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Anti-infective bile acids bind and inactivate a Salmonella virulence regulator

Abstract

Bile acids are prominent host and microbiota metabolites that modulate host immunity and microbial pathogenesis. However, the mechanisms by which bile acids suppress microbial virulence are not clear. To identify the direct protein targets of bile acids in bacterial pathogens, we performed activity-guided chemical proteomic studies. In Salmonella enterica serovar Typhimurium, chenodeoxycholic acid (CDCA) most effectively inhibited the expression of virulence genes and invasion of epithelial cells and interacted with many proteins. Notably, we discovered that CDCA can directly bind and inhibit the function of HilD, an important transcriptional regulator of S. Typhimurium virulence and pathogenesis. Our characterization of bile acid-resistant HilD mutants in vitro and in S. Typhimurium infection models suggests that HilD is one of the key protein targets of anti-infective bile acids. This study highlights the utility of chemical proteomics to identify the direct protein targets of microbiota metabolites for mechanistic studies in bacterial pathogens.

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Fig. 1: CDCA most effectively inhibits S. Typhimurium virulence in vitro.
Fig. 2: Chemoproteomic profiling of CDCA-interacting proteins in S. Typhimurium.
Fig. 3: HilD mutants confer resistance to bile and CDCA inhibition of S. Typhimurium virulence.
Fig. 4: CDCA interferes with HilD dimerization and DNA binding.
Fig. 5: The S. Typhimurium hilDQ39E,N44D,H95L mutant is resistant to microbiota suppression of virulence in vivo.
Fig. 6: The S. Typhimurium hilDQ39E,N44D,H95L mutant is more virulent in CDCA-supplemented mice.

Data availability

The proteome database (UP000002695) used in this study is available from UniProt. The MS proteomics raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034373. The Rns crystal structure (6XIV) and ToxT crystal structure (3GBG) are available on the RCSB Protein Data Bank. Source data are provided with this paper. All other data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author on reasonable request.

References

  1. Buffie, C. G. & Pamer, E. G. Microbiota-mediated colonization resistance against intestinal pathogens. Nat. Rev. Immunol. 13, 790–801 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Cai, J., Sun, L. & Gonzalez, F. J. Gut microbiota-derived bile acids in intestinal immunity, inflammation, and tumorigenesis. Cell Host Microbe 30, 289–300 (2022).

    Article  CAS  PubMed  Google Scholar 

  3. Quinn, R. A. et al. Global chemical effects of the microbiome include new bile-acid conjugations. Nature 579, 123–129 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Perino, A., Demagny, H., Velazquez-Villegas, L. & Schoonjans, K. Molecular physiology of bile acid signaling in health, disease, and aging. Physiol. Rev. 101, 683–731 (2021).

    Article  CAS  PubMed  Google Scholar 

  5. Parks Derek, J. et al. Bile acids: natural ligands for an orphan nuclear receptor. Science 284, 1365–1368 (1999).

    Article  Google Scholar 

  6. Sinha, S. R. et al. Dysbiosis-induced secondary bile acid deficiency promotes intestinal inflammation. Cell Host Microbe 27, 659–670 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Guo, C. et al. Bile acids control inflammation and metabolic disorder through inhibition of NLRP3 inflammasome. Immunity 45, 802–816 (2016).

    Article  CAS  PubMed  Google Scholar 

  8. Hang, S. et al. Bile acid metabolites control TH17 and Treg cell differentiation. Nature 576, 143–148 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Campbell, C. et al. Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells. Nature 581, 475–479 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Song, X. et al. Microbial bile acid metabolites modulate gut RORγ+ regulatory T cell homeostasis. Nature 577, 410–415 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Yang, M. et al. Bile salt-induced intermolecular disulfide bond formation activates Vibrio cholerae virulence. Proc. Natl Acad. Sci. USA 110, 2348–2353 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Alavi, S. et al. Interpersonal gut microbiome variation drives susceptibility and resistance to cholera infection. Cell 181, 1533–1546 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tam, J. et al. Intestinal bile acids directly modulate the structure and function of C. difficile TcdB toxin. Proc. Natl Acad. Sci. USA 117, 6792–6800 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hung, D. T. & Mekalanos, J. J. Bile acids induce cholera toxin expression in Vibrio cholerae in a ToxT-independent manner. Proc. Natl Acad. Sci. USA 102, 3028–3033 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kang, J. D. et al. Bile acid 7α-dehydroxylating gut bacteria secrete antibiotics that inhibit Clostridium difficile: role of secondary bile acids. Cell Chem. Biol. 26, 27–34 (2019).

