journal	Cell
Volume	186
Issue number	19
DOIs	https://doi.org/10.1016/j.cell.2023.07.038
Publication status	Published - 14 Sept 2023

Bibliographical note

Publisher Copyright:
© 2023 The Authors

Funding

This work was funded by the Netherlands Organisation for Scientific Research (NWO, grant numbers 723.014.003 and 711.018.001 to M.W. and 718.015.001 to A.M.J.J.B.). This project has received funding from the European Union's Horizon Europe grant and innovation programme under grant agreement no. 101045485 (to M.W.). Funding for T.S. K.C.L. F.G. S.D.B. T.H. K.N. U.K. and A.M.K. was provided by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project ID 398967434—TRR 261, and funding to T.S. and F.G. was provided by the German Center for Infection Research (DZIF). Funding was also provided by NIH grant AI136137 to L.L.L. NIH grant AI091224 to A.L.S. NIH grant P01AI118687 to A.L.S. and K.L. and NIH grant RO1AI170962 to K.L. NMR experiments at the 950 and 1,200 MHz instruments were supported by uNMR-NL, an NWO-Funded Roadmap NMR Facility (no. 184.032.207). Support by Instruct-ERIC (to M.L. and M.W.) is acknowledged. M.W. and M.L. acknowledge project INFRAIA-02-2020 PANACEA (H2020, contract no. 101008500). M.L. acknowledges the Fondazione CR Firenze for funding. This work has been supported BioExcel, grant numbers 675728 and 823830, funded by the Horizon 2020 program of the European Commission. The NRS strains were provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH. The authors also wish to acknowledge the help and expertise of the University of Maryland Core Genome Sequencing Facility, Micromyx LLC, Kalamazoo, MI, NeoSome Life Sciences, Billerica MA. The authors would like to thank the following scientists for their work on clovibactin at NovoBiotic Pharmaceuticals, LLC, Alysha Desrosiers, William Millett, Kelly Demeo, Ashley Zullo, and Cintia Felix. The plasmid pIJ12738 was a gift from the John Innes Centre. The authors further thank Dr. Andrei Gurinov (Utrecht University) for assistance during the DNP-ssNMR experiments and Marc Sylvester (University of Bonn) for the mass spectrometry support. K.L. T.S. D.H. L.L.L. and M.W. initiated the study. R.S. A.J.P. M.G.N.D. A.N. R.K. M.B. M.L. and M.W. did NMR experiments. A.N. isolated clovibactin. K.C.L. T.H. A.M.K. and S.D.B. did mode of action studies. R.S. and F.G. did fluorescence microscopy. A.M.K. and U.K. did single-molecule mobility measurements in supported bilayers. S.M. and W.H.R. did HS-AFM studies. A.L.S. performed the fermentations, gene knockout work, and isolated the DNA for sequencing. C.A. performed susceptibility and cytotoxicity assays. C.J.S. sequenced and annotated the genome. R.S. and M.W. did calorimetric studies. Y.L. provided research agents. R.S. M.G.N.D. and E.B. prepared ssNMR samples and lipid II. F.L. B.J.A.V. R.V.H. A.M.J.J.B. and M.W. did structure calculations. All authors contributed to data analysis and writing of the manuscript. The following authors, A.J.P. C.A. A.N. A.L.S. L.L.L. D.H. and K.L. declare competing financial interests because they are employees and consultants of NovoBiotic Pharmaceuticals. A patent US 11,203,616 B2 was issued on 12/21/2021 and describes the use of clovibactin (Novo29) and as an antibiotic, as well as the pharmaceutical composition and antibiotic use of derivatives. This work was funded by the Netherlands Organisation for Scientific Research (NWO, grant numbers 723.014.003 and 711.018.001 to M.W. and 718.015.001 to A.M.J.J.B.). This project has received funding from the European Union's Horizon Europe grant and innovation programme under grant agreement no. 101045485 (to M.W.). Funding for T.S., K.C.L., F.G., S.D.B., T.H., K.N., U.K., and A.M.K. was provided by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project ID 398967434 — TRR 261, and funding to T.S. and F.G. was provided by the German Center for Infection Research (DZIF). Funding was also provided by NIH grant AI136137 to L.L.L., NIH grant AI091224 to A.L.S., NIH grant P01AI118687 to A.L.S. and K.L., and NIH grant RO1AI170962 to K.L. NMR experiments at the 950 and 1,200 MHz instruments were supported by uNMR-NL , an NWO-Funded Roadmap NMR Facility (no. 184.032.207 ). Support by Instruct-ERIC (to M.L. and M.W.) is acknowledged. M.W. and M.L. acknowledge project INFRAIA-02-2020 PANACEA (H2020, contract no. 101008500). M.L. acknowledges the Fondazione CR Firenze for funding. This work has been supported BioExcel , grant numbers 675728 and 823830 , funded by the Horizon 2020 program of the European Commission . The NRS strains were provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH. The authors also wish to acknowledge the help and expertise of the University of Maryland Core Genome Sequencing Facility, Micromyx LLC, Kalamazoo, MI, NeoSome Life Sciences, Billerica MA. The authors would like to thank the following scientists for their work on clovibactin at NovoBiotic Pharmaceuticals, LLC, Alysha Desrosiers, William Millett, Kelly Demeo, Ashley Zullo, and Cintia Felix. The plasmid pIJ12738 was a gift from the John Innes Centre. The authors further thank Dr. Andrei Gurinov (Utrecht University) for assistance during the DNP-ssNMR experiments and Marc Sylvester (University of Bonn) for the mass spectrometry support.

