Increasing the adhesion ability of Lactobacillus to intestinal epithelial cells by heterologously expression of adhesion proteins

The adhesion ability of bacteria to intestinal epithelial cells has been considered as one of the crucial selection criteria for probiotic strains. The superior adhesion is a prerequisite for colonization of bacteria, therefore, it is an importance effect for the establishment of a stable normal flora in the mammalian intestine and host beneficial healthiness. Initial adhesion is followed by firm attachment where adhesins on the bacterial cell surface recognize receptors on the epithelial cell surface.
As the binding proteins play a key role, this study was aimed to clone the Gram-positive bacterial adhesion protein genes and transformed these genes into a poor-adhesive Lactobacillus strain to increase its adhesion ability to intestinal epithelial cells. The DNA sequences encoding collagen-binding protein (Cnb) of Lactobacillus reuteri Pg4 and the mucus-binding protein (Mub) of Listeria monocytogenes were amplified by polymerase chain reaction (PCR). The PCR fragments encoding Cnb and Mub were subcloned into the E. coli－Lactobacillus shuttle vector pNZ3004 to generate pNZ-cnb and pNZ-mub respectively, and then were used to electroporate into Lb. casei ATCC 393.
The presence of the cnb and mub gene in the Lb. casei transformants (designated Lb. casei pNZ-cnb and Lb. casei pNZ-mub) was demonstrated by direct colony PCR. The total RNA of the Lb. casei ATCC 393 transformants was extracted for estimation of the transcript levels of cnb and mub by reverse transcription-PCR. The results showed that cnb and mub were successfully expressed by Lb. casei ATCC 393 transformants. The heterologous expression of 29 kDa Cnb and 48 kDa Mub were further confirmed by Western blot analysis. Flow cytometric analysis of the Lb. casei ATCC 393 transformants indicated that Cnb and Mub were display on their cell surface. In addition, the intensity of the fluorescent signals measured on Lb. casei ATCC 393 transformants was higher than that on Lb. casei ATCC 393. In the cell adhesion test, the adhesion ability of the Lb. casei ATCC 393 transformants to Caco-2 cells was increased as compared with Lb. casei ATCC 393. Competition, exclusion and displacement of Listeria monocytogenes BCRC 15338 and Escherichia coli O157:H7 by Lb. casei ATCC 393 transformants from adhesion on Caco-2 cell surfaces were further studied. The results indicated that Lb. casei pNZ-cnb could inhibit the adhesion of Ls. monocytogenes BCRC 15338 and E. coli O157:H7 to Caco-2 cells, while Lb. casei pNZ-mub could only inhibit the adhesive ability of Ls. monocytogenes BCRC 15338.
In conclusion, this study demonstrated that Cnb and Mub could be heterologously expressed by Lb. casei ATCC393. The transformed strains Lb. casei pNZ-cnb and Lb. casei pNZ-mub could constitutively express Cnb and Mub on their cell surface, and thus showed a better adhesion ability to Caco-2 cells than Lb. casei ATCC 393 did. Furthermore, Lb. casei pNZ-cnb and Lb. casei pNZ-mub could inhibit the adhesion of pathogens to Caco-2 cells.