Engineering recombinant Lactococcus lactis as a delivery vehicle for BPC-157 peptide with antioxidant activities

Abstract

Lactic acid bacteria (LAB) are attractive hosts for the expression of heterologous proteins and can be engineered to deliver therapeutic proteins or peptides to mucosal surfaces. The gastric stable pentadecapeptide BPC-157 is able to prevent and treat gastrointestinal inflammation by reducing the production of reactive oxygen species (ROS). In this study, we used LAB Lactococcus lactis as a vector to deliver BPC-157 by surface display and trypsin shedding or by secretion to the growth medium. Surface display of BPC-157 was achieved by fusing it with basic membrane protein A (BmpA) or with the peptidoglycan binding domain of AcmA and Usp45 secretion signal. While the expression of BmpA-fusion proteins was higher than that of AcmA/Usp45-fusion protein, the surface display ability of BPC-157 was approximately 14-fold higher with AcmA/Usp45-fusion protein. Release of BPC-157 from the bacterial surface or from isolated fusion proteins by trypsinization was demonstrated with anti-BPC-157 antibodies or by mass spectrometry. The concentration of BPC-157 delivered by surface display via AcmA/Usp45-fusion was 30 ng/ml. This increased to 117 ng/ml by Usp45 signal-mediated secretion, making the latter the most effective lactococcal delivery approach for BPC-157. Secreted BPC-157 significantly decreased ROS production in 149BR fibroblast cell model, suggesting its potential benefit in the treatment of intestinal inflammations. Additionally, a comparison of different modes of small peptide delivery by L. lactis, performed in the present study, will facilitate the future use of L. lactis as peptide delivery vehicle.

Bioactive peptides produced by engineered probiotics and other food-grade bacteria: A review

“…In this sense, food-grade LAB, with probiotic potential, including L. lactis , Pediococcus spp., and Enterococcus faecalis, have been metabolically engineered to increase the production of nisin, pediocin, and enterocin, respectively ( Plavec & Berlec, 2020 ). Several gene expression systems have been developed for heterologous peptide expression in LAB, being the nisin-controlled gene expression (NICE) system one of the most widely used inducible systems, involved in the biosynthesis of nisin A, a bacteriocin produced by several L. lactis ( Škrlec et al, 2018 ). NICE P nisA promoter is induced by the NisA peptide (nisin) through the two-component systems NikS and NikR, achieving a dynamic range of expression (up to 1000-fold) by adding increasing the concentration of nisin ( Kohl et al, 2020 ).…”

Bacteria-Based Microdevices for the Oral Delivery of Macromolecules

“…Gastric stable pentadecapeptide, BPC-157, is reported to be able to prevent and treat GI inflammations [51]. In order to deliver BPC-157 orally, lactic acid bacteria, which are capable of producing and delivering diverse therapeutic proteins via genetic engineering [52,[60][61][62][63], are used to produce BPC-157 fusing with a membrane protein. The heterologous peptide is successfully displayed on the bacterial surface and orally delivered into the host as a thera-peutic agent to reduce reactive oxygen species (ROS) production [52].…”

“…In order to deliver BPC-157 orally, lactic acid bacteria, which are capable of producing and delivering diverse therapeutic proteins via genetic engineering [52,[60][61][62][63], are used to produce BPC-157 fusing with a membrane protein. The heterologous peptide is successfully displayed on the bacterial surface and orally delivered into the host as a thera-peutic agent to reduce reactive oxygen species (ROS) production [52]. Another therapeutic peptide, glucagon-like peptide-1 (GLP-1), for the treatment of non-insulin-dependent diabetes, is delivered orally by a recombinant Lactococcus lactis that is genetically modified with a plasmid-encoding GLP-1 cDNA [41].…”

Engineering Tools for the Development of Recombinant Lactic Acid Bacteria

“…[ 43 ] The antioxidant activity of heterologous BPC‐157 peptide combined with the basic membrane protein (BmpA) and peptidoglycan binding domain of aconitase gene was compared and analyzed. [ 44 ] A 14‐fold higher activity in reductive oxygen species (ROS) reduction was detected with BmpA. Further, the LysM anchor system was applied in the production of anti‐ or pro‐inflammatory molecules.…”