Funders	Funder number
BEI Resources
European Union's Horizon Europe grant and innovation programme	101045485
INFRAIA-02-2020 PANACEA
John Innes Centre
NeoSome Life Sciences
NovoBiotic
National Institutes of Health	AI136137, AI091224, 184.032.207, P01AI118687, RO1AI170962
National Institute of Allergy and Infectious Diseases
Deutsches Zentrum für Infektionsforschung
National Alliance for Research on Schizophrenia and Depression
Horizon 2020 Framework Programme	101008500
European Commission
Deutsche Forschungsgemeinschaft	398967434 — TRR 261
Universiteit Utrecht
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	711.018.001, 718.015.001, 723.014.003
Rheinische Friedrich-Wilhelms-Universität Bonn
Fondazione Cassa di Risparmio di Firenze	823830, 675728

Keywords

Anti-Bacterial Agents/isolation & purification
Bacteria
Biological Assay
Diphosphates
Soil Microbiology

Access to Document

10.1016/j.cell.2023.07.038Licence: CC BY

PIIS009286742300853XFinal published version, 17.7 MBLicence: CC BY

Cite this

Shukla, R., Peoples, A. J., Ludwig, K. C., Maity, S., Derks, M. G. N., De Benedetti, S., Krueger, A. M., Vermeulen, B. J. A., Harbig, T., Lavore, F., Kumar, R., Honorato, R. V., Grein, F., Nieselt, K., Liu, Y., Bonvin, A. M. J. J., Baldus, M., Kubitscheck, U., Breukink, E., ... Weingarth, M. (2023). An antibiotic from an uncultured bacterium binds to an immutable target. Cell, 186(19), 4059-4073.e27. https://doi.org/10.1016/j.cell.2023.07.038

