Structural and functional analyses of the multifunctional nontypeable Haemophilus influenzae SAP transporter

Lead Research Organisation: Diamond Light Source
Department Name: Science Division

Abstract

In the cells of every living organism, a myriad of tasks needs to be carried out to sustain the processes of life. Proteins do most of this work - they contribute to the structure and functioning of the cell and regulate many of its processes. Scientists can use X-rays and electrons to visualize proteins to a level which allows them to distinguish the individual atoms that make up their structures and aids in understanding their mechanisms of action. Using this knowledge we can then design molecules (drugs) that can interfere with the cell machinery to treat disease. This can significantly reduce the time required to develop a drug - examples are medicine for the treatment of flu (e.g. Tamiflu) and HIV. Methods for determining protein structures by X-ray crystallography and electron microscopy and applying them to aid design of new drugs have become faster and more reliable; which is important in the race to develop novel antibiotics to combat the worrying rise in bacterial resistance. Here, we have chosen to determine the structure and mode of action of a protein system called SAP which is vital for the survival of the bacterium Haemophilus influenzae. This bacterium is a major causative agent of respiratory disease, the third leading cause of death worldwide. This bacterium co-exists harmlessly in most individuals in the nose and throat but can become invasive and cause disease. It is particularly prevalent in young children, the elderly, cystic fibrosis sufferers and smokers. In young children middle ear infections (otitis) often precede glue ear and are a major concern, as this can lead to hearing loss, problems with speech development and educational problems. Indeed otitis is the primary cause for the prescription of antibiotics for children in developed countries.
SAP in this bacterium, assists in bacterial nutrition and as a defence mechanism to our innate immune response. Virtually all forms of life depend on iron for survival. For H. influenzae to survive and cause disease it is essential that it obtains iron from its environment but bacteria like us (our skin) have a protective coat. This protective coat, made up of lipids, is called a membrane. Membrane proteins are embedded in this coat and some of them act like gateways for the entry or exit of various molecules. These gateways are critical for life because it is through them that the cell can get essential nutrients or throw out harmful substances like antibiotics. SAP is a membrane protein that can transport iron into the bacterial cell so it can grow and multiply. SAP also transports small molecules that are produced by the human immune system. Without the SAP transporter these molecules would tear the protective bacterial membrane, which in turn would kill the invading bacteria. The overall aim of our research is to look at the structure of the SAP transporter to understand how it transports these diverse compounds and with that knowledge contribute to the design, in essence, of plugs to block its ability to function. To do this we need to obtain high resolution pictures to enable us to see the atomic interactions responsible for transport. Membrane proteins cannot easily be removed from the cell membrane without the use of harsh detergents. Additionally they do not like water and this makes it difficult to get the proteins to form crystals which are needed to visualize their structure at high resolution by the diffraction of X-rays. We have obtained crystals of a part of the SAP transporter which shows we can use this technique to obtain high resolution data. In parallel we are using the technique of electron microscopy which can also provide high resolution images of the protein structure but does not require the protein(s) to be crystallized. The structural data obtained will enable us to fully understand how SAP works and help in antibiotic discovery.

Technical Summary

No structural data is currently available for the nontypeable Haemophilus influenzae (NTHi) SAP transporter and the molecular mechanisms for the transport of haem and APs are unknown. We will use X-ray crystallography and electron microscopy to determine the molecular structure of the multi-protein SAP (and Trk) complex with the aim of identifying protein interactions that enable haem and AP transport. To obtain material for structural studies we will in parallel clone, express and purify all the components that make up the Sap/Trk complex (up to 8 individual proteins) with a focus on co-expression and co-purification to determine the structure of the complete Sap transporter. A unique collaboration will ensure that the fastest and most appropriate techniques are used - high throughput cloning/expression testing at the OPPF-UK, membrane protein focused biochemistry at the MPL , diffraction data collection at DLS beamlines, and cryo-EM and imaging at OPIC (U. Oxford) and the electron Bioimaging Facility eBIF (Diamond) when it comes online in 2015. Structural data obtained from Diamond MX beamlines and cryo-EM will be used to guide functional characterisation through use of site directed mutagenesis and will allow preliminary in silico design of small molecule inhibitors to disrupt transporter function (U. Oxford). The impact of impaired mutant Sap components will be assessed in vitro via the proteoliposome assay and in a murine model of NTHIi infection (MRC Harwell).

