Basic-alimentary tractActive delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice☆
Section snippets
Bacteria
The L. lactis strain MG1363 was used throughout this study. Bacteria were cultured in GM17 medium (i.e., M17; Difco Laboratories, Detroit, MI; supplemented with 0.5% glucose). Stock suspensions of all strains were stored at −20°C in 50% glycerol in GM17 medium. For intragastric inoculations, stock suspensions were diluted 200-fold in fresh GM17 medium and incubated at 30°C. They reached a saturation density of 2 × 109 colony-forming units (CFU) per mL within 16 hours. Bacteria were harvested by
In vitro synthesis of TFF by L. lactis
We constructed 3 recombinant L. lactis strains, LL-mTFF1, LL-mTFF2, and LL-mTFF3, that secrete mTFF1, mTFF2, and mTFF3 containing amino-terminal Myc epitope tags, respectively. Synthesis of mTFF was evaluated by Western blot (Figure 1A) and enzyme-linked immunosorbent assay (Figure 1B). After 24 hours of growth, mTFF was found in the culture supernatant at concentrations ranging from 312 ± 68 ng/mL (LL-mTFF3) to 531 ± 92 ng/mL (LL-mTFF2) (Figure 1B). Constitutive mTFF secretion did not alter
Discussion
TFF are known for their cytoprotective properties, involvement in epithelial wound healing, and reconstitution of the gastrointestinal tract. This makes them good candidate therapeutics for the treatment of acute colitis. Due to their role as structural components, however, orally administered TFF stick to the mucus of the small intestine and are removed from the lumen at the cecum26 and can therefore never reach the colon. The approach presented here, active in situ delivery of TFF by
Acknowledgements
The authors thank I. Bruggeman, H. Devlies, A. Raeymaekers, and K. Van Laer for technical assistance; J. Wells for the vector pTREX1; M. C. Rio for the mtff1 complementary DNA; J. Vandekerckhove for Edman degradation; and A. Bredan, C. Cuvelier, and F. Shanahan for critically reviewing the manuscript.
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Supported by Ghent University GOA (file no. 12050700). K.V. holds a predoctoral grant from the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen, and J.V.H. holds a predoctoral BOF grant from Ghent University (BOF011D1700). L.S. and S.N. are supported in part by Science Foundation Ireland (SFI/01/F.1/B036).