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Sulfamethoxazole drug stress upregulates antioxidant immunomodulatory metabolites in Escherichia coli

Escherichia coli is an important model organism in microbiology and a prominent member of the human microbiota1. Environmental isolates readily colonize the gastrointestinal tracts of humans and other animals, and they can serve diverse probiotic, commensal, and pathogenic roles in the host2–4. Certain strains have been associated with the severity of inflammatory bowel disease (IBD)2,5; however, 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 sub-inhibitory levels of the antifolate sulfamethoxazole (SMX), which is used to treat infections, including in IBD patients6,7. The genes folX/M and aspC/tyrB involved in monapterin biosynthesis8–10 and aromatic amino acid transamination,11 respectively, were required to initiate the colipterin pathway. We show that the colipterins are antioxidants, harbor diverse immunological activities in primary human tissues, activate anti-inflammatory interleukin-10 (IL-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.

Using the broth dilution method, 43 we first established the sub-inhibitory concentration range of SMX for Nissle1917 in LB medium (Fig. 1c). We selected four of the subinhibitory conditions for further metabolite analysis compared to controls lacking SMX. While we saw no significant difference in growth over a 24 h cultivation period (Fig. 1d), using high-resolution electrospray ionization-quadrupole-time-of-flight-mass spectrometry (ESI-Q-TOF-MS) and photodiode array spectroscopy, we observed the SMX-mediated upregulation of a family of eight metabolites in supernatants ( Fig. 1e and Extended Data Fig.  1a). These metabolites, which we named colipterins 1-8, shared pteridine-like ultravioletvisible spectra 44 (Extended Data Fig. 1b). The ESI-QTOF-MS data allowed us to propose their molecular compositions (Supplementary Figs. 1 and 2). To establish the temporal dynamics of the major 1-4, we measured their production at 12 h, 24 h, and 48 h from culture supernatants. Colipterins 1 and 2 were detected at 12 h and 24 h before being depleted at 48 h, while the levels of 3 and 4 increased over time (Extended Data Fig. 2a). In cell pellets, 3 and 4 were only detected at both 24 h and 48 h (Extended Data Fig. 2b). Quantification results of all compounds are given in Extended Data Fig. 2c. Finally, to address whether these metabolites were more broadly detected across E. coli strains, we also analyzed production of 1-8 in a representative pathogen, AIEC LF82, and a representative commensal, E. coli BW25113. While the sub-inhibitory antibiotic concentration range of SMX varied among the isolates, SMX supplementation enhanced the production of 1-5 and 8 (Extended Data Fig. 3). However, 6 and 7 were below the detection limits under these in vitro conditions. Collectively, our data suggest that select colipterins are upregulated in response to antifolate cell stress and are conserved among these probiotic, pathogenic, and commensal E. coli.
To identify the structures of colipterins 1-8, we isolated and characterized them by NMR, tandem MS, biomimetic chemical synthesis, Marfey's configurational analysis, and CP-3 computational analysis ( Fig. 1f and Supplementary Figs. 3-64 and Tables [1][2][3][4][5]. Details are provided in the Supplementary Information. All of the compounds represent previously unknown metabolites; however, 3 and 4 have been described as synthetic products with no reported activities 45 . To provide genetic support of colipterin biosynthesis, we first evaluated non-essential fol genes involved in folate and monapterin metabolism 46,47 . While folate itself is required for bacterial growth, monapterin's role in E. coli remains obscure 10,48 . Since colipterins are upregulated in antifolate SMX stress, we hypothesized that inhibition of folate metabolism in E. coli could inversely lead to the elevation of monapterin-derived products (Fig. 2a). Of the Fol enzymes, FolX catalyzes conversion of 2,3-dihydroneopterin triphosphate (DHNTP) into 2,3-dihydromonapterin triphosphate (DHMTP), which is followed by dephosphorylation to yield 2,3-dihydromonapterin (DHM) and reduction to tetrahydromonapterin (THM) by FolM (Extended Data Fig. 4a) 9,10,49 . We thus grew E. coli mutants of folX and folM for 24 h and analyzed production of 1-4, the dominant products at this time point. Relative to the parent E. coli BW25113, the ΔfolX and ΔfolM strains led to complete abolishment of 1-4 (Fig. 2b). These metabolic phenotypes could be complemented in trans with the expression of folX and folM, respectively (Extended Data Fig. 4b), further supporting that the colipterins derive from the monapterin pathway. We next focused on the aromatic amino acid transaminases AspC and TryB and their potential role in colipterin biosynthesis. Notably, it has been documented that aspC, which is responsible for the conversion of aromatic amino acids to their α-keto acid forms, is required for drug stress responses in E. coli [50][51][52] . In our analysis of single mutants, ΔaspC and ΔtyrB, we observed no significant differences in colipterin production compared to wild-type. However, we constructed two double mutants ΔaspC-tyrB in both the ΔaspC and ΔtyrB backgrounds, and the colipterins were completely abrogated (Fig. 2c). These data indicate that aromatic amino acid transamination is required for colipterin biosynthesis and that AspC and TyrB are functionally redundant in this regard. Collectively, these studies demonstrate that monapterin biosynthesis and aromatic amino acid transamination converge in colipterin production. However, potential catalysts or cellular conditions that facilitate coupling of dihydropterin (DHP) and phenylpyruvate, and to a lesser extent phenylalanine (Phe), remain undefined.
