Dereplicated MAGs and DRAM annotation.

Host-microbiome interactions are complex and hard to pinpoint; yet are essential for the physiology and fitness of ruminants. While this co-dependency is clear at a macro level, how intricate molecular interactions between the rumen microbiome and its host are linked to broader phenotypes remains poorly characterized. Here, we applied (meta)proteomic analyses of microbial, fungal, bovine, and protozoan proteins from both rumen fluid and epithelium tissue of beef cattle, which demonstrated that unique microbial and bovine proteins are differentially expressed and spatially localized. As expected, microbial proteins were largely detected in the rumen fluid compared to the epithelia, however bacterial proteins inferred in butyrate production and affiliated to Lachnospiraceae population genomes were predominantly expressed within the rumen epithelium tissue. Similarly, bovine proteins were typically expressed within epithelial tissue, although selected bovine proteins such as a gastric lipase and salivary proteins were detected at higher levels in the rumen fluid suggesting active secretion. Further analysis of co-expression and co-exclusion networks of host-microbe proteins inferred transkingdom links between host, protozoan and bacterial proteins specifically related to housekeeping functions as well as nitrogen metabolism, glucogenesis, pyruvate metabolism, and membrane binding. We additionally observed a network module enriched in protozoal-affiliated proteins that was significantly and favourably linked to feed conversion ratio of the host. Overall, our results highlight how host-microbiome proteomics can detect molecular interactions linked to host performance metrics, with potential for application at much higher resolution as the technology rapidly advances.