
==== Front
J Biol Chem
J Biol Chem
The Journal of Biological Chemistry
0021-9258
1083-351X
American Society for Biochemistry and Molecular Biology

S0021-9258(24)02115-X
10.1016/j.jbc.2024.107614
107614
Research Article
The arginine and nitric oxide metabolic pathway regulate the gut colonization and expansion of Ruminococcous gnavus
Flores Juan A. 1‡
Antonio Jayson M. 2‡
Suntornsaratoon Panan 2
Meadows Vik 12
Bandyopadhyay Sheila 1
Han Jiangmeng 12
Singh Rajbir 1
Balasubramanian Iyshwarya 1
Upadhyay Ravij 2
Liu Yue 1
Bonder Edward M. 1
Kiela Pawel 3
Su Xiaoyang 4
Ferraris Ronaldo ferraris@njms.rutgers.edu
2∗
Gao Nan ngao@newark.rutgers.edu
12∗
1 Department of Biological Sciences, Rutgers University, Newark, New Jersey, USA
2 Department of Pharmacology, Physiology and Neurosciences, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA
3 Daniel Cracchiolo Institute for Pediatric Autoimmune Disease Research, Steele Children’s Research Center, Department of Pediatrics, University of Arizona, Tucson, Arizona, USA
4 Department of Medicine, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, New Jersey, USA
∗ For correspondence: Ronaldo Ferraris; Nan Gao ferraris@njms.rutgers.edungao@newark.rutgers.edu
‡ These authors contributed equally to this work.

31 7 2024
9 2024
31 7 2024
300 9 10761418 1 2024
8 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Ruminococcus gnavus is a mucolytic commensal bacterium whose increased gut colonization has been associated with chronic inflammatory and metabolic diseases in humans. Whether R. gnavus metabolites can modulate host intestinal physiology remains largely understudied. We performed untargeted metabolomic and bulk RNA-seq analyses using R. gnavus monocolonization in germ-free mice. Based on transcriptome-metabolome correlations, we tested the impact of specific arginine metabolites on intestinal epithelial production of nitric oxide (NO) and examined the effect of NO on the growth of various strains of R. gnavus in vitro and in nitric oxide synthase 2 (Nos2)-deficient mice. R. gnavus produces specific arginine, tryptophan, and tyrosine metabolites, some of which are regulated by the environmental richness of sialic acid and mucin. R. gnavus colonization promotes expression of amino acid transporters and enzymes involved in metabolic flux of arginine and associated metabolites into NO. R. gnavus induced elevated levels of NOS2, while Nos2 ablation resulted in R. gnavus expansion in vivo. The growth of various R. gnavus strains can be inhibited by NO. Specific R. gnavus metabolites modulate intestinal epithelial cell NOS2 abundance and reduce epithelial barrier function at higher concentrations. Intestinal colonization and interaction with R. gnavus are partially regulated by an arginine–NO metabolic pathway, whereby a balanced control by the gut epithelium may restrain R. gnavus growth in healthy individuals. Disruption in this arginine metabolic regulation will contribute to the expansion and blooming of R. gnavus.

Keywords

metabolite
R. gnavus
intestine
nitric oxide
arginine
Abbreviations

ATCC American Type Culture Collection

BBE brush border expressing

DMEM Dulbecco's modified Eagle's medium

DNNoate Deta-NONOate

FBS fetal bovine serum

GF germ-free

IBD inflammatory bowel disease

LGG Lacticaseibacillus rhamnosus GG

MB Meat Glucose Broth

Neu5AC N-acetylneuraminic acid

NO nitric oxide

NOS2 nitric oxide synthase 2

qPCR quatitative polymerase chain reaction

SA sialic acid

TEER transepithelial electrical resistance

Reviewed by members of the JBC Editorial Board. Edited by Ursula Jakob
==== Body
pmcAs the human microbiome continues to be characterized by large-scale analyses, new concepts emerge from the complex relationships shared by commensal microbes and their host organisms. One important concept highlights the behaviors of some opportunistic microbes, known as pathobionts, which drive pathogenesis under certain environmental and genetic conditions. Different from pathogens that are typically absent in healthy humans, these pathobionts are normally present in the healthy population albeit at nonpathogenic levels. Their pathogenic behavior is typically associated with a noticeable expansion of colonization in the host. Ruminococcus gnavus is such a gut commensal bacterial species that has been associated with a variety of human inflammatory and metabolic diseases.

First isolated in 1976 from human feces, R. gnavus is a gram-positive obligate anaerobic bacterium of the Clostridia class (1). Fecal microbiome studies of newborn infants determined that R. gnavus is one of the first commensal bacteria that colonize the gastrointestinal tract (2). Nearly 90% of human microbiomes harbor R. gnavus, however, this species typically contributes to only about 0.01% of the total microbiota (3). Longitudinal studies of patients diagnosed with inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, reported a significant expansion of R. gnavus in posttreatment relapses of IBD patients (4, 5, 6). Moreover, fecal R. gnavus expansion in patients has been associated with nonalcoholic fatty liver disease, nonalcoholic steatohepatitis (7), liver cirrhosis (8), advanced coronary artery disease (9), as well as metabolic disorders, such as obesity (10) and type 2 diabetes (11). Despite an increasing number of reports suggesting that R. gnavus may serve as a biomarker for disease states, mechanistic details on how R. gnavus may expand during pathogenesis and how it may contribute to or regulate the disease progression remain limited.

Commensal microorganisms in the intestinal tract are separated from epithelial cells by a mucin layer barrier, which is compartmentalized into a superficial loose layer where the majority of the bacteria reside and a thicker mucosal layer devoid of microorganisms (12, 13). Most commensal bacteria utilize oligosaccharide side chains on mucin proteins for carbon and energy sources, while some species can proteolytically cleave core components of mucin proteins (14, 15). One of the terminating oligosaccharides on the mucin layer is N-acetylneuraminic acid (Neu5Ac), commonly referred to as sialic acid (SA). Genome studies of R. gnavus identified a SA-metabolizing operon (16). This operon contained genes shared by the nan operon that is utilized in the foraging of SAs on the mucosal surface by other bacteria (17, 18, 19). Interestingly, a unique mechanism for R. gnavus to effectively exploit SAs appears to be mediated by a SAT2 SA transporter and an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac. Both proteins have a higher affinity for the oxidized forms of SA (16), typically found on mucosal surfaces as an attempt by host cells to evade bacterial interaction. The ability to utilize oxidized SA suggests a unique colonization advantage by R. gnavus.

R. gnavus resides in the luminal mucin layer and does not typically attach or invade the epithelial cells. One aspect of R. gnavus that has not been fully addressed is its ability to metabolize intestinal luminal macromolecules, the metabolic products of which may affect the host gut epithelial physiology. To the best of our knowledge, R. gnavus was shown to produce the secondary bile acids ursodeoxycholic acid (20) iso-ursodeoxycholic acid (21), and tryptamine, a tryptophan metabolite (22). Because R. gnavus associates with the pathogenesis of inflammatory and metabolic disorders such as IBD, nonalcoholic fatty liver disease, and coronary artery disease, the metabolic aspects of host–R. gnavus interaction may shed light on the mechanism of intestinal and commensal microbial symbiosis as well as a potential pathogenicity by this species.

