A new antibiotic kills pathogens without detectable resistance

Journal name:
Nature
Volume:
517,
Pages:
455–459
Date published:
DOI:
doi:10.1038/nature14098
Received
Accepted
Published online
Corrected online

Abstract

Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Most antibiotics were produced by screening soil microorganisms, but this limited resource of cultivable bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this platform. Uncultured bacteria make up approximately 99% of all species in external environments, and are an untapped source of new antibiotics. We developed several methods to grow uncultured organisms by cultivation in situ or by using specific growth factors. Here we report a new antibiotic that we term teixobactin, discovered in a screen of uncultured bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin. The properties of this compound suggest a path towards developing antibiotics that are likely to avoid development of resistance.

At a glance

Figures

  1. The structure of teixobactin and the predicted biosynthetic gene cluster.
    Figure 1: The structure of teixobactin and the predicted biosynthetic gene cluster.

    a, The two NRPS genes, the catalytic domains they encode, and the amino acids incorporated by the respective modules. Domains: A, adenylation; C, condensation; MT, methylation (of phenylalanine); T, thiolation (carrier); and TE, thioesterase (Ile-Thr ring closure). NmPhe, N-methylated phenylalanine. b, Schematic structure of teixobactin. The N-methylation of the first phenylalanine is catalysed by the methyltransferase domain in module 1. The ring closure between the last isoleucine and threonine is catalysed by the thioesterase domains during molecule off-loading, resulting in teixobactin. c, Teixobactin structure.

  2. Time-dependent killing of pathogens by teixobactin.
    Figure 2: Time-dependent killing of pathogens by teixobactin.

    a, b, S. aureus were grown to early (a), and late (b) exponential phase and challenged with antibiotics. Data are representative of 3 independent experiments ± s.d. c, Teixobactin treatment resulted in lysis. The figure is representative of 3 independent experiments. d, Resistance acquisition during serial passaging in the presence of sub-MIC levels of antimicrobials. The y axis is the highest concentration the cells grew in during passaging. For ofloxacin, 256 × MIC was the highest concentration tested. The figure is representative of 3 independent experiments.

  3. Teixobactin binds to cell wall precursors.
    Figure 3: Teixobactin binds to cell wall precursors.

    a, Impact of teixobactin (TEIX) on macromolecular biosyntheses in S. aureus. Incorporation of 3H-thymidine (DNA), 3H-uridine (RNA), 3H-leucine (protein), and 3H-glucosamine (peptidoglycan) was determined in cells treated with teixobactin at 1 × MIC (grey bars). Ciprofloxacin (8 × MIC), rifampicin (4 × MIC), vancomycin (2 × MIC) and erythromycin (2 × MIC) were used as controls (white bars). Data are means of 4 independent experiments ± s.d. b, Intracellular accumulation of the cell wall precursor UDP-MurNAc-pentapeptide after treatment of S. aureus with teixobactin. Untreated and vancomycin (VAN)-treated (10 × MIC) cells were used as controls. UDP-MurNAc-pentapeptide was identified by mass spectrometry as indicated by the peak at m/z 1,149.675. The experiment is representative of 3 independent experiments. c, The effect of teixobactin on precursor consuming reactions. Experiments were performed in 3 biological replicates and data are presented as mean ± s.d. d, Complex formation of teixobactin with purified cell wall precursors. Binding of teixobactin is indicated by a reduction of the amount of lipid intermediates (visible on the thin-layer chromatogram). The figure is representative of two independent experiments. e, A model of teixobactin targeting and resistance. The teixobactin producer is a Gram-negative bacterium protected from this compound by exporting it across the outer membrane permeability barrier (upper panel). In target Gram-positive organisms lacking an outer membrane, the targets are readily accessible on the outside where teixobactin binds precursors of peptidoglycan (PG) and WTA. CM, cytoplasmic membrane; CW, cell wall; OM, outer membrane; T, teixobactin.

