Isopedopeptins A–H: Cationic Cyclic Lipodepsipeptides from Pedobacter cryoconitis UP508 Targeting WHO Top-Priority Carbapenem-Resistant Bacteria

Abstract

Pedobacter cryoconitis strain UP508 was isolated from a soil sample using a mixture of ampicillin, kanamycin, and nalidixic acid for selection. UP508 was found to produce >30 unknown antibacterial peptides, of which eight, isopedopeptins A–H ( 1 – 8 ), were isolated by bioassay-guided fractionation and characterized with respect to structures and biological properties. Compounds 1 – 8 were all composed of nine amino acid residues and one 3-hydroxy fatty acid residue, and the structures were ring-closed via an ester bond from the C-terminal aspartic acid to the 3-hydroxy fatty acid. The differences between the peptides were the size and branching of the 3-hydroxy fatty acid and the presence of a valine or a 3-hydroxyvaline residue. The isopedopeptins mainly had activity against Gram-negative bacteria, and isopedopeptin B ( 2 ), which had the best combination of antibacterial activity, in vitro cytotoxicity, and hemolytic properties, was selected for further studies against a larger panel of Gram-negative bacteria. Isopedopeptin B was found to have good activity against strains of WHO top-priority Gram-negative bacteria, i.e., carbapenem-resistant Acinetobacter baumannii , Escherichia coli , and Pseudomonas aeruginosa , with minimal inhibitory concentrations (MIC) down to 1, 2, and 4 μg/mL, respectively. Furthermore, compound 2 had activity against colistin-resistant strains of A. baumannii , E. coli , and Klebsiella pneumoniae , with a MIC down to 8, 2, and 4 μg/mL, respectively. Compound 6 was tested in an E. coli liposome system where it induced significant leakage, indicating membrane disruption as one mechanism involved in isopedopeptin antibacterial activity. Isopedopeptin B stands out as a promising candidate for further studies with the goal to develop a new antibiotic drug.

Selective Isolation of Multidrug-Resistant Pedobacter spp., Producers of Novel Antibacterial Peptides

“…Given the resistance against lipopeptides observed for P. cryoconitis UP508, it is not surprising that many of the isolated Pedobacter strains were found to produce lipopeptides. The isolate UP508 was recently described to produce isopedopeptins (Nord et al, 2020) and several other strains produced the pedopeptins (Hirota-Takahata et al, 2014), here identified by MSMS. However, a number of isolates also produced unknown compounds, including the isopedopeptin analogs present in clusters 2 and 3, here only characterized by MSMS, and linear lipopeptides (clusters 5, 6, 13, and 14), characterized by MSMS and partly by NMR (two peptides of cluster 5).…”

“…The software Compass DataAnalysis 4.3 (Bruker Daltonics) was used for calibration and data analysis. Isopedopeptins were identified by comparison with authentic compounds from previous work (Nord et al, 2020), and identification of other compounds was attempted by comparison of HRMS-data with databases (Antibase and Combined Chemical Dictionary). UHPLC-MSMS was run on the same instrument, column and UHPLCconditions, on extracts or on extracts treated with 2% NaOMe in MeOH for 30 min at room temperature, using the auto MSMS function (1+, 2+, and 3+ precursor ions, m/z 200-1100, ramped fragmentation energies: 1+: 30/40/45 eV for m/z 100/500/1000; 2+: 15/25/30 eV for m/z 100/500/1000; 3+: 10/20/25 eV for m/z 100/500/1000).…”

“…Cluster 1 in the heat map (Figure 3) contains the recently described isopedopeptins A-F (Figure 4; Nord et al, 2020), along with four additional analogous peptides. Isopedopeptins were only found in the isolate P. cryoconitis UP508.…”

“…Peptides with R 7 = 2 × OH are linear, and MSMS-data on these were obtained without NaOMe treatment. The cluster 1 peptides includes the previously published isopedopeptins (Nord et al, 2020) and the cluster 4 peptides the pedopeptins (Hirota-Takahata et al, 2014). Pedopeptin A and C were only found in cultures of two strains but are included in the cluster 4 entries.…”

“…Thus, when isolating soil bacteria for subsequent screening for new antibiotic compounds, we have combined several antibiotics in order to specifically select for MDR bacteria. The use of this approach resulted in the isolation of P. cryoconitis UP508, which was found to produce isopedopeptins, which are cyclic lipodepsipeptides with potent activity against WHO top-priority bacterial pathogens (Nord et al, 2020). In the present paper, we describe the application of this methodology to isolate several MDR strains of Pedobacter, along with UHPLC-MS based hierarchical cluster analysis (HCA) of the metabolites produced by the isolates.…”