Planned Impact

The impact on public health, especially in young children, as a result of disease caused by Nontypeable Haemophilus influenzae (NTHi) infections is significant. Occurrence of acute otitis media (AOM) is extremely common and is a leading cause of GP visits and antibiotic prescription in young children (80% of children are expected to have experienced at least one episode of AOM by their 3rd birthday). Chronic otitis media is an important cause of preventable hearing loss and the main reason for impaired speech and learning abilities. Due to this high incidence, the impact on global health is significant and directly impacts the proper use of antibiotics. Chronic respiratory diseases, such as chronic obstructive pulmonary disease (COPD) are responsible for four million deaths every year and affects hundreds of millions more. In particular the health and well-being of the patients is seriously compromised and this has a negative impact on family life and society at large. Children are particularly vulnerable, especially those in less economically developed and newly industrialised countries, due to poor environmental conditions and fossil fuel pollution. In first world countries tobacco use continues to impact these diseases and is the most important risk factor for chronic respiratory diseases. Since 1998 only four new antibiotics have been licensed for use by the Federal Drug Administration in the USA - this has been attributed to the high cost of drug development and the small market return for any approved antibiotic. Consequently, there is an urgent need for the development of new antimicrobials to combat the rise of antimicrobial resistance. Most governments now acknowledge that this needs a globally coordinated approach driven by the public sector: governments and public health institutes with engagement from industry come together to inform strategies and priority areas to be pursued. A prerequisite to drug discovery is the identification of viable targets for drug discovery. Although only the first step, without a basic understanding of the biomolecules involved in the underlying molecular mechanisms of vital life processes this cannot be achieved. The research here is aimed at understanding at the atomic level the molecular mechanism of substrate transport by the SAP ABC transporter in NTHi. It does not specifically propose to develop new antimicrobials to fight OM or COPD but the knowledge gained should inform drug development that could ultimately reduce the significant burden of respiratory disease for the human populace. However, this basic research will aid informing government policy and provides the knowledge base and research infrastructure to train the next generation of researchers. Respiratory disease impacts and affects almost everyone and thus communication of basic research programmes aimed at understanding the underlying mechanisms of disease benefits public awareness of science, aids governments in their ability to increase science budgets which helps to accelerate new discoveries that can improve the health and well-being of the general public. Development of research infrastructure like the Harwell campus should further aid viable drug development strategies in the public sector. Impact to the wider science community is also significant. Understanding vital processes for NTHi survival and persistence in the host are of wide interest to researchers with long-term aims of developing new antimicrobials and vaccines to thwart bacterial infections; in particular clinical microbiologists working in the area of infection and immunity, medicinal chemists and small and big pharma. The work proposed here will allow different strategies for disabling SAP function to be pursued and could result in a number of opportunities that can be seriously advanced for development of novel antimicrobials and their relevance for public use to be evaluated.

