Microbial colonization establishes stratified radial niches that coordinate host epithelial and immune maturation in the colon

Abstract Microbial colonization is essential for intestinal maturation, yet how spatial organization of the microbiome shapes host tissue function remains unresolved. Here, we applied Stereo-seq V2 spatial platform to simultaneously profile the host transcriptome at single- cell resolution and microbial meta-transcriptome at 5 × 5 μm resolution across the proximal, middle, and distal colon of germ-free (GF) mice and mice reconstituted by fecal microbiota transplantation (FMT), integrated with time-course fecal metagenomics. We observed that, four weeks after FMT, microbial colonization established a mature colonic architecture, increased goblet cell number and mucus layer thickness, diversified epithelial lineages, and expanded stem/transit-amplifying, myeloid, and T-cell populations. Metagenomic profiling showed succession from early colonizers to a metabolically mature, short-chain fatty acid (SCFA)-producing community that stabilized by four weeks. Distance-resolved spatial analysis resolved two reproducible strata of colonized microbiota along the radial host-lumen axis, separated at approximately 150 μm. The epithelium-proximal stratum was enriched for mucus-associated taxa such as Bacteroides thetaiotaomicron , whereas the luminal stratum harbored fiber-associated taxa such as Ruminococcus champanellensis . This radial organization was underpinned by co-occurrence networks of spatial co-localization and co-exclusion. Finally, we identified a butyrate-producing guild that preferentially colonized the epithelium-proximal stratum, localized closer to epithelial and stromal cells, and showed active butyrate- responsive transcriptional activity. Colonization therefore establishes a spatially integrated host–microbiome interface with quantifiable, stratified microbial niches in which location, and not composition alone, coordinates epithelial and immune maturation.