Identification of structurally diverse menaquinone-binding antibiotics with in vivo activity against multidrug-resistant pathogens

Abstract

The emergence of multidrug-resistant bacteria poses a threat to global health and necessitates the development of additional in vivo active antibiotics with diverse modes of action. Directly targeting menaquinone (MK), which plays an important role in bacterial electron transport, is an appealing, yet underexplored, mode of action due to a dearth of MK-binding molecules. Here we combine sequence-based metagenomic mining with a motif search of bioinformatically predicted natural product structures to identify six biosynthetic gene clusters that we predicted encode MK-binding antibiotics (MBAs). Their predicted products (MBA1-6) were rapidly accessed using a synthetic bioinformatic natural product approach, which relies on bioinformatic structure prediction followed by chemical synthesis. Among these six structurally diverse MBAs, four make up two new MBA structural families. The most potent member of each new family (MBA3, MBA6) proved effective at treating methicillin-resistant Staphylococcus aureus infection in a murine peritonitis-sepsis model. The only conserved feature present in all MBAs is the sequence 'GXLXXXW', which we propose represents a minimum MK-binding motif. Notably, we found that a subset of MBAs were active against Mycobacterium tuberculosis both in vitro and in macrophages. Our findings suggest that naturally occurring MBAs are a structurally diverse and untapped class of mechanistically interesting, in vivo active antibiotics.

Extended Data Fig. 1. Phylogenetic analysis of eSNaPD hits from six conserved A-domains found in the BGCs of the three known MBAs

Phylogenetic analysis of eSNaPD hits (a) and predicted peptide sequences of recovered clones (b). All the hits at an e-value ≤10^–45 from A-domain analysis of L-Leu-6 encoded new MBAs and formed a separate, well-defined clade with A-domains of three known MBAs, which suggested that L-Leu-6 in the proposed minimal MK-binding motif were encoded by the most highly conserved A domain in MBA-family peptides.

Extended Data Fig. 2. Three potential MBA BGCs from eSNaPD-guided soil metagenomic mining

Comparison of NRPS gene organization (a) as well as amino acid substrates (b) between the three known MBA BGCs and the three potential MBA BGCs we cloned from soil metagenomes. The blue residues represent building blocks that are conserved across all MBAs. The green circles represent residues that are shared between known and potential MBAs.

Extended Data Fig. 3. Predicted MBA peptide sequences identified in a motif search of the p-NRP database

(a) and the BGCs associated with these predicted peptides (b) The blue residues represent building blocks that are conserved across all MBAs.

Extended Data Fig. 4. The structures (a) and anti-bacterial activities (b) of the N-acylated peptides associated with known MBAs cyclized in two different ways

The (R)-3-hydroxy-octanoic acid analogs of lysocin E, WBP-29479A1 and the deoxy version of WAP-8294A1 shown here were synthesized in this study. B. subtilis 168 1A1, S. aureus USA300, S. epidemidis RP62A and M. tuberculosis H37Rv were used as tested strains. The blue residues represent building blocks that are conserved across all MBAs.

Extended Data Fig. 5. Membrane depolarization activity and resistance frequency of MBAs 1 through 6

a, The effect of each MBA on S. aureus membrane potential was measured using 3,3′-Dipropylthiadicarbocyanine iodide [DiSC3(5)]. Vancomycin (Van) and lysocin were used as the negative and positive controls, respectively. b, Resistance frequency of MBAs 1 through 6 against S. aureus USA300 in the presence of 4x the MIC of each antibiotic.

Extended Data Fig. 6

Isothermal titration of 1:1 (mol/mol) DOPC:DOPG vesicles containing MK into each MBA

Extended Data Fig. 7. Correlation between antibiotic activity and MK binding affinity for active or inactive syn-BNP MBAs

a, Isothermal titration of 1:1 (mol/mol) DOPC:DOPG vesicles containing MK into the four additional syn-BNPs we generated in Fig. 2. b, Comparison of Kd values and MICs against S. aureus USA300 for all syn-BNP MBAs in Fig. 2.

Extended Data Fig. 8. Antibiotic activity and MK binding of MBA3 with single point mutations in the proposed minimal MK-binding motif

MIC in μg/mL, highest concentration tested was 64 μg/mL.

Fig. 1 |. Identification of BGCs predicted to encode new MBAs.

Using a sequence based soil metagenome BGC discovery pipeline, three BGCs were identified that show high A-domain sequence identity and similar overall gene organization to known MBA BGCs. We predicted that each of these encoded a new MBA. Using the conserved GXLXXXW motif that we detected in structurally diverse MBAs to search an in-house database of predicted NRP (p-NRP) structures we identified three additional BGCs that we predicted would encode new MBAs. The GXLXXXW motif that we found in all MBAs is predicted to represent the minimal MK binding motif that is necessary for the antibacterial activity of this underexplored and structurally diverse class of natural antibiotics.

