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Page 2 of 13 Huang et al. Microbiome Res Rep 2024;3:30 https://dx.doi.org/10.20517/mrr.2024.14

was performed to profile 169 polar chemicals under three conditions: conventional static culture without bacteria,
GuMI without bacteria, and GuMI with F. prausnitzii. The barrier function of monolayers was measured using
transepithelial resistance.

Results: GuMI successfully cocultured patient-derived monolayers and F. prausnitzii for up to 4 days, with active
bacterial growth. Introducing flow and oxygen gradient significantly increases the barrier function, while exposure
to F. prausnitzii slightly increases the barrier function. Targeted metabolomics screened 169 compounds and
detected 76 metabolites, of which 70 significantly differed between at least two conditions. F. prausnitzii
significantly modulates the levels of nucleosides, nucleobases, and amino acids on the apical side. Further analysis
suggests that F. prausnitzii changes the mRNA level of 260 transcription factor genes in colonic epithelial cells.
Conclusion: The GuMI physiomimetic system can maintain the coculture of F. prausnitzii and colonic epithelium
from different donors. Together with metabolomics, we identified the modulation of F. prausnitzii in extracellular
chemicals and colonic epithelial cell transcription in coculture with human colonic epithelium, which may reflect its
function in gut lumen in vivo.

Keywords: Faecalibacterium prausnitzii A2-165, colonic epithelium, metabolomics, microphysiological system, host-
microbe interaction

INTRODUCTION
Microbial activities influence the host by modulating chemical structures and producing bioactive
compounds. Gut microbes ferment complex molecules such as polysaccharides to short-chain fatty acids.
Additionally, gut microbes convert compounds derived from the host and diet to their metabolites. For
example, primary bile acids, synthesized in the liver and released to the gut, are converted to secondary bile
acids by the gut bacteria and reabsorbed in the colon . Dietary amino acids such as tryptophan can be
[1]
[2,3]
metabolized by gut microbes and circulated in the bloodstream . These metabolites can target G-protein-
coupled receptors and trigger inflammation. Gut microbes can also convert dietary toxicants to metabolites
[4,5]
with lower cytotoxicity and mutagenicity .
Faecalibacterium is a genus of strictly anerobic, extremely oxygen-sensitive (EOS), Gram-positive, rod-
[6]
shaped, nonmotile, and non-spore-forming bacteria . It is highly abundant and prevalent in adult human
gut microbiota, accounting for around 5% of the human gut microbiota , and 85% of the gut samples . The
[8]
[7]
species Faecalibacterium prausnitzii (F. prausnitzii) is strongly and reversely associated with inflammatory
[9]
bowel diseases . Mechanistically, F. prausnitzii was found to have anti-inflammatory effects through several
modes of action [10-12] . Butyrate production is a hallmark of F. prausnitzii. Butyrate’s effects on modulating
host gene expression and inflammation have been extensively studied using cell lines, organoids, animal
models, and clinical trials . More recently, metabolomics has unveiled additional novel metabolic
[11]
signatures, such as 5-aminosalicylic acid, which contribute to the anti-inflammatory effects of
[13]
F. prausnitzii , suggesting that metabolomics could be an effective approach to identifying new
metabolites . F. prausnitzii was found to release amino acids in a fully defined medium . These studies
[14]
[13]
show that F. prausnitzii possesses many previously unknown metabolic functions by producing bioactive
metabolites.

Recently, gut microphysiological systems have been developed to coculture human and strictly anerobic
bacterial species under physiologically relevant oxygen gradient and flow [15-18] . These systems offer a new
tool to study the causal link between microbes and their effects on the host. GuMI has recently been shown
[15]
to culture EOS gut microbe F. prausnitzii with primary human colon epithelium of one donor .