Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

摘要

The probiotic Escherichia coli strain Nissle 1917 (EcN) is one of the few probiotics licensed as a medication in several countries. Best documented is its effectiveness in keeping patients suffering from ulcerative colitis (UC) in remission. This might be due to its ability to induce the production of human β-defensin 2 (HBD2) in a flagellin-dependent way in intestinal epithelial cells. In contrast to ulcerative colitis, for Crohn's disease (CD) convincing evidence is lacking that EcN might be clinically effective, most likely due to the genetically based inability of sufficient defensin production in CD patients. As a first step in the development of an alternative approach for the treatment of CD patients, EcN strains were constructed which were able to produce human α-defensin 5 (HD5) or β-defensin 2 (HBD2). For that purpose, codon-optimized defensin genes encoding either the proform with the signal sequence of human α-defensin 5 (HD5) or the gene encoding HBD2 with or without the signal sequence were cloned in an expression vector plasmid under the control of the T7 promoter. Synthesis of the encoded defensins was shown by Western blots after induction of expression and lysis of the recombinant EcN strains. Recombinant mature HBD2 with an N-terminal His-tag could be purified by Ni-column chromatography and showed antimicrobial activity against E. coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes. In a second approach, that part of the HBD2 gene which encodes mature HBD2 was fused with the yebF gene. The resulting fusion protein YebFMHBD2 was secreted from the encoding EcN mutant strain after induction of expression. Presence of YebFMHBD2 in the medium was not the result of leakage from the bacterial cells, as demonstrated in the spent culture supernatant by Western blots specific for β-galactosidase and maltose-binding protein. The dialyzed and concentrated culture supernatant inhibited the growth of E. coli, S. enterica serovar Typhimurium and L. monocytogenes in radial diffusion assays as well as in liquid culture. This demonstrates EcN to be a suitable probiotic E. coli strain for the production of certain defensins.