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 x 5 m 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 m. 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.