Fig 1.
Streptothricins share streptolidine and carbamoylated gulosamine sugar moieties. They are distinguished by differing numbers of β-lysines attached end-to-end through amide bonds to the ε-amino groups. Nourseothricin is the natural product mixture of several streptothricins, predominantly S-F (1 β-lysine) and S-D (3 β-lysines). Acetylation of the β-amino group blocks activity and is the major known mechanism of antimicrobial resistance to streptothricins. S-D, streptothricin D; S-F, streptothricin-F.
Fig 2.
Rapid bactericidal activity against the Klebsiella pneumoniae Nevada strain.
(A) Nourseothricin MIC 0.25 μM. (B) S-F MIC 1 μM. Data are available in S1 Data. MIC, minimal inhibitory concentration; S-F, streptothricin-F.
Fig 3.
Inhibition of prokaryotic and eukaryotic translation.
(A) Inhibition of prokaryotic in vitro translation using coupled in vitro transcription–translation extracts with readout from a nanoluciferase reporter. (B) Inhibition of eukaryotic in vitro translation using coupled in vitro transcription–translation extracts with readout from a nanoluciferase reporter. S-F, S-D, NTC, APR, and TET data represent mean and standard deviation from 3 independent experiments. Data are available in S3 Data. APR, apramycin; NTC, nourseothricin; S-D, streptothricin D; S-F, streptothricin-F; TET, tetracycline.
Fig 4.
Cytotoxicity of streptothricins against mammalian cell lines.
J774 macrophages and LLC-PK-1 renal epithelial cells were treated with 2-fold doubling dilutions of nourseothricin, S-D, and S-F for up to 5 days in the presence of SYTOX-Green. SYTOX-Green is a cell membrane-impermeant nucleic acid binding dye that fluoresces on binding to nuclear DNA. It therefore provides a real-time readout of eukaryotic cell membrane permeabilization associated with cell death that can be continuously monitored through fluorescence measurements. Cytotoxicity was minimal to absent after a single day incubation but increased on subsequent days. Nourseothricin and S-D effects were essentially indistinguishable. S-F toxicity was only observed at molar concentration at least 10-fold greater than S-D beginning at 32 μM, significantly above MIC ranges observed in activity spectrum analysis. Each data point represents mean and standard deviation for assays performed in quadruplicate. Data are available in S4 Data. MIC, minimal inhibitory concentration; S-D, streptothricin D; S-F, streptothricin-F.
Fig 5.
Delayed nephrotoxicity occurs at >10-fold higher doses of S-F than nourseothricin.
(A) S-F dosing at 100 mg/kg without obvious histological abnormality in kidney. (B) Nourseothricin dosing at 10 mg/kg showing cellular necrosis and nuclear degeneration of proximal convoluted tubule epithelial cells (arrowheads). Glomeruli and distal tubules were spared. Tissue was harvested 3 days after dosing. Size bar = 20 μM.
Fig 6.
S-F demonstrated substantial therapeutic effect against pandrug-resistant Klebsiella pneumoniae Nevada AR-0636 at doses without observable or minimal toxicity. Dotted line is assay limit of detection. Data points for 5 mice per condition are shown. * designates significant difference from untreated controls using Kruskall–Wallis nonparametric test. Data are available in S5 Data.
Table 1.
Modal MIC values for resistance mutants identified in single rrn operon E. coli strain identifies unique target in helix 34 of ribosomal 16S rRNA.
Table 2.
TetM ribosomal protection protein does not interfere with nourseothricin activity.
Fig 7.
S-F and S-D binding sites in the ribosome.
(A, B) Location of the S-F and S-D binding sites in the A. baumannii 70S ribosome with P-site tRNA, respectively. (C, D) Hydrogen-bonding interactions between the streptolidine moieties of S-F and S-D and C1054 of the 16S rRNA, respectively (E, F) Electrostatic interactions of the S-F and S-D 12-carbamoylated gulosamine moiety with A1196, respectively. (G, H) Electrostatic interactions between β-amino of the β-lysine and the O2’ atoms of C1054 and U1052. Throughout the figure, the 50S is blue, 30S is yellow, S-F is gray sticks, S-D is green sticks, and highlighted residues are cyan. The cryo-EM densities corresponding to the 2 terminal β-lysine moieties of S-D were very weak, indicating that these 2 lysine moieties are flexible. We therefore did not include these 2 terminal β-lysine moieties in panels D, F, and H. cryo-EM, cryo-electron microscopy; S-D, streptothricin D; S-F, streptothricin-F.