An essential channel in Clostridium difficile sporulation: structure and function

Clostridium difficile is one of the major causes of hospital acquired infections in the UK, a major health concern that puts a significant economic burden on healthcare systems, estimated to cost over 3000 million euros a year in the EU. Recent changes in hospital hygiene reduced the overall number of infections; however, C. difficile infections (CDI), still caused about 1500 deaths in 2012 (about 6 times more than MRSA) in England and Wales alone.

C. difficile is resistant to most antibiotics and usually grows when the normal population of gut bacteria is disturbed by these drugs. Recent outbreaks show greater antibiotic resistance, more serious disease and higher risk of repeating infections, posing an increased challenge to healthcare in the coming years. One of the main issues in fighting CDI is the limited understanding of C. difficile. In order to identify new methods to fight this serious health problem, we need to investigate the infection mechanisms at the molecular level.

A key mechanism is the bacteria's ability to form dormant cells - spores - which are responsible for CDI transmission and recurrent disease. Spores released from infected patients survive in the environment due to their resistance to common disinfectants, high temperatures and radiation. Despite its clear importance, formation of spores is poorly understood and our work focuses on one of the key aspects in this process.

During sporulation, the cell divides asymmetrically, with a smaller "daughter" cell (forespore) becoming surrounded by the mother cell with a double-membrane system, physically isolating it from the outside medium. In order for the full spore to develop, communication between the two cells has to be maintained across this physical barrier. We have identified an essential complex formed by two proteins, one from each cell: SpoIIQ, present in the forespore membrane and SpoIIIAH, a mother-cell membrane protein, that we propose creates the communication channel.

Inhibition of channel function is predicted to prevent spore formation, therefore interrupting the C. difficile transmission cycle, making it an attractive target for therapeutic intervention. Our studies will provide the necessary detailed knowledge to make sporulation, and this channel in particular, feasible targets for therapeutic research for C. difficile infections.

In this study, we will determine the structure of the channel, its functional details, and identify the molecules transported between the two cells. Our approach is two-fold: in vitro techniques will allow us to dissect the structural and biochemical properties and in vivo studies will unravel the functional details.

We will use protein X-ray crystallography to determine the structure of the whole channel formed across two membranes, the first such complex from this type of bacteria to be solved. A new fluorescence microscopy method, recently developed by our collaborators (Portugal), will be used for in vivo co-localisation of the proteins and studies of the channel effect in gene expression control. We will also elucidate the previously unidentified SpoIIQ activity in the degradation of components of the cell wall and investigate its role in correct localisation and function of the complex. Using purified proteins, we will explore a range of biochemical and biophysical techniques, complemented by in vivo studies. to determine the architecture and stability of the complete channel and identify different potential ligands.

The spore is the infectious agent of C. difficile and prevention of sporulation is an attractive therapeutic route, however molecular targets remain to be characterised. This work will elucidate key molecular details in C. difficile spore formation, which are crucial to exploit sporulation as an effective therapeutic target for treatment and control of C. difficile infections, that affect over 123,000 patients a year in Europe.

C. difficile infections (CDIs) are a major cause of morbidity and mortality in UK hospitals, with recent outbreaks of more virulent strains posing an increasing challenge to healthcare. Therefore, there is an urgent need for new, effective methods for infection control and treatment.

Spores are the transmission agents in CDIs; however, sporulation is poorly understood at the molecular level. A key aspect of spore maturation is the communication between the forespore, isolated from the medium upon engulfment, and the mother cell. We have recently identified a key forespore protein essential for effective sporulation, SpoIIQ. Our current work shows that it forms a complex with a mother cell membrane protein, SpoIIIAH, to create the necessary communication channel.

We aim to elucidate 3 key aspects of this channel:

- How does the complex form a bi-membrane channel?
We will (a) solve the X-ray structures of SpoIIQ and SpoIIIAH and their complex and (b) use NMR and single-channel studies to determine the biochemical and biophysical properties of the complex and identify potential ligands.

- What is the role of the complex in communication between forespore and mother cell?
We will use the SNAP labelling system to (a) establish the localisation of the complex, (b) identify the channel effects on gene expression control and (c) study the binding/transport of identified potential ligands.

- Is SpoIIQ endopeptidase activity required for channel formation?
We aim to (a) assess SpoIIQ endopeptidase activity in vitro (turbidity, zymogram essays, MS analysis), (b) describe catalysis at the molecular level using structural analysis and (c) investigate the effect of inactive SpoIIQ in channel localisation and formation.

Inhibition of channel function is an attractive target for therapeutic intervention. This work will provide a new understanding into sporulation of C. difficile that would open new avenues for CDI control.

This proposal focuses on understanding of a key aspect in the transmission of an important human pathogen, C. difficile. Despite being fundamental in nature, this work can have overarching impact in several aspects:

1. Scientific
We expect our work will have significant impact in several scientific communities. The most direct beneficiaries of our work are basic scientists who are interested in understanding the pathogenicity mechanisms of C. difficile, sporulation processes in important human pathogens and structural biologists tackling complex systems, particularly of membrane proteins. There are clearly large scientific communities, funded by the MRC and other public and private funding bodies, interested in these fundamental, medically important areas of research.

2. Societal
Antimicrobial resistance is an increasing concern in our societies, with severe implications in healthcare systems, as indicated by many involved in health and science policy, such as the Chief Medical Officer, who recently identified it as a 'catastrophic threat'. Fundamental research into the mechanisms of resistance, infection and transmission of such human pathogens is, therefore, of crucial importance and would have a significant societal impact. Moreover, new methods of treatment and infection control are strategic priorities of the NHS. Our work will provide such new insights and further our understanding of these 'superbugs' and, importantly, provide key new targets for the future development of new therapeutics.

3. Economic
In recent years, the pharmaceutical industry investment in basic research of new antibiotics has been reduced and new drugs often rely on key outputs from fundamental research carried out in academia. Our work would provide a detailed view of the architecture of this essential channel in C. difficile sporulation that could then be exploited by this industry to develop new drugs, specifically targeted to stall/prevent spore maturation, interrupting the infection cycle. Therefore, in the long term, this work has the potential to result in economic benefit through development and commercialisation of new drugs and a reduction in the healthcare systems budgetary burden, translated in an increased quality of life and health.

The UK economy will also benefit because this academic research will enhance its long standing reputation in biomedical discovery. Collaboration and communication between structural biologists, microbiologists and protein biophysicists on medically relevant proteins and mechanisms will ensure effective knowledge and skills transfer between these different fields of expertise within academia as well as the UK pharmaceutical industry. This will strengthen the UK's position as a world leader in fundamental biomedical science, helping to attract R&D investment. Such retention of expertise and know-how will aid the UK's capacity to remain internationally competitive.

4. Outreach
An important aspect of any publicly funded research is its potential impact to the wider public. Although our work is fundamental in essence, with potential translational developments only foreseeable in the long term and under development by industrial interested parties, we are strong advocates of the engagement of the general public(s) in our research and outputs. We will organise activities designed for both children and adults in order to develop and enhance the awareness of different scientific processes, such as protein crystallography and microbiology, as well as showcase the results of our research. This knowledge of science and research, as well as the details of the 'superbugs' and how they cause and spread disease, will form the basis for a better understanding of medical problems and treatment and an overall awareness of the societal benefits of scientific research.