Engineered Curli-Expressing Probiotic Bacteria as a Platform for Therapeutics and Diagnostics

Abstract

Extracellular matrix-associated proteins play important roles in host-microbe interactions. We and others have identified curli amyloid fibers, which are major components of the extracellular matrix produced by Enterobacteriaceae during biofilm formation, to be a highly robust and versatile platform for microbial engineering. To exploit these curli amyloid fibers, we have developed a platform called “Biofilm Integrated Nanofiber Display” (BIND), in which we genetically engineered CsgA, a major subunit of curli fibers, to have novel properties either by adding functional domains or by modifying the CsgA sequence to influence the properties of curli fibers. In this work, we demonstrate the ability of the BIND platform to influence the host-microbe interaction in vitro, the amelioration of colitis in murine models using engineered probiotics, the formation of self-renewal living hydrogels, and the non-invasive tracking of engineered bacteria in vivo. We used BIND to rationally design a bacterial strain that could interact with mucosal epithelium models. We engineered a commensal strain of E. coli to produce curli fibers fused to the trefoil factor family (TFF) peptides. These engineered microbes showed improved affinity for soluble mucins, Caco-2 intestinal cell lines, and goat intestinal explants. Some curli-TFF conjugates/fusions also enhanced cell migration of simulated Caco-2 wounds, which reflected their bioactivity comparable to their soluble TFF counterparts. Next, we engineered probiotic E. coli Nissle 1917 (EcN) to produce curli-TFFs in vitro and in vivo and confirmed that these engineered bacteria were non-pathogenic. Inoculation with the probiotic conferred improved protective effects against dextran sodium sulfate induced colitis in mouse models associated with barrier function enforcement and immunomodulation. We also found that engineered commensal E. coli expressing curli-TFFs could be fabricated into living hydrogels consisting of concentrated bacterial cultures. Such hydrogels had the ability to selectively interact with the gastrointestinal (GI) tract and to self-renew in vitro and in vivo. Finally, we demonstrated a method for tracking engineered microbes within the GI tract via non-standard amino acid incorporation into extracellular curli fibers followed by click chemistry-based labeling. Overall, this work lays the foundation for a novel and versatile platform for creating engineered probiotics with programmable therapeutic and diagnostic functions.