We previously reported that the growth and colonization of R. gnavus are sensitive to intestinal lysozyme in the lumen. Mice lacking Paneth cell lysozyme (Lyz1) have an expansion of R. gnavus in ileum and an associated enhanced goblet cell program (23). Here, we have performed a comprehensive metabolomic and transcriptomic analysis using R. gnavus (American Type Culture Collection [ATCC] 29149) monocolonized germ-free (GF) mice. We also examined the metabolome of multiple R. gnavus strains anaerobically cultured in vitro. We found that R. gnavus produces specific arginine, tryptophan, and tyrosine metabolites, some of which are dependent upon mucin and SA richness. R. gnavus transcriptionally promotes expression of intestinal amino acid transporters and enzymes involving in metabolic flux of arginine to nitric oxide (NO). R. gnavus elevated intestinal nitric oxide synthase 2 (NOS2), and several prominent R. gnavus metabolites directly regulate NOS2 protein abundances. Nos2 KO mice have a significant R. gnavus expansion, which can be reverted by chemical NO donors. Taken together, we propose that the colonization of R. gnavus is in part maintained by an arginine–NO metabolic pathway mutually modulated by host epithelial cells and R. gnavus-derived metabolites. Disruption of this delicate balance may lead to R. gnavus blooming and potential pathogenesis.

Results

R. gnavus colonization increases goblet cell, mucin production, and is associated with elevated Nos2 transcripts

To determine the specific impacts of R. gnavus on host physiology, we monocolonized GF mice with R. gnavus ATCC 29149 strain for 3 weeks. GF mice were gavaged with sterile PBS as negative controls, while GF mice monocolonized with Lacticaseibacillus rhamnosus GG (LGG) were used as a comparison (Fig. 1A). Fecal samples were collected from individual mice before the association (day 0) and subsequently collected on day 3 and day 21 post inoculation for untargeted metabolomic analysis by LC-MS. Colonization was validated by quatitative polymerase chain reaction (qPCR) analysis using specific primers for R. gnavus and LGG. All mice were sacrificed at the same time followed by ileum and colon tissue dissection for bulk RNA-seq and histology analyses.Figure 1 Ruminococcus gnavus colonization increases goblet cell number, mucin production, and distinct transcriptome profiles in the ileum.A, schematic for basic experimental design using germ-free mice. C57BL/6 GF mice were inoculated with PBS (blue), R. gnavus (red, ATCC-29149), or LGG (green). Colonization was validated by quatitative polymerase chain reaction on fecal DNA by specific primers. Mice were sacrificed 21 days after inoculation. n = 5 mice for each group. B, PBS, R. gnavus, or LGG mouse ilea were stained with Alcian blue to identify goblet cells in crypts and villi. The scale bar represents 50 μm. C, number of Alcian blue positive cells along crypt–villus axis was quantified for each mouse of different groups. N = 5 mice per group. Data are expressed as mean ± SD. Statistical significance was determined by one-way ANOVA with post hoc testing (∗∗p < 0.01, ∗p < 0.05, and ns = not significant). D, number of Muc2+ goblet cells per crypt was quantified for each mouse of different groups. N = 5 mice per group. Statistical significance was determined by one-way ANOVA with post hoc testing (∗p < 0.05, ns = not significant). E, immunofluorescence analysis and ImageJ quantification was used to determine extracellular Muc2 proteins levels on the luminal surface of cells within two-cell distances of individual goblet cells. Data with n = 25 goblet cells per condition from 5 mice. Statistical significance was determined by one-way ANOVA with post hoc testing (∗∗∗∗p < 0.0001 and ∗p < 0.05). F, immunofluorescence staining for Muc2 (green), E-cadherin (white), and 4′,6-diamidino-2-phenylindole (blue). The white arrowheads point to Muc2+ mucin at the luminal surfaces of R. gnavus mouse ileum. G, principal component analysis (PCA) of ileal transcriptome profiles from PBS, R. gnavus, and LGG-treated mice. Each point represents an individual mouse (n = 5 per group). H, heatmap of individually differentiated genes between R. gnavus and PBS treatments, with a false discovery rate (FDR) of 0.05. The red arrow indicates the Nos2 gene. Transcript per million (TPM) expression levels are normalized and color-coded (blue to red) to reflect relative levels of expression. I, molecular function and pathway analysis of upregulated genes in R. gnavus versus PBS-treated mice. Enrichment scores (–log10 p-value) are shown for significantly upregulated pathways and protein classes. J, molecular function and pathway analysis of downregulated genes in R. gnavus versus PBS-treated mice. Enrichment scores (–log10 p-value) are shown for significantly reduced pathways and protein classes. ATCC, American Type Culture Collection; LGG, Lacticaseibacillus rhamnosus GG.

R. gnavus is a mucus-foraging bacterium (16). Interestingly, R. gnavus colonization increased the number of mucin-producing goblet cells in ileum when compared to PBS or LGG-colonized mice (Fig. 1, B and C). Examination of MUC2-producing goblet cells showed that R. gnavus-colonized mice had more MUC2-expressing cells in the crypts, and there was an elevated amount of extracellular MUC2 protein covering the luminal surface of the ileum (Fig. 1, D–F). These results indicate that R. gnavus monocolonization enhances the goblet cell and mucin production program in the host intestines.

Principal component analysis of the ileal transcriptome showed a separated clustering between R. gnavus colonized mice and PBS or LGG-gavaged mice (Fig. 1G). The hierarchical clustering in the heatmap demonstrates differential gene expression including key increased genes (Als2, Nr1d1, Tef, Dbp, Nos2, Slc35f2) and decreased genes (Fam174a, Cpn1, Cldn22) (Fig. 1H). Molecular function and pathway enrichment analysis for increased (Fig. 1I) or reduced (Fig. 1J) transcriptomes revealed R. gnavus-induced cellular responses targeting cell adhesion, antigen presentation, extracellular matrix, amino acid binding, cytokine, and receptor interaction, etc. Notably, one of the most increased transcripts is Nos2, a gene encoding for NO synthase that converts arginine to citrulline while releasing NO.

