PROJECT SUMMARY/ABSTRACT Bioactive peptides, or drugs made of small proteins, are used to treat an increasing number of human diseases. However, the fact that almost no peptide drugs can be delivered orally limits their use. A long-term goal and potential solution to this unmet need is the development of bioengineered probiotics – healthy gut bacteria designed to be taken orally and deliver drugs inside the gut. This strategy has shown promise: the probiotic Limosilactobacillus reuteri has recently been engineered to produce ShK-235, an analog of an injection peptide currently in clinical trials for treatment of autoimmune disease. While oral ShK-235 peptide or L. reuteri alone are ineffective in treating disease, oral L. reuteri engineered to secrete ShK-235 (LrS235) successfully treats autoimmune arthritis in rats, circumventing the requirement for injection. Nevertheless, it is not currently understood why the microbially delivered peptide can overcome barriers to oral delivery which the peptide alone cannot. This information is necessary for the continued characterization, development, and improvement of this novel drug delivery strategy. Therefore, the overall objective of this proposal is to uncover the unique mechanisms enabling oral LrS235 to deliver ShK-235 intact into the bloodstream. The central hypothesis is that live LrS235 adheres to intestinal mucus, where secreted ShK-235 is concentrated and diffused between epithelial cells into circulation to exert its therapeutic effect. This hypothesis will be tested with two specific aims, each focusing on a distinct arm of this host-microbe interaction. For both aims, absorption studies will be performed in rats, using electrophysiology and immune cell proliferation assays as functional metrics of drug absorption. Experiments proposed in Aim 1 will determine the microbe-intrinsic factors necessary for absorption of ShK-235 from oral LrS235. Strains of LrS235 deficient in mucus adherence or secretory capacity will be used to determine what properties of LrS235 govern successful absorption. Experiments proposed in Aim 2 will determine the primary pathways by which ShK-235 from oral LrS235 permeates the gut epithelium. The permeability of microbially delivered ShK-235 across rat intestinal tissue ex vivo will be characterized with Ussing chamber assays as well as localized in vivo using immunofluorescence microscopy to tease apart what path microbially delivered ShK-235 takes from the intestine into the bloodstream. The Principal Investigator’s training environment, spanning labs with expertise in probiotic engineering and immune modulation in collaboration with experts in intestinal microscopy and absorption, is ideal for tackling this interdisciplinary research question. The integrated training plan proposed builds experience in microbiology and immunology and comprehensively prepares the Principal Investigator for a translational academic physician- scientist career. Ultimately, the proposed...