With the necessary colipterin substrate pathways defined, we turned to biomimetic synthesis studies to explain colipterin family production. We proposed that nucleophilic substitution reactions at the electrophilic C6-position of DHP could account for 1-8. To test this proposal, we prepared tetrahydropterin (THP) as previously described. 53 Individual reactions (2 h anaerobic, 2 h aerobic) with THP and phenylpyruvate or Phe yielded two groups (3-6) and (7)(8), respectively (Fig. 2d). 3 and 4 were established as degradation products of 5 and 6, and auto-oxidation of 3 to 4, and of 7 to 8, was detected (Extended Data Fig. 5a-d). To facilitate the production of 1 and 2, we reacted Phe and phenylpyruvate (1:1 ratio) in dimethylformamide (DMF) before supplementation of THP. Only these sequential conditions led to the production of 1 and 2 (Fig. 2e), supporting the order of reactivity. Lastly, the oxidation state of the pterin reactant in these studies appeared to be free DHP, as pterin and THP (anaerobic) were not competent substrates (Extended Data Fig. 5e,f). Importantly, these studies support a mechanistic model in which DHP could undergo "nucleophilic reduction" to generate C6-functionalized THP-conjugates in the absence of a biological reductant (e.g., NADH, NADPH) (Fig. 2f).
With our understanding of colipterin origins from monapterin genetic studies and biomimetic synthesis efforts, we turned to establish dominant DHP substrate origins in E. coli. Monapterin (and folate) is derived from guanosine triphosphate (GTP) through the key intermediate 2,3-dihydroneopterin triphosphate (DHNTP) by GTP cyclohydrolase I (GTPCH, folE) in E. coli 54 . Prior studies have proposed a recycling pathway in mammalian cells for the hydroxyl analog of monapterin, tetrahydrobiopterin (BH 4 ) 55 , that serves as an antioxidant and essential redox co-factor for aromatic amino acid hydroxylases and nitric oxide synthase (NOS) 56,57 . BH 4 is oxidized to the unstable quinoid dihydrobiopterin (qBH 2 ) which isomerizes to dihydrobiopterin (BH 2 ), and dihydropterin reductase (DHPR) recycles qBH 2 and BH 2 back to BH 4 57 (Extended Data Fig. 6a). Additionally, it has been shown that genetic, and metabolomic studies, we propose analogous tetrahydromonapterin (THM) recycling and degradation pathways in E. coli, in which NADPH-dependent and FolMmediated THM recycling competes with C6-side chain cleavage. However, cleaved DHP could undergo further nucleophilic pterin-core reductions to explain the colipterins (Fig. 2f).
To establish biological activities for the colipterins, we first considered the activities of other pterins. Members of the pterin family serve as xanthine oxidase inhibitors 59 , redox cofactors, 60 and as antioxidants via their radical scavenging activities 61 . Given the redox activity of pterins, we examined 1-6 (7-8 were substrate limited) for radical scavenging activity against the stable radical 1,1-diphenyl-2-picrylhydrazyl (Fig. 3d). L-Ascorbic acid and THP were used as positive controls (EC 50 values, 3.8 and 4.1 μg ml −1 , respectively, Fig.  3d). Colipterin 6 was the most active radical scavenger (EC 50 , 12.8 μg ml −1 ) with comparable activity to positive controls, whereas 1 and 2 were less active (EC 50 >70 μg ml −1 ). While 3 and 4 were inactive, chemical reduction to their THP-conjugate forms, 3a and 4a, showed good activity (13.1 μg ml −1 and 27.9 μg ml −1 , respectively). This chemical reduction is directly analogous to our nucleophilic reduction model for DHP, indicating that nucleophilic reductions could provide the organism an advantage to redox stress. Moreover, supplementation of 4, the most advanced metabolite in the colipterin pathway, to E. coli cultures using the disk diffusion method significantly reduced the zones of inhibition against hydrogen peroxide stress compared to 3, 6, pterin, THP , and solvent vehicle controls, suggesting that 4 (or reduced 4a in cells) have a cellular protective role ( Fig. 3e and Supplementary Fig. 65).
Next, we screened 1-6 against a panel of 12 human primary cell-based co-culture systems that model multiple tissues and disease states (BioMAP® Phenotypic Profiling Assay). Details are described in the methods. Briefly, 1-6 had similar effects on most of the protein biomarkers measured in these cell systems, and we observed that several of the colipterins affected IL-8 and IL-10 levels in venular endothelial and macrophage cell systems (/Mphg) (Fig. 3a). To validate selected immunomodulatory properties of the colipterins, we measured IL-8 and IL-10 (for 1-6) expression levels in human THP-1 macrophage-like cells and mouse bone marrow-derived macrophages (BMDMs), respectively, using ELISA assays. At the same BioMap® testing dose of 17 μM, 2, 3, 4, and 6 significantly stimulated IL-8 levels compared to DMSO vehicle control ( Fig. 3b) in differentiated THP-1 cells. Similarly, 1-4 and 6 upregulated IL-10 levels in BMDMs (Fig. 3c). Dose response analysis with L-ascorbic acid as an antioxidant control was comparable to most of the colipterins, suggesting that their antioxidant activities upregulate IL-10 levels. However, we observed significant IL-8 and IL-10 responses of colipterin 6 at concentrations of 1 μM or higher, whereas L-ascorbic only showed responses at an order of magnitude higher concentration (17 μM).
