Staphylococcus aureus is the leading cause of infections worldwide, and methicillin-resistant strains (MRSA) are emerging. New strategies are urgently needed to overcome this threat. Using a cell-based screen of ~45,000 diverse synthetic compounds, we discovered a potent bioactive, MAC-545496, that reverses β-lactam resistance in the community-acquired MRSA USA300 strain. MAC-545496 could also serve as an antivirulence agent alone; it attenuates MRSA virulence in Galleria mellonella larvae. MAC-545496 inhibits biofilm formation and abrogates intracellular survival in macrophages. Mechanistic characterization revealed MAC-545496 to be a nanomolar inhibitor of GraR, a regulator that responds to cell-envelope stress and is an important virulence factor and determinant of antibiotic resistance. The small molecule discovered herein is an inhibitor of GraR function. MAC-545496 has value as a research tool to probe the GraXRS regulatory system and as an antibacterial lead series of a mechanism to combat drug-resistant Staphylococcal infections.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). Source data for Figs. 1–6 are presented with the paper.
Prioritization of Pathogens to Guide Discovery, Research and Development of New Antibiotics for Drug-Resistant Bacterial Infections Including Tuberculosis (World Health Organization, 2017).
Boucher, H. W. et al. Bad bugs, no drugs: no ESKAPE! an update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48, 1–12 (2009).
Nannini, E., Murray, B. E. & Arias, C. A. Resistance or decreased susceptibility to glycopeptides, daptomycin, and linezolid in methicillin-resistant Staphylococcus aureus. Curr. Opin. Pharmacol. 10, 516–521 (2010).
Lowy, F. D. Staphylococcus aureus infections. New Engl. J. Med. 339, 520–532 (1998).
Wright, G. D. & Sutherland, A. D. New strategies for combating multidrug-resistant bacteria. Trends Mol. Med. 13, 260–267 (2007).
Maura, D., Ballok, A. E. & Rahme, L. G. Considerations and caveats in anti-virulence drug development. Curr. Opin. Microbiol. 33, 41–46 (2016).
Lee, K., Campbell, J., Swoboda, J. G., Cuny, G. D. & Walker, S. Development of improved inhibitors of wall teichoic acid biosynthesis with potent activity against Staphylococcus aureus. Bioorg. Med. Chem. Lett. 20, 1767–1770 (2010).
Sewell, E. W. & Brown, E. D. Taking aim at wall teichoic acid synthesis: new biology and new leads for antibiotics. J. Antibiot. 67, 43–51 (2014).
D’Elia, M. A., Henderson, J. A., Beveridge, T. J., Heinrichs, D. E. & Brown, E. D. The N-acetylmannosamine transferase catalyzes the first committed step of teichoic acid assembly in Bacillus subtilis and Staphylococcus aureus. J. Bacteriol. 191, 4030–4034 (2009).
D’Elia, M. A., Millar, K. E., Beveridge, T. J. & Brown, E. D. Wall teichoic acid polymers are dispensable for cell viability in Bacillus subtilis. J. Bacteriol. 188, 8313–8316 (2006).
D’Elia, M. A. et al. Lesions in teichoic acid biosynthesis in Staphylococcus aureus lead to a lethal gain of function in the otherwise dispensable pathway. J. Bacteriol. 188, 4183–4189 (2006).
Lee, S. H. et al. TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci. Sci. Transl. Med. 8, 329ra332 (2016).
Czarny, T. L. & Brown, E. D. A small-molecule screening platform for the discovery of inhibitors of undecaprenyl diphosphate synthase. ACS Infect. Dis. 2, 489–499 (2016).
Farha, M. A. et al. Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase. Proc. Natl Acad. Sci. USA 112, 11048–11053 (2015).
Santa Maria, J. P. Jr. et al. Compound-gene interaction mapping reveals distinct roles for Staphylococcus aureus teichoic acids. Proc. Natl Acad. Sci. USA 111, 12510–12515 (2014).
Falord, M., Karimova, G., Hiron, A. & Msadek, T. GraXSR proteins interact with the VraFG ABC transporter to form a five-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 56, 1047–1058 (2012).
Falord, M., Mader, U., Hiron, A., Debarbouille, M. & Msadek, T. Investigation of the Staphylococcus aureus GraSR regulon reveals novel links to virulence, stress response and cell wall signal transduction pathways. PLoS One 6, e21323 (2011).
