Abstract
The surge of antibiotic resistance in Staphylococcus aureus has created a dire need for innovative anti-infective agents that attack new targets, to overcome resistance. In S. aureus, carotenoid pigment is an important virulence factor because it shields the bacterium from host oxidant killing. Here we show that naftifine, a US Food and Drug Administration (FDA)-approved antifungal drug, blocks biosynthesis of carotenoid pigment at nanomolar concentrations. This effect is mediated by competitive inhibition of S. aureus diapophytoene desaturase (CrtN), an essential enzyme for carotenoid pigment synthesis. We found that naftifine attenuated the virulence of a variety of clinical S. aureus isolates, including methicillin-resistant S. aureus (MRSA) strains, in mouse infection models. Specifically, we determined that naftifine is a lead compound for potent CrtN inhibitors. In sum, these findings reveal that naftifine could serve as a chemical probe to manipulate CrtN activity, providing proof of concept that CrtN is a druggable target against S. aureus infections.
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References
Lowy, F.D. Staphylococcus aureus infections. N. Engl. J. Med. 339, 520–532 (1998).
DeLeo, F.R. & Chambers, H.F. Reemergence of antibiotic-resistant Staphylococcus aureus in the genomics era. J. Clin. Invest. 119, 2464–2474 (2009).
Klein, E., Smith, D.L. & Laxminarayan, R. Hospitalizations and deaths caused by methicillin-resistant Staphylococcus aureus, United States, 1999-2005. Emerg. Infect. Dis. 13, 1840–1846 (2007).
Arias, C.A. & Murray, B.E. Antibiotic-resistant bugs in the 21st century—a clinical super-challenge. N. Engl. J. Med. 360, 439–443 (2009).
Kloos, W.E. & Jorgensen, J.H. Staphylococci. in Manual of Clinical Microbiology edn. 4 (eds. Lennette, E.H., Balows, A., Hausler Jr., W.J. & Shadomy, H.J.) 143–153 (American Society for Microbiology, Washington, DC, 1985).
Liu, C.I. et al. A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence. Science 319, 1391–1394 (2008).
Clauditz, A., Resch, A., Wieland, K.P., Peschel, A. & Götz, F. Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infect. Immun. 74, 4950–4953 (2006).
Liu, G.Y. et al. Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity. J. Exp. Med. 202, 209–215 (2005).
Pelz, A. et al. Structure and biosynthesis of staphyloxanthin from Staphylococcus aureus. J. Biol. Chem. 280, 32493–32498 (2005).
Kim, S.H. & Lee, P.C. Functional expression and extension of staphylococcal staphyloxanthin biosynthetic pathway in Escherichia coli. J. Biol. Chem. 287, 21575–21583 (2012).
Bischoff, M. & Berger-Bächi, B. Teicoplanin stress-selected mutations increasing sigma(B) activity in Staphylococcus aureus. Antimicrob. Agents Chemother. 45, 1714–1720 (2001).
Palma, M. & Cheung, A.L. sigma(B) activity in Staphylococcus aureus is controlled by RsbU and an additional factor(s) during bacterial growth. Infect. Immun. 69, 7858–7865 (2001).
Lan, L., Cheng, A., Dunman, P.M., Missiakas, D. & He, C. Golden pigment production and virulence gene expression are affected by metabolisms in Staphylococcus aureus. J. Bacteriol. 192, 3068–3077 (2010).
Fey, P.D. et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio 4, e00537–12 (2013).
Ding, Y. et al. Metabolic sensor governing bacterial virulence in Staphylococcus aureus. Proc. Natl. Acad. Sci. USA 111, E4981–E4990 (2014).
Liu, Y. et al. SsrA (tmRNA) acts as an antisense RNA to regulate Staphylococcus aureus pigment synthesis by base pairing with crtMN mRNA. FEBS Lett. 584, 4325–4329 (2010).
Duthie, E.S. & Lorenz, L.L. Staphylococcal coagulase; mode of action and antigenicity. J. Gen. Microbiol. 6, 95–107 (1952).
Porretta, G.C. et al. Antifungal agents, Part 11. Biphenyl analogues of naftifine: synthesis and antifungal activities. Arch. Pharm. (Weinheim) 328, 667–672 (1995).
Gupta, A.K., Ryder, J.E. & Cooper, E.A. Naftifine: a review. J. Cutan. Med. Surg. 12, 51–58 (2008).
Wieland, B. et al. Genetic and biochemical analyses of the biosynthesis of the yellow carotenoid 4,4′-diaponeurosporene of Staphylococcus aureus. J. Bacteriol. 176, 7719–7726 (1994).
Russell, R.G. Ibandronate: pharmacology and preclinical studies. Bone 38 (suppl. 1), S7–S12 (2006).
