Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic d-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of d,d- and l,d-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.
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Typas, A., Banzhaf, M., Gross, Ca & Vollmer, W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat. Rev. Microbiol. 10, 123–136 (2012).
Egan, A. J. F., Cleverley, R. M., Peters, K., Lewis, R. J. & Vollmer, W. Regulation of bacterial cell wall growth. FEBS J. 284, 851–867 (2017).
Vollmer, W., Blanot, D. & de Pedro, M. A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167 (2008).
Lupoli, T. J. et al. Transpeptidase-mediated incorporation of d-amino acids into bacterial peptidoglycan. J. Am. Chem. Soc. 133, 10748–10751 (2011).
Wilke, M. S., Lovering, A. L. & Strynadka, N. C. β-Lactam antibiotic resistance: a current structural perspective. Curr. Opin. Microbiol. 8, 525–533 (2005).
Waxman, D. J. & Strominger, J. L. Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics. Annu. Rev. Biochem. 52, 825–869 (1983).
Tipper, D. J. & Strominger, J. L. Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-d-alanyl-d-alanine. Proc. Natl Acad. Sci. USA 54, 1133–1141 (1965).
Sauvage, E., Kerff, F., Terrak, M., Ayala, J. A. & Charlier, P. The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol. Rev. 32, 234–258 (2008).
Mainardi, J.-L. et al. A novel peptidoglycan cross-linking enzyme for a beta-lactam-resistant transpeptidation pathway. J. Biol. Chem. 280, 38146–38152 (2005).
Mainardi, J.-L. et al. Novel mechanism of β-lactam resistance due to bypass of d d-transpeptidation in Enterococcus faecium. J. Biol. Chem. 275, 16490–16496 (2000).
Hugonnet, J.-E. et al. Factors essential for l,d-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli. eLife 5, e19469 (2016).
Mazmanian, S. K., Liu, G., Jensen, E. R., Lenoy, E. & Schneewind, O. Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proc. Natl Acad. Sci. USA 97, 5510–5515 (2000).
Ton-That, H., Liu, G., Mazmanian, S. K., Faull, K. F. & Schneewind, O. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc. Natl Acad. Sci. USA 96, 12424–12429 (1999).
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).
Kuru, E. et al. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent d-amino acids. Angew. Chem. Int. Ed. 51, 12519–12523 (2012).
Kuru, E., Tekkam, S., Hall, E., Brun, Y. V. & Van Nieuwenhze, M. S. Synthesis of fluorescent d-amino acids and their use for probing peptidoglycan synthesis and bacterial growth in situ. Nat. Protoc. 10, 33–52 (2015).
Hsu, Y.-P. et al. Full color palette of fluorescent d-amino acids for in situ labeling of bacterial cell walls. Chem. Sci. 290, 30540–30550 (2017).
Hsu, Y.-P., Meng, X. & VanNieuwenhze, M. S. S. in Imaging Bacterial Molecules, Structures and Cells. Methods in Microbiology Vol. 43 (eds Harwood, C. & Jensen, G. J.) Ch. 1, 3–48 (Elsevier, 2016).
Bisson-Filho, A. W. et al. Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science 355, 739–743 (2017).
Haidekker, M. A. & Theodorakis, E. A. Environment-sensitive behavior of fluorescent molecular rotors. J. Biol. Eng. 4, 11 (2010).
Haidekker, M. A. et al. A ratiometric fluorescent viscosity sensor. J. Am. Chem. Soc. 128, 398–399 (2006).
Kuimova, M. K. Mapping viscosity in cells using molecular rotors. Phys. Chem. Chem. Phys. 14, 12671 (2012).
Hosny, N. A. et al. Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors. Proc. Natl Acad. Sci. USA 110, 9225–9230 (2013).
Goh, W. L. et al. Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions. J. Am. Chem. Soc. 136, 6159–6162 (2014).
Yu, W.-T., Wu, T.-W., Huang, C.-L., Chen, I.-C. & Tan, K.-T. Protein sensing in living cells by molecular rotor-based fluorescence-switchable chemical probes. Chem. Sci. 7, 301–307 (2016).
Dziuba, D., Jurkiewicz, P., Cebecauer, M., Hof, M. & Hocek, M. A rotational BODIPY nucleotide: an environment-sensitive fluorescence-lifetime probe for DNA interactions and applications in live-cell microscopy. Angew. Chem. Int. Ed. 128, 182–186 (2016).
Nadler, A. & Schultz, C. The power of fluorogenic probes. Angew. Chem. Int. Ed. 52, 2408–2410 (2013).
Kamariza, M. et al. Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe. Sci. Transl. Med. 10, eaam6310 (2018).
Haidekker, M. A. et al. New fluorescent probes for the measurement of cell membrane viscosity. Chem. Biol. 8, 123–131 (2001).
Sawada, S., Iio, T., Hayashi, Y. & Takahashi, S. Fluorescent rotors and their applications to the study of G–F transformation of actin. Anal. Biochem. 204, 110–117 (1992).
De, K., Legros, J., Crousse, B. & Bonnet-Delpon, D. Solvent-promoted and -controlled aza-Michael reaction with aromatic amines. J. Org. Chem. 74, 6260–6265 (2009).
Shao, J. et al. Thiophene-inserted aryl-dicyanovinyl compounds: the second generation of fluorescent molecular rotors with significantly redshifted emission and large Stokes shift. Eur. J. Org. Chem. 2011, 6100–6109 (2011).
Monnereau, C., Blart, E. & Odobel, F. A cheap and efficient method for selective para-iodination of aniline derivatives. Tetrahedron Lett. 46, 5421–5423 (2005).
