Genomic and functional characterization of a Butyricicoccus porcorum strain isolated from human gut microbiota

Abstract

The gut is the most complex microbial ecosystem in the body that greatly influences human immune and metabolic health. However, the functional understanding of gut microbiome is hampered by our limited ability to obtain bacterial cultures for experimental validation, particularly low-abundant species that may carry specific functions but are often overlooked by population-based analyses. Here, we isolated and characterized a novel strain of Butyricicoccus porcorum (named Bp 531D) from human gut microbiota, representing the first butyrate-producing human isolate within a phylogenetic clade of Butyricicoccus complex. Comparative whole-genome analysis revealed a unique capability of Bp 531D for one-carbon metabolism and a high abundance of mobile genetic elements, including six prophages and plentiful transposons, reflecting its evolutionary flexibility. Oral administration of the bacterium profoundly altered gut microbiome composition in C57BL/6 mice, leading to controlled microbial oxidative signaling and calibrated carbohydrate metabolic function in the gut. RNA sequencing (RNA-seq) analysis demonstrated notable functional programming of colonic ECs, whereupon Bp 531D primarily restricted the biosynthesis of cholesterols and activated the pathway of antigen processing and presentation. Furthermore, the expression of MHC class II was correlatively heightened in colonic dendritic cells (DCs), and the frequencies of interleukin-10- (IL-10) and IL-22-producing T helper 17 (Th17) cells were significantly elevated in mice treated with Bp 531D compared to controls. Our findings uncover the crucial roles of B. porcorum in supporting intestinal homeostasis and provide a novel functional modulator to potentially optimize microbial strategies for improving intestinal health.IMPORTANCEReduced abundance of the Butyricicoccus genus has been associated with human intestinal disorders, including inflammatory bowel diseases. While supplementation of B. pullicaecorum mitigates intestinal inflammation, it is unclear whether other Butyricicoccus species critically contribute to intestinal microbial and immune homeostasis. We identified a novel Butyricicoccus species within human gut microbiota and characterized its detailed intestinal functions using the C57BL/6 mouse model. Our findings may further highlight the genetic and functional diversities of the gut microbiome.

Keywords: Butyricicoccus porcorum; T helper 17 cell; cholesterol biosynthesis; colonic epithelial cell; gut microbiome.

Fig 1

Characterization of Bp 531D isolated from human gut microbiota. (A) Genome comparison of B. porcorum 531D (Bp 531D) with B. porcorum BB10 (Bp BB10), B. pullicaecorum 25-3T (Bpu 25-3^T), and B. intestinisimiae MSJd-7 (Bin MSJd-7). Rings with different colors indicate different bacterial genomes. Cas clusters are shown in the outermost ring. (B) Overview of mobile genetic elements. Predicted transposases, prophages (PPs), and integrative/conjugative elements (ICEs) are shown on the circles. The attachment (att) sites of PPs are indicated with different colors. (C) Chromatograms of acetate and butyrate peaks in the Bp 531D culture supernatants compared to the standards and medium control. (D) HPLC measurement of acetate and butyrate in the supernatants of bacterial cultures (n = 3 samples/time point). (E) Whole-genome phylogenetic analysis of representative strains from the Butyricicoccus genus. The host origin and country of isolation are shown. The tree was built on the protein and gene sequences for 100 single-copy genes found in selected genomes. Branch support values are obtained using 100 bootstraps, and the length of a branch is relative to the total number of changes at each site. Bacteria highlighted in colors are selected for comparative whole-genome analyses.

Fig 2

Comparative genome analysis of Butyricicoccus. (A) Heatmaps of metabolic enzymes of indicated pathways on the genomes of Bp 531D, Bp BB10, Bpu 25-3^T, and Bin MSJd-7. (B) Heatmaps of genes related to the indicated subsystems. (C) Summary table of the number of CRISPR spacers/repeats and transposons in the compared genomes. (D) The abundance of CAZymes predicted for utilizing indicated carbohydrates.