Yang, S. J. et al. The Staphylococcus aureus two-component regulatory system, GraRS, senses and confers resistance to selected cationic antimicrobial peptides. Infect. Immun. 80, 74–81 (2012).
Kraus, D. et al. The GraRS regulatory system controls Staphylococcus aureus susceptibility to antimicrobial host defenses. BMC Microbiol. 8, 85 (2008).
Li, M. et al. The antimicrobial peptide-sensing system aps of Staphylococcus aureus. Mol. Microbiol. 66, 1136–1147 (2007).
Flannagan, R. S., Kuiack, R. C., McGavin, M. J. & Heinrichs, D. E. Staphylococcus aureus uses the GraXRS regulatory system to sense and adapt to the acidified phagolysosome in macrophages. MBio 9, e01143-18 (2018).
Mannala, G. K. et al. Whole-genome comparison of high and low virulent Staphylococcus aureus isolates inducing implant-associated bone infections. Int J. Med. Microbiol. 308, 505–513 (2018).
Shanks, R. M. et al. Genetic evidence for an alternative citrate-dependent biofilm formation pathway in Staphylococcus aureus that is dependent on fibronectin binding proteins and the GraRS two-component regulatory system. Infect. Immun. 76, 2469–2477 (2008).
Campbell, J. et al. An antibiotic that inhibits a late step in wall teichoic acid biosynthesis induces the cell wall stress stimulon in Staphylococcus aureus. Antimicrob. Agents Chemother. 56, 1810–1820 (2012).
Strauss, L. et al. Origin, evolution, and global transmission of community-acquired Staphylococcus aureus ST8. Proc. Natl Acad. Sci. USA 114, E10596–E10604 (2017).
Performance Standards for Antimicrobial Susceptibility Testing 27th Edition, CLSI Supplement M100 (Clinical and Laboratory Standards Institute, 2017).
Fey, P. D. et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio 4, e00537-12 (2013).
Fridman, M. et al. Two unique phosphorylation-driven signaling pathways crosstalk in Staphylococcus aureus to modulate the cell-wall charge: Stk1/Stp1 meets GraSR. Biochemistry 52, 7975–7986 (2013).
Vestergaard, M. et al. Inhibition of the ATP synthase eliminates the intrinsic resistance of Staphylococcus aureus towards polymyxins. MBio 8, e01114-17 (2017).
Rajagopal, M et al. Multidrug intrinsic resistance factors in Staphylococcus aureus identified by profiling fitness within high-diversity transposon libraries. MBio 7, e00950-16 (2016).
Vestergaard, M. et al. Genome-wide identification of antimicrobial intrinsic resistance determinants in Staphylococcus aureus. Front. Microbiol. 7, 2018 (2016).
Martinez-Hackert, E. & Stock, A. M. Structural relationships in the OmpR family of winged-helix transcription factors. J. Mol. Biol. 269, 301–312 (1997).
Kaiser, J. C. et al. Repression of branched-chain amino acid synthesis in Staphylococcus aureus is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide. PLoS Genet. 14, e1007159 (2018).
Li, M. et al. Gram-positive three-component antimicrobial peptide-sensing system. Proc. Natl Acad. Sci. USA 104, 9469–9474 (2007).
Mandin, P. et al. VirR, a response regulator critical for Listeria monocytogenes virulence. Mol. Microbiol 57, 1367–1380 (2005).
Becker, K., Heilmann, C. & Peters, G. Coagulase-negative staphylococci. Clin. Microbiol. Rev. 27, 870–926 (2014).
del Pozo, J. L. & Patel, R. The challenge of treating biofilm-associated bacterial infections. Clin. Pharm. Ther. 82, 204–209 (2007).
Shanks, R. M., Sargent, J. L., Martinez, R. M., Graber, M. L. & O’Toole, G. A. Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces. Nephrol. Dial. Transpl. 21, 2247–2255 (2006).
Lehar, S. M. et al. Novel antibody–antibiotic conjugate eliminates intracellular S. aureus. Nature 527, 323–328 (2015).
Coady, A. et al. The Staphylococcus aureus ABC-type manganese transporter MntABC is critical for reinitiation of bacterial replication following exposure to phagocytic oxidative burst. PLoS One 10, e0138350 (2015).
Bera, A., Herbert, S., Jakob, A., Vollmer, W. & Gotz, F. Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus. Mol. Microbiol 55, 778–787 (2005).
Cheung, A. L. et al. Site-specific mutation of the sensor kinase GraS in Staphylococcus aureus alters the adaptive response to distinct cationic antimicrobial peptides. Infect. Immun. 82, 5336–5345 (2014).