Because we observed both in vitro radical scavenging activities and upregulation of antiinflammatory cytokine IL-10 in human and mouse tissue culture systems, we next investigated whether the colipterins could ameliorate severe colitis symptoms in vivo in a colitis mouse model ( Fig. 4 and Extended Data Fig. 7). We individually colonized microbiome-reduced IL-10 +/+ and IL-10 −/− mice with wild-type E. coli BW25113 and its folM mutant strain, and dextran sulfate sodium (DSS, 2.5%) was administered. We then fed SMX to the mice compared to no SMX controls (Fig. 4a). While there was no difference in E. coli colonization among the groups as expected, slight body weight loss in mice with ΔfolM was observed upon SMX administration (Fig. 4b,c). The DSS scores 62 in IL-10deficient mice were similar between wild-type and ΔfolM colonized groups; however, IL-10 +/+ mice colonized by ΔfolM exhibited significantly higher DSS scores compared to that of mice colonized with wild-type E. coli, suggesting that folM-derived metabolites, such as the colipterins, protect mice from DSS-induced colitis in an IL-10-dependent manner (Fig. 4b,c). We also observed significantly higher in vivo levels of major colipterins 3 and 6 in the stool samples of both IL-10 +/+ and IL-10 −/− mice colonized with wild-type E. coli compared to the ΔfolM-colonized groups (Extended Data Fig. 7a). Importantly, we can also detect basal levels of 3 and 6 in the ΔfolM E. coli systems, suggesting that other members of the microbiome also produce the colipterins.
To further support colipterin-mediated upregulation of IL-10 in vivo, we individually fed 3, 4, 6, and folic acid (each 10 mg kg −1 ) every other day to wild-type mice (IL-10 +/+ ) using the DSS inflammation model (Fig. 4d). 4 and 6 significantly reduced colitis severity, whereas folic acid showed no effect in our experiment. Further, we individually fed 4 and 6 (10 mg kg −1 each) to IL-10 eGFP mice, isolated immune cells from the colon, and quantified eGFP signals in CD45 + CD3 -CD11b + F4/80 + macrophages using fluorescence activated cell sorting (FACS). Relative to DMSO control, 4 and 6 significantly upregulated IL-10 production in macrophages (Fig. 4e). Finally, we examined the effects of 3, 4, 6, and folic acid on colitis severity in IL-10 −/− mice that spontaneously develop colitis after 10-weeks of age. None of compounds were protective in IL-10 −/− mice (Fig. 4f,g), further suggesting that the colipterins ameliorate colitis in an IL-10-dependent manner. Consistent with these findings, IL-10 −/− spontaneous colitis mice colonized with E. coli (wild-type BW25113 versus ΔfolM) in the presence of SMX and absence of DSS similarly showed no protective effects (Extended Data Fig. 7b). Collectively, these data link folM-derived colipterins to antiinflammatory IL-10 responses in vivo, leading to the improvement of colitis symptoms in an IL-10-dependent manner.
Modern analytical technologies have led to the realization that the vast majority of metabolites found in any microbe or animal including humans remain unknown 63 . Some of the metabolites in this so called "dark matter" of the metabolome will undoubtedly have profound functional consequences. Not only will they affect microbial compositions and activities, but they will also regulate host phenotypes, such as immunomodulatory responses. Further complicating the observed chemical smorgasbord of metabolites in the intestinal tract, antibiotics dramatically impact microbial metabolism [15][16][17][18]51 and host immune responses 27,28 through a variety of mechanisms. However, we are only beginning to understand how the microbiome regulates the immune system at the detailed molecular level and how antibiotics participate directly or indirectly in these molecular responses. Subinhibitory levels of antibiotics provide a means to study the stress responses of select microbiome members such as E. coli without significantly impacting their cell growth in vitro or intestinal colonization in vivo. In our study, application of the antifolate antibiotic SMX to intestinal E. coli strains leads to the upregulation of a previously undescribed family of pteridines termed the colipterins. In addition to harboring antioxidant roles for the E. coli, these metabolites exhibit anti-inflammatory activities in primary human tissues, in laboratory tissue cultures, and in mouse models of intestinal inflammation. Thus, these chemical biological studies provide an example that could support a broader model in which antibiotics indirectly regulate immune responses through the activation of bacterial stress responses.
E. coli strains have been linked to the severity of IBDs, including Crohn's disease and ulcerative colitis, but the mechanisms similarly remain largely undefined at the molecular level 5,64,65 . For example, AIEC LF82 has been implicated in the pathogenesis of Crohn's disease 65 , whereas human probiotic E. coli 1917 supports the remission of ulcerative colitis in clinical trials 64,66 . SMX is used to treat E. coli infections, including those in IBD patients 6,31 . Strikingly, we show that SMX antifolate stress upregulates colipterin production in E. coli and hypothesize that our nucleophilic DHP-reduction model, which accounts for colipterin production, would provide E. coli an advantage to redox stress. In principle, a reduction in NADPH levels could shift the balance from monapterin recycling to irreversible C6-fragmentation, leading to DHP. Tetrahydropterins like THP have potent antioxidant activities on par with ascorbic acid (vitamin C), and "nucleophilic reduction" of DHP to its THP-conjugates, the colipterins, in the absence of a hydride source provide a chemical strategy to enhance antioxidant activity. We support this model with radical scavenging and E. coli cell culture studies. In turn, this bacterial stress response mechanism enhances the production of anti-inflammatory IL-10 and reduces the severity of colitis in mouse models in an IL-10-dependent manner. As the human microbiome field transitions from correlation to causation 63 , the "dark matter" of the metabolome will begin to be illuminated. Illumination of the colipterins here exemplify some of the hidden functional chemical diversity of a major microbial player at the host-microbe interface.

