Cell-to-cell signaling, or quorum sensing (QS), in many Gram-negative bacteria is governed by small molecule signals (N-acyl-l-homoserine lactones, AHLs) and their cognate receptors (LuxR-type proteins). The mechanistic underpinnings of QS in these bacteria are severely limited due to the challenges of isolating and manipulating most LuxR-type proteins. Reports of quantitative direct-binding experiments on LuxR-type proteins are scarce, and robust and generalizable methods that provide such data are largely nonexistent. We report herein a Förster resonance energy transfer (FRET) assay that leverages (1) conserved tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore–AHL conjugates, and (2) isolation of the proteins bound to weak agonists. The FRET assay permits straightforward measurement of ligand-binding affinities with receptor—either in vitro or in cells—and was shown to be compatible with six LuxR-type proteins. These methods will advance fundamental investigations of LuxR-type protein mechanism and the development of small molecule QS modulators.
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The datasets generated and analyzed during this study are available from the corresponding author on request.
Miller, M. B. & Bassler, B. L. Quorum sensing in bacteria. Annu Rev. Microbiol. 55, 165–199 (2001).
Fuqua, C. & Greenberg, E. P. Listening in on bacteria: acyl-homoserine lactone signalling. Nat. Rev. Mol. Cell Biol. 3, 685 (2002).
Churchill, M. E. A. & Chen, L. Structural basis of acyl-homoserine lactone-dependent signaling. Chem. Rev. 111, 68–85 (2011).
Welsh, M. A. & Blackwell, H. E. Chemical probes of quorum sensing: from compound development to biological discovery. FEMS Microbiol. Rev. 40, 774–794 (2016).
Whiteley, M., Diggle, S. P. & Greenberg, E. P. Progress in and promise of bacterial quorum sensing research. Nature 551, 313–320 (2017).
Geske, G. D., O’Neill, J. C., Miller, D. M., Mattmann, M. E. & Blackwell, H. E. Modulation of bacterial quorum sensing with synthetic ligands: systematic evaluation of N-acylated homoserine lactones in multiple species and new insights into their mechanisms of action. JACS 129, 13613–13625 (2007).
Swem, L. R. et al. A quorum-sensing antagonist targets both membrane-bound and cytoplasmic receptors and controls bacterial pathogenicity. Mol. Cell 35, 143–153 (2009).
Amara, N. et al. Covalent inhibition of bacterial quorum sensing. JACS 131, 10610–10619 (2009).
Galloway, W. R., Hodgkinson, J. T., Bowden, S., Welch, M. & Spring, D. R. Applications of small molecule activators and inhibitors of quorum sensing in Gram-negative bacteria. Trends Microbiol 20, 449–458 (2012).
O’Loughlin, C. T. et al. A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation. Proc. Natl Acad. Sci. USA 110, 17981–17986 (2013).
Manson, D. E., O’Reilly, M. C., Nyffeler, K. E. & Blackwell, H. E. Design, synthesis, and biochemical characterization of non-native antagonists of the Pseudomonas aeruginosa quorum sensing receptor LasR with nanomolar IC50 values. ACS Infect. Dis. 6, 649–661 (2020).
Styles, M. J. et al. Chemical control of quorum sensing in E. coli: identification of small molecule modulators of SdiA and mechanistic characterization of a covalent inhibitor. ACS Infect. Dis. 6, 3092–3103 (2020).
Boursier, M. E., Manson, D. E., Combs, J. B. & Blackwell, H. E. A comparative study of non-native N-acyl l-homoserine lactone analogs in two Pseudomonas aeruginosa quorum sensing receptors that share a common native ligand yet inversely regulate virulence. Bioorg. Med. Chem. 26, 5336–5342 (2018).
McInnis, C. E. & Blackwell, H. E. Thiolactone modulators of quorum sensing revealed through library design and screening. Bioorg. Med. Chem. 19, 4820–4828 (2011).
Boursier, M. E., Combs, J. B. & Blackwell, H. E. N-Acyl l-homocysteine thiolactones are potent and stable synthetic modulators of the RhlR quorum sensing receptor in Pseudomonas aeruginosa. ACS Chem. Biol. 14, 186–191 (2019).