    Article  CAS  PubMed  Google Scholar 

  16. Buffie, C. G. et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517, 205–208 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. McKenney, P. T. et al. Intestinal bile acids induce a morphotype switch in vancomycin-resistant Enterococcus that facilitates intestinal colonization. Cell Host Microbe 25, 695–705 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sato, Y. et al. Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians. Nature 599, 458–464 (2021).

    Article  CAS  PubMed  Google Scholar 

  19. Prouty, A. M. & Gunn, J. S. Salmonella enterica serovar Typhimurium invasion is repressed in the presence of bile. Infect. Immun. 68, 6763–6769 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Eade, C. R. et al. Bile acids function synergistically to repress invasion gene expression in Salmonella by destabilizing the invasion regulator HilD. Infect. Immun. 84, 2198–2208 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Prouty, A. M., Brodsky, I. E., Falkow, S. & Gunn, J. S. Bile-salt-mediated induction of antimicrobial and bile resistance in Salmonella Typhimurium. Microbiology 150, 775–783 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Prouty Angela, M., Van Velkinburgh Jennifer, C. & Gunn John, S. Salmonella enterica serovar Typhimurium resistance to bile: identification and characterization of the tolQRA cluster. J. Bacteriol. 184, 1270–1276 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ao, T. T. et al. Global burden of invasive nontyphoidal Salmonella disease, 2010(1). Emerg. Infect. Dis. 21, 941–949 (2015).

    Article  CAS  PubMed Central  Google Scholar 

  24. LaRock, D. L., Chaudhary, A. & Miller, S. I. Salmonellae interactions with host processes. Nat. Rev. Microbiol. 13, 191–205 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Galán, J. E. & Waksman, G. Protein-injection machines in bacteria. Cell 172, 1306–1318 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Zhang, Z. J., Pedicord, V. A., Peng, T. & Hang, H. C. Site-specific acylation of a bacterial virulence regulator attenuates infection. Nat. Chem. Biol. 16, 95–103 (2020).

    Article  PubMed  CAS  Google Scholar 

  27. Lee, C. A., Jones, B. D. & Falkow, S. Identification of a Salmonella Typhimurium invasion locus by selection for hyperinvasive mutants. Proc. Natl Acad. Sci. USA 89, 1847–1851 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Schechter, L. M., Damrauer, S. M. & Lee, C. A. Two AraC/XylS family members can independently counteract the effect of repressing sequences upstream of the hilA promoter. Mol. Microbiol. 32, 629–642 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Yang, J., Tauschek, M. & Robins-Browne, R. M. Control of bacterial virulence by AraC-like regulators that respond to chemical signals. Trends Microbiol. 19, 128–135 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Narm, K. E., Kalafatis, M. & Slauch, J. M. HilD, HilC, and RtsA form homodimers and heterodimers to regulate expression of the Salmonella pathogenicity island I type III secretion system. J. Bacteriol. 202, e00012-20 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Lowden, M. J. et al. Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes. Proc. Natl Acad. Sci. USA 107, 2860–2865 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Midgett, C. R., Talbot, K. M., Day, J. L., Munson, G. P. & Kull, F. J. Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons. Sci. Rep. 11, 15663 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Golubeva, Y. A., Ellermeier, J. R., Cott Chubiz, J. E. & Slauch, J. M. Intestinal long-chain fatty acids act as a direct signal to modulate expression of the Salmonella pathogenicity island 1 type III secretion system. mBio 7, e02170-15 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Chowdhury, R., Pavinski Bitar, P. D., Keresztes, I., Condo, A. M. Jr. & Altier, C. A diffusible signal factor of the intestine dictates Salmonella invasion through its direct control of the virulence activator HilD. PLoS Pathog. 17, e1009357 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bosire, E. M. et al. Diffusible signal factors act through AraC-type transcriptional regulators as chemical cues to repress virulence of enteric pathogens. Infect. Immun. 88, e00226-20 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Hung, C. C. et al. The intestinal fatty acid propionate inhibits Salmonella invasion through the post-translational control of HilD. Mol. Microbiol. 87, 1045–1060 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sang, Y. et al. Acetylation regulating protein stability and DNA-binding ability of HilD, thus modulating Salmonella Typhimurium virulence. J. Infect. Dis. 216, 1018–1026 (2017).