@article{907b6af7c14645ef86186e36b34c12b8,

title = "An antibiotic from an uncultured bacterium binds to an immutable target",

abstract = "Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55PP, lipid II, and lipid III WTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development. ",

keywords = "Anti-Bacterial Agents/isolation & purification, Bacteria, Biological Assay, Diphosphates, Soil Microbiology",

author = "Rhythm Shukla and Peoples, {Aaron J} and Ludwig, {Kevin C} and Sourav Maity and Derks, {Maik G N} and {De Benedetti}, Stefania and Krueger, {Annika M} and Vermeulen, {Bram J A} and Theresa Harbig and Francesca Lavore and Raj Kumar and Honorato, {Rodrigo V} and Fabian Grein and Kay Nieselt and Yangping Liu and Bonvin, {Alexandre M J J} and Marc Baldus and Ulrich Kubitscheck and Eefjan Breukink and Catherine Achorn and Anthony Nitti and Schwalen, {Christopher J} and Spoering, {Amy L} and Ling, {Losee Lucy} and Dallas Hughes and Moreno Lelli and Roos, {Wouter H} and Kim Lewis and Tanja Schneider and Markus Weingarth",

note = "Publisher Copyright: {\textcopyright} 2023 The Authors",

year = "2023",

month = sep,

day = "14",

doi = "10.1016/j.cell.2023.07.038",

language = "English",

volume = "186",

pages = "4059--4073.e27",

journal = "Cell",

issn = "0092-8674",

publisher = "Elsevier BV",

number = "19",

}

Shukla, R, Peoples, AJ, Ludwig, KC, Maity, S, Derks, MGN, De Benedetti, S, Krueger, AM, Vermeulen, BJA, Harbig, T, Lavore, F, Kumar, R, Honorato, RV, Grein, F, Nieselt, K, Liu, Y, Bonvin, AMJJ , Baldus, M, Kubitscheck, U, Breukink, E, Achorn, C, Nitti, A, Schwalen, CJ, Spoering, AL, Ling, LL, Hughes, D, Lelli, M, Roos, WH, Lewis, K, Schneider, T & Weingarth, M 2023, 'An antibiotic from an uncultured bacterium binds to an immutable target', Cell, vol. 186, no. 19, pp. 4059-4073.e27. https://doi.org/10.1016/j.cell.2023.07.038

TY - JOUR

T1 - An antibiotic from an uncultured bacterium binds to an immutable target

AU - Shukla, Rhythm

AU - Peoples, Aaron J

AU - Ludwig, Kevin C

AU - Maity, Sourav

AU - Derks, Maik G N

AU - De Benedetti, Stefania

AU - Krueger, Annika M

AU - Vermeulen, Bram J A

AU - Harbig, Theresa

AU - Lavore, Francesca

AU - Kumar, Raj

AU - Honorato, Rodrigo V

AU - Grein, Fabian

AU - Nieselt, Kay

AU - Liu, Yangping

AU - Bonvin, Alexandre M J J

AU - Baldus, Marc

AU - Kubitscheck, Ulrich

AU - Breukink, Eefjan

AU - Achorn, Catherine

AU - Nitti, Anthony

AU - Schwalen, Christopher J

AU - Spoering, Amy L

AU - Ling, Losee Lucy

AU - Hughes, Dallas

AU - Lelli, Moreno

AU - Roos, Wouter H

AU - Lewis, Kim

AU - Schneider, Tanja

AU - Weingarth, Markus

PY - 2023/9/14

Y1 - 2023/9/14

N2 - Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55PP, lipid II, and lipid III WTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

AB - Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55PP, lipid II, and lipid III WTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

KW - Anti-Bacterial Agents/isolation & purification

KW - Bacteria

KW - Biological Assay

KW - Diphosphates

KW - Soil Microbiology

UR - http://www.scopus.com/inward/record.url?scp=85170683850&partnerID=8YFLogxK

U2 - 10.1016/j.cell.2023.07.038

DO - 10.1016/j.cell.2023.07.038

M3 - Article

C2 - 37611581

SN - 0092-8674

VL - 186

SP - 4059-4073.e27

JO - Cell

JF - Cell

IS - 19

ER -

An antibiotic from an uncultured bacterium binds to an immutable target

Abstract

Bibliographical note

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Cite this