R. gnavus colonization generates a specific metabolome in vivo

To identify intestinal luminal metabolites that were changed in response to R. gnavus colonization, we performed untargeted metabolomic analysis using LC-MS on longitudinally collected fecal samples (Fig. 2A). Principal component analysis of fecal metabolome at 21-day post inoculation showed distinct clustering among PBS, R. gnavus, and LGG mice (Fig. 2B). Approximately 70 polar fecal metabolites were significantly different among GF, R. gnavus, and LGG groups. Within the top 25 metabolite grouping, fecal glutamate, uracil, citrulline, ornithine, and glucose are uniquely elevated in R. gnavus monocolonized mice (Fig. 2C). Compared to PBS mice, significantly changed R. gnavus fecal metabolites of positive (Fig. 2D) and negative mode (Fig. 2E) were identified. Notably, R. gnavus promoted arginine metabolites (i.e., ornithine and citrulline); tryptophan metabolites (i.e., 5-tryptamine and hydroxytryptophan), as well as tyrosine metabolites (i.e., tyramine and homovanillic acid). We performed an independent time-course study of fecal metabolome from GF mice on day 5 and day 13 post-R. gnavus colonization and validated increases in citrulline, glucose, and glutamate (Fig. 2F). In agreement with these findings, metabolite set enrichment analysis pointed out an elevated arginine and sugar metabolism in R. gnavus mouse fecal metabolome (Fig. 2G). Closer inspection of these specific arginine, tryptophan, and tyrosine metabolites revealed that R. gnavus immediately promoted ornithine, citrulline, tryptamine, and tyramine 3 days after colonization (p < 0.05, Fig. 2H). While the abundance of tryptamine and tyramine slightly declined at 21 days, ornithine, citrulline, and homovanillic acid continued to rise at 21 days (p < 0.01, Fig. 2H). Glutamate, an arginine precursor, and argininosuccin ate, which can be converted to arginine and fumarate by argininosuccinate lyase, were also elevated at 21 days. The reduction of fecal arginine and tyrosine suggest that R. gnavus actively metabolizes these amino acids. In addition, the elevated citrulline, ornithine, tryptamine, and tyramine were not seen in LGG-colonized mice, suggesting that R. gnavus specifically contributed to the production of these metabolites (Fig. 2H). Furthermore, there was a continuous decline of 3-phenylbutrylic acid following R. gnavus colonization (Fig. 2H). These results suggest that R. gnavus differentially promotes certain amino acid metabolism.Figure 2 Ruminococcus gnavus colonization promotes specific intestinal luminal metabolites.A, fecal samples were sampled at specific time points for untargeted metabolomic analysis from GF mice inoculated with PBS, R. gnavus or LGG. n = 5 mice for each group. B, PCA analysis of fecal metabolomics of PBS, R. gnavus, and LGG mice at 21 days after inoculation. C, heatmap of top 25 differentially changed metabolites among the 3 groups. Note that R. gnavus increased glutamate, glucose, citrulline, and ornithine. D and E, comparing to PBS mice, fecal metabolites, of positive (panel D) or negative mode (panel E), which are significantly increased (red) or decreased (blue) in R. gnavus mice. Note that ornithine, citrulline, tryptamine, and tyramine are increased in R. gnavus mouse fecal samples. F, heatmap of changes in fecal metabolites of R. gnavus inoculated mice prior to inoculation (pre-RG), 5 days and 13 days after R. gnavus inoculation. n = 5 mice for each group. G, metabolic pathway analysis of fecal metabolome from PBS and R. gnavus mice. Note that arginine and proline metabolism is significantly changed in R. gnavus mice. H, time course analysis of arginine, tryptamine, and tyrosine metabolites at 3 days and 21 days after inoculation in PBS, R. gnavus, and LGG mice. Note that ornithine, citrulline, tryptamine, and tyramine are increased in R. gnavus but not LGG mice. Homovanillic acid are increased in both R. gnavus and LGG mice, while 3-phenylbutryic acid is reduced in R. gnavus colonized mice. LGG, Lacticaseibacillus rhamnosus GG; PCA, principal component analysis.

R. gnavus modulates the gut arginine metabolic enzymes and products

An integrated examination of our transcriptomic and metabolomic data suggests a regulation of arginine metabolic pathway by R. gnavus (Figs. 1, H and I and 3, C–H). Interestingly, R. gnavus colonization elevated numerous plasma membrane-localized solute carrier protein genes (Fig. 3A), including amino acid and peptide transporters for arginine and its derivative (shown in red in Fig. 3A). Some of these SLC gene promotions is also observed in LGG colonized mice (Supplementary Table), indicating a shared feature by these commensal microbes.Figure 3 Ruminococcus gnavus promotes intestinal Nos2 that potentially induces arginine metabolic flux to nitric oxide.A, heatmap of solute transporter expression levels in the ileum of R. gnavus-colonized mice compared to PBS controls. The heatmap highlights significant changes in the expression of transporter proteins involved in the transport of sugars, amino acids and peptides, nucleotides, carboxylates, and organic ions. Arginine transporters are highlighted in red. B, analysis of arginine-urea and NO cycle. Intracellular L-arginine is converted to ornithine or citrulline by arginase (ARG) or nitric oxide synthase (NOS), respectively. The biproducts for these two enzymatic reactions are urea and NO. The enzymes OTC and ASS can promote conversion of ornithine and citrulline to arginosuccinate, which can be converted to arginine by ASL. Bar graphs show RNA abundances of Nos2, Arg1, Arg2, Otc, Asl, and Ass1 in the ileum of GF (blue) and R. gnavus -colonized (red) mice. Data are expressed as mean ± SD (n = 5 mice per group). Statistical significance was determined by one-way ANOVA with post hoc testing (∗∗p < 0.01 and ∗p < 0.05). C, immunohistochemistry for Nos2 in PBS and R. gnavus mouse ileum. Low magnifications have scale bars with 50 μm. Boxed crypt regions are shown at high magnifications. The scale bars represents 30 μm. D, quantification of Nos2 protein abundance in lamina propria, villus IEC, and crypt IEC. Data points represent the average absorbance (A) per mouse, with n = 10 measurements per mouse. E–H, correlation between Nos2 expression (RPKM) and the intensities of fecal ornithine, arginosuccinate, citrulline, and arginine in GF (blue), R. gnavus (red), and LGG (green) groups. Significant inverse correlations between Nos2 expression and fecal metabolites are highlighted for the RG group. I, polarized Caco2 brush border expressing cell monolayers were treated in vitro with PBS, arginine, citrulline, and ornithine. Intracellular NO was detected by incubating cells with DAF-FM diacetate solution for 1 h at 37 °C, and fluorescent measurement at 495 to 515 nm. ATCC, American Type Culture Collection; GF, germ-free; IEC, intestinal epithelial cells; LGG, Lacticaseibacillus rhamnosus GG; RPKM, Reads Per Kilobase of transcript, per Million mapped reads.

Among the fecal metabolites increased by R. gnavus, ornithine, and citrulline belong to the L-arginine–NO pathway, where NO is synthesized from L-arginine by the NO synthases (Fig. 3B). A closer examination of enzymes of this pathway revealed that R. gnavus increased both NOS2 and ornithine transcarbamylase, which converts ornithine to citrulline (Fig. 3B), suggesting that R. gnavus stimulates NO production by promoting the transport and flux of luminal arginine and derivatives into the NO-generating NOS2 pathway in host epithelia. The role of NO in the intestinal milieu has been a focal point of debate, with conflicting reports on its impact on intestinal physiology. The source and cellular context of NO generation are critical factors in gut homeostasis. Notably, NO derived from enterocytes has been associated with protective properties in the gut, particularly in mitigating colitis. Conversely, immune cell–derived NO has been implicated in instigating inflammatory responses (24). In this context, we examined Nos2 protein distribution within the intestines of R. gnavus-colonized mice. Immunohistochemistry revealed that R. gnavus increased Nos2 protein abundance in both the intestinal crypt epithelial cells and lamina propria cells by 37.9% and 18.83%, respectively (Fig. 3, C and D).

