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Lysocin E is a new antibiotic that targets menaquinone in the bacterial membrane

Abstract

To obtain therapeutically effective new antibiotics, we first searched for bacterial culture supernatants with antimicrobial activity in vitro and then performed a secondary screening using the silkworm infection model. Through further purification of the in vivo activity, we obtained a compound with a previously uncharacterized structure and named it 'lysocin E'. Lysocin E interacted with menaquinone in the bacterial membrane to achieve its potent bactericidal activity, a mode of action distinct from that of any other known antibiotic, indicating that lysocin E comprises a new class of antibiotic. This is to our knowledge the first report of a direct interaction between a small chemical compound and menaquinone that leads to bacterial killing. Furthermore, lysocin E decreased the mortality of infected mice. To our knowledge, lysocin E is the first compound identified and purified by quantitative measurement of therapeutic effects in an invertebrate infection model that exhibits robust in vivo effects in mammals.

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Figure 1: Bactericidal effects of the new antibiotic lysocin E against S. aureus.
Figure 2: Antimicrobial effect of lysocin E on S. aureus mutants of menaquinone biosynthesis.
Figure 3: Interaction of lysocin E and menaquinone.
Figure 4: In vivo effects of lysocin E and nosokomycin A against S. aureus infection in mice.

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References

  1. Spellberg, B., Powers, J.H., Brass, E.P., Miller, L.G. & Edwards, J.E. Jr. Trends in antimicrobial drug development: implications for the future. Clin. Infect. Dis. 38, 1279–1286 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Payne, D.J., Gwynn, M.N., Holmes, D.J. & Pompliano, D.L. Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov. 6, 29–40 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Kaito, C., Akimitsu, N., Watanabe, H. & Sekimizu, K. Silkworm larvae as an animal model of bacterial infection pathogenic to humans. Microb. Pathog. 32, 183–190 (2002).

    Article  PubMed  Google Scholar 

  4. Hamamoto, H. et al. Quantitative evaluation of the therapeutic effects of antibiotics using silkworms infected with human pathogenic microorganisms. Antimicrob. Agents Chemother. 48, 774–779 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hamamoto, H., Tonoike, A., Narushima, K., Horie, R. & Sekimizu, K. Silkworm as a model animal to evaluate drug candidate toxicity and metabolism. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 149, 334–339 (2009).

    Article  PubMed  Google Scholar 

  6. Uchida, R. et al. Nosokomycins, new antibiotics discovered in an in vivo-mimic infection model using silkworm larvae. I: Fermentation, isolation and biological properties. J. Antibiot. (Tokyo) 63, 151–155 (2010).

    Article  CAS  Google Scholar 

  7. Silverman, J.A., Perlmutter, N.G. & Shapiro, H.M. Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob. Agents Chemother. 47, 2538–2544 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Carrillo, C., Teruel, J.A., Aranda, F.J. & Ortiz, A. Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin. Biochim. Biophys. Acta 1611, 91–97 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Katsu, T. et al. Mechanism of membrane damage induced by the amphipathic peptides gramicidin S and melittin. Biochim. Biophys. Acta 983, 135–141 (1989).

    Article  CAS  PubMed  Google Scholar 

  10. Kreuzer, K.N. & Cozzarelli, N.R. Escherichia coli mutants thermosensitive for deoxyribonucleic acid gyrase subunit A: effects on deoxyribonucleic acid replication, transcription, and bacteriophage growth. J. Bacteriol. 140, 424–435 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Canepari, P., Lleo, M.M., Fontana, R. & Satta, G. Streptococcus faecium mutants that are temperature sensitive for cell growth and show alterations in penicillin-binding proteins. J. Bacteriol. 169, 2432–2439 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kawai, M., Ishihama, A. & Yura, T. RNA polymerase mutants of Escherichia coli. III. A temperature-sensitive rifampicin-resistant mutant. Mol. Gen. Genet. 143, 233–241 (1976).