    Article  CAS  PubMed  Google Scholar 

  38. Wu, Y., Yang, X., Zhang, D. & Lu, C. Myricanol inhibits the type III secretion system of Salmonella enterica serovar Typhimurium by interfering with the DNA-binding activity of HilD. Front. Microbiol. 11, 571217 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Shakhnovich, E. A., Hung, D. T., Pierson, E., Lee, K. & Mekalanos, J. J. Virstatin inhibits dimerization of the transcriptional activator ToxT. Proc. Natl Acad. Sci. USA 104, 2372–2377 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Cruite, J. T. et al. Structural basis for virulence regulation in Vibrio cholerae by unsaturated fatty acid components of bile. Commun. Biol. 2, 440 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ellermeier, C. D., Ellermeier, J. R. & Slauch, J. M. HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator HilA in Salmonella enterica serovar Typhimurium. Mol. Microbiol. 57, 691–705 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Antunes, L. C. et al. Effect of antibiotic treatment on the intestinal metabolome. Antimicrob. Agents Chemother. 55, 1494–1503 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rivera-Chavez, F. et al. Depletion of butyrate-producing Clostridia from the gut microbiota drives an aerobic luminal expansion of Salmonella. Cell Host Microbe 19, 443–454 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tremblay, S. et al. Bile acid administration elicits an intestinal antimicrobial program and reduces the bacterial burden in two mouse models of enteric infection. Infect. Immun. 85, e00942-16 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Pedicord, V. A. et al. Exploiting a host-commensal interaction to promote intestinal barrier function and enteric pathogen tolerance. Sci. Immunol. 1, eaai7732 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Velazquez, E. M. et al. Endogenous Enterobacteriaceae underlie variation in susceptibility to Salmonella infection. Nat. Microbiol 4, 1057–1064 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Diaz-Ochoa, V. E. et al. Salmonella mitigates oxidative stress and thrives in the inflamed gut by evading calprotectin-mediated manganese sequestration. Cell Host Microbe 19, 814–825 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Jacobson, A. et al. A gut commensal-produced metabolite mediates colonization resistance to Salmonella infection. Cell Host Microbe 24, 296–307 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Perez-Morales, D. et al. An incoherent feedforward loop formed by SirA/BarA, HilE and HilD is involved in controlling the growth cost of virulence factor expression by Salmonella Typhimurium. PLoS Pathog. 17, e1009630 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Paik, D. et al. Human gut bacteria produce TauEta17-modulating bile acid metabolites. Nature 603, 907–912 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Yang, Y.-Y., Ascano, J. M. & Hang, H. C. Bioorthogonal chemical reporters for monitoring protein acetylation. J. Am. Chem. Soc. 132, 3640–3641 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell Proteom. 13, 2513–2526 (2014).

    Article  CAS  Google Scholar 

  53. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Soltermann, F. et al. Quantifying protein–protein interactions by molecular counting with mass photometry. Angew. Chem. Int. Ed. Engl. 59, 10774–10779 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Tsou, L. K. et al. Antibacterial flavonoids from medicinal plants covalently inactivate type III protein secretion substrates. J. Am. Chem. Soc. 138, 2209–2218 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Hung Deborah, T., Shakhnovich Elizabeth, A., Pierson, E. & Mekalanos John, J. Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. Science 310, 670–674 (2005).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank The Rockefeller Proteomics Resource Center for LC–MS analysis, Z.J. Zhang and other Hang lab members for helpful discussions. We also thank J. Hammond from the Scripps Research Biophysics and Biochemistry Core for help on mass photometry experiments. H.C.H. acknowledges support from NIH grant R01AI172915. K.R.S. acknowledges support from the Scripps Research T32 Immunology Training Grant T32AI007244. Some figures were created with BioRender.com.

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H.C.H. and X.Y. conceived the project, X.Y. performed experiments and K.R.S. performed lipocalin-2 analysis. X.Y., K.R.S. and H.C.H. analyzed the data and wrote the paper.