Furthermore, metabolome-transcriptome correlation analysis revealed a significant inverse correlation of Nos2 with fecal ornithine (Fig. 3E) and argininosuccinate (Fig. 3F), and to a lesser extent with fecal citrulline and arginine (Fig. 3, G and H). Interestingly, such correlation was found only in R. gnavus-colonized mice but not in PBS or LGG mice (Fig. 3, E–H). These findings suggest that elevated Nos2 and ornithine transcarbamylase in R. gnavus-colonized mice may stimulate the utilization of arginine metabolites for enhanced NO production. To test this, we examined the direct effects of individual arginine metabolites on NO production by intestinal epithelial cells in vitro. We added arginine, citrulline, or ornithine to polarized Caco2 brush border expressing (BBE) cell monolayers and observed a significantly increased levels of NO, compared to vehicle-treated cells (Fig. 3I). These results suggest that the arginine metabolic pathway may be modulated by both colonizing R. gnavus and host intestinal cells via a NOS2-dependent feedback mechanism (25).

R. gnavus generates specific metabolites

We next examined to what extent R. gnavus may produce any of above metabolites independent of the host. R. gnavus ATCC-29149 can metabolize SA enriched in intestinal mucin, therefore we cultured it in vitro under anaerobic condition for 3 h in its culture medium (a meat broth) with or without SA supplementation. We performed untargeted metabolomic analysis on the spent media (RG and RG + SA) and control blank medium (CT and CT + SA) (n = 3 per group, Fig. 4A). Principal component analysis analysis of a total of 155 metabolites showed distinct clustering of individual groups examined (Fig. 4B). Heatmap of the 25 most significantly changed metabolites include 12 that were elevated in R. gnavus (green) and RG+SA (purple) conditions (Fig. 4C). Arginine derivatives, ornithine, citrulline, and fumarate were elevated in both RG and RG+SA conditions (Fig. 4, C–G). Notably, the presence of SA led to a reduction in citrulline (Fig. 4F) and fumarate (Fig. 4G), indicating that SA can modulate R. gnavus metabolism of certain compounds. We also observed an active conversion of tyrosine to tyramine by R. gnavus in vitro (Fig. 4H), and the enrichment of phosphoenolpyruvate, lactate, and 2-hydroxy-4-(methylthio)butyrate in the spent media of R. gnavus (Figs. 4D and S1A).Figure 4 Ruminococcus gnavus colonization promotes specific intestinal luminal metabolites.A, R. gnavus was grown for 3 h in anaerobic condition, with or without sialic acid (SA) supplementation, and the spent media were collected for untargeted metabolomic analysis. Control media with or without SA were used as controls. B, PCA plot of metabolome of CT, CT+SA, RG, and RG+SA. n = 3 independent replicates for each condition. C, heatmap of changed metabolites among the four conditions. D, metabolites that were significantly changed by R. gnavus compared to CT blank media. E, metabolites that were significantly changed by SA supplementation comparing to R. gnavus culture without SA. Note that N-acetylneuraminc acid is SA. F–H, quantification of selected metabolites (citrulline, arginine, ornithine, fumarate, tyramine, and tyrosine) in CT, CT+SA, RG, and RG+SA conditions. Data are expressed as mean ± SD (n = 3 per group). Statistical significance was determined by one-way ANOVA with post hoc testing (∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, and ns = not significant). I–K, variable importance in projection (VIP) scores from PLS-DA analyses identifying the most discriminative metabolites in the spent media for four RG strains (AGR-2154, ATCC 35913, H2-28, ATCC 29149). VIP for metabolites in (I) plain culture medium, that is, the meat broth (+ and – mode), (J) with 1% mucin (+ and – mode), and (K) with SA (+ and – mode). Higher VIP scores indicate greater importance for group separation. ATCC, American Type Culture Collection; PCA, principal component analysis; PLS-DA, partial least squares discriminant analysis.

Furthermore, SA supplementation was found to suppress the production of specific metabolites, such as sedoheptulose, 2-deoxyuridine, citrulline, 3-phosphoglycerate, inosine monophosphate, and fumarate (Fig. 4C), suggesting homeostatic activity for SA on the metabolic pathways of R. gnavus. Moreover, we observed a depletion of sugars and nucleotide derivatives in R. gnavus spent media, which was partially restored by SA supplementation (Fig. S1A). Also, examination of the metabolites after overnight incubation showed significantly elevated levels of glutamine, tryptamine, fumarate, nicotinate, and purine in the spent medium of R. gnavus cultures (Fig. S1B). The observed dramatic increase of tryptamine (by 99.5-fold) was consistent with elevated fecal tryptamine in R. gnavus monocolonized mice (Fig. 2H).

We next expanded our study to additional R. gnavus strains due to the potentially shared or distinct metabolic adaptations. We performed similar metabolomic analysis on AGR-2154, which was isolated from calf feces, and ATCC-35913 and DSM 108212 (H2-28), two R. gnavus strains isolated from human feces. For this experiment, we cultured these strains in culture medium (meat broth, Fig. 4I), or medium supplemented with 1% porcine mucin (Fig. 4J), or SA (Fig. 4K). The choice of mucin was due to R. gnavus’ utilization of mucin sugars. When grown in meat broth, ATCC-29149 produced higher abundances of tyramine, glutamine, and glutamate, whereas ATCC-35913 produced the highest levels of tryptamine and citrulline (Fig. 4I). Mucin supplementation enhanced the ability of ATCC-29149 to produce tryptamine, tyramine, and arginine metabolites such as N-acetylornithine, NG-dimethyl-L-arginine, homoarginine, ornithine, and arginine (Fig. 4J). Mucin also significantly promoted H2-28 production of arginine-derived metabolites, making it the second highest producer. SA supplementation induced the conversion of tyrosine to tyramine in ATCC-29149 and H2-28 when compared to the other strains. These findings underscore the strain-dependent difference in in vitro metabolic profiles, while highlighting an influence of R. gnavus metabolism by luminal factors, such as the mucin and SA, with both the free and bound forms of SA appearing to have an impact. Taken together, metabolomic analysis of host-free R. gnavus showed a specific production of arginine, tryptophan, and tyrosine derivatives that can be modulated by the environmental richness of SA.

