Processing of Cargo Proteins By The Type IX Secretion System.

Abstract

Porphyromonas gingivalis is a Gram-negative oral anaerobic pathogen and is one of the key causative agents of the periodontitis. P. gingivalis utilises a range of virulence factors to drive pathogenesis that are exported and attached to the cell surface via the Type 9 Secretion System (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS via an unknown mechanism. The T9SS transports cargo proteins to the outer membrane where the CTD is remove and the cargo is anchored to the cell surface. The aim of this thesis was to provide structural characterisation of the CTD and to understand how it interacts with components of the T9SS. To achieve this, solution state NMR was used to study the structure and dynamics of the CTD from RgpB, showing it to be a seven stranded β sandwich, in alignment with the crystallographic model. The model presented here, supported by NMR relaxation data, suggests RgpB- CTD a monomer in solution, unlike the crystal structure, in which a dimeric state is observed. It has previously been shown that the outer-membrane β-barrel protein PorV is able to interact with T9SS cargo proteins. It has been possible to recombinantly express and purify the full length PorV protein and work is ongoing to demonstrate that it interacts with RpgB-CTD via a pulldown assay. In this study a predicted structure of P. gingivalis PorV was generated and simulated in a lipid bilayer using GROMACs. Representative conformations were used to generate a structural ensemble for molecular docking with the NMR model of RgpB-CTD. Binding was examined between the two proteins and v multiple binding poses were observed. From these models two possible mechanisms of CTD recognition by PorV are suggested. Future work to experimentally study this interaction will allow for the models to refined further.