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Open AccessArticle

Engineered Lactococcus lactis Secreting IL-23 Receptor-Targeted REX Protein Blockers for Modulation of IL-23/Th17-Mediated Inflammation

1
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia
2
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia
3
Laboratory of Ligand Engineering, Institute of Biotechnology of the Czech Academy of Sciences, v. v. i., BIOCEV Research Center, Průmyslová 595, 252 50 Vestec, Czech Republic
4
Laboratory of Structural Bioinformatics of Proteins, Institute of Biotechnology of the Czech Academy of Sciences, v. v. i., BIOCEV Research Center, Průmyslová 595, 252 50 Vestec, Czech Republic
*
Authors to whom correspondence should be addressed.
Microorganisms 2019, 7(5), 152; https://doi.org/10.3390/microorganisms7050152
Received: 16 April 2019 / Revised: 9 May 2019 / Accepted: 23 May 2019 / Published: 27 May 2019
(This article belongs to the Special Issue Gastrointestinal Microbiota Impacts Human Health and Disease)
Lactococcus lactis, a probiotic bacterium of food origin, has recently been demonstrated as a suitable strain for the production and in vivo delivery of therapeutically important proteins into the gut. We aimed to engineer recombinant L. lactis cells producing/secreting REX binding proteins that have been described as IL-23 receptor (IL-23R) blockers and IL-23R antagonists suppressing the secretion of cytokine IL-17A, a pivotal step in the T-helper Th17-mediated pro-inflammatory cascade, as well as in the development of autoimmune diseases, including inflammatory bowel disease (IBD). To reach this goal, we introduced cDNA sequences coding for REX009, REX115, and REX125 proteins into plasmid vectors carrying a Usp45 secretion signal, a FLAG tag sequence consensus, and a LysM-containing cA surface anchor (AcmA), thus allowing cell–surface peptidoglycan anchoring. These plasmids, or their non-FLAG/non-AcmA versions, were introduced into L. lactis host cells, thus generating unique recombinant L. lactis–REX strains. We demonstrate that all three REX proteins are expressed in L. lactis cells and are efficiently displayed on the bacterial surface, as tested by flow cytometry using an anti-FLAG antibody conjugate. Upon 10-fold concentration of the conditioned media, a REX125 secretory variant can be detected by Western blotting. To confirm that the FLAG/non-FLAG REX proteins displayed by L. lactis retain their binding specificity, cell-surface interactions of REX proteins with an IL-23R-IgG chimera were demonstrated by flow cytometry. In addition, statistically significant binding of secreted REX009 and REX115 proteins to bacterially produced, soluble human IL-23R was confirmed by ELISA. We conclude that REX-secreting L. lactis strains were engineered that might serve as IL-23/IL-23R blockers in an experimentally induced mouse model of colitis. View Full-Text
Keywords: lactococcus; binding protein; albumin-binding domain; IL-23 cytokine; IL-23R; surface display lactococcus; binding protein; albumin-binding domain; IL-23 cytokine; IL-23R; surface display
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MDPI and ACS Style

Plavec, T.V.; Kuchař, M.; Benko, A.; Lišková, V.; Černý, J.; Berlec, A.; Malý, P. Engineered Lactococcus lactis Secreting IL-23 Receptor-Targeted REX Protein Blockers for Modulation of IL-23/Th17-Mediated Inflammation. Microorganisms 2019, 7, 152.

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