Substrate Recognition by the Legionella pneumophila Type II Secretion System.

Abstract

The bacterial Type II Secretion System (T2SS) is a transmembrane multiprotein assembly that ejects folded protein substrates from the periplasm into the extracellular milieu. These substrates are essential for pathogenesis in humans, animals, and plants. In the intracellular pathogen Legionella pneumophila, the T2SS has been shown to play an important role in evading recognition by the host during infection. Although much is known about the structures of the compositional proteins of the system, recognition and recruitment of substrates for secretion by the system is a poorly understood process. Previous studies have demonstrated binding between the inner membrane protein XcpPGspC and T2SS substrates in Pseudomonas aeruginosa thus implicating its involvement in this process. In this thesis, the periplasmic linker region of the L. pneumophila homologue of XcpPGspC (LspCGspC) and the T2SS substrate Novel Type II secreted protein A (NttA) (13 kDa) were recombinantly expressed and puri ed in Escherichia coli. Using NMR spectroscopy titration experiments, a weak fast-exchange interaction between these two proteins was observed, showing similar a nities to that previously demonstrated in the study in P. aeruginosa. Solution structures of the two proteins were then solved by NMR spectroscopy. Then, through the computational docking of these two proteins and guided by NMR titration data, a recognition complex was characterised revealing residues essential in the binding process. This is the rst discovered structure of a recognition complex between the T2SS and its substrates in L. pneumophila. This study suggests a novel pharmacophore model that may be used in antibiotic drug design of inhibitors to the formation of the recognition complex.