IL-23-mediated Th-17 cell activation and stimulation of IL-17-driven pro-inflammatory axis has been associated with autoimmunity disorders such as Inflammatory Bowel Disease (IBD) or Crohn’s Disease (CD). Recently we developed a unique class of IL-23-specific protein blockers, called ILP binding proteins that inhibit binding of IL-23 to its cognate cell-surface receptor (IL-23R) and exhibit immunosuppressive effect on human primary blood leukocytes ex vivo. In this study, we aimed to generate a recombinant Lactococcus lactis strain which could serve as in vivo producer/secretor of IL-23 protein blockers into the gut. To achieve this goal, we introduced ILP030, ILP317 and ILP323 cDNA sequences into expression plasmid vector containing USP45 secretion signal, FLAG sequence consensus and LysM-containing cA surface anchor (AcmA) ensuring cell-surface peptidoglycan anchoring. We demonstrate that all ILP variants are expressed in L. lactis cells, efficiently transported and secreted from the cell and displayed on the bacterial surface. The binding function of AcmA-immobilized ILP proteins is documented by interaction with a recombinant p19 protein, alpha subunit of human IL-23, which was assembled in the form of a fusion with Thioredoxin A. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular L.lactis-ILP recombinant variants by Enzyme-Linked ImmunoSorbent Assay (ELISA). We conclude that novel recombinant ILP-secreting L. lactis strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis.

• Klíčová slova
• IL-23, albumin-binding domain, binding protein, cytokine, lactococcus, surface display,
• MeSH
• buňky Th17 účinky léků   MeSH
• ELISA MeSH
• interleukin-23 metabolismus   MeSH
• Lactococcus lactis metabolismus   MeSH
• lidé MeSH
• proteiny genetika   metabolismus   farmakologie   MeSH
• průtoková cytometrie MeSH
• rekombinantní proteiny genetika   metabolismus   farmakologie   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• interleukin-23 MeSH
• proteiny MeSH
• rekombinantní proteiny MeSH

Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine.

• MeSH
• albuminy metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• ELISA MeSH
• HeLa buňky MeSH
• imobilizované proteiny metabolismus   MeSH
• Lactococcus lactis metabolismus   MeSH
• lidé MeSH
• metody zobrazení buněčného povrchu MeSH
• podjednotky proteinů metabolismus   MeSH
• povrchová plasmonová rezonance MeSH
• proteinové domény MeSH
• průtoková cytometrie MeSH
• rekombinace genetická genetika   MeSH
• rekombinantní proteiny metabolismus   MeSH
• ribozomy metabolismus   MeSH
• sekvenční homologie aminokyselin MeSH
• shiga toxin 1 chemie   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• albuminy MeSH
• imobilizované proteiny MeSH
• podjednotky proteinů MeSH
• rekombinantní proteiny MeSH
• shiga toxin 1 MeSH