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Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus

Nature Microbiology volume 9, pages 200–213 (2024)Cite this article

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Abstract

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities.

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Fig. 1: Plasmid and epifadin BGC composition, biosynthetic modules and domain organization of the epifadin NRPS–PKS enzymes of IVK83.
Fig. 2: High instability of epifadin under in vivo-like conditions.
Fig. 3: Detection, isolation and absorption spectrum of epifadin.
Fig. 4: Molecular mass and structure of epifadin.
Fig. 5: Broad antimicrobial activity of epifadin-producing S. epidermidis IVK83.
Fig. 6: Epifadin-producing S. epidermidis IVK83 restricts S. aureus growth in vitro and in vivo in cotton rats.

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Data availability

All data supporting the findings of this study are available within the paper, its extended data or supplementary information. Whole-genome sequencing data obtained for S. epidermidis IVK83 were deposited in the NCBI Sequence Read Archive (genome available under accession number CP088002, plasmid pIVK83 under CP088003). Sequence of strain S. epidermidis B155 (Liverpool, UK) was deposited as BioSample SAMEA12384066 (BioProject PRJEB50307). Representative microscopy images are included in the extended data figures and the supplementary videos, which were deposited at Figshare (https://doi.org/10.6084/m9.figshare.24125589). NMR data were deposited at nmrXive and are available under the project identifier NMRXIV:P18 (https://doi.org/10.57992/nmrxiv.p18; https://nmrxiv.org/P18). Source data are provided with this paper.

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Acknowledgements

We thank D. Belikova, V. Augsburger and J. Straetner for excellent technical support, M. Hamburger (Pharmaceutical Biology, University of Basel, Switzerland) for providing authentic sample of militarinone C, and A. Tooming-Klunderud (Center for Ecological and Evolutionary Synthesis, Department of Biosciences, University of Oslo, Norway) for PacBio sequencing of strain IVK83. The sequencing company MicrobesNG (Birmingham, UK) is supported by the Biotechnology and Biological Sciences Research Council; grant number BB/L024209/1). The authors’ work is financed by grants from Deutsche Forschungsgemeinschaft (DFG) TRR261 (A.P., H.B.-O. and S.G.; project ID 398967434), GRK1708 (S.G., H.B.-O. and A.P.) and Cluster of Excellence EXC2124 Controlling Microbes to Fight Infection (CMFI, S.G., B.K., H.B.-O. and A.P.; project ID 390838134), TRR156 (A.P.; project ID 246807620), and ZUK 63 (N.A.S.); from the German Center of Infection Research (DZIF) to B.K., H.B.-O. and A.P.; from the Novo Nordisk Foundation (T.W., project ID NNF20CC0035580); from the German Ministry of Research and Education (BMBF) Culture Challenge to A.P.; and from the European Innovative Medicines Initiate IMI (COMBACTE) to A.P. We acknowledge support by the High Performance and Cloud Computing Group at the Zentrum für Datenverarbeitung of the University of Tübingen, the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant no. INST 37/935-1 FUGG.

Author information

Author notes

  1. Stephanie Grond

    Present address: Institute of Organic Chemistry, University of Tübingen, Tübingen, Germany

  2. These authors contributed equally: Benjamin O. Torres Salazar, Taulant Dema.

Authors and Affiliations

  1. Department of Infection Biology, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany

    Benjamin O. Torres Salazar, Daniela Janek, Ahmed M. A. Elsherbini, Sophia Krauss, Bernhard Krismer & Andreas Peschel

  2. Cluster of Excellence EXC 2124 Controlling Microbes to Fight Infections, Tübingen, Germany

    Benjamin O. Torres Salazar, Nadine A. Schilling, Daniela Janek, Ahmed M. A. Elsherbini, Sophia Krauss, José Manuel Beltrán-Beleña, Stephanie Grond, Bernhard Krismer & Andreas Peschel

  3. German Center for Infection Research (DZIF), partner site Tübingen, Tübingen, Germany

    Benjamin O. Torres Salazar, Daniela Janek, Anne Berscheid, Ahmed M. A. Elsherbini, Sophia Krauss, Heike Brötz-Oesterhelt, Bernhard Krismer & Andreas Peschel

