From left: Dr Richie Tay, Dr Dave Ow, Lim Pei Yu
Lactic acid bacteria (LAB) of the genus Lactobacillus are widely used in food and beverage fermentation and in probiotic formulations. They survive passage through the gastrointestinal tract to persist as integral members of the gut microbiota. This makes them promising delivery vehicles for protein drugs to the gut. One mode of delivery is to anchor the protein to the bacteria cell wall. With its mix of peptidoglycans and other carbohydrate polymers, the cell wall offers many sites where proteins can bind either covalently or non-covalently. Many native cell wall proteins contain specialised domains that anchor them to one of these cell wall components. We have isolated a novel anchoring domain from a LAB cell wall protein, and showed that it can be recombinantly fused to other proteins-of-interest to anchor them non-covalently to the bacteria surface.
The anchoring domain (CAD4a) comprises multiple repeats of a bacterial SH3_5 motif, which is known to interact with peptidoglycans in the bacteria cell wall. Using a fluorescent protein with a terminal fusion of CAD4a, we found the best protein display on Lactobacillus fermentum among the LAB tested. We then characterised the buffer conditions for optimal cell wall anchoring. We showed that the binding capacity of CAD4a on L. fermentum was comparable to that currently achieved with other non-covalent anchoring domains. Superoxide dismutase, an antioxidant enzyme useful for treating bowel inflammation, was successfully anchored to L. fermentum via fusion to CAD4a. By encapsulating the bacteria in a matrix of edible polymers, we showed that the displayed enzyme could be protected from gastric digestion. Taken together, our results provided a comprehensive initial assessment for using this novel anchoring domain for heterologous protein display on LAB.
There is mounting evidence for the importance of the gut microbiome in maintaining general health, and concurrent interest in engineering the gut microbiome to shape health outcomes. LAB, many of which are widely consumed as probiotics, are promising candidates for clinical applications due to their consumer recognition and safety record, and decades of industrial know-how in cell culture. LAB have been engineered to produce therapeutic proteins after oral delivery, though such strains of genetically modified bacteria may face resistance with regulators and consumers. Using our non-covalent anchoring domain (CAD4a), we can coat the surface of LAB with therapeutic proteins and employ them as drug delivery vehicles, without introducing genetic modifications to the bacteria. Such a platform could be developed for the treatment of various gut diseases, and for gut-based mucosal vaccination.
The figure shows how non-genetically modified LAB can be used as hosts for protein delivery by coating them with the protein fused to CAD4a, our anchoring domain that binds to the bacteria cell wall.
Tay PKR, Lim PY and Ow DS-W (2021) A SH3_5 Cell Anchoring Domain for Non-recombinant Surface Display on Lactic Acid Bacteria. Front. Bioeng. Biotechnol. 8:614498. doi: 10.3389/fbioe.2020.614498
Richie TAY Pei Kun, Dave OW Siak Wei (2020) Fusion protein for bacterial surface display. Singapore Patent Application 10202008968Y.