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

Lead Research Organisation: Queen Mary, University of London
Department Name: Sch of Biological and Chemical Sciences

Abstract

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 NMR
b) Study interactions between LspC TMHR residues and LspL/LspM using X-ray crystallography and/or NMR
c) 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)

Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/N50953X/1 30/09/2016 29/09/2021
1806169 Studentship EP/N50953X/1 30/09/2016 29/06/2020 Theo John Portlock
 
Description The bacterial type II secretion system (T2SS) is a 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. Although much is known about the structures of the compositional proteins of the system, recognition and recruitment of substrates by the system is a poorly understood process. Observation of binding between putative substrates and the periplasmic elements of the secretin GspD as well as the inner membrane platform protein GspC has implied their importance in performing this task. In this study, the structure of the N0 domain of the Legionella pneumophila homologue of GspD (LspD), the TMHRHR region of GspC (LspC), and the substrate NttA were solved by solution NMR. Then, through computationally docking the system elements and NttA, guided by NMR titration data, a recognition complex was characterised revealing amino acid residues essential for binding. This study suggests a novel model that can ultimately be used in the antibiotic drug design of inhibitors to the formation of the recognition complex.
Exploitation Route With a model of the recognition complex of the T2SS, the development of T2SS inhibitors is the logical next step to my investigation. This form of inhibitor may have the potential to create antimicrobial surfaces for hospitals or elsewhere.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Structural studies into novel membrane associated virulence factors of Legionella pneumophila
Amount £460,082 (GBP)
Funding ID MRR0176621 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2019 
End 04/2022
 
Description Hank Seifert 
Organisation Northwestern University
Country United States 
Sector Academic/University 
PI Contribution new work looking into Neisseria effectors
Collaborator Contribution plasmid DNA
Impact none yet
Start Year 2019
 
Description Prof. Nick Cianciotto 
Organisation Northwestern University
Department Feinberg School of Medicine
Country United States 
Sector Academic/University 
PI Contribution Structural and biochemical studies of proteins directly related to this grant
Collaborator Contribution Microbiology and mutation analysis to verify data produced in my lab
Impact Paper re currently in preparation but have not yet been submitted
Start Year 2015
 
Description Participation in an open day or visit at my research institution - Nuffield Research Placement of A-level students in the lab 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Nuffeild research project placement
Year(s) Of Engagement Activity 2019