Modulating Activity Evaluation of Gut Microbiota with Versatile Toluquinol

Abstract

Gut microbiota have important implications for health by affecting the metabolism of diet and drugs. However, the specific microbial mediators and their mechanisms in modulating specific key intermediate metabolites from fungal origins still remain largely unclear. Toluquinol, as a key versatile precursor metabolite, is commonly distributed in many fungi, including Penicillium species and their strains for food production. The common 17 gut microbes were cultivated and fed with and without toluquinol. Metabolic analysis revealed that four strains, including the predominant Enterococcus species, could metabolize toluquinol and produce different metabolites. Chemical investigation on large-scale cultures led to isolation of four targeted metabolites and their structures were characterized with NMR, MS, and X-ray diffraction analysis, as four toluquinol derivatives (1-4) through O1/O4-acetyl and C5/C6-methylsulfonyl substitutions, respectively. The four metabolites were first synthesized in living organisms. Further experiments suggested that the rare methylsulfonyl groups in 3-4 were donated from solvent DMSO through Fenton's reaction. Metabolite 1 displayed the strongest inhibitory effect on cancer cells A549, A2780, and G401 with IC₅₀ values at 0.224, 0.204, and 0.597 μM, respectively, while metabolite 3 displayed no effect. Our results suggest that the dominant Enterococcus species could modulate potential precursors of fungal origin and change their biological activity.

Keywords: Enterococcus faecalis; Enterococcus faecium; acetylation; antitumor; biotransformation; gut microbiota; toluquinol.

Figure 1

Biosynthesis and wide distribution of toluquinol as a key intermediate for a large number of fungal metabolites. (A) Biosynthetic pathway for toluquinol in fungi. (B) Structures of representative fungal metabolites using toluquinol as a key precursor. (C) Wide distribution of the key biosynthetic gene PKS for toluquinol in fungi. (D) Detection of toluquinol in the metabolic profile of Chinese steamed breads colonized with Penicillus species. (E) Detection of toluquinol in the metabolic profile of Tangerines colonized by Penicillus species.

Figure 2

Transformation of toluquinol in four gut microbes. (A–D) Comparison of metabolic profiles of E. faecalis, E. faecium, S. thermophilus, and L. lactis subsp. Lactis fed with and without toluquinol. (E–H) UV spectra of the targeted metabolites 1–4. (I–L) Mass spectra of the targeted metabolites 1–4.

Figure 3

The four targeted metabolites (1–4) derived from toluquinol in four gut microbes and the single X–ray crystallographic structure of 3. Red: oxygen; Orange: Sulfur; Blue: Hydrogen; Black: Carbon.

Figure 4

The artificial products (6–11) from benzonquinone (5) and DMSO via Fenton’ reaction and comparison of metabolic profiles of E. faecalis (E) fed with toluquinol in DMSO and MeOH.

Figure 5

Comparison of metabolic profiles of four strains, E. faecalis, E. faecium, S. thermophilus, and L. lactis subsp. Lactis, fed with and without hydroquinone (H, 5) in DMSO.

Figure 6

Effects of toluquinol-derived metabolites 1–3 and toluquinol on human tumor cell lines including non-small-cell lung cancer cell A549, skin melanoma cell A375, ovarian cancer cell A2780, kidney tumor cell G401, colorectal cancer cell HCT116, and hepatocellular carcinoma LM3.

Figure 7

Effects of toluquinol and its derived metabolites 1–2 on cell cycle, apoptosis, and necrosis of non-small lung cancer cell A549. (A,B) Analysis of A549 cell cycle distributions with flow cytometry and PI staining. (C,D) Apoptosis and necrosis analysis of A549 with flow cytometry and YO-PRO-1/PI staining. The apoptotic cells were YO-PRO-1 positive (gate Q3), and the necrotic cells were both YO-PRO-1 and PI positive (gate Q2). (E,F) Apoptosis analysis of A549 with AnnexinV-FITC/PI staining. The early apoptotic cells were AnnexinV-FITC positive (gate Q3), and late apoptotic cells were both AnnexinV-FITC and PI positive (gate Q2). Negative control: NC; T: Toluquinol.

Figure 8

Effects of E. faecalis and E. faecium on transforming versatile toluquinol from Penicillum species.