Structural and Functional Assessment of Listeria Adhesion Protein as a Tight Junction Modulator: Toward a Safer and More Effective Oral Drug Delivery System

This thesis explores the intricate mechanisms governing paracellular permeability, a process vital for maintaining homeostasis and barrier integrity in epithelial and endothelial tissues. Central to this exploration are tight junctions (TJs), multi-protein complexes that regulate substance movement between cells. The thesis begins with a comprehensive literature review synthesizing current research on how bacterial and viral proteins can enhance paracellular permeability or modulate TJs, highlighting their significance in both scientific and medical fields.

A focal point of the thesis is the detailed study of the Listeria adhesion protein (LAP), an epithelial TJ modulator (TJM). LAP’s interaction with the epithelial receptor, Hsp60, facilitates the translocation of Listeria monocytogenes across the epithelial barrier, presenting a novel approach for drug delivery. Addressing the limitations of current TJMs, such as poor bioavailability and toxic side effects, the thesis provides an in-depth analysis of LAP as an alternative biologics’ delivery vehicle. Utilizing a multidisciplinary research approach, the study reveals the cryo-EM structure of LAP at 2.9 Å resolution, elucidating its multimeric formation and interaction with Hsp60 through electrostatic forces, as supported by computational modeling.

The functional efficacy of LAP is further established through experiments demonstrating its capability to transport drug analogs across epithelial cell monolayers comparably to established TJMs. Moreover, in vivo studies using a mouse oral gavage model show that LAP significantly enhances the blood levels of peptide drugs without triggering inflammatory responses or harming tight junction architecture. This thesis ultimately positions the LAP-Hsp60 complex as a promising TJM candidate, offering an innovative means for enhancing oral drug delivery and maintaining epithelial barrier integrity.