Integrative characterization of host–microbiome-diet axes during early-life development of the murine gut

Abstract Background Early-life development of the gut microbiome plays a critical role in shaping host physiology. However, a comprehensive understanding of how diet, microbial community assembly, functional capacity, and host intestinal maturation axes evolve and coordinate over time remains lacking. Most studies focus on a single axis, rely on cross-sectional sampling, or have limited functional resolution, restricting insight into developmental dynamics. Here, we characterized early-life maturation of the gut ecosystem using longitudinal, metaproteome-level analysis in a murine model. Results Using metaproteomics, we profiled fecal samples collected at seven postnatal time points from day 10 to weaning and into early adulthood (day 48) in pups from two contemporaneously raised C57BL/6 cohorts differing only in maternal origin (a long-established local colony and newly purchased pregnant females from the same vendor). Analyses accounted for time, cohort, and sex effects. Microbial communities underwent pronounced taxonomic succession, shifting from early dominance by facultative anaerobes to obligate anaerobes after weaning, accompanied by increasing species richness and functional complexity across cohorts and sexes. Taxonomic changes were co-occurring with an increase in functional redundancy that converged by postnatal day 34 and remained stable into early adulthood. KEGG clustering revealed patterns consistent with changes in metabolic pathway representation alongside maintenance of core functions. Direct detection of low-abundant dietary proteins provided evidence for dietary transitions coinciding with microbial maturation: milk proteins were detected only before weaning, while solid food components predominated over time. Maternal origin was significantly associated with microbial engraftment trajectories, leading to cohort-specific taxonomic and functional differences despite identical housing and diet. In parallel, host intestinal proteome maturation mirrored microbial succession, with concomitant shifts in metabolic, absorptive, regulatory, and effector pathways, including antimicrobial peptides and carbohydrate-modifying enzymes. Conclusions By directly integrating microbial, dietary, functional, and host axes within a longitudinal framework, this study provides a comprehensive view of murine gut ecosystem maturation during early life and offers a reference for interpreting developmental microbiome dynamics and improving experimental design and reproducibility in mouse studies.