Christopher E. Morgan, Yoon-Suk Kang ,Alex B. Green ,Kenneth P. Smith ,Matthew G. Dowgiallo,Brandon C. Miller, Lucius Chiaraviglio, Katherine A. Truelson, Katelyn E. Zulauf, Shade Rodriguez, Anthony D. Kang, Roman Manetsch, Edward W. Yu, James E. Kirby .
Abstract
The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 μM, and 0.25 and 0.5 μM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D.
Introduction
The rapid emergence of antimicrobial resistance presents a significant challenge for treatment of bacterial infections. Carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii are of particular concern. Almost all approved antimicrobials that can overcome the gram-negative permeability barrier are natural products or synthetic or semisynthetic derivatives of natural products. Small molecules commonly available in high-throughput screening libraries rarely share similar physicochemical properties associated with gram-negative penetrance and activity [1]. Therefore, high-throughput screening efforts to identify novel antimicrobials using synthetic chemical libraries with rare exceptions have been nonproductive [1]. As a result, there is a significant antimicrobial discovery void.
Material and methods
Streptothricin purification
Purification of separate streptothricin compounds from nourseothricin sulfate (Gold Biotechnology, St. Louis, MO) was performed through modification of a previously reported method [9]. A glass column (150 cm × 2.4 cm) was packed with Sephadex LH-20 size exclusion gel (GE Healthcare, Chicago, IL) using a mobile phase of 10% methanol/H2O. The flow rate was adjusted using compressed air to 0.6 mL/min. Purifications were run in batches of approximately 300 mg of nourseothricin sulfate, which was diluted in 0.6 mL of H2O and loaded dropwise directly onto the top of the column. A mobile phase of 10% methanol/H2O was used for elution and fraction sizes of 3 mL were collected. Fractions testing positive for the ninhydrin stain were analyzed for purity by LC–MS. Pure S-D began eluting after approximately 120 mL of mobile phase, followed by mixed fractions of streptothricin D/E/F, and, finally, pure streptothricin F. Pure fractions for S-D were combined, frozen, and lyophilized to give a powdery, off-white solid.
Results
Activity spectrum studies
Our original studies with streptothricins were motivated by the observation that a nourseothricin resistance cassette served as a reliable genetic marker for experiments in otherwise highly drug-resistant gram-negative pathogens [16,17]. This led to the question of whether nourseothricin or its constituent streptothricin homologues had more general activity against multidrug-resistant gram-negative pathogens such as CRE and A. baumannii. We, therefore, adapted a previously reported method to purify S-F (1 β-lysine) and S-D (3 β-lysines) to homogeneity using Sephadex chromatography [9]. Confirmation of purity and precise molar content was determined by elemental analysis, NMR, and LC–MS as described in S1 Results; S1 Materials and Methods; S1–S3 Figs; and S1 and S2 Tables.
Discussion
We demonstrate that streptothricins, in particular S-F, S-D, and the natural product mixture, nourseothricin, are highly active against contemporary, carbapenem-resistant E. coli, Klebsiella, Enterobacter, and A. baumannii. Their activity spectra are therefore highly relevant to the emerging gap in gram-negative antimicrobial coverage, even more so since they appear to act in a unique manor to kill bacterial cells. Most of the existing description of streptothricin class antibiotic activity was from several decades ago and made use of unstandardized microbiological and physicochemical methods. In general, the natural product mixture was characterized as a whole without quantitative delineation of contributions from constituent streptothricins.
Acknowledgments
The HP D300 digital dispenser and TECAN M1000 were provided for our use by TECAN (Morrisville, NC). Tecan had no role in study design, data collection/interpretation, manuscript preparation, or decision to publish.
Citation: Morgan CE, Kang Y-S, Green AB, Smith KP, Dowgiallo MG, Miller BC, et al. (2023) Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome. PLoS Biol 21(5): e3002091.
https://doi.org/10.1371/journal.pbio.3002091
Academic Editor: Csaba Pál, Biological Research Centre, HUNGARY
Received: May 30, 2022; Accepted: March 22, 2023; Published: May 16, 2023
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the paper and its Supporting Information files with the following exception: Atomic coordinates and EM maps for SF bound to the A. baumannii P-site tRNA 70S, E-site tRNA 70S and Empty 70S have been deposited to the RCSB Protein Data Bank (PDB) under accession codes 7UVV, 7UVW and 7UVX and the Electron Microscopy Data Bank (EMDB) under accession codes EMD-26817, EMD-26818 and EMD-26819, respectively. Complexes of S-D with the A. baumannii P-site tRNA 70S, E-site tRNA 70S and Empty 70S have been deposited in the PDB under accession codes 7UVY, 7UVZ and 7UW1 and EMDB under codes EMD-26820, EMD-26821 and EMD-26822, respectively.
Funding: This work was supported by R21 AI140212 to R.M. and J.E.K, and R01AI157208 to J.E.K., E.W.Y. and R.M. from National Institutes of Health. K.P.S. and K.E.Z. were supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers F32 AI124590 and T32 AI007061, respectively. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRE, carbapenem-resistant Enterobacterales; cryo-EM, cryo-electron microscopy; IP, intraperitoneally; MIC, minimal inhibitory concentration; MTD, maximum tolerated dose; S-D, streptothricin D; S-F, streptothricin F; wt, wild-type; 2-DOS, 2-deoxystreptamine.
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002091#abstract0
