Discovery of Novel Glycosidase-Derived Cell-Penetrating Peptides Encoded by Human Gut Commensals

Intracellular delivery of therapeutics remains a major challenge for modern medicine. To enhance intracellular uptake, therapeutics can be delivered with carrier proteins possessing an inherent cell-penetrating activity. There is an increasing need for new cell-penetrating carriers with diverse biophysical properties and mechanisms of action to transport a wide range of therapeutic cargo. As many cell-penetrating proteins and peptides derive from natural proteins, we sought to mine a previously unexplored community, the human gut microbiome, for cell-penetrating sequences. Here, we performed a high-throughput functional metagenomic screen to identify cell-penetrating protein fragments from the human gut microbiome. We identified protein fragments encoded within glycosidase enzymes from members of the Bacteroidetes phylum that mediate internalization into human cell lines when displayed on the surface of nonpathogenic, noninvasive Escherichia coli. We investigate one fragment, dubbed Gh_112, that adheres to human fibronectin, activates multiple endocytic pathways, and specifically promotes uptake of E. coli into multiple cancerous epithelial cell lines rather than healthy epithelial tissue in vitro. Overall, this work demonstrates that the human gut microbiome is a source of cell-penetrating sequences and expands the known repertoire of cell-penetrating carrier systems.