Leejae, S., Hasap, L. & Voravuthikunchai, S.P. Inhibition of staphyloxanthin biosynthesis in Staphylococcus aureus by rhodomyrtone, a novel antibiotic candidate. J. Med. Microbiol. 62, 421–428 (2013).
Pantoliano, M.W. et al. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J. Biomol. Screen. 6, 429–440 (2001).
Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).
Raisig, A. & Sandmann, G. 4,4′-diapophytoene desaturase: catalytic properties of an enzyme from the C30 carotenoid pathway of Staphylococcus aureus. J. Bacteriol. 181, 6184–6187 (1999).
Furubayashi, M., Li, L., Katabami, A., Saito, K. & Umeno, D. Construction of carotenoid biosynthetic pathways using squalene synthase. FEBS Lett. 588, 436–442 (2014).
David, M.Z. & Daum, R.S. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin. Microbiol. Rev. 23, 616–687 (2010).
Kuroda, M. et al. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet 357, 1225–1240 (2001).
El-Gohary, M. et al. Topical antifungal treatments for tinea cruris and tinea corporis. Cochrane Database Syst. Rev. 8, CD009992 (2014).
Belter, A. et al. Squalene monooxygenase—a target for hypercholesterolemic therapy. Biol. Chem. 392, 1053–1075 (2011).
Volkman, J.K. Sterols in microorganisms. Appl. Microbiol. Biotechnol. 60, 495–506 (2003).
Alsterholm, M., Karami, N. & Faergemann, J. Antimicrobial activity of topical skin pharmaceuticals—an in vitro study. Acta Derm. Venereol. 90, 239–245 (2010).
Nolting, S. & Bräutigam, M. Clinical relevance of the antibacterial activity of terbinafine: a contralateral comparison between 1% terbinafine cream and 0.1% gentamicin sulphate cream in pyoderma. Br. J. Dermatol. 126 (suppl. 39), 56–60 (1992).
Ciftci, E., Guriz, H. & Aysev, A.D. Mupirocin vs terbinafine in impetigo. Indian J. Pediatr. 69, 679–682 (2002).
Koning, S. et al. Interventions for impetigo. Cochrane Database Syst. Rev. 1, CD003261 (2012).
Hammond, R.K. & White, D.C. Inhibition of vitamin K2 and carotenoid synthesis in Staphylococcus aureus by diphenylamine. J. Bacteriol. 103, 611–615 (1970).
Raisig, A. & Sandmann, G. Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways. Biochim. Biophys. Acta 1533, 164–170 (2001).
Bae, T. et al. Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing. Proc. Natl. Acad. Sci. USA 101, 12312–12317 (2004).
Schaub, P. et al. On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase. PLoS One 7, e39550 (2012).
Lineweaver, H. & Burk, D. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658–666 (1934).
Acknowledgements
We thank M. Wu for helpful discussion, S.F. Reichard for editing the manuscript, and F. Götz (University of Tübingen) for providing the S. carnosus strains TM300∷pTX15 and TM300∷pTXcrtMN. This work was supported by the National Natural Science Foundation of China (21472207 and 31270126 to L.L.; 21222211 and 91313303 to J.L.), Hundred Talents Program of the Chinese Academy of Sciences (L.L.), Shanghai Institute of Materia Medica Foundation (CASIMM0120152018 to L.L.), Shanghai Municipal Education Commission and Shanghai Education Development Foundation (14SG28 to J.L.) and Foundation of the State Key Laboratory of Drug Research (SIMM1302KF-01 to J.L.). We acknowledge the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program” (2013ZX09507-004 to C.-G.Y.) and Shanghai Committee of Science and Technology (12ZR1453200 to F.C.).
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F.C. performed the biological experiments and drafted the biology part of the manuscript. H.D. performed the biological experiments. Y.W. synthesized naftifine derivatives. F.L. and N.Z. performed one-dimensional NMR spectroscopy analysis. Q.C., B.X., X.Z. and N.Y. participated in the mouse infection experiment; G.L. and C.-G.Y. contributed to protein purification; Y.X. and H.J. contributed to data analysis. J.L. designed the chemical synthesis and drafted the chemistry part of the manuscript. L.L. conceived the study, designed the biological experiments and wrote the manuscript. All authors contributed to interpretation of data, and read and approved the final manuscript.
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L.L., J.L., F.C., Y.W. and H.J. are named inventors of pending patent applications (CN201410190672 and PCT/CN2014/079565, to the Chinese Patent Office) related to the work described.
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Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–18 (PDF 3365 kb)
Supplementary Data Set 1
A collection of 412 marked drugs used for the screening in this study (PDF 528 kb)
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Synthetic Procedures (PDF 3633 kb)
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Chen, F., Di, H., Wang, Y. et al. Small-molecule targeting of a diapophytoene desaturase inhibits S. aureus virulence. Nat Chem Biol 12, 174–179 (2016). https://doi.org/10.1038/nchembio.2003
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DOI: https://doi.org/10.1038/nchembio.2003
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