Two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation

Abstract

Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1-carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2-carbon-transporting folate in the form of 5,10-ethenyl-tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2-carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP-1 cells with ethionine, their transcription of TNF-α was inhibited and cell proliferation reduced. Mass spectrometry of THP-1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone-methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2-carbon transporting variant of the folate cycle and modify human chromatin via ethylation.-Röth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.

Keywords: ethenyltetrahydrofolate; lysine ethylation; microbiome; posttranslational modification; probiotic bacteria.

Figure 1

L. reuteri produces novel folates. A) MALDI-TOF spectrum of L. reuteri 5,10-methenyl-THF-polyglutamates (MGlu_n); arrows point to signals MGlu_n with 4–8 glutamates. The novel folate has a 14-Da increased mass (M’Glu_n). B) Arrows indicate the potential localization of an additional methyl group within 5,10-methenyl-THF.

Figure 2

The additional methyl group in the novel folate form is not derived from histidine, serine, or glucose. A–C) Representative MALDI-TOF mass spectra of folylpolyglutamates from L. reuteri cultured in LDM containing unlabeled (top) or isotopically labeled (bottom) ¹⁵N₃[¹³C]₆-histidine (A), [¹³C]₃-serine (B), or [¹³C]₆-glucose (C). D) Structures representing the synthesis of folic acid from GTP. Red dots show the 5 glucose [¹³C] atoms that form the ribose part of GTP and are incorporated into folic acid. E–G) MALDI-TOF mass spectra of folylpolyglutamates from L. reuteri cultured in LDM containing increasing amounts of folic acid (E), with or without pABA (F), or isotopically labeled guanine (G). H) Structures representing the synthesis of folic acid from guanine. Colored dots highlight [¹³C] and ¹⁵N atoms in guanine and the synthesized folic acid.

Figure 3

L. reuteri produces the novel folate, 5,10-ethenyltetrahydrofolyl polyglutamate, from acetate. A, C, D) MALDI-TOF mass spectrometric data of folylpolyglutamates from L. reuteri cultured in LDM containing 0 mM, 18.3 mM (0.1×), and 183 mM (1×) acetate (A), isotopically labeled acetate (C), or 183 mM of the indicated SCFAs or 18.3 mM formate (D). B) Ratios of 5,10-ethenyl-THF to 5,10-methenyl-THF after culturing in varying concentrations of formate or acetate at 3 distinct time points.

Figure 4

L. reuteri produces 5,10-methylmethenyl (ethenyl) THF. A–C) MALDI-TOF/TOF MS/MS spectra (A) of SAX purified ethenyl THF Glu₆ localizes the acetate-derived methyl group into the folate head group (B, C). D–F) NMR experiments. D) In the 3D HMCMCO experiment, the magnetization is transferred from methyl proton to methyl carbon, and then to the imidine carbon (blue arrows). After chemical shift labeling of the imidine carbon (t1), the magnetization is transferred back to methyl carbon (red arrows) followed by the chemical shift labeling (t2), and then back to methyl proton with proton detection (t3). E) Overlay of the ¹H-[¹³C] HSQC spectra of the methyl groups from acetate with 5,10-ethenyl-THF (in blue) and 5,10-ethenyl-THF alone (in red). The signal splittings are from carbon-carbon 1-bond J couplings. F) Cross peak between methyl proton and carbonyl carbon in acetate, and imidine carbon in 5,10-ethenyl-THF. Red characters in E and F mark the ¹H and [¹³C] nuclei from which the cross peaks were obtained.

Figure 5

L. reuteri produces the immunomodulator ethionine. A) Triple quadrupole LC/MS multiple reaction monitoring (MRM) chromatograms of ethionine-d₅ standard (left panels) and ethionine (right panels). The top row in each panel gives the m/z of the MRM transitions. Peak area counts of valid ethionine signals that have both quantifier and qualifiers are given in colored numbers. B) Quantification of ethionine in LDM3 after 7 d of culture. The LDM3 contained 0, 6.1, 18.3, 61.3, and 183 mM acetate or 6.1, 18.3, and 61.3 mM formate. C, D) mRNA levels of IκBα (C) and TNF-α (D) in THP-1 cells pretreated with 2.5 mM ethionine for 48 h and stimulated with 100 ng/ml LPS for 2 h. Expression was calculated with the ΔΔC_t method and normalized to untreated samples. E) Cell viability of THP-1 cells after incubation with ethionine at given time points as determined by flow cytometry using forward scatter/side scatter exclusion.

Figure 6

Ethionine can replace methionine during protein biosynthesis. A) Relative quantitation of histone 3 peptides containing oxidized and non-oxidized methionine, ethionine, and ethionine-d₅. Each line represents 1 peptide. B) Extent of incorporation of ethionine and ethionine-d₅ into histone 3. C) Representative spectra and ion tables of the peptide KTVTAMDVVYALAK. B₆-b₁₃ ions and y₉-y₁₃ ions demonstrate the incorporation of ethionine (+14 Da) and ethionine-d₅ (+19 Da). THP-1 cells were grown in the presence of 2.5 mM ethionine or ethionine-d₅ for 48 h. Histones were isolated and ethionine incorporation was determined by LS-MS/MS. “K(+prop)” indicate propionylated lysine residues (see text).

Figure 7

Histone 3 lysine ethylation is a novel posttranslational modification. A, B) Representative MS/MS spectra and ion tables of the unmodified (A) and the ethylated (B) peptide KSAPATGGVKKPHR. B₁₀-b₁₃ ions and y₅-y₁₃ ions demonstrate the ethylation of K10 (+33 Da). C) N-terminal sequence of histone 3. Black letters mark peptides with identified lysine ethylation and red Ks mark the localization of the modified lysines. Lower panels: Quantitation of peptides containing ethionine-d₅. Histone 3 peptides were from THP-1 cells grown in the presence of 2.5 mM ethionine or ethionine-d₅ for 48 h.