General Chemistry Analysis.
Analytical HPLC-MS spectra of metabolites were collected using an Agilent 1260 Infinity HPLC with an Agilent 6120 Quadrupole low-resolution (LR) electrospray ionization (ESI) mass spectrometer (Agilent, Santa Clara, CA, USA). Liquid column chromatography was carried out using either LiChroprep ® RP-18 (

Computational Analysis for Absolute Structure Elucidation of 1 and 2.
Conformational searches were conducted using the Macromodel (version 2015-2, Schrödinger LLC) program with "mixed torsional/low mode sampling" in the MMFF94 force field. The searches were implemented in the gas phase with a 50 kJ mol −1 energy window limit and 10,000 maximum number of steps to completely explore low-energy conformers. The Polak−Ribiere conjugate gradient method was used for minimization processes with 10,000 maximum iterations and a 0.001 kJ (mol Å) −1 convergence threshold on the rms gradient. Conformers within 10 kJ mol −1 of each global minimum of plausible epimers addressed in this study were provided for the GIAO shielding constant calculations without geometry optimization using the Gaussian 09 package (Gaussian Inc) at the B3LYP/6-31G(d,p) level in the gas phase. These calculated chemical shift values were averaged based on their Boltzmann populations (Supplementary Table 2) and utilized for CP3 analysis facilitated by the applets available at http://www-jmg.ch.cam.ac.uk/tools/nmr/ CP3.html. In particular, calculated and experimental chemical shift values of diastereotopic protons were compared with their closest matches based on the original author's recommendation 67 .

