The Bile Stress Response Mechanism And The Functional Role Of Two-component System In Bile Stress Response In Bifidobacterium Longum BBMN68

Bifidobacteria are natural inhabitants of the human intestinal tract. Some bifidobacterial strains are well known for their health-promoting effects and used as probiotics in fermented dairy production. Following consumption, bifidobacteria finally colonize the lower intestinal tract where the concentration of bile salts remains nearly0.05%to2.0%. Bile salts are detergent-like antimicrobial compounds which can disrupt the cellular membrane of bacteria, induce protein misfolding and oxidative damage to DNA. Therefore, tolerance to physiological bile stress is indeed essential for bifidobacteria to survive and perform the probiotic effects. In this study, RNA-Seq transcriptomic analysis complemented with2-DE proteomic analysis was used to investigate the cellular response to bile in Bifidobacterium longum BBMN68. The main contents and results of this dissertation are as followed.(1) Construction of the transcriptomic and proteomic profiles in B. longum BBMN68under bile stress conditions. The physiological bile stress was simulated by adding0.075%ox-bile into MRSc medium. The Next-Generation Sequencing (NGS) based RNA-Seq reflected the transcript levels of236genes were significantly changed (≥3.0-fold, p<0.001), meanwhile2-DE and mass spectrometry showed that44intracellular soluble proteins were differentially abundant (≥1.6-fold, p<0.01), including15genes whose expression were up-or down-regulated at both mRNA and protein level. These results demonstrated that the bile stress response was a complex physiological regulation network.(2) Bioinformatics analysis of the mechanisms of bile stress response in B. longum BBMN68. The GO and COG cluster analysis revealed that the differentially expressed genes and proteins in the presence of bile were related with many metabolic processes. The bile stress response mechanisms were suggested on the basis of functional annotation according to NCBI, UniPort, and KEGG database. On one hand, bile salt hydrolase, bile efflux transporters and some barrier proteins on the membrane directly conferred bile resistance on BBMN68; on the other hand, BBMN68adapted to bile stress conditions by induction of general stress response, alterations in central metabolism processes (such as bidid shunt sugar metabolism, biosynthesis of amino acid, nucleotide and fatty acid), transmembrane transport, gene transcription and translation, cell proliferation and so on.(3) Bile salts act as gut signal and promote the interaction between B. longum BBMN68and the host When BBMN68exposed to bile, the express of sortase was up-regulated for anchoring more pili to the cell wall, which could increase the adhesion of BBMN68to the intestinal epithelial cells. This hypothesis was verified by the decreased autoaggregation level and5-fold increased adhesion ability to HT-29cells of BBMN68in the presence of bile. And bile enhanced the synthesis of autoinducer-2which stimulated the communication between BBMN68and other enterobacteria. Moreover, the up-regulation of an aminoglycoside nucleotidyltransferase conferred resistance to aminoglycosidic antibiotics, contributing to the long-term colonization of BBMN68in the gut of healthy centerian.(4) Functional characterization of two-component system SenX3-RegX3in response to bile in B. longum BBMN68. The DNA-binding site (GARRACY) of response regulator RegX3was determined using bacterial one-hybrid and MEME tool. Target Explorer anaylsis resulted9putative target genes of SenX3-RegX3, and the DNA-binding specificity of RegX3to target gene pstS was assessed by EMSA in vitro. The function of pstS was further evaluated by heterologous overexpression using NICE system in Lactococcus lactis, and the recombinant strain showed improved bile tolerance. These results demonstrated that SenX3-RegX3could sense the bile stress signal and then activate the expression of pstS leading to accelerated Pi uptake for more production of ATP which contributed to the bile resistance of BBMN68.In summary, this study represented the first combined transcriptomic and proteomic analysis of bile stress response in B. longum. The results enriched the knowledge of bifidobacterial response to bile stress and raised new insights for comprehensive exploration of the bile stress response mechanisms.