Zhu, J. & Winans, S. C. Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR turnover rates in whole cells. Proc. Natl Acad. Sci. USA 96, 4832–4837 (1999).
Bottomley, M. J., Muraglia, E., Bazzo, R. & Carfì, A. Molecular insights into quorum sensing in the human pathogen Pseudomonas aeruginosa from the structure of the virulence regulator LasR bound to its autoinducer. J. Biol. Chem. 282, 13592–13600 (2007).
Lintz, M. J., Oinuma, K.-I., Wysoczynski, C. L., Greenberg, E. P. & Churchill, M. E. A. Crystal structure of QscR, a Pseudomonas aeruginosa quorum sensing signal receptor. Proc. Natl Acad. Sci. USA 108, 15763–15768 (2011).
Schaefer, A. L., Hanzelka, B. L., Eberhard, A. & Greenberg, E. P. Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs. J. Bacteriol. 178, 2897–2901 (1996).
Schuster, M., Urbanowski, M. L. & Greenberg, E. P. Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR. Proc. Natl Acad. Sci. USA 101, 15833–15839 (2004).
Oinuma, K.-I. & Greenberg, E. P. Acyl-homoserine lactone binding to and stability of the orphan Pseudomonas aeruginosa quorum-sensing signal receptor QscR. J. Bacteriol. 193, 421 (2011).
Kim, T. et al. Structural insights into the molecular mechanism of Escherichia coli SdiA, a quorum-sensing receptor. Acta Crystallogr. D. Biol. Crystallogr. 70, 694–707 (2014).
Poulter, S., Carlton, T. M., Spring, D. R. & Salmond, G. P. The Serratia LuxR family regulator CarR 39006 activates transcription independently of cognate quorum sensing signals. Mol. Microbiol. 80, 1120–1131 (2011).
Welch, M. et al. N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia. EMBO J. 19, 631–641 (2000).
Wu, P. & Brand, L. Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1–13 (1994).
Lee, M. M. & Peterson, B. R. Quantification of small molecule–protein interactions using FRET between tryptophan and the pacific blue fluorophore. ACS Omega 1, 1266–1276 (2016).
Mattmann, M. E., Shipway, P. M., Heth, N. J. & Blackwell, H. E. Potent and selective synthetic modulators of a quorum sensing repressor in Pseudomonas aeruginosa identified from second-generation libraries of N-acylated l-homoserine lactones. Chem. Bio. Chem. 12, 942–949 (2011).
Eibergen, N. R., Moore, J. D., Mattmann, M. E. & Blackwell, H. E. Potent and selective modulation of the RhlR quorum sensing receptor by using non-native ligands: an emerging target for virulence control in Pseudomonas aeruginosa. ChemBioChem 16, 2348–2356 (2015).
Slinger, B. L., Deay, J. J., Chandler, J. R. & Blackwell, H. E. Potent modulation of the CepR quorum sensing receptor and virulence in a Burkholderia cepacia complex member using non-native lactone ligands. Sci. Rep. 9, 13449 (2019).
Styles, M. J. & Blackwell, H. E. Non-native autoinducer analogs capable of modulating the SdiA quorum sensing receptor in Salmonella enterica serovar Typhimurium. Beilstein J. Org. Chem. 14, 2651–2664 (2018).
Wysoczynski-Horita, C. L. et al. Mechanism of agonism and antagonism of the Pseudomonas aeruginosa quorum sensing regulator QscR with non-native ligands. Mol. Microbiol. 108, 240–257 (2018).
Gerdt, J. P., McInnis, C. E., Schell, T. L., Rossi, F. M. & Blackwell, H. E. Mutational analysis of the quorum-sensing receptor LasR reveals interactions that govern activation and inhibition by nonlactone ligands. Chem. Biol. 21, 1361–1369 (2014).
Tonge, P. J. Quantifying the interactions between biomolecules: guidelines for assay design and data analysis. ACS Infect. Dis. 5, 796–808 (2019).
Pollard, T. D. A guide to simple and informative binding assays. Mol. Biol. Cell 21, 4061–4067 (2010).
Zhang, J. H., Chung, T. D. & Oldenburg, K. R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen 4, 67–73 (1999).
