The Bile Stress Response Mechanism And The Functional Role Of Transcription Factor TF0225 In Bile Stress Response In Lactobacillus Salivarius Ren

Lactobacillus salivarius is a member of the indigenous microbiota of the human gastrointestinal tract (GIT), and a variety of probiotic properties have been proposed for some L. salivarius strains. Bile salts in the intestine are detergent-like antimicrobial compounds which can disrupt cellular membranes, cause protein unfolding and induce oxidative damage to DNA. Therefore, tolerance to physiological bile stress is critical for L. salivarius to survive in the GIT and exert their beneficial effects. In this study, RNA-Seq transcriptomic analysis complemented with 2-DE proteomic analysis was employed to investigate the cellular response to bile in L. salivarius Ren, a centenarian-originated strain. The main contents and results of this dissertation are as follows.(1) Construction of the transcriptomic and proteomic profiles in L. salivarius Ren under bile stress conditions. L. salivarius Ren was grown in MRS supplemented with 0.07% oxgall. The Next-Generation Sequencing (NGS) based RNA-Seq revealed that the transcript levels of 192 genes were significantly changed (log2 fold_change≥1.5, p<0.001), including 47 genes up-and 145 genes down-regulated. Meanwhile,2-DE and mass spectrometry showed that 44 intracellular soluble protein spots were differentially abundant (fold_change≥1.25-fold, p<0.05), of which 15 proteins were more abundant and 27 proteins were less abundant under bile stress. Protein abundance changes correlated with transcriptome level changes for 31 proteins. These results demonstrated that the bile stress response in L. salivarius Ren was a complex physiological regulation network.(2) Bioinformatics analysis of the mechanisms of bile stress response in L. salivarius Ren. The bile stress response mechanisms were predicted on the basis of functional annotation of differentially expressed genes according to UniPort, KEGG and NCBI database. The results indicated that proteins involved in glycolysis pathway, maltose and glycerol metabolism were up-regulated, suggesting an alteration in carbohydrate utilization to enhance engery production. Bile also induced the expression of enzymes involved in the biosynthesis of lysine and threonine, which could be used to decorate the cell envelope and enhance the stability of cell membrane. The transcription of three genes encoding ABC transporters was up-regulated, which may play a role in the extrusion of bile that accumulated in the cytoplasm. The abundance of several chaperones and oxidordeuctase were up-regulated, which are likely to protect cells against protein misfolding and oxidative stress induced by bile. In contrast, enzymes involved in transcription, translation, cell proliferation and fatty acids biosynthesis were down-regulated, which may result in the reduced growth rate of cells under bile stress. In addition, the expression level of 9 transcription factors and a two-component system was also affected by bile, which may parcitipate in the regulation of response to bile in L. salivarius Ren.(3) Functional characterization of transcription factor TF0225 in response to bile in L. salivarius Ren.First, homologous overexpression of TF0225 in L. salivarius Ren was carried out to investigate its functional role in bile tolerance. The results indicated that the TF0225 over-producing strain displayed 23-fold higher tolerance to 0.5% oxgall compared to the control strain. Then the DNA-binding site of TF0225 (STKGMCA) was determined by using bacterial one-hybrid and MEME tool, and 16 putative target genes were predicted by using Target Explorer in L. salivarius Ren. Furthermore, real-time qPCR analysis showed that the transcription level of the target gene oppA was down-regulated in the transcription factor overexpression strain, suggesting that the transcription of oppA was negatively regulated by TF0225.(4) Functional analysis of oppA in bile salt resistance in L. salivarius Ren. Homologous overexpression of the oppA gene was applied to investigate its functional role in L. salivarius Ren. The results indicated that the OppA over-producing strain displayed 20-fold higher tolerance to 0.5% oxgall compared to the control strain. Moreover, different individual bile salts (GCA, GDCA, TCA and TDCA) were selected to assess the bile tolerance speciicity, and a substrate preference for taurine-conjugated bile salts was observed in the recombinant strain. In addition, the overexpression of OppA increased resistance to heat (55℃) and salt (7.5% NaCl) stress in L. salivarius Ren, suggesting that OppA plays important roles in protection against multiple stresses.In summary, this study represented the first combined transcriptomic and proteomic analysis of bile stress response in L. salivarius. The results will provide new insights into the mechanisms that enable L. salivarius Ren to cope with bile stress.