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Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling

Abstract

Addressing the ongoing antibiotic crisis requires the discovery of compounds with novel mechanisms of action that are capable of treating drug-resistant infections1. Many antibiotics are sourced from specialized metabolites produced by bacteria, particularly those of the Actinomycetes family2. Although actinomycete extracts have traditionally been screened using activity-based platforms, this approach has become unfavourable owing to the frequent rediscovery of known compounds. Genome sequencing of actinomycetes reveals an untapped reservoir of biosynthetic gene clusters, but prioritization is required to predict which gene clusters may yield promising new chemical matter2. Here we make use of the phylogeny of biosynthetic genes along with the lack of known resistance determinants to predict divergent members of the glycopeptide family of antibiotics that are likely to possess new biological activities. Using these predictions, we uncovered two members of a new functional class of glycopeptide antibiotics—the known glycopeptide antibiotic complestatin and a newly discovered compound we call corbomycin—that have a novel mode of action. We show that by binding to peptidoglycan, complestatin and corbomycin block the action of autolysins—essential peptidoglycan hydrolases that are required for remodelling of the cell wall during growth. Corbomycin and complestatin have low levels of resistance development and are effective in reducing bacterial burden in a mouse model of skin MRSA infection.

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Fig. 1: Phylogeny-guided discovery of complestatin and corbomycin.
Fig. 2: Corbomycin and complestatin affect peptidoglycan metabolism.
Fig. 3: Corbomycin and complestatin inhibit autolysins.
Fig. 4: Corbomycin and complestatin bind to peptidoglycan.

Data availability

Phylogenetic trees, including those of 84 GPA genes and gene segments from 71 BGCs that were analysed in this study, are available at https://github.com/waglecn/GPA_evolution. Streptomyces sp. WAC01529 and Streptomyces sp. WAC01325 genome sequences are available in GenBank with accession numbers NZ_CP029617.1 and NZ_QHKK00000000.1, respectively. Source Data for the animal experiments shown in Extended Data Fig. 8 are included online.

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Acknowledgements

We thank S. French for help with the imaging of vancomycin–BODIPY stained cells, and V. Rao and V. Yarlagadda for MIC measurement of N. gonorrhoeae. This research was funded by a Canadian Institutes of Health Research grant (FRN-148463), the Ontario Research Fund, and by a Canada Research Chair to G.D.W.; the work was also supported by National Institute of Health grants R35GM122556 to Y.V.B. and 5R01GM113172 to M.S.V. and Y.V.B., and a Canada 150 Research Chair in Bacterial Cell Biology to Y.V.B. E.J.C. was supported by a CIHR Vanier Canada Graduate Scholarship. N.W. was supported by a CIHR Canada Graduate Scholarship Doctoral Award.

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Contributions

E.J.C., N.W. and G.D.W. conceived the study and designed experiments. N.W. performed phylogenetic analysis, structural predictions and resistant mutant genome analysis. W.W. performed compound purification and structural elucidation. E.J.C., A.A.F.-C. and B.K.C. designed animal studies and A.A.F.-C. performed animal experiments. E.J.C. and D.S. performed peptidoglycan and enzyme purification. E.J.C. and K.K. performed the synthesis of BODIPY derivatives. Y.-P.H. and Y.V.B. designed FDAA studies, M.S.V. provided FDAAs and Y.-P.H. performed experiments. E.J.C. performed all other experiments. E.J.C. and G.D.W. prepared the manuscript.

Corresponding author

Correspondence to Gerard D. Wright.

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Competing interests

E.J.C., N.W., W.W. and G.D.W. are inventors on a provisional patent application that covers the use of complestatin and corbomycin as antimicrobial therapies.

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Extended data figures and tables

Extended Data Fig. 1 crb and com biosynthetic gene clusters.

a, Labelling scheme for the position of the condensation domains with respect to the centrally encoded 4-hydroxyphenylglycine. b, Full maximum-likelihood phylogenetic tree of condensation domains with labels and colours according to the scheme in a. Figure 1a is an enlargement of the C-2 region. The tree is shown as a rectangular cladogram with a midpoint root. Labels indicate well-supported clades (SH-like support values > 0.89) of condensation domains for the true GPAs, complestatin (comp)-type BGCs and corbomycin (corb)-type BGCs. c, d, Schematic and gene annotations of com, the BGC of Streptomyces sp. WAC01325 corresponding to complestatin (c) and crb, the BGC of Streptomyces sp. WAC01529 corresponding to corbomycin (d). Gene names in c are based on the previously published complestatin BGC in Streptomyces lavendulae10, the gene synteny of which matches exactly except for comE, an MbtH-like protein that appears fused to the C terminus of comD in the sequence of Streptomyces sp. WAC01325. This may be due to sequencing error or incorrect calling of the open reading frame, which we performed using Prodigal as part of the antiSMASH workflow. e, Proposed biosynthetic pathway for corbomycin. A full description is provided in the Supplementary Discussion.

