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A broad-spectrum lasso peptide antibiotic targeting the bacterial ribosome

Nature volume 640, pages 1022–1030 (2025)Cite this article

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An Author Correction to this article was published on 11 September 2025

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Abstract

Lasso peptides (biologically active molecules with a distinct structurally constrained knotted fold) are natural products that belong to the class of ribosomally synthesized and post-translationally modified peptides1,2,3. Lasso peptides act on several bacterial targets4,5, but none have been reported to inhibit the ribosome, one of the main targets of antibiotics in the bacterial cell6,7. Here we report the identification and characterization of the lasso peptide antibiotic lariocidin and its internally cyclized derivative lariocidin B, produced by Paenibacillus sp. M2, which has broad-spectrum activity against a range of bacterial pathogens. We show that lariocidins inhibit bacterial growth by binding to the ribosome and interfering with protein synthesis. Structural, genetic and biochemical data show that lariocidins bind at a unique site in the small ribosomal subunit, where they interact with the 16S ribosomal RNA and aminoacyl-tRNA, inhibiting translocation and inducing miscoding. Lariocidin is unaffected by common resistance mechanisms, has a low propensity for generating spontaneous resistance, shows no toxicity to human cells, and has potent in vivo activity in a mouse model of Acinetobacter baumannii infection. Our identification of ribosome-targeting lasso peptides uncovers new routes towards the discovery of alternative protein-synthesis inhibitors and offers a novel chemical scaffold for the development of much-needed antibacterial drugs.

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Fig. 1: LAR and its BGC.
Fig. 2: LAR exhibits bactericidal activity and targets bacterial protein synthesis.
Fig. 3: Structures and electron density maps of ribosome-bound LAR and LAR-B.
Fig. 4: Structure of LAR in complex with the T. thermophilus 70S ribosome.
Fig. 5: Therapeutic efficacy of LAR in a mouse neutropenic thigh infection model.

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Data availability

Data supporting the findings of this study are available within this paper and its Supplementary Information or have been deposited to the indicated databases. Coordinates and structure factors were deposited in the RCSB Protein Data Bank (PDB) with the following accession codes: 9DFC for the wild-type T. thermophilus 70S ribosome in complex with LAR, mRNA, aminoacylated A-site Phe-tRNAPhe, aminoacylated P-site fMet-tRNAiMet, and deacylated E-site tRNAPhe; 9DFD for the wild-type T. thermophilus 70S ribosome in complex with LAR-B, mRNA, aminoacylated A-site Phe-tRNAPhe, aminoacylated P-site fMet-tRNAiMet, deacylated E-site tRNAPhe; and 9DFE for the wild-type T. thermophilus 70S ribosome in complex with LAR and protein Y. All previously published structures used in this work for structural comparisons were retrieved from the RCSB Protein Data Bank: PDB entries 6XHW, 6XHX, 6CAE, 4G5K, 4YBB and 4W2I. The complete genome sequence of Paenibacillus sp. M2 is available in NCBI GenBank under accession no. CP169648. Phylogenetic tree and the DNA segments containing lrc-like BGC are available at https://github.com/jangrm1/LAR-BGC. Source data are provided with this paper.

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Acknowledgements

The authors thank M. Cook for determining mycobacterial MICs; A. Guitor for help with breseq analysis of resistant mutants; M. Surette for MIC determination against gut microbiota; E. Brown for E. coli and B. subtilis gene-deletion strains; the McMaster Biointerfaces Institute for MALDI–MSMS analysis; the Centre for Microbial Chemical Biology for mammalian toxicity experiments; and the McMaster Centre for Advanced Light Microscopy facility for confocal microscopy. This work is based on research conducted at the Center for BioMolecular Structure beamlines (17ID-1 and 17ID-2), which are primarily supported by the National Institute of General Medical Sciences from the National Institutes of Health (P30-GM133893) and by the DOE Office of Biological and Environmental Research (KP1605010). NSLS2 is a US DOE Office of Science User Facility operated under contract no. DE-SC0012704. This publication resulted from the data collected using the beamtime obtained through NECAT BAG proposal 311950. This work was supported by the Canadian Institutes for Health Research (project grant PJT190298 to G.D.W.), National Institute of General Medical Sciences of the National Institutes of Health (grant R35-GM127134 to A.S.M.; grant R35-GM151957 to Y.S.P.; grant R01-GM132302 to Y.S.P.), National Institute of Allergy and Infectious Diseases of the National Institutes of Health (grant R01-AI162961 to A.S.M., N.V.-L. and Y.S.P.), National Science Foundation (MCB-2345351 to N.V.-L.) and the Illinois State startup funds (to Y.S.P.). The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.

