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  • Letter
  • Published:

Sulfamethoxazole drug stress upregulates antioxidant immunomodulatory metabolites in Escherichia coli

Abstract

Escherichia coli is an important model organism in microbiology and a prominent member of the human microbiota1. Environmental isolates readily colonize the gastrointestinal tract of humans and other animals, and they can serve diverse probiotic, commensal and pathogenic roles in the host2,3,4. Although certain strains have been associated with the severity of inflammatory bowel disease (IBD)2,5, the diverse immunomodulatory phenotypes remain largely unknown at the molecular level. Here, we decode a previously unknown E. coli metabolic pathway that produces a family of hybrid pterin–phenylpyruvate conjugates, which we named the colipterins. The metabolites are upregulated by subinhibitory levels of the antifolate sulfamethoxazole, which is used to treat infections including in patients with IBD6,7. The genes folX/M and aspC/tyrB involved in monapterin biosynthesis8,9,10 and aromatic amino acid transamination,11 respectively, were required to initiate the colipterin pathway. We show that the colipterins are antioxidants, harbour diverse immunological activities in primary human tissues, activate anti-inflammatory interleukin-10 and improve colitis symptoms in a colitis mouse model. Our study defines an antifolate stress response in E. coli and links its associated metabolites to a major immunological marker of IBD.

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Fig. 1: Colipterins upregulated by SMX antifolate drug stress in E. coli Nissle1917.
Fig. 2: Genetic and biomimetic synthesis support for the colipterin pathway.
Fig. 3: Immunomodulatory activities of the colipterins.
Fig. 4: Colipterins reduce colitis severity in an IL-10-dependent manner.

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Data availability

HR–ESI–Q–TOF–MS/MS datasets for colipterins are available from GNPS public MassIVE under accession no. MSV000085621. CP3 analysis was facilitated by applets provided by the Goodman Group (http://www-jmg.ch.cam.ac.uk/tools/nmr/CP3.html). Supplementary Information and Source data are provided with this Letter. Additional data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

No custom code was used.

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Acknowledgements

This work was supported by the National Institutes of Health (nos. 1DP2-CA186575 and R00-GM097096 to J.M.C.), the Burroughs Wellcome Fund (no. 1016720 to J.M.C.), the Camille & Henry Dreyfus Foundation (no. TC-17-011 to J.M.C.), the Howard Hughes Medical Institute (to R.A.F.) and the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (no. 2019R1A6A3A12033304 to C.S.K.). Z.W. was supported by the China Scholarship Council.

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Authors and Affiliations

Authors

Contributions

H.B.P. and J.M.C. conceived the study and designed the metabolism experiments. H.B.P. characterized the colipterin pathway from E. coli by drug stress, metabolome analysis, isolation, structure characterization, synthesis, genetic complementation and antioxidant assays. Z.W. performed ELISA assays for IL-8/-10 and all mouse studies. J.O. performed CP3 computational analysis on colipterins 1/2 and contributed to colipterin accumulation and NMR measurements. H.X. maintained the IL-10eGFP mice, isolated immune cells from the small intestine and analysed FACS data for IL-10 expression. C.S.K. constructed double-mutant strains of aspC and tyrB. R.W., T.P.W. and G.P. contributed to BioMap analysis of colipterins. R.A.F. conceived and supervised in vivo mouse studies. H.B.P. and J.M.C. wrote the manuscript with input from all authors. All authors reviewed and edited the manuscript.

Corresponding authors

Correspondence to Richard A. Flavell or Jason M. Crawford.

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Competing interests

R.A.F. is a recipient of a grant from AbbVie, Inc. R.W., T.P.W. and G.P. are employees of Merck Exploratory Science Center, Merck & Co., Inc., Kenilworth, NJ, USA. Employees may hold stocks and/or stock options in Merck & Co., Inc., Kenilworth, NJ, USA. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 UV-LC-MS detection of colipterins 1-8 produced by the probiotic E. coli Nissle1917.

a, Dose-responses of 1-8 from the probiotic E. coli Nissle1917 exposed to the sub-lethal levels of sulfamethoxazole (SMX). Intensity was determined by extracted ion counts (EICs) with m/z corresponding to each colipterin within a 10 ppm error window. High-resolution ESI-QTOF-LC-MS spectra of samples were analyzed using Phenomenex Kinetex C18 (100 Å) 5 μm (250 × 4.6 mm) column with a gradient from 10%-100% aqueous acetonitrile in 0.1% formic acid over 30 min and with a 0.7 ml min-1 flow rate. Data were collected in biological triplicate, and representative EIC chromatograms are shown. b, UV-Vis spectra of colipterins 1-8 and pterin.