NO suppresses R. gnavus growth

NO is known to mediate the host's defense against pathogens. The observed increase in intestinal Nos2 expression induced by R. gnavus and the promotion of epithelial NO production by arginine metabolites prompted us to investigate the existence of a potential reciprocal regulation between the host and R. gnavus. To examine if NO affects the growth or colonization of R. gnavus, individual strains were exposed to different concentrations (0 mM, 0.05 mM, 0.5 mM, 5 mM) of the NO donor Deta-NONOate (DNNoate). We observed inhibited growth with increasing NO donor concentration, most notably at 0.5 and 5 mM for all R. gnavus strains (Fig. 5, A–D). ATCC-29149 exhibited the highest sensitivity to NO (Fig. 5A), with notable growth inhibition at the lowest NO donor concentration of 0.05 mM. ATCC-35913 (Fig. 5B) and AGR-2254 (Fig. 5C) displayed the highest resistance to NO exposure. These differences in NO sensitivity suggest that R. gnavus strains may possess varying capacities to tolerate nitrosative stress. None of the R. gnavus strains could grow in the presence of 0.5 mM DNNoate, while the growth of LGG (Fig. S1C) or Escherichia coli (26) were not affected.Figure 5 NO suppresses Ruminococcus gnavus growth, which is expanded in Nos2−/− mice.A–D, growth curves of four R. gnavus strains (ATCC 29149, ATCC 35913, AGR 2154, H2-28) in the presence of varying concentrations of the NO donor, DNNate (Veh, 0.05 mM, 0.5 mM, 5 mM) over 24 h. Insets show cultures turbidity at the end of the experiment with the indicated NO donor concentrations. E, relative abundance of R. gnavus in fecal samples from WT and Nos2 KO mice, showing a significant increase in R. gnavus abundance in Nos2 KO mice, ∗∗p < 0.01. n = 5 to 6 per genotype. F, fecal R. gnavus abundance was determined by quatitative polymerase chain reaction. Note that feeding NO donor to Nos2−/− mice reduced R. gnavus abundance (day 0–4), which recovered during a washout phase of NO donor (day 4–15). G, Alcian blue staining of goblet cells for WT and Nos2−/− mouse ileum. Quantification of Alcian blue positive cells per individual crypt-villus units of WT and Nos2−/− mouse ileum. The scale bars represent 100 μm. H, Mptx2 staining for WT and Nos2−/− mouse ileum. Quantification of Mptx2 abundance per crypt unit of WT and Nos2−/− mouse ileum. The scale bars represents 100 μm. I, Dclk1 staining for WT and Nos2−/− mouse ileum. Quantification of Dclk1 positive cells along individual crypt-villus units of WT and Nos2−/− mouse ileum. The scale bars represents 100 μm. J, lysozyme staining for WT and Nos2−/− mouse ileum. Quantification of lysozyme abundance per crypt unit of WT and Nos2−/− mouse ileum. The scale bars represents 100 μm. Data are expressed as mean ± SD (n = 3 per group). Statistical significance was determined by one-way ANOVA with post hoc testing (∗∗∗∗p < 0.0001). ATCC, American Type Culture Collection; DNNoate, Deta-NONOate; NO, nitric oxide.

We then examined R. gnavus abundances in Nos2 KO mice, which lack of systemic NO production. Fecal microbial DNAs were extracted and quantified for R. gnavus using specific qPCR primer set. There was a significantly increased R. gnavus abundance in Nos2−/− mice compared to WT mice (all C57BL/6) (Fig. 5E). To explore the impact of exogenous addition of NO on R. gnavus colonization, Nos2−/− mice were subjected to a 4-day oral administration of DNNate followed by an 11-day withdrawal phase. Fecal microbiota was longitudinally analyzed for R. gnavus by qPCR. Remarkably, feeding mice with the NO donor rapidly decreased R. gnavus abundance, whereas the subsequent withdrawal of the NO donor led to recovery in R. gnavus population size (Fig. 5F). These data suggest that R. gnavus is sensitive to luminal concentration of NO, and the levels of luminal NO could determine the colonization efficacy of R. gnavus.

Because R. gnavus colonization increased ileal goblet cells (Fig. 1B), we next examined if the increased R. gnavus in Nos2−/− mice may also correlate with a goblet cell differentiation. Alcian blue staining demonstrated that goblet cells (Fig. 5G) were not significantly changed in Nos2−/− mouse ileum, suggesting that the observed R. gnavus effects on goblet cells may require Nos2 signaling. Likewise, insignificant changes were seen for Mptx2 or Dclk1, products of Paneth or Tuft cells (Fig. 5, H and I). However, lysozyme abundance is significantly increased in Nos2−/− mouse crypts (Fig. 5J), suggesting that lack of Nos2 signaling may somehow induce lysozyme production by Paneth cells.

R. gnavus metabolites modulate intestinal epithelial cell Nos2 expression

To test if R. gnavus metabolites directly regulate intestinal epithelial cell Nos2 expression, we examined NOS2 protein abundances in polarized Caco2 BBE cells treated by individual metabolites. Arginine, the direct substrate of NOS2, significantly boosted NOS2 protein levels, with a peak observed at 12 h (Fig. 6A). Citrulline, which can be converted back to arginine via the urea cycle, also increased NOS2 levels with a delay at 3 h but sustained up to 24 h (Fig. 6B), suggesting that citrulline supplementation may provide a continuous supply of arginine thereby maintaining elevated Nos2 expression. Additionally, fumarate, a product linked to the arginine biosynthesis pathway, rapidly upregulated Nos2, with an effect that lasted for 12 h (Fig. 6C). Ornithine did not rapid change NOS2 abundance and led to a decline at later time points (Fig. 6D). These results indicate that arginine availability may boost intestinal epithelial cell Nos2 abundances.Figure 6 Effect of Ruminococcus gnavus metabolites on NOS2 protein expression in Caco-2 cells.A–F, Western blot analysis of Nos2 protein expression in response to (A) arginine (1 mM), (B) citrulline (1 mM), (C) fumarate (10 μM), (D) ornithine (1 mM), (E) tyramine (30 nM), and (F) tryptamine (30 μM) treatments over a time course (0, 1, 3, 6, 12, 24 h). Actin is used as a loading control. Quantification of Nos2 expression relative to actin is presented in the corresponding line graphs below each blot. Data are expressed as mean ± SD (n = 3 independent experiments). Statistical significance was determined by one-way ANOVA with post hoc testing (∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05). G, TEER analysis using Caco2 brush border expressing cell monolayer. Cells were grown into confluency for 3 weeks and were apically treated with individual metabolites of indicated concentrations. TEER readings were collected before treatment and monitored daily for 8 consecutive days. The 3 chosen concentrations represent 0.1, 1, and 10-fold of physiological concentrations based on literature report. All experimental procedures and conditions conducted on the same multiwell culture plate, thus the PBS wells serve as controls for all experiments. Data represent mean ± SEM. H, a schematic model shows that at steady state, basal level of NO production via host cell Nos2 restrains the growth of R. gnavus. When R. gnavus is expanded in lumen, luminal accumulation of arginine metabolites, ornithine and citrulline may be transported into cells to boost Nos2-dependent NO production, further suppressing R. gnavus growth. During pathologic conditions such as impaired Nos2 machinery, elevated levels of R. gnavus produces produce increased levels of Nos2-suppressing metabolites that, in turn, lead to loss of cell–cell barrier function and increased susceptibility to inflammation. NO, nitric oxide; NOS2, nitric oxide synthase 2; TEER, transepithelial electrical resistance.

