Christensenella minuta interacts with multiple gut bacteria

Abstract

Introduction: Gut microbes form complex networks that significantly influence host health and disease treatment. Interventions with the probiotic bacteria on the gut microbiota have been demonstrated to improve host well-being. As a representative of next-generation probiotics, Christensenella minuta (C. minuta) plays a critical role in regulating energy balance and metabolic homeostasis in human bodies, showing potential in treating metabolic disorders and reducing inflammation. However, interactions of C. minuta with the members of the networked gut microbiota have rarely been explored.

Methods: In this study, we investigated the impact of C. minuta on fecal microbiota via metagenomic sequencing, focusing on retrieving bacterial strains and coculture assays of C. minuta with associated microbial partners.

Results: Our results showed that C. minuta intervention significantly reduced the diversity of fecal microorganisms, but specifically enhanced some groups of bacteria, such as Lactobacillaceae. C. minuta selectively enriched bacterial pathways that compensated for its metabolic defects on vitamin B1, B12, serine, and glutamate synthesis. Meanwhile, C. minuta cross-feeds Faecalibacterium prausnitzii and other bacteria via the production of arginine, branched-chain amino acids, fumaric acids and short-chain fatty acids (SCFAs), such as acetic. Both metagenomic data analysis and culture experiments revealed that C. minuta negatively correlated with Klebsiella pneumoniae and 14 other bacterial taxa, while positively correlated with F. prausnitzii. Our results advance our comprehension of C. minuta's in modulating the gut microbial network.

Conclusions: C. minuta disrupts the composition of the fecal microbiota. This disturbance is manifested through cross-feeding, nutritional competition, and supplementation of its own metabolic deficiencies, resulting in the specific enrichment or inhibition of the growth of certain bacteria. This study will shed light on the application of C. minuta as a probiotic for effective interventions on gut microbiomes and improvement of host health.

Keywords: Christensenellaceae; Faecalibacterium prausnitzii; Klebsiella pneumoniae; co-occurrence network; intestinal microorganism; nutrient cross-feeding.

Figure 1

DNA sequencing data obtained in this study. (A) Experimental design for resource collections and numbers of metadata and isolates of each treatment. (B) Binning of metagenomic data and assembled MAGs from each sample.

Figure 2

Impacts of C. minuta SJ-2 on the species composition of the fecal microbiomes. (A) The top 20 abundant taxa at the family level (annotated for MAGs) in fecal, GAMe, and CMe samples. Sequences for Christensenellaceae were removed for this analysis. (B) The Linear discriminant analysis Effect size (LefSe) distribution of bacterial taxa between the GAMe group and CMe group. The annotated bacterial species names were shown beside the bin numbers.

Figure 3

Impacts of C. minuta SJ-2 on the functionality of fecal microbiomes. (A) Relative abundance of L2 metabolic pathways in three groups. (B) Principle component analysis (PCA) score plot comparing the relative abundance of L3 metabolic pathways between GAMe and CMe. (C) The heatmap of relative abundance composed of the modules of amino acid metabolism with significant differences (Linear discriminant analysis, LDA ≥ 2) between the GAMe and CMe groups. (D) The heatmap of relative abundance composed of the modules of vitamin metabolism with significant differences (LDA ≥ 2) between the GAMe and CMe groups. (E) The heatmap of relative abundance composed of L3-level pathways with significant differences (LDA ≥ 2) between the GAMe and CMe groups. The color bars from red to blue indicated the relative abundance from high to low.

Figure 4

The co-occurrence network diagram with C. minuta SJ-2 intervention. The pink node labeled with bin.92 represented C. minuta SJ-2, while yellow, green, orange, and purple nodes represented Bacillota, Bacteroidetes, Actinobacteria, and Proteobacteria, respectively. The size of each node corresponded to the status of MAGs in the network, with larger nodes indicating more associations with other bacteria. The connecting lines between nodes were colored red for positive correlations and blue for negative correlations, with thicker lines reflecting stronger correlations. The right side of the network displayed bacteria indirectly associated with C. minuta SJ-2. The cluster at the bottom represented bacteria with no direct correlation with C. minuta SJ-2. The table displayed the correlation of bacteria in the co-occurrence network with C. minuta SJ-2: red, positive; blue, negative. All bacteria in the table were arranged in descending order of correlation intensity.

Figure 5

The phylogenetic tree of medium to high quality MAGs was obtained from three different groups. A phylogenetic tree was constructed based on the genome sequences of 91 MAGs using GTDBtk. The tree was built using p_Proteobacteria (GCF_000006765.1) as an outgroup to determine the evolutionary relationships among the MAGs, the outer ring represents the isolation status of the corresponding MAGs at the species level. Cycles from inside to outside: (1) phylum-level assignments (Bacteroidota, orange; Thersmodesulfobacteria, yellow; Proteobacteria, green; Actinobacteriota, pink; Bacillota, blue; Outgroup, gray); (2) isolation/culture status of the 91 (cultivated in this study, purple; strains from previous studies, black; no representative culture, gray).

Figure 6

Phylogenetic tree of bacteria isolated and cultured from intestinal samples. Based on the 16S rRNA gene sequences of 121 bacterial strains, a phylogenetic tree (NJ tree) was constructed using MEGA11 with Clustal W algorithm for multiple sequence alignment, using strain Pseudomonas aeruginosa DSM 50071 (NR_026078.1) as an outgroup. The Bar charts were the abundance of cultivated bacterial strains corresponding to MAGs in three treatments (red, Feces; green, GAMe; blue, CMe).

Figure 7

C. minuta SJ-2 interacts with other bacteria. (A) The pairwise co-cultivation on agar plates. (B) The growth of bacteria in mmGAM broth (red curves) and broth that was conditioned with C. minuta SJ-2 (blue curves). (a) Klebsiella pneumoniae; (b) Phocaeicola vulgatus; (c) Bacteroides caccae; (d) Bifidobacterium bifidum; (e) Bifidobacterium longum; (f) Bacteroides uniformis; (g) Bacteroides fragilis; (h) Bacteroides stercoris; (i) Eubacterium ventriosum; (j) Ruminococcus gnavus; (k) Clostridium innocuum; (l) Parabacteroides distasonis; (m) Finegoldia magna; (n) Anaerostipes hadrus; (o) Romboutsia timonensis; (p) Alistipes putredinis; (q) Flavonifractor plautii; (r) Sellimonas intestinalis; (s) Parabacteroides merdae; (t) Phocaeicola coprocola; (u) Limosilactobacillus fermentum; (v) Dorea formicigenerans; (w) Faecalibacterium prausnitzii (same number as in solid culture medium).

Figure 8

Description of the interaction mechanisms between C. minuta SJ-2 and other bacteria. (A) Assimilation of substrates. Dark green: highly utilized; light green: moderately utilized; no color: none utilized. (B) Substrate utilization experiment of F. prausnitzii to explore the mechanism of cross-feeding: (a) lysine; (b) tryptophan; (c) cysteine; (d) proline; (e) leucine; (f) isoleucine; (g) arginine; (h) methionine; (i) valine; (j) histidine; (k) sodium acetate; (l) fumaric acid.

Figure 9

Demonstration of metabolic cross-feeding between C. minuta SJ-2 and F. prausnitzii.