Extended Data Fig. 2 FabI and FabL are not the target of complestatin or corbomycin.

a, b, Dose–response curves of B. subtilis growth against complestatin, corbomycin and triclosan. a, B. subtilis has two enoyl-ACP reductase orthologues, FabI and FabL. fabI and fabL gene deletions (BKK library strains BKK11720 and BKK08650, respectively41) do not affect susceptibility to complestatin or corbomycin, but sensitize cells to triclosan—a strong fabI inhibitor—when fabL is deleted. b, Overexpression of fabI and fabL does not affect susceptibility towards complestatin or corbomycin but protects cells from triclosan when fabL is overexpressed. Genes were expressed from the pSWEET integrative plasmid under the control of a xylose-inducible promoter46. The average and standard deviation of biological triplicates is shown for a and b. c, Supplementing exogenous fatty acid was previously reported to modestly reduce susceptibility to complestatin15, so was repeated here using 2D checkerboards of fatty acids against complestatin, corbomycin or triclosan in S. aureus ATCC 29213. There is no robust reduction in susceptibility to complestatin or corbomycin in contrast to triclosan. Colour is scaled to OD600 with white representing no growth and dark blue representing growth. d, Bright-field microscopy of S. aureus ATCC 29213 grown in 0.6 × MIC triclosan (0.004 μg ml−1), complestatin (0.6 μg ml−1) or corbomycin (0.6 μg ml−1) was used to examine the effect of these antibiotics on phenotype. Whereas complestatin and corbomycin cause a distinct clumping phenotype, triclosan shows no gross morphological defect. All experiments were performed on two independent occasions with similar results.

Extended Data Fig. 3 Peptidoglycan biosynthesis is not inhibited by complestatin or corbomycin.

a, b, Incorporation of fluorescent HADA into peptidoglycan of B. subtilis treated with complestatin or corbomycin for 5 min before continued exposure with HADA for 1 h, or with ampicillin for 1 min before FDAA incubation for 5 min. For a, samples left to right, the number of individual cells measured was n = 243, 242, 299, 271. Whiskers show the minimum and maximum values in the dataset, the box shows the upper and lower quartiles and the line shows the median value. The results in b are quantified in Fig. 2d. c, Incorporation of HALA or HADA, as in a. Cells were treated with 0.6 × MIC and HALA incorporation is shown to represent transpeptidase activity. d, Enumeration of cfu after treatment of B. subtilis 168 for 6 h shows that complestatin and corbomycin are bacteriostatic, similar to the control antibiotic chloramphenicol. Mean and standard deviation cfu values are shown for four independent experimental replicates. e, Washing and subculturing antibiotic-treated cells as prepared in d similarly shows successful outgrowth, indicating a bacteriostatic antibiotic. Mean and standard deviation is shown for three biological replicates. All experiments were performed twice with similar results.

Extended Data Fig. 4 Complestatin and corbomycin have a novel mode of action.

a, Two-dimensional checkerboards in E. coli BW25113 (polymyxcin B nonapeptide; PMBN) or B. subtilis 168 (remainder). For the complestatin versus corbomycin checkerboard, the fractional inhibitory concentration index is 0.75. The colour is scaled to OD600, with white representing no growth and dark blue representing growth. b, Phenotype of B. subtilis 168 grown in 2 × MIC (8 μg ml−1) complestatin or corbomycin for 2 h, or treated with sub-MIC fosfomycin (100 μg ml−1), ampicillin (0.2 μg ml−1), vancomycin (1 μg ml−1) and triclosan (0.5 μg ml−1) until growth to mid-log phase. All experiments were performed on two independent occasions with similar results.

Extended Data Fig. 5 Mutants raised with complestatin or corbomycin.

a, Results of serially passaging S. aureus in sub-MIC concentrations of antibiotic over 25 days. Passaging was performed on two independent lines with similar results. b, MICs of strains COM14 and COR20 against various antibiotics, with respect to B. subtilis 168. The mbl mutation in strain COR14 renders it not viable in 40 μg ml−1 ZnCl2 used for bacitracin, so MIC determination was not possible. c, Susceptibility of B. subtilis strains to moenomycin was assessed by a Kirby–Bauer assay on Mueller–Hinton agar, which results in a zone of sub-lethal growth inhibition in the wild-type strain. d, Bright-field, fluorescence and transmission electron microscopy of resistant mutants COM20 and COR14 in comparison to wild-type and independently generated mutants in relevant genes. e, Fold increase in MIC as compared with wild-type B. subtilis 168 of various independently generated mutants. All knockouts were obtained from a non-essential gene knockout library (see Extended Data Table 2). ND, not determined. *The MIC of the mbl mutant was measured in the presence of 25 mM MgCl2. f, The effects of CwlO knockout and overexpression, either full length or the truncated version identified in COM20, on antibiotic susceptibility was determined. MICs were measured in the presence of 3% xylose to induce expression from Pxyl. Results are described in the Supplementary Discussion. All experiments were performed on at least two independent occasions with similar results.