Suzuki, A. Recent advances in the cross-coupling reactions of organoboron derivatives with organic electrophiles, 1995–1998. J. Organomet. Chem. 576, 147–168 (1999).
Delcour, A. H. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta 1794, 808–816 (2009).
Sampson, B. A., Misra, R. & Benson, S. A. Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability. Genetics 122, 491–501 (1989).
Zhou, F. et al. Molecular rotors as fluorescent viscosity sensors: molecular design, polarity sensitivity, dipole moments changes, screening solvents and deactivation channel of the excited states. Eur. J. Org. Chem. 2011, 4773–4787 (2011).
Haidekker, M. A. et al. Molecular rotors—fluorescent biosensors for viscosity and flow. Org. Biomol. Chem. 5, 1669–1678 (2007).
Randich, A. M. & Brun, Y. V. Molecular mechanisms for the evolution of bacterial morphologies and growth modes. Front. Microbiol. 6, 580 (2015).
Lupoli, T. J. et al. Lipoprotein activators stimulate Escherichia coli penicillin-binding proteins by different mechanisms. J. Am. Chem. Soc. 136, 52–55 (2014).
Bertsche, U., Breukink, E., Kast, T. & Vollmer, W. In vitro murein (peptidoglycan) synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli. J. Biol. Chem. 280, 38096–38101 (2005).
Born, P., Breukink, E. & Vollmer, W. In vitro synthesis of cross-linked murein and its attachment to sacculi by PBP1A from Escherichia coli. J. Biol. Chem. 281, 26985–26993 (2006).
Qiao, Y. et al. Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams. Nat. Chem. Biol. 13, 793–798 (2017).
Navratna, V. et al. Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance. J. Bacteriol. 192, 134–144 (2010).
Wyke, A. W., Ward, J. B., Hayes, M. V. & Curtis, N. A. A role in vivo for penicillin-binding protein-4 of Staphylococcus aureus. Eur. J. Biochem. 119, 389–393 (1981).
Memmi, G., Filipe, S. R., Pinho, M. G., Fu, Z. & Cheung, A. Staphylococcus aureus PBP4 is essential for β-lactam resistance in community-acquired methicillin-resistant strains. Antimicrob. Agents Chemother. 52, 3955–3966 (2008).
Swenson, J. M. et al. Correlation of cefoxitin MICs with the presence of mecA in Staphylococcus spp. J. Clin. Microbiol. 47, 1902–1905 (2009).
Kocaoglu, O. & Carlson, E. E. Profiling of β-lactam selectivity for penicillin-binding proteins in Escherichia coli strain DC2. Antimicrob. Agents Chemother. 59, 2785–2790 (2015).
Wehrli, R., von Graevenitz, A. & Lüthy, R. Susceptibility and tolerance of β-lactamase-producing, methicillin-sensitive strains of Staphylococcus aureus towards seven broad-spectrum penicillins. Infection 11, 322–325 (1983).
Uri, J. V. Detection of beta-lactamase activity with nitrocefin of multiple strains of various microbial genera. Acta Microbiol. Hung. 32, 133–145 (1985).
Pitkälä, A., Salmikivi, L., Bredbacka, P., Myllyniemi, A.-L. & Koskinen, M. T. Comparison of tests for detection of beta-lactamase-producing staphylococci. J. Clin. Microbiol. 45, 2031–2033 (2007).
Cava, F., de Pedro, M. A., Lam, H., Davis, B. M. & Waldor, M. K. Distinct pathways for modification of the bacterial cell wall by non-canonical d-amino acids. EMBO J. 30, 3442–3453 (2011).
Guo, Y., Cai, S., Gu, G., Guo, Z. & Long, Z. Recent progress in the development of sortase A inhibitors as novel anti-bacterial virulence agents. RSC Adv. 5, 49880–49889 (2015).
Oh, K.-B. et al. Discovery of diarylacrylonitriles as a novel series of small molecule sortase A inhibitors. J. Med. Chem. 47, 2418–2421 (2004).
Lorand, L. & Graham, R. M. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat. Rev. Mol. Cell Biol. 4, 140–156 (2003).
Kunjapur, A. M. et al. Engineering posttranslational proofreading to discriminate nonstandard amino acids. Proc. Natl Acad. Sci. USA 115, 619–624 (2018).
The authors thank K. C. Huang for providing strain E. coli imp4213, S. Walker for providing S. aureus PBP4 plasmid, D. Kysela for help with image processing and analysis and J. Rittichier for providing the enzyme substrates used in the in vitro assays and his advice on RfDAA synthesis. This study is supported by NIH grants 5R01GM113172 to M.S.V. and Y.V.B. and R35GM122556 to Y.V.B., and by a Canada 150 research Chair in Bacterial Cell Biology to Y.V.B.
The authors declare no competing interests.
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The protocol for RfDAA synthesis, characterization data and bacteria labelling and imaging experiments. The file also includes supplementary discussions, figures, tables and NMR spectra of the probes.
The time-lapse microscopy of PG synthesis labelled by Rf470DL in S. venezuelae.
The time-lapse microscopy of PG synthesis labelled by Rf470DL in B. subtilis.
The time-lapse microscopy of PG synthesis labelled by Rf470DL in B. subtilis. The effect of RfDAA labeling in dead cells was highlighted.
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Hsu, YP., Hall, E., Booher, G. et al. Fluorogenic d-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays. Nat. Chem. 11, 335–341 (2019). https://doi.org/10.1038/s41557-019-0217-x
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