As R. gnavus produces a noticeable amount of biogenic amines, such as tyramine and tryptamine, however the link to modulation of intestinal epithelial cell Nos2 by biogenic amines is not fully understood. We thus performed similar experiments on Caco2 BBE cells and found that tyramine caused a gradual decrease in NOS2 abundance most evidently at 12 h (Fig. 6E). In contrast, tryptamine induced a transient increase in NOS2 at 3 h (Fig. 6F). These findings highlight the complex inter-regulation between the biogenic amines and the arginine-NO–metabolizing enzyme NOS2. In addition, we analyzed the impact of these metabolites on intestinal epithelial barrier function by transepithelial electrical resistance (TEER) analysis on Caco2 BBE monolayer. Based on literatures of physiological concentrations of the tested metabolites, apical loading of low, medium, and high concentrations of fumarate, tryptamine, and tyramine led to a different extent of barrier impairment, with higher sensitivities observed for fumarate and tyramine (Fig. 6G).

Discussion

Despite numerous reports of R. gnavus association with inflammatory or metabolic disorders (6, 7, 8, 9), knowledge of how R. gnavus may metabolically regulate host physiology remains limited. Our experimental approach used gnotobiotic mice as a model coupled to transcriptome and metabolome analysis to uncover molecular regulatory pathways in the R. gnavus–host colonization axis. Specifically, we revealed in vivo and in vitro that R. gnavus generates a considerable amount of ornithine, citrulline, and fumarate, metabolites of the arginine-urea and NO cycle. These metabolites can be made from amino acids or catecholamines in bacteria as reported in prior studies (27, 28, 29). At the transcriptomic level, R. gnavus promoted the expression of transporters for amino acids and sugars, indicating an elevated epithelial uptake of these nutrients. Interestingly, Nos2 is one of the most responsive genes in R. gnavus-colonized mouse ileum. As Nos2 expression is regulated by TLR4–MyD88 microbial sensing pathway (30), the elevated Nos2 is certainly not unique to R. gnavus. However, our in vitro and in vivo experiments suggest that R. gnavus appears especially sensitive to NO that is generated by NOS2 from arginine. Host gut epithelial cells may utilize this mechanism to constrain R. gnavus growth, while abnormal expansion of R. gnavus may accumulate NOS2-regulating and barrier-impairing metabolites, such as tryptamine and tyramine. The combined findings from our multiomic and functional studies point to a reciprocal crosstalk between R. gnavus and the host (Fig. 6H).

One of the most notable pathways affected by gut microbiota is arginine-proline metabolism (31, 32, 33, 34), which is linked to various physiological processes such as tissue regeneration (35, 36) and immune regulation (37). NOS2 converts arginine to citrulline and concurrently produces NO. NO is a critical gaseous signaling molecule that plays a pivotal role in maintaining the integrity and function of the gastrointestinal mucosal lining (38). For example, NO has been shown to act as a potent vasodilator and to promote angiogenesis, vascular perfusion, and nutrient delivery, thereby supporting tissue regenerative capacities (39). Additionally, NO activates the production and secretion of mucin in gastric and respiratory mucosa via cGMP, protein kinase G, PKC, and mitogen-activated protein kinase pathways (40, 41, 42). Based on our observed Nos2 elevation, increased goblet cells and surface mucin in R. gnavus-colonized mice, we speculate that the arginine–NOS2–NO axis may play a role in these mice. Despite the R. gnavus expansion in Nos2 KO mice, we did not observe increased goblet cells, which may echo the finding that NOS2 regulates mucin production (43). Thus, R. gnavus appears to promote goblet cell mediated mucin production in a NOS2-dependent fashion.

In our study, excessive NO appears to suppress the in vitro growth of multiple R. gnavus strains. This NO-sensitive feature is interesting as we found that Lactobacillus and Salmonella strains exhibited resistance to the same concentrations of NO used in our experiments. E. coli and Enterococcus faecalis that are associated with intestinal dysbiosis have also been reported to be NO-resistant (44, 45). Among the R. gnavus strains tested, sensitivity to NO varies, indicating potentially different mechanisms regulating NOS2 or NO tolerance. For instance, ornithine was shown to inhibit the host CAT-1, an arginine transporter that interacts with NOS2, thereby reducing arginine uptake (46) while high levels of ornithine and citrulline may act as competitive inhibitors of NOS2 (47, 48, 49). Along this line, we found that several metabolites, such as homoarginine, NG-dimethyl-L-arginine (49, 50), and aminoguanidine (50, 51), which exhibit NOS2-inhibiting properties are predominantly produced by the most NO-sensitive R. gnavus strains, ATCC 29149, and H2-28, when cultured in media with mucin supplementation. These results suggest that NO-sensitive R. gnavus, when fed on mucin, may produce NOS2-inhibiting metabolites to minimize NO generation by the host, suggesting a symbiotic relationship between the host and R. gnavus.

The observed modulation of NOS2 protein abundances by metabolites in Caco2 BBE cells is consistent with the report that arginine and citrulline increase the expression of Nos2 and NO production in enterocytes (24). The induction of Nos2 by arginine was also observed in ovine trophectoderm cells (52) and astrocytes (53). In the latter case, NO production depends on the extracellular availability of arginine. The observed rapid elevation of NOS2 in our time course experiments could reflect the reported translational regulation of NOS2 by the GCN2 kinase–mediated phosphorylation of eIF-2α in cytokine-stimulated astrocytes (53), or control of NOS2 protein stability via S-nitrosylation (54). These and our studies suggest that extracellular metabolites, such as arginine and its derivatives, may promote NO production by elevating NOS2 through posttranscriptional control. Our observations also indicated a regulation of epithelial cell NOS2 abundance by biogenic amines, such as tyramine and tryptamine that are produced by R. gnavus. These results demonstrated complicated cross-regulations of NOS2 by arginine and nonarginine metabolites, the balance of which may be important for the abundance and activity of intestinal NOS2 machinery.

In addition to the arginine related metabolites, we also revealed that R. gnavus produces potential neuromodulators, tyramine, homovanillic acid, and tryptamine (27, 28, 29). Recent work by Natalie and colleagues has reported a potential gut–brain axis signaled through tryptamine (22). Molecular mechanisms mediated by microbiome on behavior, stress, memory, and nervous system development are being explored in GF and antibiotic-treated mice (55, 56, 57, 58). Tyramine production by microbiome bacteria Lactobacillus bulgaricus, E. faecalis, and Lactobacillus plantarum has been reported (29), and excessive tyramine accumulation in host organisms may lead to a variety of disorders including hypertension but has been traditionally linked to the consumption of fermented foods including wine and cheese (59, 60). Future work will determine the biological effects of major metabolites produced by R. gnavus strains to understand how they may exhibit various degrees of virulence in homeostasis and disease conditions.

