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

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.

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).

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.