  4. Teixobactin is efficacious in three mouse models of infection.
    Figure 4: Teixobactin is efficacious in three mouse models of infection.

    a, Single dose treatment (i.v., 1 h post-infection, 6 mice per group) with teixobactin and vancomycin in septicemia protection model using MRSA. Survival is depicted 48 h after infection. b, Single dose (i.v., 2 h post-infection, 4 mice per group) treatment with teixobactin and vancomycin in neutropenic mouse thigh infection model using MRSA. For drug-treated animals, thigh colony-forming units (c.f.u.) were determined at 26 h post-infection. For controls, c.f.u. in thighs were determined at 2 h and 26 h post-infection. c, Two dose treatment, 5 mice per group, with teixobactin (i.v., 24 h and 36 h post-infection) and single dose treatment with amoxicillin (subcutaneous, 24 h post-infection) in immunocompetent lung infection model using S. pneumoniae. Lung c.f.u. were determined at 48 h post-infection. The c.f.u. from each mouse are plotted as individual points and error bars represent the deviation within an experimental group. *P < 0.05, ***P < 0.001 (determined by non-parametric log-rank test).

  5. The iChip.
    Extended Data Fig. 1: The iChip.

    ac, The iChip (a) consists of a central plate (b) which houses growing microorganisms, semi-permeable membranes on each side of the plate, which separate the plate from the environment, and two supporting side panels (c). The central plate and side panels have multiple matching through-holes. When the central plate is dipped into suspension of cells in molten agar, the through-holes capture small volumes of this suspension, which solidify in the form of small agar plugs. Alternatively, molten agar can be dispensed into the chambers. The membranes are attached and the iChip is then placed in soil from which the sample originated.

  6. 16S rRNA gene phylogeny of Eleftheria terrae.
    Extended Data Fig. 2: 16S rRNA gene phylogeny of Eleftheria terrae.

    a, The phylogenetic position of E. terrae within the class β-proteobacteria. The 16S rRNA gene sequences were downloaded from Entrez at NCBI using accession numbers retrieved from peer-reviewed publications. b, The phylogenetic position of E. terrae among its closest known relatives. The sequences were downloaded from NCBI using accession numbers retrieved from the RDP Classifier Database. For both trees, multiple sequence alignments (MSA) were constructed using ClustalW2, implementing a default Cost Matrix, the Neighbour-Joining (NJ) clustering algorithm, as well as optimized gap penalties. Resulting alignments were manually curated and phylogenetic trees were constructed leveraging PhyML 3.0 with a TN93 substitution model and 500 Bootstrap iterations of branch support. Topology search optimization was conducted using the Subtree–Pruning–Regrafting (SPR) algorithm with an estimated Transition–Transversion ratio and gamma distribution parameters as well as fixed proportions of invariable sites.

  7. NMR assignment of teixobactin.
    Extended Data Fig. 3: NMR assignment of teixobactin.

    a, 13C-NMR of teixobactin (125 mHz, δ in p.p.m.). b, Structure of teixobactin with the NMR assignments.

  8. NMR spectra of teixobactin.
    Extended Data Fig. 4: NMR spectra of teixobactin.

    a, 13C NMR spectrum of teixobactin. b, 1H NMR spectrum. c, HMBC NMR spectrum. d, HSQC NMR spectrum. e, COSY NMR spectrum.

  9. Hypothetical biosynthesis pathway of teixobactin.
    Extended Data Fig. 5: Hypothetical biosynthesis pathway of teixobactin.

    The eleven modules of the non-ribosomal peptide synthetases Txo1 and Txo2 are depicted with the growing chain attached. Each module is responsible for the incorporation of one specific amino acid in the nascent peptide chain. The N-methylation of the first amino acid phenylalanine is catalysed by the methyltransferase domain in module 1. The ring closure (marked by a dashed arrow) between the last isoleucine and threonine is catalysed by the thioesterase domains during molecule off-loading, resulting in teixobactin.

  10. Teixobactin activity against vancomycin-resistant strains.
    Extended Data Fig. 6: Teixobactin activity against vancomycin-resistant strains.

    a, Vancomycin intermediate S. aureus (VISA) were grown to late exponential phase and challenged with vancomycin or teixobactin. Cell numbers were determined by plating for colony counts. Data are representative of 3 independent experiments ± s.d. b, Complex formation of teixobactin with cell wall precursor variants as formed by vancomycin-resistant strains. Purified lipid intermediates with altered stem peptides were incubated with teixobactin at a molar ratio of 2:1 (TEIX:lipid II variant). Reaction mixtures were extracted with BuOH/PyrAc and binding of teixobactin to lipid II variants is indicated by its absence on the thin-layer chromatogram. Migration behaviour of unmodified lipid II is used for comparison. The figure is representative of 3 independent experiments.