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Correspondence to Howard C. Hang.

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Nature Chemical Biology thanks Victor Bustamante and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Chemoproteomic analysis of bile acid reporter-interacting proteins in S. Typhimurium.

a,b,c, LFQ proteomic analysis of bile acid reporters-labeled proteins in S. Typhimurium under UV irradiation (biologically independent samples, n = 4). S. Typhimurium cell lysates were reacted with az-biotin for the enrichment of bile acid reporters-labeled proteins with streptavidin beads and identification by LC-MS/MS. Volcano plots were presented for alk-X-CDCA (a), alk-X-LCA (b) and alk-X-UDCA (c). d, Gene ontology analysis of the shared 129 protein targets identified by LFQ proteomic reveals their association with pathogenesis (analyzed by The Database for Annotation, Visualization and Integrated Discovery (DAVID)). The names of the 16 proteins specifically assigned to the functional cluster of “virulence” are shown. e, Cellular component analysis of protein hits in a by gene ontology (GO).

Source data

Extended Data Fig. 2 Identification of bile acid resistant HilD mutations.

a, Three amino acids (N260, K264, R267) which are adjacent to potential binding pocket are highlighted in Robetta predicted HilD dimer. Green: DNA binding domain, orange: “jelly roll” motif, the putative binding domain. b. An overnight culture of S. Typhimurium (hilD-HAWT or hilD-HAmutants generated by CRISPR-Cas9) were diluted 1/50 into 4 mL SPI-1 inducing LB aliquots containing DMSO or CDCA (0.5 mM) and incubated for 4 h at 37 °C with 220 rpm shaking. SPI-1 effectors and flagella components levels in S. Typhimurium growth media were monitored by SDS-PAGE followed by Coomassie blue staining55. Experiments were repeated at least two times with similar results. c, Schematic for screening of mutant hilD library. Generally, hilD mutant library was generated from pTXB1-HilD by error prone PCR. The mutant library was transformed to reporter strain (S. Typhimurium tetRA-hilD-3XFLAG attλ::pDX1::hilA’-lacZ) and screened by plates (IPTG, carbenicillin, X-gal and 2.5 % bile acids)56. Plasmids from blue colonies were sequenced.

Source data

Extended Data Fig. 3 Investigation of HilD and CDCA interaction.

a, HilA-HA expression of HilD-mutant strains in presence of CDCA (500 µM). SPI-1 transcriptional factor HilA-HA expression of hilDWT or hilDmutant S. Typhimurium (hilA-HA) strains grown with or without 0.5 mM of CDCA. Western blot was quantified by grayscale analysis. Two-way ANOVA followed by Sidak’s multiple comparisons test, adjusted P value. Centerline, average. Error bar, SD. (biologically independent samples, n = 3). Comparisons with P > 0.05 were not considered significant. b,c, Docking of CDCA into HilD model. HilD protein model was generated based on AraC family protein Rns (PDB 6XIV32) by SWISS-MODEL. The structure model of HilD was generated with the protein preparation wizard using Maestro software (12.4, Schrödinger, LLC), and Q39, N44, H95 were chosen as centroid of selected residues and the Ligand Docking protocol was used to model potential binding modes. Green: DNA binding domain, orange: “jelly roll” motif, the putative binding domain.

Source data

Extended Data Fig. 4 HilD Asparagine 44 mutations affect Salmonella response to bile acids and long chain fatty acid.

a,c, HilA-HA expression of hilDWT or hilDmutant S. Typhimurium (HilA-HA) strains grown with or without 2% bile acids (a) and 25 μM HDA (cis-2-hexadecenoic acid) (c). Western blot was quantified by grayscale analysis. b, Gentamicin protection assay of S. Typhimurium grown with 2% bile acids infecting HT-29 cells at MOI = 10. Statistical analysis for a,b,c, (a, biologically independent samples, n = 3; b, biologically independent samples, n = 6; c, biologically independent samples, n = 3) Two-way ANOVA followed by Sidak’s multiple comparisons test, adjusted P value. Centerline, average. Error bar, SD. Comparisons with P > 0.05 were not considered significant. d, Western blot for c.