Extended Data Fig. 2 Production profiles of colipterins from E. coli Nissle1917 cultures.

a, Time-course analyses of major colipterins 1-4 from E. coli Nissle1917 cultures. Peaks were extracted using the EIC method corresponding to the m/z of 1-4 within a 10 ppm window. The mean and s.d. (error bars) from three biological experiments (n = 3) are shown. Statistical significance (two-tailed unpaired t-test) is compared to 12 h; nd, not detected. b, Detection of col253 (3) and col267 (4) from the cell pellet extracts. SMX was supplemented into E. coli cultures with a sub-lethal concentration of 200 μg ml-1. Representative data from 24 h cultures are given from three biological replicates (n = 3). The mean and s.d. (error bars) are presented. A two-tailed unpaired t-test was used to calculate P values. Concentrations of 3 and 4 in cell pellet extracts without (and with SMX 200 μg ml-1) drug stress are as follows: 3, ~ 0.7 (~ 4.0) μM; 4, ~ 0.3 (~ 4.8) μM. c, Calibration curves for the colipterin quantification. Concentrations of 1-8 in culture without (and with SMX 400 μg ml-1) drug stress are as follows: 1, ~ 0.2 (~ 1.0) μM; 2, ~ 0.3 (~ 1.5) μM; 3, ~ 2.0 (~ 7.1) μM; 4, ~ 1.3 (~ 5.3) μM; 5, ~ 0.06 (~ 0.2) μM; 6, nd (~ 0.6) μM; 8, ~ 0.07 (~ 0.2) μM. 7 oxidized to 8 in the methanol solution during the acquisition of Q-TOF-MS experiments. Data are shown as the mean ± s.d., n = 3 from 3 technical triplicates.

Source data

Extended Data Fig. 3 Sulfamethoxazole (SMX) drug stress responses of colipterins 1–8 in the pathogenic E. coli LF82 and commensalistic E. coli BW25113.

Yellow area for optical density (OD600) measurement represents a range of sub-lethal concentrations of SMX against both E. coli LF82 (a) and BW25113 (b) strains. Peaks were extracted using EIC method corresponding to the m/z of colipterins 18 within a 10 ppm window. Red and black lines indicate Ctrl (Control, no SMX treatment) and SMX, respectively, in the MS chromatogram. Intensities in the bar graphs indicate integration value of EIC peak area. The mean and s.d. (error bars) from three biological experiments (n = 3) are shown. P values were analyzed by a two-tailed unpaired t-test; ns, not significant.

Source data

Extended Data Fig. 4 Functional characterization of FolX and FolM involved in colipterin production.

a, Folate and monapterin pathway are shown. b, Genetic complementation of folX and folM. Note that the production of colipterins is abolished in ΔfolX and ΔfolM strains. While the colipterins were not detected in both ΔfolX and ΔfolM carrying pBAD empty vector, complementation of folX and folM in pBAD into ΔfolX and ΔfolM strains, respectively, resulted in the recovery of major colipterins 3 and 4 production. 24 h E. coli cultures were analyzed by high-resolution ESI-QTOF-LC-MS using Phenomenex Kinetex C18 (100 Å) 5 μm (250 × 4.6 mm) column with a gradient from 10%-100% aqueous acetonitrile in 0.1% formic acid over 30 min and with a 0.7 ml min-1 flow rate. Data are mean ± s.d. from three biological replicates (n = 3); nd, not detected.