  11. Model for the mechanism of action of teixobactin.
    Extended Data Fig. 7: Model for the mechanism of action of teixobactin.

    Inhibition of cell wall synthesis by teixobactin. Lipid II, precursor of peptidoglycan, is synthesized in the cytoplasm and flipped to the surface of the inner membrane by MurJ48 or FtsW49. Lipid III, a precursor of wall teichoic acid (WTA), is similarly formed inside the cell and WTA lipid-bound precursors are translocated across the cytoplasmic membrane by the ABC-transporter TarGH50. Teixobactin (TEIX) forms a stoichiometric complex with cell wall precursors, lipid II and lipid III. Abduction of these building blocks simultaneously interrupts peptidoglycan (right), WTA (left) biosynthesis as well as precursor recycling. Binding to multiple targets within the cell wall pathways obstructs the formation of a functional cell envelope. Left panel, teixobactin targeting and resistance. The producer of teixobactin is a Gram-negative bacterium which is protected from this compound by exporting it outside of its outer membrane permeability barrier. The target Gram-positive organisms do not have an outer membrane. CM, cytoplasmic membrane; CW, cell wall; OM, outer membrane; LTA, lipoteichoic acid; WTA, wall teichoic acid.

  12. Pharmacokinetic analysis of teixobactin.
    Extended Data Fig. 8: Pharmacokinetic analysis of teixobactin.

    a, The mean plasma concentrations of teixobactin after a single i.v. injection of 20 mg per kg teixobactin (3 mice per time point). Data are the mean of plasma concentration, and error bars represent the standard deviation from 3 animals in each time point. b, Pharmacokinetic parameters of teixobactin calculated with a non-compartmental analysis model based on WinNonlin.

Tables

  1. Antibacterial spectrum of teixobactin
    Extended Data Table 1: Antibacterial spectrum of teixobactin
  2. Antagonization of the antimicrobial activity of teixobactin by cell wall precursors
    Extended Data Table 2: Antagonization of the antimicrobial activity of teixobactin by cell wall precursors

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Change history

Corrected online 21 January 2015
Two minor typos were corrected in the main text and Methods.

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Author information

  1. These authors contributed equally to this work.

    • Losee L. Ling &
    • Tanja Schneider

Affiliations

  1. NovoBiotic Pharmaceuticals, Cambridge, Massachusetts 02138, USA

    • Losee L. Ling,
    • Aaron J. Peoples,
    • Amy L. Spoering,
    • Dallas E. Hughes,
    • Douglas R. Cohen,
    • Cintia R. Felix,
    • K. Ashley Fetterman,
    • William P. Millett,
    • Anthony G. Nitti &
    • Ashley M. Zullo
  2. Institute of Medical Microbiology, Immunology and Parasitology—Pharmaceutical Microbiology Section, University of Bonn, Bonn 53115, Germany

    • Tanja Schneider,
    • Ina Engels &
    • Anna Mueller
  3. German Centre for Infection Research (DZIF), Partner Site Bonn-Cologne, 53115 Bonn, Germany

    • Tanja Schneider,
    • Ina Engels,
    • Anna Mueller &
    • Till F. Schäberle
  4. Antimicrobial Discovery Center, Northeastern University, Department of Biology, Boston, Massachusetts 02115, USA

    • Brian P. Conlon,
    • Chao Chen &
    • Kim Lewis
  5. Institute for Pharmaceutical Biology, University of Bonn, Bonn 53115, Germany

