Developing Heterologous Protein Secretion Tools for Bacteroides Species

Bacteroides species, one of the most abundant and prevalent bacterial populations in the human gut, are capable of long-term, stable colonization of the gastrointestinal tract, making them a promising chassis for developing long-term treatment and prevention for chronic diseases. However, a lack of efficient heterologous protein secretion tools prevents their use as engineered, on-site delivery vehicles for protein-based biologic drugs or disease-responsive reporters. To address this limitation, we systematically investigated methods to enable heterologous protein secretion using both endogenous and exogenous secretion systems in Bacteroides thetaiotaomicron. Here, we revealed a group of signal peptides and full-length proteins that can be used as secretion carriers to export functional antibody fragments and reporter proteins across multiple Bacteroides species at high titers. To provide a more complete understanding of these secretion tools, we characterized the post-secretion extracellular fate and the cargo size limit of several secretion carriers. Finally, we validated the activity of our secretion carriers in vivo by observing the production and secretion of reporter proteins from engineered Bacteroides strains in the mouse gut. This toolkit expands the potential therapeutic impact of stably-colonizing commensal bacterial strains, enabling them to deliver protein-based therapeutics from within the gut over long periods of time, which may support more effective treatment strategies for chronic gastrointestinal disease.