Fig 3

16S rRNA sequencing of fecal microbiome. (A) qPCR quantification of Bp 531D-specific 16S rRNA copies in the feces of mice (n = 7) after a single oral gavage (10⁹ CFU/mouse), or 14 days after 7 gavages (n = 8/group). (B) Experimental scheme. Groups of C57BL/6 mice were orally gavaged with Bp 531D (10⁹ CFU/mouse) or PBS every 2 days. Feces were collected from the two groups of mice on day 14 for 16S rRNA and metagenomics sequencing. The transcriptomes of colonic ECs and colonic immune response were also analyzed on day 14. (C) Shannon index derived from 16S rRNA analysis of the two groups of fecal microbiotas (n = 4/group). (D) The weighted UniFrac principal coordinate analysis (PCoA) plot depicts the bacterial community structures. (E) Top bacterial class abundance. *P < 0.05, unpaired Student t-test. (F) The relative abundance of top bacterial families. (G) LEfSe plot showing differentiating taxa (species level; LDA score >2) between Bp 531D- and PBS-gavaged mice. (H) HPLC chromatograms of acetate and butyrate peaks in the extracts of the standards (top) and cecum contents (bottom). (I) HPLC quantification of acetate and butyrate in the cecal contents of mice gavaged with Bp 531D or PBS (n = 9-10/group). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, unpaired Student t-test.

Fig 4

Functional metagenomic profiling of fecal microbiota in mice treated with Bp 531D. (A) Bacterial richness in the fecal metagenomes of Bp 531D- and PBS-gavaged mice (n = 4/group). (B) PCoA plot based on Bray-Curtis distances between the two groups of metagenomes. (C) LEfSe plot showing differentiating taxa at the species level. Annotations using the Genome Taxonomy Database (GTDB) are included in parentheses. SGB, species-level genome bin. (D) The abundances of bacterial functional pathways are differentially enriched in Bp 531D-gavaged mice compared to PBS controls. (E) Volcano plot of CAZymes detected in the metagenomes. The horizontal dashed red line denotes the P-value of 0.05. GH, glycoside hydrolase; CBM, carbohydrate-binding module; CE, carbohydrate esterase; PL, polysaccharide lyase; GT, glycosyltransferase. (F) The total abundance of CGCs predicted to utilize the indicated substrates. TPM (Transcripts Per Million) is used as a measurement of normalized sequence reads mapped to CAZymes. *P < 0.05 and **P < 0.01, unpaired Student t-test.

Fig 5

Transcriptomic programming of colonic ECs. (A) Principal component analysis (PCA) plot of transcriptomes of colonic ECs derived from Bp 531D- and PBS-gavaged mice (n = 4-5/group). (B) RNA-seq results depicted as a volcano plot showing differentially expressed genes (DEGs) (fold change >1.5, FDR < 0.05) between the two groups of ECs. The number of upregulated (red) and downregulated (blue) DEGs in the Bp 531D group is shown in parentheses. (C) Significant DAVID Gene Ontology (GO) terms related to biological process. (D) Representative GSEA plots showing differentially enriched KEGG pathways in colonic ECs of Bp 531D-gavaged mice versus controls. (E) Heatmap of DEGs. (F) qPCR analysis of DEG expression in the Bp 531D group versus PBS or the control bacterium group (Fp UT1). *P < 0.05 and **P < 0.01, One-way ANOVA plus Tukey’s post-test.

Fig 6

Colonic immune response in mice gavaged with Bp 531D. (A) Upregulation of MHC-II expression, as shown by increased mean fluorescence intensity (MFI), in colonic DCs and macrophages (Macs) isolated from mice gavaged with Bp 531D vs. PBS or Fp UT1 controls (n = 9/group). (B) Representative FACS plots and frequencies of colonic IL-17A⁺ Th17 cells (IL-10⁺, IL-22⁺, and IFNγ⁺ Th17 subsets), IL-17A^- IFNγ⁺ Th1 cells, and FoxP3⁺ Tregs in mice gavaged with Bp 531D or PBS for 14 days (n = 10/group). ns, not significant; *P < 0.05, **P < 0.01, One-way ANOVA plus Tukey’s post-test (A) or unpaired Student t-test (B).