Fig. 2 |. Synthesis and antibacterial activity of syn-BNPs based on BGCs predicted to encode MBAs.

The (R)-3-hydroxy-octanoic acid derivatized linear peptides that are predicted to be encoded by MBA BGCs were cyclized through either the hydroxyl group of the fatty acid (cFA) or through a nucleophilic amino acid side-chain (cSC). When the first amino acid was predicted to contain a nucleophilic side chain (i.e., a serine or threonine) both the cFA and cSC analogs were synthesized (MBA2, MBA5 and MBA6). In the case of MBA5-cSC2, the serine at position 2 was also used for cyclization. If the first amino acid of peptide did not contain a nucleophilic side chain, only a cFA derivative was produced (MBA1, MBA3 and MBA4). syn-BNPs marked with an asterisk were the most active to arise from each BGC and assumed to be the “naturally cyclized” versions of the potential MBA. All MIC assays were done in duplicate (n=2).

Fig. 3 |. Bactericidal effects and mode-of-action analysis of six new MBAs.

a, Bactericidal activity of MBA1–6 against S. aureus USA300. Cultures were incubated with each antibiotic at 2x its MIC. The number of viable cells was counted (n=3). Cultures were plated at defined times points to determine CFU per mL. b, Effect of MBA1–6 on S. aureus membrane lysis was determined using SYTOX fluorescence assay. c, The S. aureus antibacterial activity of MBA1–6 was determined in the presence of different concentrations of menaquinone (MK) (blue) or ubiquinone (UQ) (orange) (n=2). d, The MICs (μg/mL) of MBA1–6 against S. aureus mutants deficient in MK biosynthesis (ΔmenA or ΔmenB) (n=2). Lysocin and Van (vancomycin) were included as positive (MK binding) and negative (non-MK binding) antibiotic controls. The different shades of yellow represent MBA potency with darker color indicating higher activity.

Fig 4. |. Anti-Mtb activity and mode of action analysis of new MBAs.

a, MIC (μg/mL) values of all known and new MBAs against a panel of Mtb strains, including two BSL2 strains (mc² 6206 and mc² 7901), a wild-type strain (H37Rv) and four multidrug-resistant (MDR) clinical isolates (n=2). The four MDR strains (800, 4557, 10571 and 116) are resistant to rifampicin, rifampicin, ethambutol/isoniazid/rifampicin/streptomycin and ethambutol/isoniazid/para-aminosalicylic acid, respectively. MIC in μg/mL. b, Effects of MK (blue) or UQ (orange) on the antibacterial activity of MBA1–3 against Mtb mc² 6206 (n=2). c, The ability of Mtb-active MBAs to permeabilize the Mtb membrane was examined using a 3’-dipropylthiadicarboncyanine iodide [DiSC₃(5)] fluorescence assay. Verapamil and rifampicin were used as positive and negative depolarization controls, respectively. d, Activity of the three Mtb-active MBAs against Mtb mc² 6206/mLux itself (MIC, n=2) and Mtb mc² 6206/mLux in a macrophage infection assay (IC₅₀, n=2). The different shades of yellow represent MBA potency with darker color indicating higher activity.

Fig. 5. |. Structures of six new MBAs grouped by structural family.

a, Phylogenetic tree of linear MBA peptide sequences. The branches on the tree are labeled with the name of the MBA and the source of its BGC. In this study we identified congeners of two known MBAs (b) as well as two new MBA structural families (c, d). All MBAs share the conserved GXLXXXW motif (blue) that is predicted to be the minimal sequence that is associated with MK-binding as a mode of action. The conserved residues within each MBA family are highlighted. In accordance with the long standing tradition of giving bioactive natural products trivial names, we have given these six syn-BNPs the following names: wameb (MBA1, WBP-29479A1-like menaquinone-binding antibiotic), lysomeb (MBA2. lysocin E-like menaquinone-binding antibiotic), metameb (MBA3, metagenome menaquinone-binding antibiotic), alcameb (MBA4, P. alcaliphilus menaquinone-binding antibiotic), tabameb (MBA5, D. tabacisoli menaquinone-binding antibiotic) and mobimeb (MBA6, D. mobilis menaquinone-binding antibiotic). The blue residues represent building blocks that are conserved across all MBAs. The brown, green and red residues represent building blockings that are conserved acrosss the known MBA family (b), the first new MBA family (c) or the second new MBA family (d), respectively.

Fig 6. |. MBA3 (a) and MBA6 (b) are effective against S. aureus infections in mice.

Either MBA3 or MBA6 was subcutaneously injected 1 h after intraperitoneal administration of S. aureus COL into mice (n=5). 30% solutol was used as the vehicle.