Experimental procedures

Mice

Gnotobiotic adult C57BL/6J mice were purchased from Charles River, housed at the Newark Gnotobiotic Core Facility at Rutgers New Jersey Medical School, and handled in a sterile gnotobiotic isolator. GF status was monitored throughout the experiments by IDEXX Laboratories, and by the genomics core facility using in-house protocols. NOS2 KO (Nox2tm1Lau/J) mice were purchased from Jackson Laboratory (Stock number 007072), and acclimated for 1 week in the Rutgers Newark animal facility. All mice were maintained on a 12:12 light-dark cycle and fed an autoclaved nonpurified diet. Mice were randomly divided into control and experimental groups, subjected to experimental procedures on the same day, and euthanized at the same end point. For specific pathogen-free experiments, cage effects were considered by using littermates from various breeders. All animal experiments were conducted in accordance with NIH guidelines and US federal laws and approved by the Institutional Animal Care and Use Committee at Rutgers University and Rutgers New Jersey Medical School Comparative Medicine Resources.

R. gnavus growth culture

R. gnavus strains ATCC 29149, ATCC 35913, DSM 108212 (H2-28), AGR 2154, and LGG (ATCC, 53103) were acquired from the ATCC, USA or Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH were cultured anaerobically using an inoculum from established stocks. Cultures were grown in 14 ml tubes containing 10 ml of AnaeroGRO Chopped Meat Glucose Broth (MB) (Hardy Diagnostics, AG19H) at 37 °C. NO stress was induced using DNNoate (Thermo Fisher Scientific, 328651000) at concentrations of 0.05, 0.5, and 5 mM, and a DNNoate-free control. Absorbance at 600 nm (A600) was monitored via spectrophotometry at various time points to measure bacterial growth. Data were expressed as mean ± SD from three independent experiments.

Bacterial strains for gnotobiotic work

R. gnavus (ATCC, 29149) was anaerobically cultured in AnaeroGRO Chopped MB (Hardy Diagnostics, AG19H) at 37 °C. LGG (ATCC, 53103) was cultured at 37  °C in Lactobacilli MRS broth (Hardy Diagnostics, K15) as previously described (61, 62, 63).

Bulk RNA-seq of GF, LGG, and RG mice

For the GF, LGG, and R. gnavus experiments, the LGG and RG cultured broth was centrifuged at 1200g for 10 min to pellet the bacteria. On day 0, a cohort of GF mice was gavaged with PBS (n = 5), another cohort with 200 μl of LGG (n = 5) and a third with RG (n = 5) (108 colony-forming units/ml) resuspended in the PBS solution. On day 21, animals were sacrificed, and whole ileal tissue was used for RNA extraction using the RNeasy Plus Mini kit (Qiagen #74034). The bulk RNA-seq transcriptome datasets were analyzed on the CLC Workbench version 20.04. Mapping was performed using the Mus musculus reference genome and transcript sequence from Ensembl (GRCm38) on trimmed reads (80 bp average read length). Reads Per Kilobase of transcript, per Million mapped reads counts were generated and used for heatmap generations by utilizing the pheatmap package on R.

Real-time PCR

Genomic DNA was isolated using Qiagen QIAamp DNA Stool Mini Kit (Catalog No. 51604). To detect bacterial colonization in fecal samples, PCR cycling conditions were 3 min preheating at 98 °C followed by 50 cycles of denaturing (98 °C for 10 s), annealing (60 °C for 30 s), and extension (72 °C for 30 s). In addition, melting curves were analyzed to ensure PCR specificities. Water was included as a negative control. Primers for the gene encoding universal 16S rRNA were as follows: 16S rRNA forward: 5′- ACTACGTGCCAGCAGCC-3′ and reverse 5′- GGACTACCAGGGTATCTAATCC-3′. Primers for RG-specific detection are as follows: RG forward: 5′-GGACTGCATTTGGAACTGTCAG-3′ and RG reverse 5′-AACGTCAGTCATCGTCCAGAAAG-3′. Primers for LGG-specific detection are as follows: LGG forward: 5′-GCCCTTAACAGCAGTCTTC-3′ and LGG reverse 5′- GCCCTCCGTATGCTTAAACC-3.

Polar metabolite collection

Frozen feces were weighed on a calibrated scale and placed on ice. The polar metabolites were extracted with a mixture of acetonitrile/methanol/water (40:40:20) containing 0.1 M formic acid (extraction buffer). The volume of extraction buffer was adjusted by fecal weight prior to the extraction procedure. After addition of the extraction buffer to the feces, samples were then sonicated (4  °C, 30 s) and centrifuged (17,000g, 2 min). After centrifugation, 150 ul of supernatant was diluted 4-fold with extraction buffer prior to LC-MS analysis.

For spent media from R. gnavus cultures grown in AnaeroGRO Chopped MB, or MB supplemented with 1% porcine mucin (Sigma M1778), or MB with SA, samples were collected after 3 h of incubation. Metabolites were extracted using the same acetonitrile/methanol/water (40:40:20) method, and the aqueous phase was analyzed by LC-MS in both positive and negative ionization modes. Data processing was performed with MetaboAnalyst software (https://www.metaboanalyst.ca/). Partial least squares discriminant analysis identified key metabolites contributing to group separation, with variable importance in projection scores ranking their importance.

NO detection

To assess the impact of the specified metabolites on intracellular NO levels, Caco-2 BBE (10,000 cells per well) were seeded in a 96-well plate and incubated for 24 h in a CO2 incubator. The cells were then cultured for a period of 14 days, with daily changes of Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum (FBS). Following the maintenance period, cell cultures were treated for 48 h with metabolites (arginine, citrulline, ornithine) at a concentration of 3 μM in PBS or in PBS alone (control). Subsequently, the cells were washed twice with PBS and incubated with 100 μl of a 5 μM 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM diacetate, Thermo Fisher Scientific, D-23841) solution in Hank's Balanced Salt Solution per well. The DAF-FM diacetate solution was allowed to incubate for 1 h at 37 °C, and the fluorescence was measured at 495 to 515 nm using a GloMax plate reader.

NO donor administration in mice

Fecal samples were collected from individual WT and Nos2−/− mouse before the experiment to establish a baseline for RG quantification. DNNoate (Cayman Chemicals, 82120) was administered at a dosage of 80 mg/kg through gavage for four consecutive days, with daily fecal sample collection during this period. After the treatment phase, DNNoate was discontinued for an 11-day period, followed by another round of fecal sampling. RG abundance was determined by qPCR.

Caco2 BBE cell culture and TEER assay

Detailed methods were described in a previous report (64). Briefly, Caco2 BBE cells were cultured in DMEM supplemented with 20% FBS (Thermo Fisher, SH30070.03), 1% Pen-strep (Thermo Fisher Scientific, 15140-122), and 0.2% Primocin (Thermo Fisher Scientific, NC9141815). The cells were grown at 37 °C with 5% CO2. The culture medium was replenished every 2 days. Cells were passaged at 80% confluence. For TEER analysis, 2 × 105 Caco2 BBE cells were seeded in 96-well 0.4 μm pore size Transwell plate (STEMCELL, 1000419). After 22 days, cell cultures were a differentiated monolayer and TEER readings were stable for 3 consecutive days. Treatments were applied to the monolayer from apical side of the cells; apical compartments held 70 μl medium while basolateral compartment contained 250 μl of culture media. Every day, cells were washed with PBS and fresh medium and treatment were changed in both chambers. TEER readings were then collected by EVOM (World Precision Instruments, EVM-MT-03-01) at a consistent time of a day. Three concentrations were used for each metabolite, corresponding to 0.1, 1, and 10-fold of physiological concentration based on literature for fumarate (65, 66), tyramine (67), tyramine (68).

