Immunotargeting of E.coli TolC outer membrane channel

Abstract

TolC is an outer-membrane channel of Gram-negative bacteria, responsible for extrusion of antibiotics and other toxic compounds from the cell and plays a determinant role in bacterial resistance. Together with two other proteins, an inner membrane efflux pump and a periplasmic fusion protein, TolC forms a complex efflux system. Interestingly the specificity of this complex is defined by the type of efflux pump to which TolC binds to, thus making TolC a common and critical component in a wide range of efflux systems. This channel, composed by 428 residues, creates a duct in the outer-membrane given its homotrimeric conformation. Structurally, it is composed by a α-helix motif in the periplasmic side and a β-barrel inserted in the outer-membrane. This later harbors an extracellular loop that is a central feature for the purpose of this work. The main goal of this work was to generate antibodies against E.coli TolC channel with the intention of blocking or modulate its extrusion activity. In order to achieve this, three recombinant forms of E.coli TolC, two full-length forms and a linear epitope one, were produced and characterized by SDS-PAGE, size exclusion chromatography (SEC), western blot and differential scanning calorimetry (DSC). These results revealed the purity, robustness and stability of the produced proteins, ideal for the immunizations protocols. Produced proteins where then used to immunize experimental model birds, namely quails (Coturnix japonica), making using of the unique setup of CNC’s Avian Facility. Antibodies produced were then purified from the egg yolk of hyperimmune birds and biochemical characterized by ELISA and western blot in terms of their targetspecificity and reactivity. These antibodies presented good reactivity against both recombinant TolC proteins, specially recognizing the extracellular region of TolC channel. This work confirmed that the E.coli TolC is suitable for immunotargeting using avian antibodies, particularly the extracellular region of the channel. We believe that our results are a good starting point for subsequent characterization of the anti-TolC antibodies through functional efflux assays. Ultimately this will allow the development of therapeutic monoclonal anti-TolC antibodies to be used to fight bacterial resistance.