Publications

10 25 50
 
Description Mass Spectrometry analysis of SapA 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Structural and functional analysis of the substrate binding protein SapA of the SAP transporter from Haemophilus influenzas
Collaborator Contribution Native mass spectrometry analysis
Impact Work completed and publication in progress. This was a multi-disciplinary collaboration. The team at Oxford brought expertise in mass spectrometry to the project. The project also used X-ray diffraction, X-ray solution scattering and biophysical characterisation (thermofluor, ITC)
Start Year 2018
 
Description Molecular Dynamics and modelling 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We have determined the crystal structure of the substrate binding protein SapA which forms part of the SAP transporter system which is an ABC transporter system that has been implicated in transport of iron (in the form of haem) and antimicrobial peptides in non-typeable Haemophilus influenzae (NTHi). We have thus far failed to obtain structural data for sapA bound to ligand. The Group of Prof Phi Biggin at Oxford are developing and applying computational methods to examine conformational changes and properties of ligand-binding. By producing a model of sapA:haem complex allows us to probe potential amino acid residues responsible for ligand binding in sapA and assess their importance on NTHi disease in a mouse model of NTHi infection.
Collaborator Contribution The group of Biggins are using molecular modelling/dynamics to produce models of SapA in open (ligand free) and closed (ligand bound - haem) conformations.
Impact This is currently work in progress
Start Year 2015
 
Description Gordon Research Conference for 3DEM, Hong Kong 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gordon Conference series 3DEM focused on cryoEM "Advancing and Reshaping Structural Biology with Electron Cryo-Microscopy". Conference aims were to discuss the latest technological advances, progress in applying the methods to addressing important and critical biological questions, methodologies and criteria in validating critical results, as well as data deposition and accessibility. It also provided a forum for thorough discussions among both established experts and junior scientists from different disciplines, with either academia or industrial backgrounds. We presented preliminary work through poster presentation but our main aim in participating was to gather new methodology for the analysis of membrane proteins with CryoEM. The conference was extremely useful and informative
Year(s) Of Engagement Activity 2016
URL https://www.grc.org/programs.aspx?id=10865
 
Description BBSRC Interdisciplinary Bioscience Doctoral Training Partnership training visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Provides an overview of techniques available at Diamond Light Source and the Research Complex at Harwell - Detailed overviews of Macromolecular crystallography, biological solutions scattering, X-ray Cryo-transmission microscopy, CryoEM and protein production and purification for structural studies. This training event is organised and coordinated by Martin Walsh and includes direct participation of his research group.
Year(s) Of Engagement Activity 2017
URL http://www.biodtp.ox.ac.uk
 
Description CCP-EM annual meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at the CCP-EM spring symposium describing to the EM community how to access EM facilities at eBIC. The workshop was attended by over 100 delegates working in the cryoEM area
Year(s) Of Engagement Activity 2016
URL http://www.ccpem.ac.uk/courses.php
 
Description CCP4/Diamond workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Participant in CCP4/Diamond workshop - gave a talk to students on structural biology infrastructure
Year(s) Of Engagement Activity 2016
URL http://www.ccp4.ac.uk/schools/DLS-school/
 
Description Correlative imaging workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact We participated in a practical workshop dedicated to demonstrations and practical training sessions in correlative microscopy techniques. Correlative microscopy workflows to be demonstrated include cryo-TEM, cryo-X-ray microscopy, cryo-fluorescence and super-resolution microscopy, and sample marking using multi photon techniques. Our group provided 2 days of tutorials and training in the use of cryo-TEM.
Year(s) Of Engagement Activity 2017
URL https://www.clf.stfc.ac.uk/Pages/Octopus-CorrelativeMicroscopySymposium.aspx
 
Description CryoEM Sample Preparation Workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Responsible for organising and designing a practical workshop to train novice users how to prepare samples for primarily single particle cryoEM. 16 students from across the UK and Europe attended the workshop which consisted of two parts 1) a set of introductory (theory and best practice) lectures on CryoEM technique, with a special emphasis on practicalities of how to prepare samples and current methods; 2) Intensive tutoring practical sessions on sample preparation and sample characterisation by state-of-the-art microscopes. Students got hands on experience in sample preparation with current leading plunge freezers for CryoEM sample preparation and sessions on the Titan Krios Microscopes at eBIC to characterise and assess sample grid quality. Students indeed were even able to solve test sample from their own prepared samples
Year(s) Of Engagement Activity 2017
URL http://www.diamond.ac.uk/Home/Events/2017/cryoEM-Worskhop-October-2017.html
 
