pNZ8148

Dysregulated production of interleukin (IL)-6 has been implicated in the pathology of inflammatory bowel disease (IBD). Safe probiotics that neutralize IL-6 in the gut may help relieve intestinal inflammation. Here, researchers developed Lactococcus lactis with potent and selective IL-6 binding activity by displaying an IL-6-specific affinity body on its surface. The anti-IL-6 affibody (called ZIL) was expressed as a fusion with the lactococcal secretory peptide Usp45 and the anchoring protein AcmA. A large number of ZIL fusion proteins were detected on the bacterial surface, and their functionality was verified by confocal microscopy and flow cytometry. ZIL-displaying L. lactis sequestered recombinant human IL-6 from solution in a concentration-dependent manner with up to 99% efficacy and did not bind other proinflammatory cytokines, thus demonstrating high specificity for IL-6. The ability of engineered bacteria to capture IL-6 from cell culture supernatants was evaluated using immunostimulatory human monocyte cell lines (THP-1 and U-937) differentiated into macrophage-like cells. L. lactis exhibiting ZIL reduced IL-6 content in the supernatants of both cell lines by up to 94% in a concentration-dependent manner.

In this study, the gene encoding the IL-6 binding protein ZIL was codon-optimized for L. lactis, synthesized, and cloned into a lactococcus plasmid for surface display. A labeling tag sequence (DYKDDDDK) is attached to the N terminus to facilitate antibody detection. This binder is fused to the Usp45 signal peptide and AcmA anchor to enable its secretion into the growth medium and subsequent binding to the bacterial surface (Figure 1a). By Coomassie blue staining and Western blot analysis, a band with an apparent molecular weight of ~35 kDa was detected in the entire lysates of host cells, whereas no signal was present in the cell lysates of control cells containing the empty plasmid pNZ8148 (Figure 1b). To confirm that the nisin promoter was activated, the researchers compared protein expression in induced and uninduced bacterial cultures. Bands representing ZIL fusion proteins were present in whole-cell lysates from induced cultures, whereas they were absent from uninduced cultures.

Figure 1. The IL-6 binding affibody ZIL is expressed in L. lactis. a Genetic construct for expression of IL-6 binding affibody ZIL on the surface of L. lactis. b Coomassie brilliant blue stained SDS-PAGE gel (left) and Western blot analysis (right) of L. lactis whole lysate containing plasmid pSD-ZIL or pSD-ZIL-flag. Cont., L. lactis containing empty plasmid pNZ8148. (Zahirović, Abida, and Aleš Berlec. 2022)