Bacillus subtilis Produces Amino Acids to Stimulate Protein Synthesis in Ruminal Tissue Explants via the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta–Serine/Threonine Kinase–Mammalian Target of Rapamycin Complex 1 Pathway

Abstract

Background: Bacillus subtilis is a probiotic strain that is widely used as a feed supplement for ruminants. In this study, one B. subtilis strain isolated from the ruminal fluid of Holstein dairy cows was used for an ex vivo study with ruminal tissue explants. The main goal was to assess the potential endosymbiotic links between B. subtilis and the ruminal epithelium using molecular analyses and amino acid profiling. The explant culture protocol was first optimized to determine the ideal conditions in terms of tissue viability before performing the actual experiments involving active and inactive bacteria with or without protein synthesis inhibitors, such as LY294002 (phosphatidylinositol 3-kinase inhibitor) or rapamycin [mammalian target of rapamycin (mTOR) inhibitor].

Results: The mRNA levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB), serine/threonine kinase (AKT), mTOR, P70S6K1, and eukaryotic translation initiation factor 4E binding protein 1 were the highest (p < 0.01), while those of programmed cell death 4 were the lowest when the tissue was incubated with 107 of B. subtilis. Compared with the inactivated bacteria, the expression levels of PIK3CB and AKT, and overall changes in mTOR and P70S6K1 were greater in rumen explants with living bacteria (p < 0.05). With an increase in B. subtilis concentration, the trends of protein and corresponding gene changes were consistent. There were differences in the concentrations of individual amino acids in the supernatants of living and inactivated bacterial culture groups, with most amino acids enriched in pathways, such as aminoacyl tRNA biosynthesis, cyanoamino acid metabolism, monobactam biosynthesis, or glycine, serine, and threonine metabolism. The addition of psilocybin upregulated the expression levels of PIK3CB and AKT. A significant decrease (p < 0.05) in PIK3CB and mTOR protein expression levels was detected after the addition of LY294002 and rapamycin. In addition, These responses were associated with the downregulation (p < 0.05) of AKT and P70S6K protein expression levels.

Conclusions: We confirmed that the in vivo ruminal tissue culture system is a suitable model for studying probiotic-induced alterations in tissue function. As such, this study provides a means for future mechanistic studies related to microbial regulation and the dietary supply of proteins. In addition, living and inactivated B. subtilis can promote protein synthesis in ruminal tissue explants by altering the expression levels of related factors in the PIK3CB-AKT-mTORC1 pathway, which could further aid in optimizing the feed efficiency and increasing the use of inactivated bacteria as additives in dairy cow farming.

Keywords: Bacillus subtilis; amino acids; mTOR; protein expression; rumen explants.