Rule-based omics mining reveals antimicrobial macrocyclic peptides against drug-resistant clinical isolates

The study addresses the urgent need for new antibiotics to combat multidrug-resistant gram-positive pathogens. Macrocyclic peptides, particularly RiPPs, offer protease resistance and high target affinity. Despite abundant microbial genomes, prioritizing biosynthetic gene clusters (BGCs) and linking them to metabolites remains challenging. The authors focus on aminovinyl-(methyl)cysteine (Avi(Me)Cys)-containing peptides (ACyPs), whose conserved biosynthetic logic and favorable drug-like properties make them attractive antibiotic leads. The research aims to systematically mine actinobacterial and firmicute genomes to identify Avi(Me)Cys RiPP BGCs, connect them to their products using rule-based metabolomics, and evaluate antibacterial potency, especially against drug-resistant clinical isolates.

Macrocyclic peptides, including RiPPs and NRPs, have yielded notable antibiotics (e.g., nisin, ruminococcin C1, thiostrepton, nosiheptide, darobactin, dynobactin, vancomycin, teixobactin, brevicidine, corbomycin, clovibactin). Genome mining has enabled discovery of RiPP antibiotics but suffers from rediscovery and prioritization bottlenecks. Prior work from the authors uncovered antagonistic archaeal lanthipeptides and human microbiome bacteriocins and identified hidden RiPP peptidases/PTM enzymes via sequence and structure-based strategies. ACyPs occur across several RiPP families (lanthipeptides, lipolanthins, lanthidins, thioamitides, linaridins) and share a conserved formation of Avi(Me)Cys via a flavoprotein-catalyzed oxidative decarboxylation of a C-terminal cysteine followed by thioether cyclization to Dha/Dhb. Microbisporicin and lexapeptide exemplify potent Avi(Me)Cys lanthipeptides targeting lipid II and resistant strains. These precedents motivate targeted mining of ACyP BGCs using biosynthetic rules.

- Genome mining: Applied the rule-based SPECO pipeline to 52,577 bacterial genomes (21,911 actinobacteria; 30,666 firmicutes) to identify ACyP BGCs using constraints: small peptides 20–100 aa ending with cysteine and bearing a T/S–Xn–C motif (2≤n≤6), presence of a PF02441 flavoprotein within ≤15 kb, and precursor–flavoprotein pairing. Clustered precursor sequences via MMseqs2 for sequence similarity networks (SSN) and generated sequence logos and phylogenies to prioritize novel families (e.g., mat and sis). - Mass mapping workflow: Built theoretical libraries of intact and fragment ion m/z values by enumerating post-translational modifications expected for ACyPs (dehydrations, decarboxylation, thioether cyclizations, hydrogenations, methylations). Matched theoretical m/z to UPLC-HRMS data of crude extracts with ppm error <10 to target candidate peptides. - Strain cultivation and extraction: Cultured wild-type and heterologous Streptomyces strains on optimized media (GYM, R5A, MS). Extracted with methanol, partitioned with n-butanol, and analyzed by UPLC-HRMS. - Heterologous expression: Cloned entire mat (from Streptomyces leeuwenhoekii DSM42122), sis (from S. kasugaensis DSM40819), and keb (from S. kebangsaanensis DSM42048) BGCs into vector pJTU2554 and expressed in Streptomyces albus J1074 via conjugation. Constructed a matR1 deletion to probe reductase function. Performed leader-core chimeras with keb maturase (keb-syn1/2) to assess enzyme promiscuity. - Structural elucidation: Assigned modifications and ring patterns via tandem MS matching to calculated fragments; purified compounds for 1D/2D NMR (HMBC/HSQC) to confirm Avi(Me)Cys and lanthionine linkages and geometries; determined absolute configurations via advanced Marfey’s analysis (L/D-FDLA) to identify D-amino acids formed by reductases. - Antibacterial assays: Kirby-Bauer agar diffusion assays and MIC determinations against gram-positive and gram-negative strains; compared to nisin and vancomycin. Tested clinical isolates (MRSA, LRSA, VMSA, E. faecalis, VRE) with levofloxacin as control. - In vitro properties: Assessed bactericidal kinetics vs S. aureus, protease and thermal stability, and resistance development via serial passaging at sub-MIC. - In vivo efficacy and safety: Acute toxicity in mice (single i.p. doses 10–50 mg/kg). MRSA septicemia mouse model with post-infection dosing (10–50 mg/kg) and survival monitoring over 7 days.