    Article  CAS  PubMed  Google Scholar 

  13. Inoue, R. et al. Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus. Mol. Genet. Genomics 266, 564–571 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Kaito, C., Kurokawa, K., Hossain, M.S., Akimitsu, N. & Sekimizu, K. Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene. FEMS Microbiol. Lett. 210, 157–164 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Matsuo, M. et al. Isolation and mutation site determination of the temperature-sensitive murB mutants of Staphylococcus aureus. FEMS Microbiol. Lett. 222, 107–113 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Li, Y. et al. Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication. Mol. Genet. Genomics 271, 447–457 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Murai, N., Kurokawa, K., Ichihashi, N., Matsuo, M. & Sekimizu, K. Isolation of a temperature-sensitive dnaA mutant of Staphylococcus aureus. FEMS Microbiol. Lett. 254, 19–26 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Ishibashi, M. et al. Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus. FEMS Microbiol. Lett. 274, 204–209 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Bentley, R. & Meganathan, R. Biosynthesis of vitamin K (menaquinone) in bacteria. Microbiol. Rev. 46, 241–280 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Wakeman, C.A. et al. Menaquinone biosynthesis potentiates haem toxicity in Staphylococcus aureus. Mol. Microbiol. 86, 1376–1392 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Samuelsen, O. et al. Staphylococcus aureus small colony variants are resistant to the antimicrobial peptide lactoferricin B. J. Antimicrob. Chemother. 56, 1126–1129 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Kurokawa, K. et al. Evaluation of target specificity of antibacterial agents using Staphylococcus aureus ddlA mutants and d-cycloserine in a silkworm infection model. Antimicrob. Agents Chemother. 53, 4025–4027 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Proctor, R.A. et al. Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat. Rev. Microbiol. 4, 295–305 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Hayakawa, M. & Nonomura, H. Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. J. Ferment. Technol. 65, 501–509 (1987).

    Article  CAS  Google Scholar 

  25. Tamura, T. & Hatano, K. Phylogenetic analysis of the genus Actinoplanes and transfer of Actinoplanes minutisporangius Ruan et al. 1986 and 'Actinoplanes aurantiacus' to Cryptosporangium minutisporangium comb. nov. and Cryptosporangium aurantiacum sp. nov. Int. J. Syst. Evol. Microbiol. 51, 2119–2125 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Jukes, T.H. & Cantor, C.R. in Mammalian Protein Metabolism (Academic Press, New York, 1969).

  29. Saitou, N. & Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425 (1987).

    CAS  PubMed  Google Scholar 

  30. Felsenstein, J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791 (1985).

    Article  PubMed  Google Scholar 

  31. Hamase, K. et al. Simultaneous determination of hydrophilic amino acid enantiomers in mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional high-performance liquid chromatographic concept. J. Chromatogr. A 1217, 1056–1062 (2010).

    Article  CAS  PubMed  Google Scholar 

  32. Miyoshi, Y., Oyama, T., Itoh, Y. & Hamase, K. Enantioselective two-dimensional high-performance liquid chromatographic determination of amino acids; analysis and physiological significance of d-amino acids in mammals. Chromatography 35, 49–57 (2014).

    Article  CAS  Google Scholar 

  33. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard—Eighth Edition (CLSI document M07–A8) (Clinical and Laboratory Standards Institute, Wayne, PA, 2009).

  34. Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard (CLSI document M27-A) (Clinical and Laboratory Standards Institute, Wayne, PA, 1997).

  35. National Committee for Clinical Laboratory Standards. Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline (NCCLS document M26-A) (National Committee for Clinical Laboratory Standards, Wayne, PA, 1999).

  36. Maki, H., Miura, K. & Yamano, Y. Katanosin B and plusbacin A3, inhibitors of peptidoglycan synthesis in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 45, 1823–1827 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Paudel, A. et al. Identification of novel deoxyribofuranosyl indole antimicrobial agents. J. Antibiot. (Tokyo) 65, 53–57 (2012).

