Atomic insight into virulence factor translocation by Gram-negative bacterial type-II secretion systems

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Many Gram-negative bacterial pathogens use a type-II secretion system (T2SS), a syringe-like mechanism, to transport protein substrates into their surroundings. These exported substrates have diverse roles including the formation/establishment of biofilms, subversion of host responses to infection and promoting intracellular host invasion/survival. Substrates are first transported into the periplasm where they fold into their native states. In P. aeruginosa, substrates interact with the TMHR of its LspC equivalent (an unstructured linker between the transmembrane helix (TM) and homologous region (HR))2,3, causing conformational changes within and entry to its T2SS. It is thought that cargo is then transported through the pore by the pseudopilus, driven by ATP hydrolysis1,3. Mutational analyses of several substrates belonging to different bacteria indicate that residues essential for secretion are distantly located and come together to form a conformational structure upon folding4. However, with mutation-based approaches it is difficult to uncouple these events and as the T2SS targeting motif is a 3D one, a high-resolution structural strategy is essential. My group is currently focussing on the Legionella pneumophila T2SS5-8, which is highly amenable to structural studies. We have shown that several substrates of this T2SS, as in P. aeruginosa, also recognize residues within the TMHR linker. Interactions between LspC and LspD connect the inner and outer membrane components of T2SSs9, and we observe that the TMHR also provides interactions with LspD.Therefore, substrate recognition by LspC may be coupled to displacement of the LspC-LspD complex, allowing entry into the system. Moreover, the LspC-TMHR sequence adjacent to the substrate recognition site is in close proximity with LspL/LspM and likely interacts with these two proteins. Therefore substrate recognition of LspC may also trigger a conformational change in LspL/LspM that could provide a pathway for signal transduction resulting in active substrate export. The specific aims of this project will be to:a) Determine the atomic structures of one or more L. pneumophila T2SS substrates in complex with the TMHR of LspC using X-ray crystallography and/or NMRb) Study interactions between LspC TMHR residues and LspL/LspM using X-ray crystallography and/or NMRc) Determine whether the interactions identified in (a) and (b) are unique to L. pneumophila or are also observed other bacteria (P. aeruginosa, E. coli and D. dadantii) by analysing in vitro protein:protein interactions (e.g. NMR, chemical cross-linking, ITC, BiaCore)