The antimicrobial resistance crisis requires the introduction of novel antibiotics. The use of conventional broad-spectrum compounds selects for resistance in off-target pathogens and harms the microbiome. This is especially true for Mycobacterium tuberculosis, where treatment requires a 6-month course of antibiotics. Here we show that a novel antimicrobial from Photorhabdus noenieputensis, which we named evybactin, is a potent and selective antibiotic acting against M. tuberculosis. Evybactin targets DNA gyrase and binds to a site overlapping with synthetic thiophene poisons. Given the conserved nature of DNA gyrase, the observed selectivity against M. tuberculosis is puzzling. We found that evybactin is smuggled into the cell by a promiscuous transporter of hydrophilic compounds, BacA. Evybactin is the first, but likely not the only, antimicrobial compound found to employ this unusual mechanism of selectivity.
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M. tuberculosis genome data (NC_000962.3) were used as a reference of genome sequencing. All data supporting the findings of this study are available within the paper and its Supplementary Information or have been deposited to the indicated databases. The genome of P. noenieputensis DSM 25462 has been deposited to GenBank with accession number RCWC00000000.1. Structural data have been deposited in the PDB with PDB identifier 7UGW. All other data are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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This work was supported by National Institutes of Health grants P01AI118687 (K.L.), R01CA077373 (J.B.) and R35263778 (J.B.). Part of this work was conducted at the AMX beamline of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under contract number DE-SC0012704. The AMX beamline is a member of the the Center for BioMolecular Structure, which is primarily supported by the National Institute of General Medical Sciences through a Center Core P30 grant (P30GM133893) and by the DOE Office of Biological and Environmental Research (KP1607011). We thank D. J. Slotboom (University of Groningen) for providing BacA/BacA-like transporter protein sequences. We thank S. Abbatiello and A. Iinishi (Northeastern University) and Y.-S. Hong (Korea Research Institute of Bioscience and Biotechnology) for help with LC–MS experiments. We acknowledge the Korea Basic Science Institute for providing NMR data. We appreciate discussions with G.E. Martin (Seton Hall University) and J. Oh (Yale University) about the 1,1- ADEQUATE NMR experiment.
The authors declare no competing interests.
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a, 1H. b, 13C. c, COSY. d, ROESY. e, 1H-13C HSQC. f, 1H-13C HMBC.
All correlations were measured in DMSO-d6 except for the HMBC correlation from H-41 to C-2 was recorded in D2O.
Mice were infected by E. coli ATCC 25922 through intraperitoneal injection, and antibiotics were administrated 1 h later. Survival was monitored over 5 days. The experiment was repeated three times (n=4 biologically independent mice); lines are the mean of experiments. Gentamicin (Gen) was used as a positive control. All treatment are in mg kg−1.
BacA phylogenic tree was generated by using the Maximum Likelihood method based on the JTT matrix-based model52. The tree with the highest log likelihood (−8349.22) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 17 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 274 positions in the final dataset. Evolutionary analyses were conducted in MEGA753. Protein sequences were obtained from a previous study54.
Effect of evybactin on macromolecular biosyntheses in E. coli WO153. Incorporation of 14C-thymidine (DNA), 14C- uridine (RNA), 14C -L-amino acid mixture (protein), 14C-Acetic acid (fatty acid) and 14C-acetyl-glucosamine (peptidoglycan) was determined in cells treated with 8x MIC of evybactin (grey bars). Ciprofloxacin (8x MIC), rifampicin (8x MIC), chloramphenicol (8x MIC), triclosan (8xMIC) and fosfomycin (8x MIC) were used as controls (white bars). Values are plotted as mean values ±SD, n=3 independent biochemical experiments.
a, Electron density omit maps for evybactin contoured at 1σ. Gyrase is depicted as a blue cartoon and evybactin as magenta sticks. b, Comparison of the evybactin-binding pocket (top panels) with the thiophene-binding pocket (bottom panels – PDB ID: 5NPK14). Gyrase subunits are colored in dark blue (GyrA) and light blue (GyrB) with evybactin and the thiophene colored magenta and green, respectively. Hydrophobic residues forming the shared evybactin and thiophene binding pocket are labeled (left panels). A glutamate residue in GyrB that is critical for thiophene binding to S. aureus gyrase (E634) is a threonine (T664) in M. tuberculosis gyrase (right panels). c, Electron density omit maps (1σ) for the evybactin-bound M. tuberculosis gyrase structure (left) and the thiophene-bound S. aureus gyrase structure (right, PDBID: 5NPK). Gyrase is colored in green and DNA is orange.
Extended Data Fig. 7 Mutations at the evybactin binding site effect evybactin and moxifloxacin induced cleavage.
a, Plots represent quantitation of evybactin- and moxifloxacin-induced cleavage in the presence of ATP. Fraction of linearized plasmid plotted at indicated concentrations of compound (0-500 µM). Cleavage was conducted with 20 nM wild-type MtbGyrase or MtbGyrase GyrA mutants and 6 nM DNA. Lines represent non-linear fits to the data, as in Fig. 4, values are plotted as mean values ±SD, n=3 independent biochemical experiments. b, Representative cleavage assays used for quantitation. Samples were separated on agarose gels run in the presence of ethidium bromide. The positions of nicked, linear, and uncleaved plasmid are indicated.
Extended Data Fig. 8 Evybactin and moxifloxacin stimulated cleavage activity of M. tuberculosis gyrase mutants.
Native agarose gel-based cleavage assay conducted with 125 nM gyrase, 6 nM plasmid DNA, and indicated amounts of evybactin or moxifloxacin (0-20 µM). Mutations in GyrA or GyrB are indicated above each panel. The positions of nicked, linear, and supercoiled plasmid DNA are indicated. Note that high protein concentrations are used to see activity in the resistance mutants; as a result, the DNA in the WT MtbGyrase reactions becomes degraded and disappears as evybactin concentrations are increased due to the presence of multiple, randomly spaced cleavage complexes. All assays repeated at least 2 times with similar results.
Native agarose gel analysis of supercoiling activity using indicated amounts of M. tuberculosis gyrase and resistance mutants (0-20 nM). The migration positions of relaxed starting material and supercoiled products are indicated. All assays repeated at least 3 times with similar results.
Extended Data Fig. 10 The evybactin binding pocket is concealed in the M. tuberculosis gyrase ‘ATPase open’ state.
Structure of S. cerevisiae TOP2 bound to DNA and nonhydrolyzable ATP analog, illustrating the ‘ATPase closed’ conformation of type-II topoisomerases (left, PDBID: 4GFH55). The ATPase and transducer domains are colored yellow and light green, the nucleolytic core is illustrated in orange and blue, and DNA is shown in grey. Structure of M. tuberculosis gyrase in an ‘ATPase open’ state (right, PDBID: 6GAV38), in which the ATPase regions are folded down from the position shown at left. The binding site for evybactin is illustrated as a black outline and shown as a purple surface in the inset. The inset shows the loop within the GyrB ATPase domain that is specific to Corynebacteriales gyrases and how this loop occludes the evybactin binding site in the ‘ATPase open’ conformation of the enzyme.
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Imai, Y., Hauk, G., Quigley, J. et al. Evybactin is a DNA gyrase inhibitor that selectively kills Mycobacterium tuberculosis. Nat Chem Biol 18, 1236–1244 (2022). https://doi.org/10.1038/s41589-022-01102-7
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