Moore, J. D., Rossi, F. M., Welsh, M. A., Nyffeler, K. E. & Blackwell, H. E. A comparative analysis of synthetic quorum sensing modulators in Pseudomonas aeruginosa: new insights into mechanism, active efflux susceptibility, phenotypic response, and next-generation ligand design. JACS 137, 14626–14639 (2015).
O’Reilly, M. C. et al. Structural and biochemical studies of non-native agonists of the LasR quorum-sensing receptor reveal an L3 Loop ‘Out’ conformation for LasR. Cell Chem. Biol. 25, 1128–1139.e3 (2018).
Boursier, M. E. et al. Structure-function analyses of the N-butanoyl l-homoserine lactone quorum-sensing signal define features critical to activity in RhlR. ACS Chem. Biol. 13, 2655–2662 (2018).
Zarkan, A., Liu, J., Matuszewska, M., Gaimster, H. & Summers, D. K. Local and universal action: the paradoxes of indole signalling in bacteria. Trends Microbiol. 28, 566–577 (2020).
Lee, J., Jayaraman, A. & Wood, T. K. Indole is an inter-species biofilm signal mediated by SdiA. BMC Microbiol. 7, 42 (2007).
Chu, W. et al. Indole production promotes Escherichia coli mixed-culture growth with Pseudomonas aeruginosa by inhibiting quorum signaling. Appl. Environ. Microbiol. 78, 411–419 (2012).
Hidalgo-Romano, B. et al. Indole inhibition of N-acylated homoserine lactone-mediated quorum signalling is widespread in Gram-negative bacteria. Microbiology 160, 2464–2473 (2014).
Sabag-Daigle, A., Soares, J. A., Smith, J. N., Elmasry, M. E. & Ahmer, B. M. The acyl homoserine lactone receptor, SdiA, of Escherichia coli and Salmonella enterica serovar Typhimurium does not respond to indole. Appl. Environ. Microbiol. 78, 5424–5431 (2012).
Kim, J. & Park, W. Indole inhibits bacterial quorum sensing signal transmission by interfering with quorum sensing regulator folding. Microbiology 159, 2616–2625 (2013).
Wu, H. et al. Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice. J. Antimicrob. Chemother. 53, 1054–1061 (2004).
Müh, U. et al. Novel Pseudomonas aeruginosa quorum-sensing inhibitors identified in an ultra-high-throughput screen. Antimicrob. Agents Chemother. 50, 3674–3679 (2006).
Zou, Y. & Nair, S. K. Molecular basis for the recognition of structurally distinct autoinducer mimics by the Pseudomonas aeruginosa LasR quorum-sensing signaling receptor. Chem. Biol. 16, 961–970 (2009).
Venturi, V. & Ahmer, B. M. Editorial: LuxR solos are becoming major players in cell–cell communication in bacteria. Front. Cell. Infect. Microbiol. 5, 89 (2015).
Hudaiberdiev, S. et al. Census of solo LuxR genes in prokaryotic genomes. Front. Cell Infect. Microbiol. 5, 20 (2015).
Nguyen, Y. et al. Structural and mechanistic roles of novel chemical ligands on the SdiA quorum-sensing transcription regulator. MBio. 6, 2 (2015).
Niesen, F. H., Berglund, H. & Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2, 2212–2221 (2007).
Financial support for this work was provided by the National Institutes of Health (NIH) (grant no. R35 GM131817). M.J.S. was supported in part by the UW–Madison NIH Biotechnology Training Program (grant no. T32 GM008349) and a National Science Foundation (NSF) Graduate Research Fellowship (grant no. DGE-1747503). B.L.S. was supported in part by an NIH Ruth Kirschstein National Research Service Award (grant no. F32 AI138918). The authors made use of facilities at the UW–Madison including the Biophysics Instrumentation Facility (supported by the NSF (grant no. BIR-9512577) and NIH (grant no. S10 RR13790)) and at the UW–Madison Paul Bender Chemical Instrument Center.
The authors declare no competing interests.
