SURFACE DISPLAY OF EVASINS ON RECOMBINANT LACTIC ACID BACTERIUM LACTOCOCCUS LACTIS FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASE
Škrlec K., Štrukelj B., Berlec A.
Jozef Stefan Institute, Ljubljana, Slovenia
Chemokines, small chemoattractant cytokines, are key signals in the intestinal immune system. They play an important role in the recruitment and activation of immune cells in mucosa, which is fundamental event in the pathogenesis of both forms of inflammatory bowel disease (IBD), Crohn’s disease and ulcerative colitis. The development of chemokine binding proteins as potential therapeutic agents in IBD is therefore of great interest.
Recombinant lactic acid bacteria (LAB) are considered interesting candidates for use in therapy, and could be engineered to bind chemokines on their surface, and thereby prevent chemokine proinflammatory action.
We used model LAB Lactococcus lactis for the surface display of chemokine binding proteins, produced by the salivary gland of the brown tick Rhipicephalus sanguineus, named evasins. Evasins are small proteins, which have the ability to bind and neutralize chemokines of different families (CC and CXC) and inhibit the chemokine-mediated recruitment of leukocytes.
We designed genes for evasin-1, evasin-3 and evasin-4. Evasin genes were cloned into lactococcal surface display vector pSDLBA3b in fusion with secretion signal, B domain and surface anchor, and over-expressed in L. lactis NZ9000.
Expressed fusion proteins were detected with SDS-PAGE and Western blot. The surface localization of evasins was assessed with flow cytometry and confocal microscopy, using fluorescein (FITC)-conjugated human IgG, or anti-protein A antibody, both recognizing B domain part of the fusion protein. Evasin fusion proteins functionality was tested by the ability to remove the individual chemokines from the solution with ELISA and Luminex. The influence of evasin-3-displaying L. lactis on the CXCL8 secretion by Caco-2 intestinal epithelial cells was evaluated.
Flow cytometry of evasin-displaying cells showed a distinct shift in mean fluorescence intensity in comparison to the control which indicates that evasins were successfully displayed on the surface.
The significant binding of chemokines after incubation with 2×109 evasin-displaying L. lactis cells was observed for CCL3 by evasin-1, CCL5 by evasin-4 and murine CCL1, CXCL2, murine CXCL2 and CXCL8 by evasin-3. The binding depended on the quantity of the bacterial cells, and was decreased by lowering the number of cells. By decreasing the concentration of the chemokine that was added to the constant number of the evasin-displaying bacterial cells, the portion of removed chemokine also decreased.
The ability of evasin-3-displaying L. lacits to decrease IL-1β-induced secretion of CXCL8 from Caco-2 cells was demonstrated. Significant time-dependent decrease of CXCL8 secretion was observed by increasing the bacterial concentration.
In the present study we developed evasin-displaying L. lactis with the ability to bind chemokines as a promising approach for the treatment of IBD. Developed bacteria will be tested in an animal model of IBD.
Keywords: Lactococcus lactis, Evasins, Probiotics, Chemokine binding, Inflammatory bowel disease, Caco-2 cell model
Škrlec K., Štrukelj B., Berlec A. (2016). Surface display of evasins on recombinant lactic acid bacterium Lactococcus lactis for the treatment of inflammatory bowel disease. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 116.). IPC2016