[en] Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid II-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. In addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.
Disciplines :
Microbiology
Author, co-author :
Boes, Adrien ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'ingénierie des protéines
Olatunji, Samir
Mohammadi, Tamimount
Breukink, Eefjan
Terrak, Mohammed ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'ingénierie des protéines
Language :
English
Title :
Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ.
Vollmer, W., Blanot, D. & de Pedro, M. A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167 (2008).
Sham, L.-T. et al. Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis. Science 345, 220–2 (2014).
Mohammadi, T. et al. Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane. EMBO J. 30, 1425–32 (2011).
Liu, X., Meiresonne, N. Y., Bouhss, A. & den Blaauwen, T. FtsW activity and lipid II synthesis are required for recruitment of MurJ to midcell during cell division in Escherichia coli. Mol. Microbiol., 10.1111/mmi.14104 (2018).
Ruiz, N. Lipid Flippases for Bacterial Peptidoglycan Biosynthesis. Lipid Insights 8, 21–31 (2015).
Meeske, A. J. et al. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature 537, 634–638 (2016).
Taguchi, A. et al. FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein. Nat. Microbiol. 4, 587–594 (2019).
Sjodt, M. et al. Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis. Nature 556, 118–121 (2018).
Sauvage, E. & Terrak, M. Glycosyltransferases and Transpeptidases/Penicillin-Binding Proteins: Valuable Targets for New Antibacterials. Antibitics 5, 12 (2016).
Typas, A., Banzhaf, M., Gross, C. A. & Vollmer, W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nature Reviews Microbiology, 10.1038/nrmicro2677 (2011).
Cho, H. et al. Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously. Nat. Microbiol. 1, 16172 (2016).
Boes, A., Olatunji, S., Breukink, E. & Terrak, M. Regulation of the Peptidoglycan Polymerase Activity of PBP1b by Antagonist Actions of the Core Divisome Proteins FtsBLQ and FtsN. MBio 10 (2019).
Leclercq, S. et al. Interplay between Penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis. Sci. Rep. 7 (2017).
Grein, F., Schneider, T. & Sahl, H. G. Docking on Lipid II—A Widespread Mechanism for Potent Bactericidal Activities of Antibiotic Peptides. Journal of Molecular Biology, 10.1016/j.jmb.2019.05.014 (2019).
Chamakura, K. R. et al. A viral protein antibiotic inhibits lipid II flippase activity. Nat. Microbiol. 2, 1480–1484 (2017).
Welzel, P. Syntheses around the transglycosylation step in peptidoglycan biosynthesis. Chem. Rev. 105, 4610–4660 (2005).
Butaye, P., Devriese, L. A. & Haesebrouck, F. Influence of different medium components on the in vitro activity of the growth-promoting antibiotic flavomycin against enterococci. J. Antimicrob. Chemother. 46, 713–6 (2000).
Oppedijk, S. F., Martin, N. I. & Breukink, E. Hit’em where it hurts: The growing and structurally diverse family of peptides that target lipid-II. Biochim. Biophys. Acta, 10.1016/j.bbamem.2015.10.024 (2015).
Malin, J. J. & de Leeuw, E. Therapeutic compounds targeting Lipid II for antibacterial purposes. Infect. Drug Resist. 12, 2613–2625 (2019).
Bolla, J. R. et al. Direct observation of the influence of cardiolipin and antibiotics on lipid II binding to MurJ. Nat. Chem. 10, 363–371 (2018).
Derouaux, A. et al. Small molecule inhibitors of peptidoglycan synthesis targeting the lipid II precursor. Biochem. Pharmacol. 81, 1098–105 (2011).
Bury, D. et al. Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase. Biochem. Pharmacol. 93, 141–50 (2015).
Egan, A. J. F. & Biboy, J. van’t Veer, I., Breukink, E. & Vollmer, W. Activities and regulation of peptidoglycan synthases. Philos. Trans. R. Soc. B Biol. Sci. 370, 20150031 (2015).
Breukink, E. et al. Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes. J Biol Chem 278, 19898–19903 (2003).
van Dam, V. et al. Transmembrane transport of peptidoglycan precursors across model and bacterial membranes. Mol. Microbiol. 64, 1105–14 (2007).
Terrak, M. et al. The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli. Mol. Microbiol. 34 (1999).
Roehrl, M. H. A., Wang, J. Y. & Wagner, G. A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization. Biochemistry 43, 16056–16066 (2004).
Dandliker, W. B., Hsu, M. L., Levin, J. & Rao, B. R. Equilibrium and Kinetic Inhibition Assays Based upon Fluorescence Polarization. Methods Enzymol. 74, 3–28 (1981).
Moerke, N. J. Fluorescence Polarization (FP) Assays for Monitoring Peptide-Protein or Nucleic Acid-Protein Binding. Curr. Protoc. Chem. Biol. 1, 1–15 (2009).
Nikolovska-Coleska, Z. et al. Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. Anal. Biochem. 332, 261–273 (2004).
Offant, J. et al. Optimization of conditions for the glycosyltransferase activity of penicillin-binding protein 1a from Thermotoga maritima. FEBS J. 277, 4290–4298 (2010).
