Expression of Cell-Adhesion Molecules in E. coli: A High Throughput Screening to Identify Paracellular Modulators

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Cell-adhesion molecules (CAMs) mediate crucial cellular interactions.
• Tight junctions (TJs) regulate paracellular permeability, with no current therapeutic modulators.
• Claudins (CLDNs) are key protein components of TJs.

Purpose of the Study:

• To develop a novel high-throughput screening (HTS) method for identifying TJ modulators.
• To investigate the functional expression of CLDNs in a bacterial system.
• To screen for small molecules that modulate CLDN2-mediated paracellular permeability.

Main Methods:

• Engineered *E. coli* to express CLDN proteins in its outer membrane.
• Utilized Flow Cytometry (FC) to quantify bacterial aggregation induced by CLDN expression.
• Developed the iCLASP (inspection of cell-adhesion molecules aggregation through FC protocols) HTS method.
• Validated identified compounds in the mammalian A549 cell line.

Main Results:

• Induced CLDN expression in *E. coli* shifted behavior from unicellular to multicellular aggregation.
• The iCLASP method successfully enabled HTS for CAM interactions.
• Identified potential paracellular modulators for CLDN2.
• Demonstrated proof-of-concept for iCLASP in a mammalian cell line.

Conclusions:

• Bacterial expression of CLDNs provides a novel platform for studying CAMs.
• iCLASP is an effective HTS method for discovering TJ and paracellular permeability modulators.
• This approach holds promise for developing new therapeutics targeting TJs.