Bacterial colonization factors control specificity and stability of the gut microbiota

Abstract

Mammals harbour a complex gut microbiome, comprising bacteria that confer immunological, metabolic and neurological benefits. Despite advances in sequence-based microbial profiling and myriad studies defining microbiome composition during health and disease, little is known about the molecular processes used by symbiotic bacteria to stably colonize the gastrointestinal tract. We sought to define how mammals assemble and maintain the Bacteroides, one of the most numerically prominent genera of the human microbiome. Here we find that, whereas the gut normally contains hundreds of bacterial species, germ-free mice mono-associated with a single Bacteroides species are resistant to colonization by the same, but not different, species. To identify bacterial mechanisms for species-specific saturable colonization, we devised an in vivo genetic screen and discovered a unique class of polysaccharide utilization loci that is conserved among intestinal Bacteroides. We named this genetic locus the commensal colonization factors (ccf). Deletion of the ccf genes in the model symbiont, Bacteroides fragilis, results in colonization defects in mice and reduced horizontal transmission. The ccf genes of B. fragilis are upregulated during gut colonization, preferentially at the colonic surface. When we visualize microbial biogeography within the colon, B. fragilis penetrates the colonic mucus and resides deep within crypt channels, whereas ccf mutants are defective in crypt association. Notably, the CCF system is required for B. fragilis colonization following microbiome disruption with Citrobacter rodentium infection or antibiotic treatment, suggesting that the niche within colonic crypts represents a reservoir for bacteria to maintain long-term colonization. These findings reveal that intestinal Bacteroides have evolved species-specific physical interactions with the host that mediate stable and resilient gut colonization, and the CCF system represents a novel molecular mechanism for symbiosis.

Figure 1. Bacteroides species occupy species-specific niches in the gut via an evolutionarily conserved genetic locus

a-c, Germ-free mice were mono-associated with B. fragilis and challenged orally with (a) B. thetaiotaomicron; (b) B. vulgatus; or (c) B. fragilis. d, Mice were mono-associated with erythromycin sensitive B. fragilis, and subsequently challenged with erythromycin resistant B. fragilis. Erythromycin was administered where indicated. e, Genomic organization of the ccf locus. f, Mice were mono-associated with either WT B. fragilis, mutant strains deleted in ccfC, ccfD, ccfE and ccfC-E (BFΔCCF), or complemented strain (BFΔCCF∷CCF) and challenged with WT B. fragilis. CFUs were determined after 30 days. g, Mice were mono-associated with WT B. vulgatus or a mutant strain deleted in ccfC-E genes (BVΔCCF), and challenged with WTB. vulgatus. CFUs were determined after 30 days. In all sequential colonization studies, results are representative of at least 2 independent trials (n=3-4 animals/group). h, Cross-colonization between WT B. fragilis and BFΔCCF mono-associated mice at 7 days after encounter measured by CFUs of the initially colonizing and the horizontally transmitted (challenge) strains. (n=2 animals/encounter, 5 independent trials). All graphs: Dashed line indicates the limit of detection at 100 CFU/g feces, and error bars indicate standard deviation (SD).

Figure 2. B. fragilis colonization of the colonic crypts is mediated by the CCF system

a, qRT-PCR of ccf gene expression levels normalized to 16S rRNA (n=3 animals, 2 trials). b, Mice were mono-associated with either WT B. fragilis or B. fragilisΔCCF, and challenged with WT B. fragilis. The percentage of challenge strain was determined in the lumen (feces) and colon after 1 day (n=8 animals/group). c, Confocal micrographs of germ-free, WT B. fragilis or B. fragilisΔCCF mono-associated mice colon whole-mount. Crypts are visualized by DAPI (nuclei, blue) and phalloidin (F-actin, green). Bacteria (red) are stained with IgY polyclonal antibody raised against B. fragilis. Images are representative of 7 different sites analyzed from at least 2 different colons. Scale bar: 5 μm. d, 3D reconstructions of colon crypts from WT B. fragilis or B. fragilisΔCCF mono-associated mice. Bacteria are detected on the apical surface of the epithelium (arrows) and in the crypt space (arrowhead). Scale bar: 10 μm. e, Quantification of bacterial penetration, measured as distance from the epithelial surface per crypt. Error bars indicate standard error of the mean (SEM). NS: not significant. ND: not detected. **p<0.01.****p<0.0001.

Figure 3. B. fragilis requires the ccf genes for stable and resilient colonization of mice

a, Groups of SPF Rag-/- mice were gavaged with either WT B. fragilis or B. fragilisΔCCF. b, SPF Rag-/- mice were given a 1:1 co-inoculum of WT B. fragilis and B. fragilisΔCCF by single gavage. c, SPF NOD mice were gavaged with either WT B. fragilis or B. fragilisΔCCF. d, SPF NOD mice were given a 1:1 co-inoculum of WT B. fragilis and B. fragilisΔCCF by single gavage. e, SPF mice were co-associated with WT B. fragilis and B. fragilisΔCCF, and infected with Citrobacter rodentium. f, SPF mice were co-associated with WT B. fragilis and B. fragilisΔCCF, and given ciprofloxacin in drinking water for the time period shown. For all analyses, bacterial colonization levels were assessed by real-time qRT-PCR from stool DNA (n=4 animals/group). Results are representative of at least 2 independent trials per experiments. Error bars indicate SEM.*p<0.05. ***p<0.001.