Tissue fixation and immunostaining

Mouse intestinal and liver tissues were fixed in 10% neutral formalin overnight, transferred to 70% ethanol the next day, and embedded in paraffin at the Histology Core Facility at Rutgers New Jersey Medical School. Afterward, 5 μm thick sections were prepared from the paraffin blocks, rehydrated, and subjected to H&E staining. For immunostaining, tissue sections were rehydrated and underwent antigen retrieval using a 0.1 M citrate acid buffer (pH 6), with slides immersed in the buffer at sub-boiling temperature for 20 min before being transferred to running water. The slides were then incubated in blocking buffer (PBS containing 0.1% Triton X-100, 2% normal serum, and 2% bovine serum albumin) at room temperature for 1 h. For immunofluorescence, slides were probed with the primary antibody Muc2 (1:200, sc-15334, Santa Cruz) at 4 °C overnight. The following day, the slides were washed three times with PBS and incubated with fluorescent dye-conjugated secondary antibodies for 1 h at room temperature. After additional washes and 4′,6-diamidino-2-phenylindole nuclear counterstaining, the slides were air-dried and mounted with Prolong Gold Antifade (Invitrogen). Fluorescent images were captured using a Zeiss LSM980 confocal microscope and analyzed with Image J (https://imagej.net/ij/). For immunohistochemistry, slides were probed overnight with primary antibodies against Mptx2 (1:200, ab238123, Abcam), lysozyme (1:500, PU024-5UP, BioGenex), Nos2 (1:200, ab15323, Abcam), and DCLK1 (1:250, ab37994, Abcam), followed by standard immunohistochemical processing. The staining was visualized at 10× magnification with three representative sections per mouse using a Nikon TE2000 microscope. Quantification of Mptx2, lysozyme, and Nos2 intensity was conducted by detecting diaminobenzidine (DAB) staining within regions of interest, specifically per crypt or crypt-villus unit. Semiquantification analysis of DCLK1 (1:250, ab37994, Abcam) and Alcian Blue was determined as the average number of DCLK1 or Alcian Blue-positive cells per crypt-villus unit per image.

Western blotting

Caco-2 BBE cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% nonessential amino acids at 37 °C with 5% CO2 and polarized for 21 days. Cells were treated with arginine (1 mM) (SIGMA A8094-100G), citrulline (1 mM) (SIGMA C7629-1G), fumarate (10 μM) (ACROS Organic 215531000), ornithine (1 mM) (SIGMA 02375-25G), tyramine (30 nM) (SIGMA T90344-5G), and tryptamine (30 μM) (SIGMA 193747-10G) for 0, 1, 3, 6, 12, and 24 h. Protein lysates were prepared using radioimmunoprecipitation assay buffer buffer with protease and phosphatase inhibitors, and protein concentrations were determined via the Bradford assay. Equal protein amounts were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk in Tris buffered saline with Tween-20 and then incubated overnight at 4 °C with primary antibodies against Nos2 (1:1000) (ab15323, Abcam) and Actin (1:5000) (sc-47778, Santa Cruz). Following secondary antibody incubation and washing, protein bands were detected using enhanced chemiluminescence and imaged. Densitometry analysis of band intensities was performed using ImageJ, normalizing Nos2 expression to Actin.

Statistical analysis

All graphic data and statistical analyses were conducted using GraphPad Prism 9 (https://www.graphpad.com) and Microsoft Excel Office 365. The data were reported as mean ± SD or SEM. Statistical comparisons for two groups were performed by one-tailed Student's t test and for multiple groups either one-way or two-way ANOVA. The significant level of enrichment score in the gene set enrichment analysis was computed by an empirical phenotype-based permutation test conducted to reserve the complex correlation structure of gene expression patterns. The phenotype labels were permuted, and then the enrichment score of the gene set was recalculated for the permuted data, thus resulting in a null distribution for the enrichment score. The nominal p value of the resulting enrichment score was empirically computed relative to this null distribution as previously described (Subramanian et al., 2005).

Data availability

The metabolomic data have been uploaded to MassIVE (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp), and can be accessed using the following accession numbers: MSV000095089 for in vitro cultured R. gnavus ATCC 29149; MSV000095093 and MSV000095090 for various R. gnavus strains cultured in plain meat broth culture medium, with sialic acid, or with porcine mucus supplementation; and MSV000095115 and MSV000095105 for mouse fecal metabolites from PBS or R. gnavus colonized mice. Bulk RNA-seq data is available on NCBI under Bioproject number PRJNA993709.

Supporting information

This article contains supporting information.

Conflict of interests

The authors declare that they have no conflicts of interest with the contents of this article.

Supporting information

Suplemental Figure legends

SFig1

STable

Acknowledgments

We are grateful to Paula Lopez, Niyati Boghani, Nisarg Baboo, Aditi Mandaliya, and Eric Chiles for help in experiments, and to the staff of Comparative Medicine Resources, Rutgers University, for help in establishing our germ-free colonies.

Author contributions

J. A. F. and J. M. A. writing–original draft; J. A. F., J. M. A., R. F., and N. G. visualization; J. A. F., J. M. A., P. S., V. M., S. B., J. H., R. B., I. B., R. U., Y. L., E. M. B., P. K., X. S., R. F., and N. G. methodology; J. A. F., J. M. A., V. M., J. H., J. K., and N. G. formal analysis; J. A. F., J. M. A., P. S., V. M., S. B., J. H., R. B., I. B., R. U., Y. L., E. M. B., P. K., and X. S. data curation; J. A. F., J. M. A., R. F., and N. G. conceptualization; J. A. F., J. M. A., P. S., V. M., S. B., J. H., R. B., I. B., R. U., Y. L., E. M. B., and X. S., software; V. M., S. B., E. M. B., R. F., and N. G. funding acquisition; J. H., E. M. B., R. F., and N. G. writing–review and editing; E. M. B., R. F., and N. G. resources; R. F. and N. G. supervision; R. F. and N. G. investigation; R. F. and N. G. validation; N. G. project administration.

Funding and additional information

This was supported by 10.13039/100000002 NIH Grants R01AT010243 (N. G. and R. P. F.), R01DK102934 , R01DK119198 , and 10.13039/100000001 NSF IIS-2128307 (N. G.), F31DK121428 (S. B.), 10.13039/100000001 NSF Grant No. 217484 (E. M. B.), 10.13039/100000001 NSF Grant No. IOS 1754783 (R. P. F.), and 10.13039/100000002 NIH -funded Rutgers INSPIRE IRACDA Postdoctoral Program GM093854 (V. M.). Services, results, and/or products in support of the research project were generated by the Rutgers Cancer Institute of New Jersey Metabolomics Shared Resource, supported, in part, with funding from NCI-CCSG P30CA072720-5923. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Present address for Panan Suntornsaratoon: Department of Physiology, Faculty of Science, Mahidol University, Bangkok Thailand.
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