Improved adhesive properties of recombinant bifidobacteria expressing the Bifidobacterium bifidum-specific lipoprotein BopA

Abstract

BackgroundBifidobacteria belong to one of the predominant bacterial groups in the intestinal microbiota of infants and adults. Several beneficial effects on the health status of their human hosts have been demonstrated making bifidobacteria interesting candidates for probiotic applications. Adhesion of probiotics to the intestinal epithelium is discussed as a prerequisite for colonisation of and persistence in the gastrointestinal tract.ResultsIn the present study, 15 different strains of bifidobacteria were tested for adhesion. B. bifidum was identified as the species showing highest adhesion to all tested intestinal epithelial cell (IEC) lines. Adhesion of B. bifidum S17 to IECs was strongly reduced after treatment of bacteria with pronase. These results strongly indicate that a proteinaceous cell surface component mediates adhesion of B. bifidum S17 to IECs. In silico analysis of the currently accessible Bifidobacterium genomes identified bopA encoding a lipoprotein as a B. bifidum-specific gene previously shown to function as an adhesin of B. bifidum MIMBb75. The in silico results were confirmed by Southern Blot analysis. Furthermore, Northern Blot analysis demonstrated that bopA is expressed in all B. bifidum strains tested under conditions used to cultivate bacteria for adhesion assays. The BopA gene was successfully expressed in E. coli and purified by Ni-NTA affinity chromatography as a C-terminal His6-fusion. Purified BopA had an inhibitory effect on adhesion of B. bifidum S17 to IECs. Moreover, bopA was successfully expressed in B. bifidum S17 and B. longum/infantis E18. Strains overexpressing bopA showed enhanced adhesion to IECs, clearly demonstrating a role of BopA in adhesion of B. bifidum strains.ConclusionsBopA was identified as a B. bifidum-specific protein involved in adhesion to IECs. Bifidobacterium strains expressing bopA show enhanced adhesion. Our results represent the first report on recombinant bifidobacteria with improved adhesive properties.

Mechanisms of Bifidobacteria Resistance to Lipolytic Enzymes of Staphylococcus Aureus

“…Изначально, до обработки B. breve липо-литическими ферментами стафилококка, в со-став фосфолипидов мембраны входило боль-шое количество ненасыщенных (олеиновая, линолевая) и разветвленных жирных кислот (изоформы), присутствие которых увеличива-ет текучесть данной оболочки микроорганиз-мов и способствует формированию высокой гидрофобности и аутоагглютинации [5,6,19].…”

“…Только при определенных параметрах теку-чести и вязкости данной оболочки у бактерий осуществляется рост и размножение, транс-порт веществ, функционирование мембран-ных ферментов [3,4]. Состав фосфолипидов определяет также гидрофобность клеток, ад-гезивные свойства, чувствительность к анти-бактериальным препаратам, устойчивость к перекисному окислению [5,6,7]. Бактериаль-ная цитоплазматическая мембрана является ключевой мишенью для стрессовых факторов микросреды биотопа.…”

Expression of Fluorescent Proteins in Bifidobacteria for Analysis of Host-Microbe Interactions

“…Thus, adhesion to intestinal epithelial cells or mucus is one of the properties frequently tested for potential probiotic strains (6,(54)(55)(56). Until now, most studies on adhesion of bifidobacteria to cultured epithelial cells have used methods involving bacteria labeled by radioisotopes or by classical plate counting (6,39,(57)(58)(59). The results obtained in this study demonstrate that the use of fluorescent bifidobacteria in adhesion assays is a promising application yielding results comparable to those obtained with classical assays, e.g., by plate counting or using radioactively labeled bacteria.…”

“…The pMDY23 backbone was chosen for the generation of further plasmids based on successful transformation into a total of 15 strains belonging to 7 different species with 4 subspecies (see Table S2 in the supplemental material) (39,50,51). To our knowledge, this represents the widest range of bifidobacterial hosts for any E. coli/Bifidobacterium shuttle vector described so far (2,21,52).…”

“…Adhesion to Caco-2 cells was determined by the classical plate counting method described previously (39 …”