Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

• Published in: PloS one 2016-09, Vol.11 (9), p.e0162625-e0162625
• Main Authors: Zadravec, Petra, Marečková, Lucie, Petroková, Hana, Hodnik, Vesna, Perišić Nanut, Milica, Anderluh, Gregor, Štrukelj, Borut, Malý, Petr, Berlec, Aleš
• Format: Article
• Language: English
• Subjects: Acids; Albumin; Albumins - metabolism; Bacteria; Binding; Biology; Biology and Life Sciences; Biotechnology; Cell Surface Display Techniques; Combinatorial analysis; Cytometry; E coli; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Flow Cytometry; Food contamination & poisoning; Gene expression; Genetic engineering; Golgi apparatus; Health aspects; HeLa Cells; Humans; Immobilized Proteins - metabolism; Immunoglobulins; Infections; Intestine; Laboratories; Lactic acid; Lactic acid bacteria; Lactococcus lactis; Lactococcus lactis - metabolism; Libraries; Ligands; Medicine and Health Sciences; Peptidoglycans; Pharmacy; Probiotics; Protein Binding; Protein Domains; Protein Subunits - metabolism; Protein Transport; Proteins; Recombinant Proteins - metabolism; Recombination, Genetic - genetics; Research and analysis methods; Retrograde transport; Ribosomes - metabolism; Secretion; Sequence Homology, Amino Acid; Shiga toxin; Shiga Toxin 1 - chemistry; Shiga Toxin 1 - metabolism; Shigella dysenteriae; Surface Plasmon Resonance; Thermophoresis; Toxins
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0162625

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.