Structural and Functional Investigation of Meningococcal TonB Dependent Transporters

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

Neisseria meningitidis (the meningococcus) is a major worldwide health concern causing septicaemia and bacterial meningitis. Meningococcal septicaemia develops rapidly and is fatal in 10% of cases and survivors can face permanent disability. There is currently no approved vaccine that protects against all meningococcal strains.

As a defence mechanism against invading bacteria, the human iron transport proteins transferrin and lactoferrin restrict iron availability. The meningococcus, with a requirement for iron, has two specialised iron transporter proteins on its surface that bind transferrin and lactoferrin and strip iron from them by an unknown mechanism. This project aims to investigate the structures of these transporters bound to their target human proteins, thus revealing the mechanism by which they collect iron.
This project will also investigate a) the structure of a related iron transporter that is currently in clinical trials as a vaccine component and b) the function and target uptake molecule of a similar transporter protein shown to be important in the initial meningococcal colonisation of the nasal pharynx. Results from these studies will improve our understanding of meningococcal infection, help in the development of meningococcal vaccines and potentially lead to novel antibiotic targets.

Technical Summary

Neisseria meningitidis (the meningococcus) is a human host specific pathogen and a major cause of bacterial meningitis and sepsis. Meningococcal septicaemia develops rapidly and is fatal in about 10% of cases with survivors facing the possibility of permanent disability. There is currently no approved vaccine that protects against all circulating meningococcal strains.
An essential feature of meningococcal survival and replication is its ability to extract iron from the human iron transport proteins transferrin and lactoferrin. The mechanism of this iron extraction is unknown but the key players are the outer membrane TonB dependent transporters (TBDTs) TbpA and LbpA (Transferrin/Lactoferrin binding protein A). Other interesting meningococcal TBDTs studied herein include the siderophore receptor FetA, a potential vaccine candidate, and NMB1829, which has an unidentified transport substrate but aids host colonisation.
This project will investigate the structural basis of iron uptake from host proteins by TbpA and LbpA. Initial investigations will study the formation of complexes between the TBDT, its target host protein and secondary lipoprotein with the aim of establishing a stable, monodispersed complex suitable for structural studies. The ultimate aim of this part of the work is to elucidate a high-resolution structure of binary or tertiary complexes to explain the iron uptake mechanism.
This project also aims to solve the structure of FetA to aid its development as a vaccine component. A collaborating meningococcal vaccine consortium has incorporated FetA into a vaccine formulation due to enter Phase I clinical trials and are therefore interested in its structure.
An additional TonB dependent receptor (NMB1829) has been identified as important for meningococcal colonisation of the nasal pharynx and will also be targeted with the aim of not only solving its structure, but more importantly revealing its target molecule by analysing protein expression and bacterial survival in the presence or absence of potential nutrients.
The same screen that identified NMB1829‘s role in colonisation also identified three soluble proteins as being important colonisation factors. This project will also structurally characterise these virulence factors and use the resulting structures and specific mutations to test predictions of their functions using biochemistry and microbiology experiments. Deciphering the roles of these proteins and other uncharacterised virulence factors is an important goal as it could lead to the identification of new virulence pathways and antibiotic targets in the meningococcus and potentially in other bacterial pathogens.
 
Description EQUIPMENT COMPETITION FOR MRC-FUNDED MID-CAREER RESEARCHERS IN UK UNIVERSITIES
Amount £51,584 (GBP)
Funding ID C0493 
Organisation Medical Research Council (MRC) 
Department Medical Research Foundation
Sector Charity/Non Profit
Country United Kingdom
Start 03/2014 
End 10/2014
 
Description Mutational analysis of HpuA 
Organisation Virginia Commonwealth University
Country United States 
Sector Academic/University 
PI Contribution We solved the HpuA:haemoglobin complex structure and analysed interaction biochemically.
Collaborator Contribution Our partners are conducting mutagenesis in live gonococci and analysing the effect on hemoglobin binding and heme uptake.
Impact This collaboration is leading ot a greater understanding of heme uptake by pathogenic neisseria. It is multi-disciplinary involving structural biology, biophysics and microbiology
Start Year 2014
 
Description School visits to CMBI open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact Approximately 100 pupils attended the open day. As part of this we discussed with them how using x-rays can help us 'see' things not visible under light microscopes and gave them hands on experience with microscopes and setting up crystallisation experiments.

Pupils were excited by the science on display and being able to get their hands on some equipment.
Year(s) Of Engagement Activity 2013