Recombinant production of selected autotransporters from proteus mirabilis for biochemical and biophysical studies

Autotransporters are a family of outer membrane proteins in Gram-negative bacteria. This family of proteins has a conserved architecture consisting of the membrane domain and the extracellular domain which contains the effector function of the protein. The membrane domain anchors the protein to the cellular surface and has a pore-like structure. The extracellular domain is connected to the anchor domain by means of an α-helix, which permits translocation of the extracellular domain. This family of proteins are referred to as autotransporters due to their ability to be transported to the extracellular surface of the cell without the need of external factors such as ATP, ion gradients or other proteins suggesting that the membrane domain is solely responsible for translocation across the outer membrane. Although highly conserved, autotransporters have numerous functionalities and are primarily involved in cell adhesion. The membrane domain is highly conserved across all bacterial species, however the extracellular domain has been found to vary greatly and have a diverse range of activities such as; protease, lipase, adhesin, phosphatase and haemolysin. Pathogenic Gram-negative bacteria contain typically several of these proteins to attach to host cells and evade an immune response. Proteus mirabilis, a causative agent in urinary tract infections of patients with indwelling catheters, is one such pathogenic bacteria. This project will investigate a selection of autotransporters involved in virulence of P. mirabilis as outlined by Alamuri and Mobley 2008. The conditions needed for expression of five predicted autotransporters from P. mirabilis were investigated, specifically: 0844 an acid phosphatase, 2575 an adhesin, 2341 a serine protease, 2174 an adhesin and 2126 a serine protease. Expression and purification of 2575, as well as the passenger domains of 0844 and 2575, was successful, enabling characterisation of both of these proteins using a variety of techniques such as circular dichroism, florescence spectroscopy as well as activity assays in the case of the passenger domain of 0844