Expression of cell-adhesion molecules in E. coli: a high-throughput method to identify paracellular modulators

Abstract

Cell-adhesion molecules (CAM) are membrane proteins responsible for cell-cell interactions or cell-extracellular matrix interactions. Among these proteins, claudins (CLDN), occludin (OCLN), and junctional adhesion molecules (JAM) are components of the tight junction (TJ), the single proteic structure tasked with safeguarding the paracellular space. The TJ is responsible for controlled permeability of blood-tissue barriers, regulating the passage of molecule passage by size and charge. Currently there is no translational solution to manipulate the TJ with the exception of Focused Ultra-sound (FUS) and Micro bubbling (MB) techniques, still in clinical trials. Here we describe the expression of TJ proteins in the outer membrane of E. coli and report its consequences. When expression is induced, the unicellular behavior of E. coli is replaced with multicellular aggregations that can be quantified using Flow Cytometry (FC). The adhesion properties of the aggregates are representative of the individual membrane proteins expressed. This method, called iCLASP (inspection of cell-adhesion molecules aggregation through FC protocols), allows the high-throughput interrogation of small-molecules influence on paracellular permeability, enabling for the first time the discovery of its modulators for therapeutic strategies.