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Page 8 of 13 Huang et al. Microbiome Res Rep 2024;3:30 https://dx.doi.org/10.20517/mrr.2024.14

Correspondingly, guanine, the hydrolysis product of guanosine, increased by ~9 times in GuMI-FP
compared to GuMI-NB (P < 0.0001, Figure 3C). The lower increase in guanine may hint that F. prausnitzii
consumes it during the growth. On the other hand, uridine was at a higher level in Static culture than
GuMI-NB, indicating that it is derived from epithelial cells. In GuMI-FP, the uridine level decreased to an
undetectable level (zeros in all samples, Figure 3D, Supplementary Tables 1 and 2), while uracil, the
hydrolysis product of uridine, increased by ~4 times compared to that in GuMI-NB. These results suggest
that F. prausnitzii converted medium-derived guanosine and epithelium-derived uridine to their hydrolysis
products, guanine and uracil. In agreement, only one nucleoside hydrolase-encoding gene was found in the
genome of F. prausnitzii A2-165 [Figure 3E]. Other nucleosides and nucleobases were consumed entirely by
F. prausnitzii, evidenced by the deficient levels of adenosine, adenine, and cytidine in GuMI-FP compared
to GuMI-NB [Supplementary Tables 1 and 2]. In agreement, adenine, inosine, xanthosine, and
5’-methylthioadenosine were found to be consumed by F. prausnitzii in a Caco-2-F. prausnitzii coculture
[24]
compared to Caco-2 monoculture .

Beyond nucleoside and nucleobases, F. prausnitzii modified amino acids and amino acid-related
metabolites in the apical compartment. The levels of proline, N-acetylglutamate, itaconic acid,
2-hydroxyglutarate, N-acetylglutamine, and N6-acetyllysine in GuMI-FP were significantly higher than that
in GuMI-NB [Figure 3C]. Recently, it was shown that proline is essential to maintain gut homeostasis by
[25]
activating lymphoid tissue inducer (LTi) cells . Disruption of proline uptake in LTi cells impairs LTi cell
[25]
activation and promotes DSS-induced colitis . In addition, several intracellular intermediates of glucose
and lipid metabolism, such as glucose-5-phosphate, F6P/G1P, and glycerol-3-phosphate [Figure 3C],
increased significantly in GuMI-FP compared to those in other conditions, indicating that bacterial cell lysis
occurred.

The modulation of nucleosides and amino acids was observed previously for F. prausnitzii. In a defined
medium, F. prausnitzii consumed guanine, uracil, xanthine, orotic acid, histidine, leucine, tryptophan,
phenylalanine, and nicotinamide while producing L-glutamine and L-threonine. In the GuMI experiment,
14
histidine, leucine, tryptophan, phenylalanine, and nicotinamide were also detected in GuMI-NB but not
significantly changed by F. prausnitzii in GuMI-FP. This disagreement might be due to different
experimental settings, i.e., a static culture of F. prausnitzii alone in a defined medium vs. a fluidic coculture
of F. prausnitzii with epithelial cells in an undefined YCFA medium. Nevertheless, most of the metabolites
detected in vitro in the GuMI platform were also observed in the human intestine in vivo. For instance,
guanosine, guanine, uracil, adenosine, cytidine, amino acids, and amino acid metabolites [Supplementary
Tables 1 and 2] were detected in human fecal samples [26,27] . Together, these results indicate F. prausnitzii may
play an essential role in modulating chemicals in the human intestinal lumen. Whether these modifications
by F. prausnitzii translate into clinical settings warrants more well-controlled clinical trials .
[28]

Transcriptional changes of transcription factors in colonic epithelium linked to their ligands
metabolized by F. prausnitzii
Transcription factors (TFs) in the host cells are master regulators of host-microbe, host-virus, and host-
pathogen interactions because transcription factors profoundly influence gene transcription in the
downstream pathways. As F. prausnitzii changed the levels of many metabolites beyond butyrate, we
hypothesize that more transcription factors are modulated by F. prausnitzii and the microenvironment in
GuMI (e.g., oxygen gradient and flow). Hence, identifying the F. prausnitzii-transcription factor gene
interaction may offer new insights into the beneficial effects of F. prausnitzii. To test this, we re-analyze the
transcriptomic data in our previous study . Among 1,665 known human transcription factors obtained
[15]
from Human Transcription Factor Database (http://bioinfo.life.hust.edu.cn/HumanTFDB), 1,351 were
detected, and 260 were significantly changed (|log2 fold change| ≥ 1, adjusted P ≤ 0.05) in the epithelial cells