Genetic Complementation of folX and folM mutants.
The genes, folX and folM, were PCR amplified from E. coli BW25113. Primers used for the amplification are shown in Supplementary Table 6. The purified PCR products and the pBAD18 vector were independently digested using SacI and KpnI and were ligated using T4 DNA ligase (NEB) according to the manufacturer's protocol. These products, pBAD-empty vector, pBAD-folX, and pBAD-folM, were then transformed by heat-shock into chemically competent E. coli DH5α followed by sequence validation. Electrocompetent E. coli BW25113, ΔfolX, and ΔfolM strains were generated, and the empty vector, pBAD-folX, or pBAD-folM were electroporated into the corresponding electrocompetent strains. Cells were grown on LB agar plates supplemented with ampicillin at 100 μg ml -1 . 5 ml overnight cultures in biological triplicates were sub-cultured (1:1000) into 5 ml aliquots of LB supplemented with 100 μg ml −1 ampicillin and grown at 37 °C until the OD 600 = 0.4-0.5. The cultures were induced with 0.1% L-arabinose and further incubated at 37 °C with agitation (250 r.p.m) for 48 h. After centrifugation, the supernatants were extracted with 6 ml n-butanol and the extracts were dried under reduced pressure on a HT-4X evaporation system. The samples were analyzed as described in "Metabolome Analysis of E. coli in Response to SMX stress."

Construction of aspC and tyrB Double Mutants.
E. coli BW25113 ΔtyrB/ΔaspC double mutant strains were constructed using the Red recombinase system of bacteriophage lambda. The FTR-flanked spectinomycin resistance cassette of pIJ778 was amplified (primers, Supplementary Table 6) with short sequence extensions homologous to the flanking regions of aspC or tyrB. Purified PCR products were desalted and respectively transformed into E. coli JW4014-2 (ΔtyrB747::kan) or JW0911-1 (ΔaspC745::kan) carrying plasmid pKD46. Transformants were selected by plating on LB agar supplemented with spectinomycin (50 μg ml −1 ). Colonies were analyzed with overspanning PCR and the resulting products were sequence validated and cured of pKD46.

Biomimetic Synthesis.
Pterin, L-phenylalanine (L-Phe), phenylpyruvic acid sodium salt (PP), and phenylacetic acid (PAA) were purchased from Millipore Sigma. To prepare tetrahydropterin (THP), pterin (500 mg, 3.0 mmol) was suspended in anhydrous methanol (200 ml) following addition of the platinum oxide (50 mg, 0.22 mmol) as a catalyst. Hydrogenation of pterin was initiated in the presence of hydrogen gas. After overnight reaction at room temperature, the resulting materials were filtered through Celite ® 545, and the filtrates were dried under nitrogen gas purge in a glove bag to yield THP (200 mg, 38%). The three substrates (PP for 5 and 6, L-Phe for 7 and 8, and PAA for 3 and 4) were individually prepared, dissolved in water with a concentration of 1 mg ml −1 , and stirred at room temperature for 10 min. For 1 and 2, L-Phe (1 mg) and PP (1 mg) were mixed in the dimethylformamide (DMF, 2 ml) and incubated at room temperature for 5 h. The dried THP was re-suspended in water (~1 mg ml −1 ), and 50 μl of THP solution was dispensed into 96 well-plate, which was followed by the supplementation of 50 μl of pre-mixed L-Phe+PP for 1 and 2, PAA for 3 and 4, PP for 5 and 6, and L-Phe for 7 and 8. The reaction materials were anaerobically incubated for 2 h in the glove bag with nitrogen purge and were exposed to air for additional 2 h. The 10 μl of reaction samples were monitored by HPLC system with low-resolution ESI-MS spectrometer.