- Genome mining: Identified 1172 putative ACyP precursor–flavoprotein pairs across actinobacteria and firmicutes, grouped into 67 families; many precursors were novel, indicating large unexplored chemical space. - mat family discovery (class V lanthipeptide): Linked mat BGC to massatide A (1) with [M+2H]2+ = 817.8963 by heterologous expression. Structural analysis indicated six dehydrations, one decarboxylation, three hydrogenations, two methylations, with AviCys rings and D-amino acids (D-Abu6, D-Ala9, D-Ala16) inferred by MS and confirmed by Marfey’s. NMR supported Avi(Me)Cys Z-geometry and presence of N,N-dimethyl MeLan. Deletion of matR1 yielded massatide B (1a) 2 Da lighter with Dhb at Thr6, establishing MatR1 role in D-Abu6 formation and MatR2 likely for D-Ala9/16 formation. - sis family discovery: From S. kasugaensis, identified sistertide A1 (2) ([M+3H]3+ = 1085.2083) with three thioether rings (including C-terminal AviMeCys) and N-terminal methylation, and sistertide A2 (3) ([M+3H]3+ = 816.7487) with two rings. A1 contained D-Ala residues derived from dehydrated/reduced Ser. Heterologous expression confirmed ribosomal origin and activity against M. luteus. - keb family discovery: From S. kebangsaanensis, heterologous expression produced kebanetide A1 (4) ([M+3H]3+ = 921.4331) with one AviMeCys ring and two thioether crosslinks, and kebanetide A2 (5) ([M+3H]3+ = 793.7395) with AviMeCys plus an N-terminal thioether ring. Chimeric leader-core experiments showed keb maturases accept SisA cores and can produce new methylated analogs, indicating leader-selective but core-tolerant tailoring enzymes. - Antibacterial activity (MIC, µg/mL): Massatide A showed strong gram-positive activity: S. aureus ATCC25923 (0.5), M. luteus DSM1790 (0.06), B. subtilis 168 (2), E. faecalis OG1RF (4), E. faecium MCC2763 (2), outperforming nisin and comparable or superior to vancomycin on several strains. Massatide B similar but slightly weaker. Kebanetides showed moderate activity (e.g., vs M. luteus 8 for 4) and outperformed nisin against Enterococcus spp. No activity against gram-negative bacteria. - Clinical isolates: Massatide A MICs (µg/mL): LRSA 20-1 (0.25), MRSA ATCC43300 (0.25), MRSA 15-1 (2), MRSA ATCC33591 (1), vancomycin-intermediate S. aureus ATCC700788/700699 (4), E. faecalis 19-1 (4), linezolid-resistant E. faecalis 20-1 (4), VREfa ATCC51575 (4). Gram-negative isolates showed poor activity (≥64 or >64). - Pharmacology and resistance: Massatide A is bactericidal against S. aureus, protease-resistant, heat-stable; low resistance development in serial passaging (MIC from 0.5 to 4 µg/mL over 25 days, then stable). Cytotoxicity: HeLa IC50 136.3 µg/mL; moderate toxicity to HEK293T. Mouse acute toxicity: no deaths at 10–50 mg/kg i.p. MRSA septicemia model: survival improved to 80% at 10 mg/kg and 100% at 50 mg/kg single dose.

The study demonstrates that a rule-based omics strategy anchored in conserved Avi(Me)Cys biosynthetic logic can efficiently prioritize BGCs, map them to metabolites, and deliver bioactive macrocyclic peptide antibiotics. Discoveries across mat, sis, and keb families validate the approach and reveal enzyme features (dual F420-dependent reductases in mat; leader-selective but core-tolerant maturases in sis/keb) that generate structural diversity, including D-amino acid incorporation and varied crosslinking patterns. Massatide A’s potent in vitro and in vivo efficacy against gram-positive and drug-resistant S. aureus/Enterococcus underscores ACyPs as promising therapeutic leads. The lack of gram-negative activity is consistent with many RiPPs. The approach’s success in linking genomics to chemistry and bioactivity addresses rediscovery challenges and suggests that exploring the broad distribution of flavoprotein-linked BGCs can expand RiPP antibiotic space. However, the rule-based metabolomics may miss metabolites with unanticipated or unknown modifications, highlighting the need to enhance enzyme annotation and predictive MS/MS mapping. The observed maturase promiscuity offers opportunities for engineering simplified, potent, and safer analogs.

By integrating rule-based genome mining, PTM-informed metabolomics, and heterologous expression, the authors uncovered five Avi(Me)Cys-containing RiPPs and established massatide A as a potent, stable, and comparatively safe gram-positive antibiotic with in vivo efficacy and low resistance risk. The work expands known ACyP families, reveals novel biosynthetic features (dual reductases; D-amino acids in Avi(Me)Cys ring), and provides a generalizable platform to connect BGCs to active compounds. Future efforts should refine enzyme function prediction and mass mapping to capture unexpected modifications, elucidate the mode of action of massatide A (e.g., lipid II engagement), optimize pharmacological properties and safety (particularly renal cell toxicity), and leverage maturase promiscuity to generate new-to-nature analogs with improved efficacy and spectrum.

- Rule-based metabolomic matching may overlook compounds with unanticipated or complex modifications not covered by the rules, risking false negatives. - Limited material for complete structural elucidation of some peptides (e.g., insufficient sistertide A2 for NMR), leaving some structures suggested rather than fully confirmed. - Activity was confined to gram-positive bacteria; no efficacy against gram-negative strains was observed. - Moderate cytotoxicity in HEK293T cells and HeLa IC50 of 136.3 µg/mL for massatide A indicate the need for further safety optimization. - Resistance assessment was limited to laboratory passaging over several weeks; long-term or clinical resistance evolution remains unknown. - In vivo evaluations were limited to acute toxicity and a single septicemia model; broader pharmacokinetic, pharmacodynamic, and chronic toxicity studies are needed.