    Article  CAS  Google Scholar 

  38. Breeuwer, P. & Abee, T. Assessment of the Membrane Potential, Intracellular pH and Respiration of Bacteria Employing Fluorescence Techniques (Kluwer Academic Publishers, Netherlands, 2004).

  39. Komagoe, K., Kato, H., Inoue, T. & Katsu, T. Continuous real-time monitoring of cationic porphyrin-induced photodynamic inactivation of bacterial membrane functions using electrochemical sensors. Photochem. Photobiol. Sci. 10, 1181–1188 (2011).

    Article  CAS  PubMed  Google Scholar 

  40. Nishida, S. et al. Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus. J. Biol. Chem. 281, 1714–1724 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Kaito, C. et al. Silkworm pathogenic bacteria infection model for identification of novel virulence genes. Mol. Microbiol. 56, 934–944 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Tao, L., LeBlanc, D.J. & Ferretti, J.J. Novel streptococcal-integration shuttle vectors for gene cloning and inactivation. Gene 120, 105–110 (1992).

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, L., Rozek, A. & Hancock, R.E. Interaction of cationic antimicrobial peptides with model membranes. J. Biol. Chem. 276, 35714–35722 (2001).

    Article  CAS  PubMed  Google Scholar 

  44. Dartois, V. et al. Systemic antibacterial activity of novel synthetic cyclic peptides. Antimicrob. Agents Chemother. 49, 3302–3310 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was supported by a grant from Genome Pharmaceuticals Institute Co., Ltd. for K.S., the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) for K.S., a Grant-in-Aid for Young Scientists (A; 24689008) for H.H., a Grant-in-Aid for Scientific Research (25460036) for T. Katsu from Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Scientific Research on Innovative Areas–Chemical Biology of Natural Products to K.S. (24102510) and to H.H. (26102714) from MEXT, and it was supported in part by a Grant-in-Aid for JSPS Fellows (24-11042) for K.I. from JSPS and the Japan Science and Technology Agency (JST) A-STEP (High-Risk Challenging Type). We thank A. Noguchi, F. Aihara and S. Nishida (Genome Pharmaceutical Institute, Co., Ltd.) for screening and production of lysocin E. We thank K. Sakamoto (Hirosaki University) for advice regarding the menaquinone analysis. We thank M. Hyodo (Okayama University), K. Kyogoku (University of Tokyo) and Y. Matsuzawa (Genome Pharmaceutical Institute, Co., Ltd.) for technical assistance. We thank Shiseido Co. Ltd. for their technical support in 2D-HPLC analyses.

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Authors and Affiliations

Authors

Contributions

H.H. performed screening and purification, chemical structure analysis, mechanism analysis, acute toxicity analysis and manuscript drafting. M.U. performed chemical structure analysis and mechanism analysis, and drafted the supplementary results. K.I. performed bacterial genetic analysis, mechanism analysis and manuscript drafting. J.Y. performed bacterial genetic analysis and mechanism analysis. A.P. performed screening and purification and mechanism analysis. M.M., T. Kaji, T. Kuranaga and M.I. critically discussed and confirmed the lysocin E chemical structure. K.H. performed chemical structure analysis. T. Katsu and J.S. performed mechanism analysis. T.A. performed bacterial genetic analysis. R.U. performed analysis of nosokomycin A. H.T. performed analysis of nosocomycin A and critically revised the article for important intellectual content. M.Y. and H.K. performed acute toxicity analysis. M.S. performed chemical structure analysis. K.S. critically revised the article for important intellectual content and final approval of the article.

Corresponding author

Correspondence to Kazuhisa Sekimizu.

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Competing interests

K.S. is a consultant for Genome Pharmaceutical Institute Co., Ltd.

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Hamamoto, H., Urai, M., Ishii, K. et al. Lysocin E is a new antibiotic that targets menaquinone in the bacterial membrane. Nat Chem Biol 11, 127–133 (2015). https://doi.org/10.1038/nchembio.1710

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