Extended Data Fig. 5 UV-LC-MS profiles of biomimetic synthesis of colipterins in various conditions.

a, Differential production of 5 and 6 from the tetrahydropterin (THP)-phenylpyruvate (PP) coupling reaction. 5 and 6 were observed to be major products from the reaction in pH 5.0 and pH 6.0 in water, respectively. b, 3 (m/z 254.1042) and 4 (m/z 268.0834) from the reaction with THP in the presence of initially expected substrate, phenylacetic acid (PA), were not observed. c, Individual incubation of 3, 4, 5, and 6 in methanol at room temperature for 12 h was monitored by HPLC. While 4 was most stable, 5 and 6 were interconvertible and irreversibly degraded to 3 and 4 as anticipated. Image for 4 is not shown. d, 7 (~0.5 mg) in 2 ml methanol was incubated at room temperature for 96 h and analyzed at five different time-points (6, 24, 48, 72, and 96 h). Auto-oxidation of 7 to 8 was observed over time. Image for the color change of 7 (colorless) to 8 (yellow) is shown in inset. e, Demonstration of the oxidation state of the pterin reactant. Independent reactions with pterin-PP and THP-PP were performed. While colipterins were not detected in reactions with pterin as expected, a reaction with THP in the presence of oxygen led to the colipterins. Representatively, 3 and 4 are shown. f, Reaction with THP under anaerobic conditions in vitro yielded either no detectable levels or basal levels of production. The reaction materials were analyzed by LC-MS with Phenomenex Kinetex C18 (100 Å) 5 μm (250 × 4.6 mm) column with a gradient from 10%-100% acetonitrile in water containing 0.1% formic acid for 30 min and with a 0.7 ml min-1 flow rate. All reaction was analyzed by UV (254 and 310 nm) and MS using EIC method.

Extended Data Fig. 6 Pterin profile for the demonstration of DHP substrate origins.

a, Previously described recycling and side-chain cleavage mechanism of biopterin in mammalian cells. Pterin and 7,8-dihydroxanthopterin (XPH2) are known as break-down products of biopterin. Similarly, we observed pterin (m/z 164.0572) and XPH2 (m/z 182.0678) in the BH4 in vitro chemical reaction via hydrogenation of commercial BH2 followed by aerobic incubation in water, but not in the BH2 solution. Additionally, colipterins were detected in BH4 solution supplemented with PP. Representatively, col327 (6) (m/z 328.1046) is shown. b, Upregulation of pterin and XPH2 in E. coli Nissle1917 in response to the sub-lethal levels of SMX. Dose-responses of pterin and XPH2 in antifolate SMX stress. The mean and s.d. (error bars) from three biological experiments (n = 3) are shown. Statistical significance was accessed using a two-tailed unpaired t-test. MS intensity of EIC chromatogram was determined by ion counts corresponding to pterin ((M + H)+ m/z 164.0572) and XPH2 ((M + H)+ m/z 182.0678) within a 10 ppm error window. High-resolution ESI-QTOF-LC-MS spectra of samples were analyzed using Phenomenex Kinetex C18 (100 Å) 5 μm (250 × 4.6 mm) column with a gradient from 10%-100% aqueous acetonitrile in 0.1% formic acid over 30 min and with a 0.7 ml min-1 flow rate.

Source data

Extended Data Fig. 7 E. coli BW25113 exposed to SMX drug stress reduce colitis severity in an IL-10-dependent manner.

a, Primary evaluation on the reduction of colitis severity in SMX-treated colitis mice (IL-10+/+ and IL-10-/-) that were colonized with E. coli (wild-type E. coli BW25113 and its folM mutant strain). CFUs, body weight, and clinical scores were obtained as the same procedure that we described in Fig. 4. HR-ESI-QTOF-MS quantification of colipterins 3 and 6 in stool samples. P values were determined by a two-tailed unpaired t-test. Error bars represent mean ± s.e.m.; ns, not significant. b, Effects of SMX-treated E. coli (wild-type E. coli BW25113 and its folM mutant strain) in the IL-10-/- male and female mice that spontaneously develop colitis. IL-10+/+ male and female mice that do not develop spontaneous colitis were used as control groups. E. coli was colonized at week-3. CFUs were measured at 3 weeks after colonization. Body weight was monitored weekly. SMX was mixed in the drinking water at 0.3 mg ml−1 after the E. coli colonization. At week-10, endoscopy was performed and clinical scores were used as a metric of colitis severity. Data are presented by mean ± s.e.m. (error bars).

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Park, H.B., Wei, Z., Oh, J. et al. Sulfamethoxazole drug stress upregulates antioxidant immunomodulatory metabolites in Escherichia coli. Nat Microbiol 5, 1319–1329 (2020). https://doi.org/10.1038/s41564-020-0763-4

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