Chen, F. et al. Small-molecule targeting of a diapophytoene desaturase inhibits S. aureus virulence. Nat. Chem. Biol. 12, 174–179 (2016).
Gao, P., Davies, J. & Kao, R. Y. T. Dehydrosqualene desaturase as a novel target for anti-virulence therapy against Staphylococcus aureus. MBio 8, e01224-17 (2017).
Nielsen, A. et al. Solonamide B inhibits quorum sensing and reduces Staphylococcus aureus mediated killing of human neutrophils. PLoS One 9, e84992 (2014).
Wang, L. et al. The therapeutic effect of chlorogenic acid against Staphylococcus aureus infection through sortase a inhibition. Front Microbiol 6, 1031 (2015).
Li, W. et al. Analysis of the Staphylococcus aureus capsule biosynthesis pathway in vitro: characterization of the UDP–GlcNAc C6 dehydratases CapD and CapE and identification of enzyme inhibitors. Int. J. Med. Microbiol. 304, 958–969 (2014).
Ejim, L. et al. Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy. Nat. Chem. Biol. 7, 348–350 (2011).
El-Halfawy, O. M. & Brown, E. D. High-throughput screening for inhibitors of wall teichoic acid biosynthesis in Staphylococcus aureus. in Bacterial Polysaccharides: Methods in Molecular Biology vol. 1954 (ed. Brockhausen, I.) 297–308 (Humana Press, 2019).
Zlitni, S., Blanchard, J. E. & Brown, E. D. High-throughput screening of model bacteria. Methods Mol. Biol. 486, 13–27 (2009).
Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically Approved Standard Ninth Edition CLSI Document M07-A9 (Clinical and Laboratory Standards Institute, 2012).
Bae, T. & Schneewind, O. Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 55, 58–63 (2006).
Charpentier, E. et al. Novel cassette-based shuttle vector system for gram-positive bacteria. Appl. Environ. Microbiol. 70, 6076–6085 (2004).
Grosser, M. R. & Richardson, A. R. Method for preparation and electroporation of S. aureus and S. epidermidis. Methods Mol. Biol. 1373, 51–57 (2016).
Whitmore, L. & Wallace, B. A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32, W668–W673 (2004).
Merritt, J. H., Kadouri, D. E. & O’Toole, G. A. Growing and analyzing static biofilms. Curr. Protoc. Microbiol. Chapter 1, Unit 1B.1 (2005).
Harding, C. R., Schroeder, G. N., Collins, J. W. & Frankel, G. Use of Galleria mellonella as a model organism to study Legionella pneumophila infection. J. Vis. Exp. 81, e50964 (2013).
Flannagan, R. S., Heit, B. & Heinrichs, D. E. Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination. Cell Microbiol. 18, 514–535 (2016).
We thank A. Keddie from the University of Alberta for providing valuable advice on breeding Galleria and supplying the first batch of larvae, and B. Weber and A. Khaled for their help in breeding Galleria. We thank G. Wright from McMaster University for providing S. aureus clinical isolates. We also thank S. French for preparing the graphical abstract. This work was supported by grants from the Canadian Institutes of Health Research (foundation grant FRN-143215), the Canadian glycomics network (GlycoNet, https://doi.org/10.13039/501100009056, a National Centre of Excellence) and a Tier I Canada Research Chair award to E.D.B. D.E.H. acknowledges operating grant support from Cystic Fibrosis Canada. Studies performed in the laboratory of M.G.O. were funded by the Ontario Research Foundation. O.M.E.-H. was supported by a Michael G. DeGroote Fellowship Award in Basic Biomedical Science.
E.D.B., O.M.E.-H., T.L.C., J.D., M.G.O., R.S.F. and D.E.H. are inventors on a patent application on the use of MAC-545496 and analogs thereof, alone and in combination with other antibiotics, for the treatment of MRSA infections.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
El-Halfawy, O.M., Czarny, T.L., Flannagan, R.S. et al. Discovery of an antivirulence compound that reverses β-lactam resistance in MRSA. Nat Chem Biol 16, 143–149 (2020). https://doi.org/10.1038/s41589-019-0401-8
Polymyxins, the last-resort antibiotics: Mode of action, resistance emergence, and potential solutions
Journal of Biosciences (2021)
Nature Reviews Microbiology (2020)
Nature Reviews Drug Discovery (2020)