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Each member of the FRET-probe library was assayed at 10 μM for antagonism with each receptor listed on the top of the heatmap in a heterologous reporter strain (see Supplementary Table 5 for strain details and main text) with the EC50 of the native ligand present (EC90 for SdiAse and SdiAec). Each receptor:FRET-probe pair has its own square in the heatmap. The heatmap is organized vertically by linker length (left of the heatmap), horizontally by receptor, and within a column by fluorophore (coumarin (CU) on the left and dansyl (DA) on the right). Receptor inhibition is normalized for each strain as the activity of the reporter with native ligand only (0%) and no ligand added (100%). See Supplementary Table 3 for native ligand identities and EC50 and EC90 concentrations. Each value is an average of two independent biological replicates, each with three technical replicates (a total of six independent replicates).
For each receptor, the native ligand (OdDHL and OOHL were used for the orphan receptors QscR and SdiA, respectively) and several FRET-probes identified as agonists (Fig. 2) were characterized by performing the cell-based transcriptional reporter assays at several concentrations to obtain dose-response plots. All assays were performed as three independent biological replicates, each with six technical replicates (a total of 18 independent replicates). Activation reported as the percentage (%) of activity normalized to the maximum activity of the native ligand (100% activation) and DMSO (0% activation). Error bars show s.d. Data were fit using GraphPad Prism software using a four parameter (varied slope) dose-response equation (see Supplementary Table 2 for parameter values from these fits).
These compounds have been identified in previous studies by the Blackwell laboratory27,31. Compounds that are able to activate QscR to greater than 85–100% activity (relative to OdDHL) are categorized as agonists. Compounds able to reduce the activity of QscR in antagonism assays (with the EC50 of OdDHL present) are categorized as antagonists. Note, all the antagonists listed here are also able to partially activate QscR in agonism assay formats (10–70% relative to OdDHL).
Extended Data Fig. 4 Cell-based reporter assay dose-response curves for small molecule modulators of QscR.
Cell-based reporter assays were performed as described above for each compound (listed on x-axis of each plot) in an agonism format (data in black) and an antagonism format (data in red). Data was fit to either an activation or inhibition four parameter (varied slope) dose response equation in GraphPad Prism (model fits shown as lines). For compounds with a non-monotonic antagonism profile (OHHL, D6, C10, and S5)36, the upturn concentrations were not used in the model fit. All data points represent three independent biological replicates, each with three technical replicates; error bars indicate s.d.
Extended Data Fig. 5 In-cell FRET binding dose-response curves for select receptor:FRET-probe pairs.
In-cell FRET was performed as described above for each compound in a binding format (receptor and fluorophore listed above each plot). Data were fit to an activation four parameter (varied slope) dose-response equation in GraphPad Prism (model fits shown as lines and compiled in Supplementary Table 4). All data points represent at least two independent biological replicates, each with four technical replicates; error bars indicate s.d.
Extended Data Fig. 6 In-cell FRET displacement dose-response curves for select receptor:FRET-probe pairs.
In-cell FRET was performed as described above for each compound in a displacement format (receptor, fluorophore, and competing ligand listed above each plot). Data were fit to an inhibition four parameter (varied slope) dose-response equation in GraphPad Prism (model fits shown as lines and compiled in Supplementary Table 4). Fluorophore concentration was 10 μM in each case except for RhlR, for which 50 μM fluorophore was used. All data points represent at least two independent biological replicates, each with four technical replicates; error bars indicate s.d.
An SdiAec cell-based reporter assay was performed as described above for indole in an agonism format. Data were fit to an activation four parameter (varied slope) dose-response equation in GraphPad Prism (model fit shown as a line). All data points represent three independent biological replicates, each with three technical replicates; error bars indicate s.d.
a, Representative DSF thermal melt profiles for QscR with no compound (DMSO, green), an agonist (OdDHL, black), and two representative examples of inhibitors (the partial agonist Q9, red; the non-monotonic partial agonist C10, blue). b, The first derivatives of the RFU data shown in part a.
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Styles, M.J., Boursier, M.E., McEwan, M.A. et al. Autoinducer-fluorophore conjugates enable FRET in LuxR proteins in vitro and in cells. Nat Chem Biol 18, 1115–1124 (2022). https://doi.org/10.1038/s41589-022-01089-1