Characterization of bifidobacterial adhesion to intestinal epithelial cells
Auch gedruckt in der BibliothekW: W-H 12.898
FakultätFakultät für Naturwissenschaften
Ressourcen- / MedientypDissertation, Text
Datum der Freischaltung2012-05-07
Adhesion of probiotics is discussed as a prerequisite for the persistence and the colonization of the gut. Based on previous studies of our group, the strain B. bifidum S17 could be identified as promising candidate to investigate adhesion properties (Riedel et al., 2006a; Preising et al., 2010). Several E. coli-Bifidobacterium shuttle vectors with different antibiotic resistances were generated. Using a gusA reporter assay the promoter Pgap was shown to have detectable transcriptional activity in bifidobacteria. Competitive adhesion tests with cell fractions strongly indicate that the structures mediating adhesion of B. bifidum S17 to IECs are located on the cell surface. Adhesion of B. bifidum S17 was reduced about 50 % after treatment with pronase suggesting that a protein is involved in adhesion to IECs. Adhesion studies with 15 different strains of bifidobacteria identified B. bifidum as the species showing the highest adhesion to the tested cell lines. In silico analysis with several Bifidobacterium genomes allowed the identification of a sequence with high homology to bopA, encoding an adhesion previously identified in B. bifidum MIMBb75, exclusively in B. bifidum strains. The in silico results were corroborated by Southern Blot analysis. Furthermore, Northern blot analysis demonstrated that bopA is expressed in B. bifidum strains under conditions used to cultivate the strains for adhesion assays. BopA was successfully purified by Ni-NTA affinity chromatography as a C-terminal His6-fusion after heterologous expression in E. coli BL21. Purified BopA had an inhibitory effect on adhesion of B. bifidum S17 to T84, Caco-2 and HT29 cells. Moreover, bopA was successfully expressed in B. bifidum S17 and B. longum/infantis E18. Strains overexpressing bopA showed improved adhesion to T84, Caco-2 and HT29 cells, clearly demonstrating a role of BopA in adhesion of B. bifidum strains.