DPPH Radical Scavenging Assay.
Radical scavenging assays were performed as previously described in triplicate experiments 53 .

Cellular Protection Assay.
E. coli Nissle1917 was streaked onto an LB agar plate. Single colonies were selected and inoculated into 5 ml LB broth and incubated at 37 °C with agitation (250 r.p.m.) overnight. Cell densities were adjusted to OD 600 = 0.05, and 100 μl of diluted cells were spread onto fresh LB agar plates. Compounds were prepared in DMSO (10 mg ml −1 ) and 10 μl of solution was soaked onto paper disk. DMSO was used for control. The disk was placed on the agar plate containing E. coli and incubated at 37 °C for 3 h. 1 μl of 30% hydrogen peroxide was then added to the disks and the cultures were further incubated overnight. Zones of inhibition were measured after overnight incubation. This assay was performed in biological triplicates. . Systems are derived from either single cell types or co-culture systems. Adherent cell types were cultured in 96 or 384-well plates until confluence, followed by the addition of PBMC (SAg and LPS systems). The BT system consists of CD19+ B cells co-cultured with PBMC and stimulated with a BCR activator and low levels of TCR stimulation. Compounds 1-6 were prepared in DMSO (final concentration ≤ 0.1%) and added at a final concentration of 17 μM, 1 h before stimulation and remained in culture for 24 h or as otherwise indicated (48-hrs: MyoF system; 72-hrs: BT system (soluble readouts); 168 h: BT system (secreted IgG)). Each plate contained drug controls, negative controls (e.g., nonstimulated conditions) and vehicle controls (e.g., 0.1% DMSO) appropriate for each system. Direct ELISA was used to measure biomarker levels of cell-associated and cell membrane targets. Soluble factors from supernatants were quantified using either HTRF® detection, bead-based multiplex immunoassay or capture ELISA. Overt adverse effects of test agents on cell proliferation and viability (cytotoxicity) were detected by sulforhodamine B (SRB) staining, for adherent cells, and alamarBlue® reduction for cells in suspension. For proliferation assays, individual cell types were cultured at subconfluence and measured at time points optimized for each system (48-hrs: 3C and CASM3C systems; 72-hrs: BT and HDF3CGF systems; 96-hrs: SAg system). Cytotoxicity for adherent cells was measured by SRB (24-hrs: 3C, 4H, LPS, SAg, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, lMphg systems; 48-hrs: MyoF system), and by alamarBlue staining for cells in suspension (24-hrs: SAg system; 42-hrs: BT system) at the time points indicated.

Mouse Studies.
C57BL/6 and IL-10 eGFP mice were used in this study 68 . IL-10 −/− mice were a gift from Sandy Morse (002251-B6.129P2-Il10tm1Cgn/J, Jackson Laboratory, Bar Harbor, ME). Experimental groups of wild-type and IL-10 −/− mice were generated by heterozygote-byheterozygote breeding. Sample sizes were chosen in line with pervious experimental experience and consistent with the broader literature. 8-10-week-old equal male and female mice were used if not otherwise specified. All experiments were performed using cohoused mice littermate controls. Iso-sexual male or female littermates were cohoused at 21-24°C with 40-60% humidity. A 12 light/12 dark cycle was used. All mouse studies were performed in compliance with Yale Institutional Animal Care and Use Committee protocols. No formal blinding or randomization was conducted; however, control and treated groups were chosen arbitrarily for each experiment. Mice weights and CFUs were measured in a blinded manner.

E. coli Mouse Colonization.
Before colonization, 8-10-week-old mice were fasted for 4 h followed by gavage of kanamycin (20 mg

DSS-Induced Colitis Model.
After E. coli colonization, 8-10-week-old mice were administered 2.5% DSS (MP Bio) for 5 days and returned to regular drinking water and monitored daily. Sulfamethoxazole (SMX) was orally administrated by 100 mg per kilogram body weight at day-1, day-3 and day-5. Weight loss was measured daily. On day-6, endoscopy was performed and clinical scores were used as a metric of colitis severity. On day-7, fecal pellets were collected for the quantification of colipterins.