  4. Institute of Organic Chemistry, University of Tübingen, Tübingen, Germany

    Taulant Dema, Nadine A. Schilling & José Manuel Beltrán-Beleña

  5. Department of Microbial Bioactive Compounds, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany

    Jan Bornikoel, Anne Berscheid & Heike Brötz-Oesterhelt

  6. Institute of Pharmaceutical Sciences, University of Tübingen, Tübingen, Germany

    Simon J. Jaag & Michael Lämmerhofer

  7. Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China

    Min Li

  8. Department of Infection Biology and Microbiomes, University of Liverpool, Liverpool, UK

    Norah Alqahtani & Malcolm J. Horsburgh

  9. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark

    Tilmann Weber

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Contributions

B.O.T.S. performed and analysed most of the bacteriological, molecular and compound isolation experiments with help by D.J., S.K. and B.K., who originally isolated strain IVK83; animal experiments were performed by B.O.T.S. and B.K.; T.D., N.A.S. and S.G. elucidated the structure of epifadin with support from J.M.B.-B.; T.D. performed total syntheses of all tetrapeptides, their purification and chemical analyses; A.B. analysed epifadin toxicity and membrane potential effects; J.B. performed all microscopic experiments; A.M.A.E. performed the bioinformatic search for epifadin-like BGCs; M. Li., M.J.H. and S.K. identified and provided epifadin-producing S. epidermidis strains.; N.A. and M.J.H. performed the experimental evolution and analysed the epifadin-resistant mutants; S.J.J. and M. Lämmerhofer confirmed the absolute configuration of the tetrapeptide via chiral HPLC; T.W. analysed the potential epifadin biosynthesis enzymes; H.B.-O., S.G., B.K. and A.P. supervised the experiments and wrote the manuscript.

Corresponding authors

Correspondence to Stephanie Grond or Bernhard Krismer.

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Nature Microbiology thanks Rolf Müller, James O’Gara and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Comparison of MS/MS spectra of the synthetic and natural peptide amide fragments of epifadin.

a, MS/MS spectrum of the natural peptide amide after decomposition of epifadin. b, MS/MS spectrum of the synthetic peptide amide 2. c, Fragmentation pattern of the synthetic and natural peptide amides 2. Fragmentation pattern for the peptide amide 2 is shown in black. F, phenylalanine; D, aspartate; N, asparagine; CO, carbon monoxide; NH3, ammonia.

Extended Data Fig. 2 Proton NMR spectrum of the synthetic peptide amide FfDn-NH2 (DMSO-d6, 600MHz, 303K).

The integrals of the proton signals are depicted as black curves. The scale shows the chemical shift δ in parts per million (ppm).

Extended Data Fig. 3 1H-1H-ROESY NMR spectrum of the purified epifadin in DMSO-d6 (700 MHz, 303 K).

The red circles highlight coupling between the NH-proton and the protons of the methyl group of the alanine residue (9.14 ppm/1.95 ppm) and to the proton of the adjacent methine group (9.14 ppm/6.77 ppm). Also, the coupling of the protons of the methyl group from the alanine residue to the methine group is shown (6.77 ppm/1.95 ppm).

Extended Data Fig. 4 1H NMR spectrum (DMSO-d6, 700 MHz, 303 K) of purified epifadin and its decomposition analyzed by HPLC-MS.

a, DMSO-d6 signal at 2.50 ppm as reference. The integrals of the proton signals are depicted as black curves. The scale shows the chemical shift δ in parts per million (ppm). b,c, The epifadin-enriched material was dissolved in a mixture of acetonitrile and water (1:1) with 0.05% trifluoroacetic acid, resulting in a concentration of 0.2 mg/mL and analyzed by HPLC-ESI-TOF-high resolution MS. The extracted ion chromatograms (EICs) of epifadin (C51H61N7O12 [M+H]+, m/z 964.4451 ± 0.005) are depicted in red (retention time 15.2 min and 15.6 min) and the base peak chromatograms (BPCs) in gray. EICs of the peptide amide (C26H32N6O7 [M+H]+, m/z 541.2405 ± 0.005) are depicted in blue (retention time 7.4 min) accumulating by strong decomposition of epifadin in the mentioned solvent after storage at −20 °C. b, analyzed after purification. c, Analyzed after 14 days of storage at −20 °C.

Extended Data Fig. 5 Deduced fragmentation pattern for the peptide amide and the PKS/NRPS moiety.

From a six-membered transition state a rearrangement results in a neutral loss of the peptide amide moiety. The newly formed allene (m/z 424.2131) decomposes into further fragments.

