Engineering the bacterial flagellum: tissue bioglue

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Matthew Hicks, P.C. Wright, R. Ridley and G.P. Stafford

Department of Oral Pathology, United Kingdom

Bacterial flagella are extensively characterised extracellular appendages used for locomotion. Being long proteinaceous fibres, they present great potential as vehicles to display bioactive peptides. We have targeted the field of poorly healing wounds as an application of flagellar display technology. Such wounds are characterised by poor vasculature, a dearth of adhesive molecules – such as collagen IV, VII and laminin – and a surfeit of collagen I and degradative enzymes. The E. coli flagellar filament is comprised of a protein called flagellin (FliC) consisting of four domains denoted D0, D1, D2 and D3. Domains D0 and D1 are formed from two peptides each, one located at the N-terminus and the other at C-terminus of the FliC peptide, and form the core of the polymerised flagellin filament. The D2 and D3 domains face the exterior of the molecule, and can be removed without affecting polymerisation. We have produced a set of modular peptide presentation plasmids that enable replacement of varying lengths of the D2 and D3 domains with candidate bioglue molecules. These include collagen and laminin binding proteins from bacterial and human origin. All fusion proteins effectively polymerise and can be produced and purified from E. coli. In 2D models, these “bioglues” effectively bind collagen I and laminin and furthermore promote attachment of keratinocytes to surfaces. Several E. coli strains have been engineered for their ability to allow optimal production of a flagellin fusion protein under the control of an artificial promoter. Optimum production parameters for an apparatus that requires growth under aerobic, low-shear conditions are also defined.