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Author notes

  1. These authors contributed equally: Manoj Jangra, Dmitrii Y. Travin

Authors and Affiliations

  1. David Braley Centre for Antibiotics Discovery, McMaster University, Hamilton, Ontario, Canada

    Manoj Jangra, Manpreet Kaur, Lena Darwish, Kalinka Koteva, Wenliang Wang, Maya Tiffany, Akosiererem Sokaribo, Xuefei Chen, Brian K. Coombes & Gerard D. Wright

  2. M. G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada

    Manoj Jangra, Manpreet Kaur, Lena Darwish, Kalinka Koteva, Wenliang Wang, Maya Tiffany, Akosiererem Sokaribo, Xuefei Chen, Brian K. Coombes & Gerard D. Wright

  3. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

    Manoj Jangra, Manpreet Kaur, Lena Darwish, Kalinka Koteva, Wenliang Wang, Maya Tiffany, Akosiererem Sokaribo, Xuefei Chen, Brian K. Coombes & Gerard D. Wright

  4. Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Dmitrii Y. Travin, Dorota Klepacki, Nora Vázquez-Laslop, Yury S. Polikanov & Alexander S. Mankin

  5. Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Dmitrii Y. Travin, Dorota Klepacki, Nora Vázquez-Laslop, Yury S. Polikanov & Alexander S. Mankin

  6. Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Elena V. Aleksandrova & Yury S. Polikanov

  7. State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

    Zixin Deng & Meifeng Tao

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  1. Manoj Jangra
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Contributions

M.J., M.K. and G.D.W. conceived the study and planned initial experiments. M.K. isolated the soil strains and performed preliminary antimicrobial screening. M.J. isolated LAR and conducted initial chemical characterization. W.W. performed NMR experiments. M.J. and M.T. performed heterologous expression studies in Streptomyces. M.J. and K.K. did the chemical analysis and synthesized fluorophore conjugates. M.J., L.D. and B.K.C. designed animal studies, and L.D. performed animal experiments. B.K.C. supervised animal studies. M.K. and A.S. performed DNA preparation and whole-genome sequencing. M.J., D.Y.T., M.K. and D.K. performed the biochemical and microbiological experiments. E.V.A., D.Y.T. and Y.S.P. designed and performed X-ray crystallography experiments. M.J. and D.Y.T. performed bioinformatics analysis. A.S.M., N.V.-L., Y.S.P., X.C., Z.D, and M.T. developed and supplied Streptomyces strains for heterologous expression, G.D.W. designed and supervised the experiments. All authors interpreted the results. M.J., D.Y.T., A.S.M., N.V.-L., Y.S.P. and G.D.W. wrote and edited the manuscript with input from L.D. and K.K. All authors approved the manuscript before submission.

Corresponding authors

Correspondence to Yury S. Polikanov, Alexander S. Mankin or Gerard D. Wright.

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Extended data figures and tables

Extended Data Fig. 1 Paenibacillus sp. M2 strain produces colistin and LAR.

(a) The antibacterial activity of partially fractionated extract from Paenibacillus M2 against A. baumannii C0286 (‘Ab’) and the following E. coli BW25113 strains: wild type (WT), colistin-resistant expressing mcr-1, and the antibiotic hypersusceptible ΔtolC/ΔbamB mutant (ΔT/ΔB). (b) Bioactivity assay of RP-HPLC fractions of the pre-fractionated (on SP-sepharose column) Paenibacillus sp. M2 extract against A. baumannii C0286 strain, showing the presence of two distinct antibiotics. (c) Liquid chromatography-mass-spectrometry analysis of fractions 7 and 21 (see panel b) shows the presence of LAR and colistin, respectively. The upper panel shows the extracted ion chromatogram, and the bottom panels represent mass spectra of corresponding fractions. LAR, lariocidin; LAR-B, lariocidin B, and LAR-C, lariocidin C.

Extended Data Fig. 2 LAR does not affect the bacterial cell envelope.

(a) Inner membrane permeabilization assay in E. coli TOP10 cells with pUC19 plasmid (containing the lacZ gene) using membrane-impermeable dye Ortho-nitrophenyl-β-D-galactopyranoside (ONPG). LAR (40 μg/ml) did not facilitate the uptake of ONPG, unlike colistin (5 μg/ml), which creates membrane pores. Data are representative of two independent experiments. (b) Membrane depolarization assay using DiOC2(3) dye. The fluorescence of the dye quenches when it enters depolarized cells (due to membrane potential disruption). LAR was used at 10xMIC (40 μg/ml). Protonophore, CCCP (20 μM) served as a positive control. Data represent three biological experiments, with error bars indicating SD of three replicates (c) Scanning electron microscopy images of E. coli treated with 10xMIC of LAR, showing no obvious changes in morphology or defects in the cell envelope. The images are representative of two independent samples.