Extended Data Fig. 6 Complestatin and corbomycin block autolysins in vitro and in vivo.

a, Effect of chloramphenicol on the lysis of B. subtilis. Cells were treated with ampicillin (100 μg ml−1), fosfomycin (50 μg ml−1) or sodium azide (75 mM) to induce lysis, in addition to chloramphenicol, MgSO4 or solvent control. Excess Mg2+ inhibits azide-induced lysis by a mechanism that is not well-understood but is thought to be partially through the modulation of autolysin activity50,51. b, Complestatin and corbomycin antagonism of S. aureus lysis induced by fosfomycin (50 μg ml−1) or ramoplanin (50 μg ml−1). Average values and standard deviation from biological triplicates are plotted for a and b. c, Activity of hen egg white lysozyme on peptidoglycan incubated with 400 μg ml−1 complestatin or corbomycin. The assay was performed in technical triplicate with similar results. The legend refers to both b and c. d, Vancomycin–BODIPY FL (van–BODIPY) staining shows nascent peptidoglycan containing d-Ala-d-Ala in cells treated with 10 × MIC antibiotic for 30 min. All experiments were performed twice with similar results.

Extended Data Fig. 7 Tools used to measure peptidoglycan binding.

a, Activity of CwlO on peptidoglycan that was preincubated with 100 μg ml−1 antibiotic, then washed and resuspended in fresh buffer. The assay was performed in technical triplicate with similar results each time. b, HPLC chromatograms of antibiotic left unbound after incubation with either B. subtilis or S. aureus peptidoglycan. Additional negative control drugs rifampicin (absorbing at 330 nm) and daptomycin (absorbing at 280 nm) were tested under conditions identical to those described in Fig. 4a. Incubation with peptidoglycan does not reduce peak height, indicating the absence of non-specific binding. c, Chemical structures of corbomycin–BODIPY FL (Corb–BODIPY) and complestatin–BODIPY FL (Comp–BODIPY). d, BODIPY derivatives of corbomycin and complestatin induce the same twisted cell chaining as the parent compounds, so retain the same mechanism of action. All experiments were performed on two independent occasions with comparable results.

Extended Data Fig. 8 Complestatin and corbomycin are effective in a mouse skin infection model.

A topical application of antibiotic in petroleum jelly (%w/w) was used to treat MRSA in a skin infection model using neutropenic mice. Mice were assessed 33 h after infection and compared to those receiving the vehicle control (petroleum jelly containing 10% DMSO). a, The cfu per gram of tissue was determined. Significance was tested by one-way ANOVA on ranks with Kruskal–Wallis test, P values versus vehicle as follows: 1% complestatin, 0.0062; 1% corbomycin, 0.0157; 0.25% fusidic acid, 0.0003. b, The change in weight (%) after infection compared to pre-infection was measured for each mouse. Significance was tested by a two-sided unpaired Mann–Whitney t-test, P-values versus vehicle as follows: 1% complestatin, 0.0016; 1% corbomycin, 0.016; 0.25% fusidic acid, 0.0028. For a, b, * denotes significance (P < 0.05), and whiskers show the minimum and maximum values of the dataset, the box shows upper and lower quartiles and the line shows the median value. c, Visual inspection of the wounds of representative mice in each treatment group. Each treatment condition was tested with replicate mice (n = 6 complestatin, corbomycin, fusidic acid; n = 9 vehicle) divided between two independent experiments.

Source data

Extended Data Table 1 MICs of complestatin and corbomycin
Extended Data Table 2 Strains used in this study

Supplementary information

Supplementary Information

This file contains supplementary discussion, Fig. 1-6 and table 1. Discussion is of the structural characterization of corbomycin, the biosynthetic pathway and gene cluster for corbomycin, and further analysis of resistant mutants raised to complestatin and corbomycin. Figures and table are for NMR spectra of corbomycin.

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Supplementary Data

This file contains complestatin and corbomycin structures.

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Culp, E.J., Waglechner, N., Wang, W. et al. Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling. Nature 578, 582–587 (2020). https://doi.org/10.1038/s41586-020-1990-9

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