Source data

Extended Data Fig. 5 CDCA inhibits hilD transcription and has little effect on HilD stability.

a,c,e, Overnight culture of Salmonella Typhimurium (hilD-HA for a,e; hilD-HA, lon:: cat for c) was subcultured 1:50 to 20 mL LB (300 mM NaCl). Bacteria was cultured for 3.5 h and another half an hour with treatment of CDCA or DMSO control (a,c) or cultured for 4 h with treatment of CDCA or DMSO (e). Then these cultures were treated with rifampin (100 ug/ml), streptomycin (200 ug/ml), and spectinomycin (50 ug/ml) to halt gene transcription and protein translation and kept at 37 oC with 220 rpm. Samples are picked at indicated time point. HilD-HA protein level was analyzed by Western blot. b,d,f, western blot was quantified by grayscale analysis for a,c,e correspondingly. For b,d Data was analyzed by two-way ANOVA and Sidak’s multiple comparisons test. Comparisons with P > 0.05 were not considered significant. Centerline, average. Error bar, SD (biologically independent samples, n = 3). e,f was repeated twice with similar results. g,h, Overnight culture of Salmonella (HilD-HA) was subcultured 1:50 to 20 mL LB (300 mM NaCl). Bacteria was cultured for 4 h with treatment of CDCA or DMSO. HilD-HA and GroEL protein level was analyzed by Western blot (biologically independent samples, n = 6). Unpaired t test (two-sided). Centerline, average. Error bar, SD. i. Expression of SPI-1 and control genes of S. Typhimurium grown with DMSO, CDCA (concentration = 0.5 mM) measured by qRT-PCR (biologically independent samples, n = 6, results are pooled from two independent experiments, one independent experiment was carried out together with Fig. 1f, three replicates of control gene ftsZ were shared with 1 f). Unpaired t test (two-sided). Centerline, average. Error bar, SD. Comparisons had P > 0.05 were not considered significant.

Source data

Extended Data Fig. 6 HilDQ39E+N44D+H95L Salmonella is resistant to other gut microbiota metabolized small molecules.

a, SPI-1 transcriptional factor HilA-HA expression of S. Typhimurium (hilA-HA, hilDWT or Q39E+N44D+H95) grown with propionic acid (10 mM), palmitic acid (0.1 mM) and cis-2-hexadecenoic acid (0.025 mM). b, Western blot was quantified by grayscale analysis, normalized with OD 600 value. All metabolites were compared to DMSO with one-way ANOVA and Dunnett’s multiple comparisons test, adjusted P value. Centerline, average. Error bar, SD. (biologically independent samples, n = 3). Comparisons with P > 0.05 were not considered significant. c, chemical structures of propionic acid, palmitic acid and cis-2-hexadecenoic acid.

Source data

Extended Data Fig. 7 Mice fecal metabolites inhibit Salmonella virulence.

a, feces were collected from C57BL/6 mice one day post with or without 20 mg streptomycin treatment. Feces from 4 mice were pooled together and added to lysing matrix D (Methanol: H2O = 9: 1, 100 mg/mL). Feces were then homogenized by Fastprep-24 5 G (MP Biomedicals, 4 m/s, 3 cycles, 5 s for each cycle). Supernatant after centrifugation (18 000 g, 4 oC, 30 min) were lyophilized and the corresponding residues were redissolved in LB for bacterial culture (metabolites from 50 mg of feces were dissolved in 1 mL of LB). Overnight culture of Salmonella (hilA-HA, hilDWT or Q39E+N44D+H95) was subcultured 1:50 to LB with or without metabolites. Bacteria were cultured for 4 h at 37 oC. Expression of HilA-HA and GroEL was analyzed by western blot analysis. b,c, Western blot was quantified by grayscale analysis, normalized with GroEL expression. Bacteria treated with metabolites were compared to bacteria treated with LB by one-way ANOVA and Dunnett’s multiple comparisons test, adjusted P value. Centerline, average. Error bar, SD. (biologically independent samples, n = 3). Comparisons with P > 0.05 were not considered significant.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–13, Tables 1–4, materials, synthesis of bile acid reporters and NMR spectra of bile acid reporters.

Reporting Summary

Supplementary Data 1

Proteomics dataset.

Supplementary Data 2

Robetta-predicted HilD dimer structure model.

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Yang, X., Stein, K.R. & Hang, H.C. Anti-infective bile acids bind and inactivate a Salmonella virulence regulator. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01122-3

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