Extended Data Fig. 3 Synthesis of lariocidin-fluorophore conjugates and confocal microscopy.

(a) Synthesis of lariocidin-fluorophore conjugates via click-chemistry. TEA, triethylamine; PyBOP benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; TFA, trifluoroacetic acid; DMSO, dimethylsulfoxide; DCM, dichloromethane. (b) MIC of LAR-fluorophores against E. coli BW25113 strain in MOPS minimal medium. (c, d) Confocal microscopy images of E. coli BW25113 cells treated with LAR-BODIPY (20 μg/ml) (c) or LAR-rhodamine (20 μg/ml) (d) indicating intracellular accumulation of probes. Fm-4-64 was used to stain the membrane, and Hoechst 33342 for DNA visualization. The images are representative of three biological replicates. Scale bar is 5 μM.

Extended Data Fig. 4 Lariocidin utilizes membrane potential to enter the bacterial cytoplasm.

E. coli BW25113 was used in all the experiments. (a) Anaerobic conditions result in an increase of LAR MIC determined in different media. MOPS MM is MOPS minimal medium and MHB is cation-adjusted Mueller-Hinton Broth. (b) Addition of bicarbonate, known to potentiate certain antibiotics like macrolides and aminoglycosides by enhancing the active membrane potential38, reduces LAR MIC in the MOPS and MHB media. MIC is also reduced in RPMI medium, which mimics physiological conditions better than MHB. (c) Effect of lower pH (known to decrease the membrane potential) on MIC. The experiment was conducted in MOPS MM. (d) The protonophore, CCCP, which eliminates the membrane potential, protects the cells from the killing action of LAR. cfu were enumerated 1 h after treatment with LAR (40 μg/ml) and/or CCCP (20 μM) in MOPS MM. However, no significant change in MIC of LAR was observed at this concentration of CCCP, suggesting that cells may overcome the effect of CCCP during prolonged growth. Data are plotted and mean ± SD of three biological replicates (e) LAR-BODIPY uptake under various treatment conditions using confocal microscopy showing that lowing pH or pretreatment of cells with the protonophore CCCP significantly reduces the accumulation of the LAR-BODIPY fluorescence inside the cells. Fm-4-64 was used to stain the membrane, and Hoechst 33342 for DNA visualization. The images are exemplary of two biological experiments. Scale bar is 5 μM.

Extended Data Fig. 5 LAR-resistance mutations in the 16S rRNA.

(a) Characteristics of LAR-resistant mutants selected in E. coli SQ110 ΔtolC. (b) Location of the mutations conferring increased resistance to LAR in the 16S rRNA helices h31 and h34. (c) Odilorhabdins (NOSO-95719) and LAR have different resistance profiles, as evidenced by the lack of LAR MIC changes in most NOSO-95719-resistant strains with point mutations in the 16S rRNA gene. (d) Spatial arrangement of the LAR-resistance mutations in the 16S rRNA shown in the structure of the E. coli small ribosomal subunit (PDB ID: 4YBB)69.

Extended Data Fig. 6 Intramolecular H-bonds of LAR and comparison of LAR and LAR-B structures.

(a, b) Structure of ribosome-bound LAR is shown from two opposite sides, highlighting intramolecular H-bonds that stabilize its fold. Carbon atoms are colored yellow, nitrogens are blue, oxygens are red. (c, d) Superposition of the structures of ribosome-bound LAR and LAR-B. Residues whose positions differ between the two isoforms are labeled. Nitrogen and oxygen atoms in the isopeptide bonds are colored blue and red, respectively.

Extended Data Fig. 7 LAR effectively kills A. baumannii C0286 in vitro and ex vivo.

(a) In vitro time-kill assay in MHB medium showing the bactericidal effect of LAR as denoted by reduction in viable cfu counts. Data are plotted as the mean of three biological replicates with the error bars indicating SD. (b) Cidality of LAR in the ex vivo model. A. baumannii C0286 was inoculated in human blood, and bacterial cfu were enumerated after 4 h treatment with LAR. Data are plotted as mean ± SD of three biological experiments. Significance was determined using one-way ordinary ANOVA with Dunnett’s multiple comparisons test. (*P = 0.0118; **P = 0.0017). (c) MIC of LAR against A. baumannii C0286 in MHB with or without the addition of serum. FBS=fetal bovine serum; HS=human serum (heat inactivated).

Extended Data Fig. 8 lrc-like biosynthetic gene clusters in various bacterial genomes.