Extended Data Fig. 6 MS/MS spectra of epifadin showing fragmentation products from ionization in MS.

a, The mass of 964 Da corresponds to the intact proton adduct (m/z 964.4) of epifadin. 524 Da (m/z 524.2) corresponds to the proton adduct of the tetrapeptide EfiA product, and the mass of 441 Da (m/z 441.2) is assigned to the proton adduct of the EfiBCDE product (expansion shows also minor signals of peptide fragments). [M+H]+, monoisotopic positively charged ion; F, phenylalanine; D, aspartate; N, asparagine; CO, carbon monoxide. b, The fragmentation pattern for the peptide moiety in epifadin is shown. Numbering of amino acids and carbon atoms of PKS chain in red.

Extended Data Fig. 7 Epifadin is bactericidal for susceptible bacterial cells but does not inhibit mammalian cells.

a, Time-dependent elimination of S. aureus by epifadin. Incubation of S. aureus USA300 LAC with epifadin concentrations of 24 µg/mL and 12 µg/mL led to a fast decline of CFUs reaching the detection limit of 1 × 103 CFU/mL after 210 min. Data represent means with SEM of three independent experiments. b,c, Cell viability assay. HeLa cells incubated with epifadin do not show increased cell death compared to mock-treated cells (DMSO treatment set as 100%) even at high concentrations of 12 µg/mL. Cycloheximide (CHM) was included as a positive control. Only at concentration of 24 µg/mL, epifadin shows a significant effect on cell viability, still leaving 84% of HeLa cells intact. Data points represent the mean ± SD of three independent experiments. Significant differences between lowest compound concentrations and higher concentrations were analyzed by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Exact p values for the CHM treated HeLa cells were 0.0192 (6.25 × 10−2 µg/ml), 0.0009 (0.125 µg/ml), 0.0001 (0.25 µg/ml).

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Extended Data Fig. 8 Epifadin-producing S. epidermidis IVK83 restricts S. aureus growth in vitro.

a–c, in vitro competition assays in TSB. a, S. aureus growth is inhibited by IVK83 wild type (grey or light blue bars, respectively) already after 24 h of incubation in TSB inoculated at ratios of ~50:50. b, in contrast, the mutant IVK83 ΔefiTP is overgrown by S. aureus over time when inoculated at a 50:50 ratio. c, Complemented strain overgrew S. aureus for 48 h, after 72 h, ratio of complemented strain and S. aureus were similar to starting conditions. Data points represent mean value ± SD of three independent experiments. Significant differences between the starting condition and the indicated time points were analyzed by one-way ANOVA (**P < 0.01; ***P < 0.001; ****P < 0.0001).

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Extended Data Fig. 9 Structures of semi-synthetic derivatives of peptide amide and MS/MS spectra.

a) methyl ester of natural peptide amide and b) acetylated natural peptide amide.

Extended Data Fig. 10 Epifadin leads to depolarization of the bacterial membrane and rapid cell lysis of S. aureus.

(a) S. aureus USA300 JE2 cells were applied to an agarose pad, onto which 2 µL of extracts (50 mg/mL) of the epifadin producer IVK83 (left) or ΔefiTP (right) had been previously spotted. Image acquisition was started in the surrounding of the respective extract spot 15 min after S. aureus application. The agarose contained FM4-64 (red, 0.25 µg/mL, membrane dye) and Sytox Green (green, 0.25 µM, only visible upon membrane barrier malfunction). Representative micrographs are depicted, all adjusted in the Sytox green channel to the same settings for qualitative comparison. White scale bar, 5µm. (b) Time-resolved effects of extracts of IVK83 wild type and ΔefiTP on the membrane potential of S. aureus NCTC8325 as monitored by DiOC2(3) staining. CCCP (5 µM), positive control; DMSO, untreated control. Mean and s.d. of two biological with two technical replicates (n = 4). Black arrow, time point of compound addition.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–8, information and Tables 1–7.

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Supplementary Video 1

Agarose-embedded S. aureus cells lyse within 15 min after contact with epifadin-containing DMSO extract.

Supplementary Video 2

Agarose-embedded S. aureus cells after contact with an epifadin-free DMSO extract. Growth and cell division is seen within 4 h.

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Torres Salazar, B.O., Dema, T., Schilling, N.A. et al. Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus. Nat Microbiol 9, 200–213 (2024). https://doi.org/10.1038/s41564-023-01544-2

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