| Zusammenfassung |
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. |
