Tight Junction-like structures in E. coli
Tight Junctions are the master regulators of the paracellular space and the gatekeepers of the blood-tissue barriers. Claudins are the membrane proteins responsible for cell-cell contacts at the Tight Junctions. Absorption enhancers (AE) are molecules that disrupt the paracellular space at the Tight Junction level, allowing for increased transport of material between cells, thus, greatly enhancing the translational potential for drug delivery and even for enabling new drug discovery. Currently, no AE have been identified for clinical use because the study of Tight Junctions involves mammalian cell monolayers in a very low throughput setting. We engineered Tight Junction-like structures in E. coli, driving cell-to-cell interactions. This new approach provides us with a high-throughput method to investigate large libraries of compounds to identify AE.
The role of Tight Junctions in Mechanotransduction
Cells have three sources of energy: chemical, electrical, and mechanical. We know very little about the third. The study of mechanical transduction is difficult because the stimulus at the site of a transducer is generally unknown. Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Using a recently described optogenetic tool that upregulates and downregulate such forces with high spatiotemporal accuracy, we aim to interrogate epithelial monolayers that contain Tight Junctions. The anchoring of Tight Junctions to the actin cytoskeleton has not been studied in depth. There are many indirect evidence of Tight Junction role in mechanotransduction. We seek to clarify some of those findings and further our understanding of the role of Tight Junctions in mechanotransduction.
The role of Tight Junctions in Host pathogen interactions
For many bacterial pathogens, surface capsules are the first line of contact with the host. Over the past 16 years, the occurrence of a β-(1→6)–linked polymeric-N-acetylglucosamine (PNAG) polysaccharide has been discovered as a common component of serious and diverse prokaryotic and eukaryotic pathogens. The conserved production of PNAG suggests that it is a critical factor in microbial biology. We are using E coli outer membrane vesicles (OMV) coated with PNAG to represent pathogens interacting with epithelial cells of the host to study the role of Tight Junctions. An indispensable role of Tight Junctions involved in pathogen infection has been widely demonstrated since disruption of Tight Junctions leads to a distinct increase in paracellular permeability and polarity defects, which facilitate viral or bacterial entry and spread. The use of OMV-PNAG results in a great tool to begin understanding the role of Tight Junctions in host pathogen interactions.
Wound healing and Inosculation
Among the many things we know of Tight Junctions, little is known about the process of strengthening them. The main focus of laboratories around the world study absorption enhancers to disrupt the Tight Junctions. We are looking to identify molecules, and engineer proteins, that may bring cells together to improve the barrier properties of the paracellular space. Bridging gaps between cells can be beneficial in organ and tissue engineering, wound healing and prevention of metastasis in tumors, among others. Inosculation is the matching of a graft and an organ, at the cellular and molecular level, specifically the blood vessels. The endothelium in blood vessels is organized largely through Tight Junctions. Using engineered proteins, we are beginning to understand basic aspects of bridging the paracellular space to bring cells together.
Tight Junctions are composed of soluble and membrane proteins. Claudins are the membrane proteins responsible for cell-cell contacts. Claudins are organized as a belt around the apical domain of the cell. This organization contains lateral contacts among claudin proteins (cis interactions) and also trans interactions to bring together neighboring cells. The key elements of this organization are not well understood. Additionally, claudin proteins can interact with other claudin proteins of the same type (homomeric interaction) or between different types (heteromeric interaction). A main focus is to understand these native interactions (observed under physiological conditions) as well as to identify non-native interactions. For example, we know of existing claudin pairs because they have been described as expressed in the same tissue. Others are not co-expressed in the same tissue, yet the characteristics of these new interactions could be of great worth to tissue engineering, translational solutions, nanotechnology and drug design.
The Atlas particle for drug delivery
We have engineered a proteic structure that behaves as a soluble pore. We have endowed this particle with absorption enhancers that interact with the Tight Junction. The interior pore can accommodate nanoparticles for drug delivery. The separate elements in this particle ensure selectivity as the absorption enhancers bind specifically to given Tight Junctions, and the pore allows the direct delivery of the cargo into the paracellular space. The absorption enhancers establish contact through claudin proteins in the Tight Junction, pushing cells apart but maintaining the connection through the proteic structure of the particle. This particle, called the ATLAS, is under development and further study for drug delivery.
The role of Tight Junctions in Inflammatory Bowel Disease (IBD)
IBD are characterised by inflammation that compromises the integrity of the epithelial barrier. The intestinal epithelium is not only a static barrier but has evolved complex mechanisms to control and regulate bacterial interactions with the mucosal surface. The characterisation of alterations in Tight Junction proteins as key players in epithelial barrier function in inflammatory bowel diseases is rapidly enhancing our understanding of critical mechanisms in disease pathogenesis as well as novel therapeutic opportunities. Currently many changes to the balance between claudin-2 and claudin-4 in IBD have been reported. IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of Tight Junctions has not been explored. Recent literature has shown that IQGAP1 modulates Tight Junction formation by controlling the expression and recruitment of claudin 2 and recruitment of claudin 4. This key fact places IQGAP1 in a central role to be altered during IBD.
Why we collaborate?
We truly believe that people will benefit from our research. We also believe people don’t have decades to wait for our research to advance to translational solutions. We call upon all those who are interested in helping people, to collaborate, and further our designs in order for the research to become clinical solutions at a speedy pace.