    • Till F. Schäberle
  6. Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA

    • Slava Epstein
  7. Selcia, Ongar, Essex CM5 0GS, UK

    • Michael Jones,
    • Linos Lazarides &
    • Victoria A. Steadman

Contributions

K.L. and T.S. designed the study, analysed results, and wrote the paper. L.L.L. designed the study and analysed results. A.J.P. designed the study, performed compound isolation and structure determination and analysed data. B.P.C. designed the study, performed susceptibility experiments and wrote the paper. D.E.H. oversaw preclinical work including designing studies and analysing data. S.E. designed cultivation experiments and analysed data. M.J., L.L. and V.A.S. designed and performed experiments on structure determination and analysed data. I.E. and A.M. designed and performed experiments on mechanism of action. A.L.S., D.R.C., C.R.F., K.A.F., W.P.M., A.G.N., A.M.Z. and C.C. performed experiments on compound production, isolation, susceptibility testing and data analysis. T.F.S. identified the biosynthetic cluster.

Competing financial interests

The following authors, L. L. Ling, A. J. Peoples, A. L. Spoering, D. E. Hughes, D. R. Cohen, C. R. Felix, K. A. Fetterman, W. P. Millett, A. G. Nitti, A. M. Zullo, K. Lewis, and S. Epstein, declare competing financial interests as they are employees and consultants of NovoBiotic Pharmaceuticals.

Corresponding author

Correspondence to:

The biosynthetic gene cluster for teixobactin has been deposited with GenBank under accession number KP006601.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: The iChip. (183 KB)

    ac, The iChip (a) consists of a central plate (b) which houses growing microorganisms, semi-permeable membranes on each side of the plate, which separate the plate from the environment, and two supporting side panels (c). The central plate and side panels have multiple matching through-holes. When the central plate is dipped into suspension of cells in molten agar, the through-holes capture small volumes of this suspension, which solidify in the form of small agar plugs. Alternatively, molten agar can be dispensed into the chambers. The membranes are attached and the iChip is then placed in soil from which the sample originated.

  2. Extended Data Figure 2: 16S rRNA gene phylogeny of Eleftheria terrae. (191 KB)

    a, The phylogenetic position of E. terrae within the class β-proteobacteria. The 16S rRNA gene sequences were downloaded from Entrez at NCBI using accession numbers retrieved from peer-reviewed publications. b, The phylogenetic position of E. terrae among its closest known relatives. The sequences were downloaded from NCBI using accession numbers retrieved from the RDP Classifier Database. For both trees, multiple sequence alignments (MSA) were constructed using ClustalW2, implementing a default Cost Matrix, the Neighbour-Joining (NJ) clustering algorithm, as well as optimized gap penalties. Resulting alignments were manually curated and phylogenetic trees were constructed leveraging PhyML 3.0 with a TN93 substitution model and 500 Bootstrap iterations of branch support. Topology search optimization was conducted using the Subtree–Pruning–Regrafting (SPR) algorithm with an estimated Transition–Transversion ratio and gamma distribution parameters as well as fixed proportions of invariable sites.

  3. Extended Data Figure 3: NMR assignment of teixobactin. (371 KB)

    a, 13C-NMR of teixobactin (125 mHz, δ in p.p.m.). b, Structure of teixobactin with the NMR assignments.

  4. Extended Data Figure 4: NMR spectra of teixobactin. (224 KB)

    a, 13C NMR spectrum of teixobactin. b, 1H NMR spectrum. c, HMBC NMR spectrum. d, HSQC NMR spectrum. e, COSY NMR spectrum.

  5. Extended Data Figure 5: Hypothetical biosynthesis pathway of teixobactin. (220 KB)

    The eleven modules of the non-ribosomal peptide synthetases Txo1 and Txo2 are depicted with the growing chain attached. Each module is responsible for the incorporation of one specific amino acid in the nascent peptide chain. The N-methylation of the first amino acid phenylalanine is catalysed by the methyltransferase domain in module 1. The ring closure (marked by a dashed arrow) between the last isoleucine and threonine is catalysed by the thioesterase domains during molecule off-loading, resulting in teixobactin.