Fecal Colipterin Quantification.
Fecal pellets were collected and resuspended in methanol (HPLC grade) followed by centrifugation. Supernatants were dried in vacuo and resuspended in 100 μl methanol and analyzed by high-resolution ESI-Q-TOF-MS.

Colipterin Administration.
Colipterins were orally gavaged by 10 mg per kilogram body weight every other day for two weeks. Mice were euthanized 2 days after the last colipterin administration for intestinal immune cell analysis.

Immune Cell Isolation from the Small Intestine.
Intestinal tissues were first incubated with 1 mM DTE at 37 °C for 30 min and then further digested with collagenase from Clostridium histolyticum (Sigma, #2139) and DNase I at 37 °C for another 1 h. Cells from both fractions were pooled and further separated with Percoll gradient.

Flow Cytometry and Staining Antibodies.
We

IL-10 −/− Spontaneous Colitis Model.
After weaning, body weight of mice was monitored weekly. For compound administration, 3, 4, 6, folic acid, and an equivalent amount of DMSO as a solvent vehicle control were individually mixed in the drinking water at 0.03 mg ml −1 after 6 weeks of age. This concentration was equivalent to 5 mg kg −1 body weight oral dose per day based on the assumption that the average daily consumption of water for an adult 25 g mouse was around 4 ml. For E. coli colonization experiments, the colonization was performed directly after weaning using the protocol described above, then SMX was mixed in the drinking water at 0.3 mg ml −1 after the colonization. This concentration was equivalent to 5 mg kg −1 body weight oral dose per day based on the assumption that the average daily consumption of water for an adult 25 g mouse was around 4 ml. At week-10, endoscopy was performed and clinical scores were used as a metric of colitis severity.

Data and Statistical analysis.
All graphical and statistical analysis were performed using GraphPad Prism 8. Statistical significance was calculated using a two-tailed unpaired t-test or a one-way ANOVA with Dunnett's test. P values and the statistical parameters are indicated in the figure and figure legends, respectively. Agilent OpenLAB CDS ChemStation (Version C.01.04) and MassHunter Workstation Data Acquisition (Version B.05.01) were used for low-resolution UV/ESI-MS and high-resolution ESI-QTOF-MS data collection, respectively. The highresolution mass spectrometry datasets were analyzed by MassHunter Qualitative Analysis (Version B.06.00). Conformational analyses of compounds were conducted using the Macromodel (version 2015-2, Schrödinger LLC), and Gaussian 09 package (Gaussian Inc) was used for Geometry optimization analysis of compounds. BD FACSDiva Software (V8.0.1) was used to collect raw data files from all flow cytometry experiments. All resultant data files were analyzed using FlowJo version 10 or newer. All experiments were conducted at least two times with reproducibility, and colipterin compounds were qualitatively screened via singleton BioMAP® phenotypic profiling.

Reporting Summary.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
High-resolution ESI-QTOF-MS/MS data sets for colipterins are available from GNPS public MassIVE under accession number MSV000085621. CP3 analysis was facilitated by the 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.
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 C 18 (100Å) 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. 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

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. A two-tailed unpaired t-test was used to calculate P values; ns, not significant.
Corresponding extracted ion counts (EIC) chromatograms are shown in Extended Data Fig.  1a. f, Chemical structure of colipterins 1-8. Detailed NMR-based structure elucidation of the colipterins is described in the Supplementary Discussion. THM, Tetrahydromonapterin; and qDHM, quinoid dihydromonapterin. Solid blue and red arrows indicate coupling of PP and L-Phe to DHP, respectively. Dashed arrows represent the formation of PP-Phe conjugate (purple) and coupling of this conjugate to DHP (green), respectively. female (g) mice that spontaneously develop colitis. IL-10 +/+ mice administered with DMSO were used as control. Body weight was monitored weekly. Compounds were mixed in the drinking water at 0.03 mg ml −1 individually after the mice were 6 weeks old. At week-10, endoscopy was performed and clinical scores were used as a metric of colitis severity. All P values were calculated by a two-tailed unpaired t-test. All error bars are represented as mean values ± s.e.m.; ns, not significant.