(a) Gene composition of the representative set of lrc-like BGCs from different bacterial phyla. Each gene is represented by an arrow, and the proposed functions of the encoded proteins are listed on the right. The lrc BGC from Paenibacillus sp. M2 is labeled in bold. Note that BGCs from Actinomycetota (including the BGC of triculamin from S. griseocarneus ATCC 29818 (ref. 35)) lack the homologs of lrcB1B2 genes as the encoded precursors contain the unusual C-terminal follower peptide instead of the N-terminal leader peptide typical for other LPs. (b) Phylogenetic tree of lrc-like BGCs (n = 29) built based on the amino acid sequence similarity between LrcC (lasso cyclase) homologs. The lasso cyclase from the biosynthetic gene cluster of the lasso peptide paeninodin served as the outgroup. The alignment of the amino acid sequences of the precursor peptide(s) core parts is shown on the right. The amino acids are colored according to their physico-chemical properties. The consensus sequence and the extent of sequence conservation for each position are shown below. Note that for three triculamin-like peptides from Actinomycetota containing a follower rather than the leader peptide, the actual cleavage site for the follower-peptide (marked with black asterisks) could not be identified unambiguously. RRE – RiPP recognition element, GNAT – GCN5-Related N-acetyltransferase, OM – outer membrane.

Extended Data Fig. 9 Common mechanisms conferring resistance to clinically relevant ribosome-targeting antibiotics do not impact LAR antibacterial activity.

The graph shows the MIC increase of LAR and corresponding control antibiotics (Ab) upon overexpression of the designated resistance determinants in E. coli BW25113 ΔtolCΔbamB. The strain design and details of plasmids are described by Cox et al.52 The color of the gene name reflects the mechanism of resistance: antibiotic modification/inactivation – black, rRNA modification – orange, ribosome protection – blue. Functions of specific genes are- aad(3′′)(9)= spectinomycin adenyltransferase; apmA, aac(2′)-Ia, aac(6′)-Ib, and aac(2′)-IIa= aminoglycoside N-acetyltransferases; aph(4)-Ia, aph(3′)-IIIa, aph(3′)-IVa, and aph(6)-Ia= aminoglycoside phosphotransferases; sat= streptothricin acetyltransferase; tetX= tetracycline inactivation; vph= viomycin phosphotransferase; cat= chloramphenicol acetyltransferase; linB= lincosamide nucleotidyltransferase; vatD= streptogramins acetyltransferase; vgb= streptogramin B lyase; kamB, npmA, armA and rmtB = 16S rRNA methyltransferases; cfrA and ermC = 23S rRNA methyltransferases. Control antibiotics (Ab)- apramycin (apmA, kamB, npmA), gentamicin (aac(2′)-IIa, armA, rmtB), hygromycin B (aph(4)-Ia), kanamycin (aac(6′)-Ib, aph(3′)-IIIa), kasugamycin (aac(2′)-IIa), ribostamycin (aph(3′)-IVa), streptomycin (aph(6)-Ia), clindamycin (cfrA, linB, ermC), flopristin (vatD), quinupristin (vgb), nourseothricin (sat), oxytetracycline (tetM, tetX), viomycin (vph). For the controls marked with asterisks (*), the actual MIC change value is higher than that of the one presented; this reflects the growth of bacteria in the presence of the antibiotic in the highest concentration tested. Antibiotics targeting the small ribosomal subunit are in the light blue background, large ribosomal subunit – is in the light orange.

Extended Data Table 1 X-ray data collection and refinement statistics
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Supplementary information

Supplementary Information

This file contains Supplementary Figs. 1–14 and Tables 1–6. These figures and tables are for the chemical and biological characterization of LAR(s), plasmid pIJ10256-lrc, details of spontaneous resistant mutants, 70S ribosome crystal structure with LAR and protein Y, raw blots and list of strains, plasmids and primers used.

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Supplementary Video 1

Illustration of LAR binding to the bacterial ribosome. The video consecutively shows: (1) zoom-out and (2) close-up views of the LAR-binding site adjacent to the A site on the small subunit of the T. thermophilus 70S ribosome; (3) lasso-like structure of the LAR peptide; (4) 2Fo − Fc Fourier electron density map of ribosome-bound LAR (blue mesh); and (5) details of LAR interactions with the 16S rRNA and the A-site tRNA. The E. coli numbering of the 16S rRNA nucleotides is used. Hydrogen bonds between LAR, rRNA and A-site tRNA are indicated with dashed lines.

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Jangra, M., Travin, D.Y., Aleksandrova, E.V. et al. A broad-spectrum lasso peptide antibiotic targeting the bacterial ribosome. Nature 640, 1022–1030 (2025). https://doi.org/10.1038/s41586-025-08723-7

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