  6. Extended Data Figure 6: Teixobactin activity against vancomycin-resistant strains. (267 KB)

    a, Vancomycin intermediate S. aureus (VISA) were grown to late exponential phase and challenged with vancomycin or teixobactin. Cell numbers were determined by plating for colony counts. Data are representative of 3 independent experiments ± s.d. b, Complex formation of teixobactin with cell wall precursor variants as formed by vancomycin-resistant strains. Purified lipid intermediates with altered stem peptides were incubated with teixobactin at a molar ratio of 2:1 (TEIX:lipid II variant). Reaction mixtures were extracted with BuOH/PyrAc and binding of teixobactin to lipid II variants is indicated by its absence on the thin-layer chromatogram. Migration behaviour of unmodified lipid II is used for comparison. The figure is representative of 3 independent experiments.

  7. Extended Data Figure 7: Model for the mechanism of action of teixobactin. (182 KB)

    Inhibition of cell wall synthesis by teixobactin. Lipid II, precursor of peptidoglycan, is synthesized in the cytoplasm and flipped to the surface of the inner membrane by MurJ48 or FtsW49. Lipid III, a precursor of wall teichoic acid (WTA), is similarly formed inside the cell and WTA lipid-bound precursors are translocated across the cytoplasmic membrane by the ABC-transporter TarGH50. Teixobactin (TEIX) forms a stoichiometric complex with cell wall precursors, lipid II and lipid III. Abduction of these building blocks simultaneously interrupts peptidoglycan (right), WTA (left) biosynthesis as well as precursor recycling. Binding to multiple targets within the cell wall pathways obstructs the formation of a functional cell envelope. Left panel, teixobactin targeting and resistance. The producer of teixobactin is a Gram-negative bacterium which is protected from this compound by exporting it outside of its outer membrane permeability barrier. The target Gram-positive organisms do not have an outer membrane. CM, cytoplasmic membrane; CW, cell wall; OM, outer membrane; LTA, lipoteichoic acid; WTA, wall teichoic acid.

  8. Extended Data Figure 8: Pharmacokinetic analysis of teixobactin. (239 KB)

    a, The mean plasma concentrations of teixobactin after a single i.v. injection of 20 mg per kg teixobactin (3 mice per time point). Data are the mean of plasma concentration, and error bars represent the standard deviation from 3 animals in each time point. b, Pharmacokinetic parameters of teixobactin calculated with a non-compartmental analysis model based on WinNonlin.

Extended Data Tables

  1. Extended Data Table 1: Antibacterial spectrum of teixobactin (586 KB)
  2. Extended Data Table 2: Antagonization of the antimicrobial activity of teixobactin by cell wall precursors (82 KB)

Supplementary information

PDF files

  1. Supplementary Information (187 KB)

    This file contains a Supplementary Discussion.

Comments

  1. Report this comment #65059

    Lorraine Draper said:

    We note with interest the novel antibiotic identified by Ling and co-authors (Ling, L.L. et al. Nature (2015)) and the elegant manner in which the producing strain was sourced. However, we urge caution with respect to suggestions that it reveals ?a path towards developing antibiotics that are likely to avoid development of resistance?. Teixobactin may be structurally distinct from lantibiotics, but they share similar potency (against, for example, MRSA and VISA) and often have a common binding moiety i.e. the pyrophosphate linker of lipid II and lipid III (Wiedemann, I. et al. J. Biol. Chem (2001); Muller, A. et al. Microbial drug resistance (2012)). While compiling a review of mechanisms that confer resistance to lantibiotics (Draper, L.A. et al. MMBR (2015) (in press)), we noted that while resistance to these peptides is somewhat rare, there are nonetheless multiple mechanisms through which it can occur. In some cases, these resistance mechanisms specifically target the antimicrobial itself; for example, nisinase is a specific reductase that inactivates the prototypical lantibiotic nisin. Even more frequently, non-specific resistance mechanisms, involving modification of cell envelope charge, composition or rigidity as well as increased expression of drug resistance transporters can contribute to resistance. These mechanisms have been revealed despite the fact that nisin has not been employed in clinical settings (although extensively used for decades as a food preservative). We might expect that even broader variety of resistance mechanisms would be revealed were it to be used in clinical settings. This is also likely to be true for teixobactin. We do not discount the possibility that teixobactin can become an important tool in the battle against multi-drug resistant Gram positive bacteria but urge caution as over-confidence with respect to the power of antibiotics to control all pathogens has contributed to current problems.

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