Novel cross-feeding human gut microbes metabolizing tryptophan to indole-3-propionate

Abstract

Tryptophan-derived indoles produced by the gut microbiota, particularly indole-3-propionate (IPA), are key compounds associated with gastrointestinal balance and overall health. Reduced levels of IPA have been associated with inflammatory bowel disease, type 2 diabetes, and colorectal cancer. Since fiber-rich diets have been shown to promote IPA, we aimed to decipher fiber-specific effects and identify associated IPA-producing taxa in a range of healthy individuals. We cultured fecal microbiota from 16 adults with tryptophan and eight different dietary fibers and monitored community shifts by 16S rRNA gene amplicon sequencing and tryptophan-derived indoles using targeted liquid chromatography with diode array detection. The concentrations and types of indoles produced were donor-specific, with pectin strongly promoting IPA production in certain donors. IPA production was not associated with any known IPA producer but with the pectin-utilizing species Lachnospira eligens, which produced indole-3-lactate (ILA) in vitro, the IPA precursor. Supplementation of ILA in additional fecal microbiota cultures (n = 6) revealed its effective use as a substrate for IPA production. We identified a novel IPA producer, Enterocloster aldenensis, which produced IPA exclusively from ILA but not from tryptophan. Co-culture of L. eligens and E. aldenensis resulted in IPA production, providing new evidence for an ILA cross-feeding mechanism that may contribute to the IPA-promoting effects observed with pectin. Overall, we highlight the potential for targeted dietary interventions to promote beneficial gut taxa and metabolites.

Keywords: Enterocloster aldenesis; Indolepropionate; Lachnospira eligens; anaerobic cultivation; dietary fiber; fecal microbiota; indole; indolelactate; pectin; prebiotics.

Figure 1.

Effect of fiber supplementation on indoles production during fecal cultivation with healthy donor microbiota. (a) Overview of the tested fibers (3 gl⁻¹; starch, xylan, AG, bGlc, inulin, dextrin, pectin, pea or H₂O), the detected indoles and respective metabolic pathways. Red arrows highlight the oxidative, and blue arrows the reductive pathway. (b) The specific concentrations (relative to endpoint OD₆₀₀) of indoles detected in supernatants of fecal microbiota cultures (n = 16 donors) at the end of the fermentation (48 h, 37°C). Limit of detection: 2.5 µM across all metabolites. Bars represent means, and error bars represent standard deviations. Comparisons of fiber-supplemented cultures and negative controls (H₂O) were done using a paired Wilcoxon test. Statistically significant results are marked by stars, with *p ≤ 0.05, **p ≤ 0.01. (c) Metabolite-ASV correlations in fecal cultures fecal of healthy donor microbiota supplemented with tryptophan (5 mM). Positive ASV-metabolite correlations (p ≤ 0.05) detected in at least three donor microbiotas were assigned to the corresponding taxonomic group at species level. Due to the large number of comparisons, no multiple testing correction was applied. Each circle represents the correlation result for one ASV in one donor microbiota, with color indicating the Pearson correlation coefficient (R).

Figure 2.

Conversion of ILA to IPA in fecal microbiota cultures from healthy donors and pure intestinal strains. (a) Fecal microbiota (n = 6 donors) were incubated (37°C, 48 h) in bYCFA supplemented with 5 mM ILA and two different C-source combinations (6C+muc, pectin) or H₂O as control. Controls without ILA were included for 6C+muc and H₂O. Fecal microbiota cultures were analyzed for community composition, growth (OD₆₀₀ in 200 μl) and metabolite concentrations. b) Indoles production and growth (OD₆₀₀ in 200 μl) in pure batch cultures supplemented with 1 mM tryptophan or ILA (in bYCFA-3C, 48 h, 37°C). All 17 strains were tested in biological replicates (n = 3). C) Indole metabolite production from tryptophan in single and co-cultures of L. eligens DSM 3376 and E. aldenensis FBT_C. The data represent biological replicates (n = 3).

Figure 3.

C. sporogenes genes in the reductive pathway of tryptophan metabolism and corresponding homologs in E. aldenesis and L. eligens genomes. (a) The reductive pathway starts with the conversion of indole-3-pyruvate to ILA by the phenyllactate dehydrogenase (fldH: CLOSPO_RS01525), followed by the conversion of ILA to indole-3-acrylate by the phenyllactate dehydratase enzyme complex (fldLAIBC: CLOSPO_RS01525-CLOSPO_RS01545), and finally the conversion of indole-3-acrylate to IPA by the acyl-CoA dehydrogenase (acdA: CLOSPO_RS01550). (b) The reductive pathway genes (CLOSPO_RS01525 to CLOSPO_RS01570) are arranged in a gene cluster in C. sporogenes ATCC 15579 (NCBI: GCF_000155085.1). The homolog with high amino acid sequence similarity (AA similarity %) for fldH in L. eligens DSM 3376 (NCBI: GCF_000146185.1) co-localizes with a transaminase (highlighted in red). Genes for homologous enzymes in E. aldenesis (HumGut: GUT_GENOME001547) form a similar cluster.