Description CryoEM sample handling workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact A practical hands on workshop to train new users to CryoEM in sample preparation techniques. The workshop provided access to state of the art facilities for sample preparation and characterisation for 12 students, 6 from the UK and 6 from EU member countries with 50% of funding coming from H2020 funding. Our group were responsible for leading the workshop (concept, securing funding and contributing to training)
Year(s) Of Engagement Activity 2017
URL http://www.diamond.ac.uk/Home/Events/2017/EM-Workshop-2017.html
 
Description Diamond Open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Diamond open day events offer the chance for members of the public to visit the facility and see inside our particle accelerator and experimental laboratories. It also gives us the opportunity to summarise our research activities into antimicrobial resistance and how we use Diamond to understand bacterial disease
Year(s) Of Engagement Activity 2016
URL http://www.diamond.ac.uk/Public/VisitUs.html
 
Description Harwell Campus open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Harwell campus was opened to the general public and schools in July 2015. Our group participated in tours of our labs in the Research complex at Harwell. The purpose was to raise public awareness and generally show people the amazing instruments and capabilities at the Harwell campus
Year(s) Of Engagement Activity 2015
URL http://harwellcampus.com/open-days/
 
Description Introduction to Research at Diamond for Wellcome Doctoral Training programme 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact 15 undergraduates interested in doing PhD research at Diamond
Year(s) Of Engagement Activity 2020
URL https://www.medsci.ox.ac.uk/study/graduateschool/courses/dtc-structured-research-degrees/cellular-st...
 
Description MRC Medical Sciences DTP 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact A series of talks describing the work at Diamond Light Source followed by a tour with which our group were involved (1 talk and tours)
Year(s) Of Engagement Activity 2018
 
Description MRC festival Harwell 2019 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 50-100 school children and teachers - interactive demo - showing how to extract DNA from strawberries and general introduction to molecular biology
Year(s) Of Engagement Activity 2019
URL https://har.mrc.ac.uk/research/news/20930/mrc-harwell-festival-%E2%80%93-19-20-june-2019
 
Description Oxford Interdisciplinary bioscience DTP 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact introductory talk and then visits to a number of instruments at Diamond, general introductions to MX, CryoEM, SAXS and imaging
Year(s) Of Engagement Activity 2019
URL https://www.ox.ac.uk/admissions/graduate/courses/interdisciplinary-bioscience?wssl=1
 
Description Oxford Interdisciplinary bioscience DTP 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Lecture and tours to 50 postgraduates
Year(s) Of Engagement Activity 2018
 
Description Protein Crystallography Summer school 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact St. Andrews summer school in crystallogrpahy - series of lectures and tutorials from theoretical background to working with experimental data (data collection, structure solution, phasing, molecular replacement and structure refinement.
Year(s) Of Engagement Activity 2019
URL https://synergy.st-andrews.ac.uk/proteincrystallography/
 
Description Students carrying out a Masters in Natural Sciences at University of Southampton - visit to Diamond to complete Drug Design Course 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact A group of 9 students (2 PhD students) from the University of Southampton given an overview of structural biology - with focus on x-ray fragment screening approach. Students got first hand overviews of biological solution scattering SAXS and relevance to drug design/discovery, new state of the art VMXi beamline at Diamond and role in drug discovery, the X-Chem facility at Diamond and the national CryoEM centre eBIC. The one-day workshop was organised and coordinated by Martin Walsh
Year(s) Of Engagement Activity 2018
 
Description White Rose DTP 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact David Owen gave a flash and poster presentation to a group of visiting White Rose DTP Students describing the work that the Martin Walsh group does in the Research Complex at Harwell. This was followed by a meet and greet session so they could learn what it is like to be a post doc.
Year(s) Of Engagement Activity 2018