Molecular Mechanism Of Enhanced Intestinal Adhesion Of Lactobacillus Plantarum WCFS1 By Bile Salt Hydrolase

At present, there are many reports about the function of bile salt hydrolase ?BSH?,but the study of its functional mechanism is still lacking. Two strains were constructed in the early researches of our laboratory, one of which was bile salt hydrolase gene ?bshl?mutant strain ?A? of lactobacillus plantarum WCFS1, and the other strain B was strain A with a recombinant expression plasmid containing the exogenous bile salt hydrolase gene.The results showed that the recombinant strain B could enhance the intestinal colonization ability and the in vitro adhesion ability to Caco-2 cell, which indicated that bile salt hydrolase could enhance the adhesion of lactobacillus plantarum WCFS1. In this paper, the molecular mechanism of enhanced adhesion ability of lactobacillus plantarum WCFS1 by bile salt hydrolase was studied.Firstly, the proteomic differences between strain A and B under different bile salts treatment were analyzed by iTRAQ proteomics, and 1712 proteins were identified. Three possible adhesion proteins, F9UP14, F9UM21 and F9USM7 ?accession number in Uniprot database?, were found in the differentially expressed proteins by searching databases and alignment, and their coding genes were lp₁643, lp₀800 and lp₃114.According to the proteomic analysis, the expression of these three genes was increased by 2.9, 3.5 and 2.6 folds in the environment of bile salts, respectively, which were positively correlated with bsh gene. Compared with strain A without bile salts, in the case of bile salt environment and BSH expression, lp₁643 upregulated by 6.9 folds in strain B with bile salts. Finally, lp₁643 was selected as the target gene, and its adhesion function was verified. This paper selected lp₁643 to verify its adhesion function.After the target gene lp₁643 was selected, six corresponding sequences in the lp₁643 gene were identified by searching sequences of MUB domain in other genes.Based on these six repetitive sequences, the upstream and downstream homologous arms were designed so that the expression of the six repeats could be interrupted after knockout successfully. Then the knockout plasmid pNZ5319/lp₁643 was constructed.The knockdown plasmid pNZ5319 / Ip₁643 was transformed into competent cells of lactobacillus plantarum WCFS1 to knock out the target fragment of lp₁643 gene, and the expression of 6 repeats was interrupted.Finally, by comparing the difference in the adhesion ability of lactobacillus plantarum WCFS1 wild-type strain C and lp₁643 gene mutant strain D to colon cancer cells, the adhesion rate was about 0.74% before knockout and 0.12% after knockout. The adhesion rate of strain D was significantly lower than that of strain C. Therefore, it is concluded that lp₁643 gene is an important factor affecting the adhesion of lactobacillus plantarum WCFS1. Combined with the results of previous proteomics analysis, it was concluded that bile salt hydrolase indirectly enhanced the adhesion of the strain by promoting the expression of lp₁643 gene.In summary, the molecular mechanism of increased intestinal adhesion of lactobacillus plantarum WCFS1 by bile salt hydrolase was studied by means of iTRAQ proteomics and gene knockout, which promoted the study of the function mechanism of bile salt hydrolase.