Characterisation of the essential C. difficile S-layer secretion system

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Clostridium difficile is an important human pathogen, causing serious illness and even death. C. difficile associated disease (CDAD) occurs most commonly in a hospital setting, affecting patients who are already suffering ill health, but infections in the community are an increasing problem. C. difficile is naturally resistant to many common antibiotics so whilst these antibiotics kill the beneficial bacteria in the human gut they have no effect on C. difficile and actually benefit the bacterium by removing competition. There is an urgent need to develop new therapies to combat CDAD and, in particular, to develop therapies which kill C. difficile but do not cause damage to the beneficial gut bacteria. A new therapy would ideally target a distinctive part of the bacteria which is essential for the bacteria's lifecycle.The proteins which coat the C. difficile surface are the appendages through which the bacterium interacts with its human host. Our research focuses on how these surface structures are assembled. A critical early step in this assembly is the transport of the proteins from the interior of the cell, where they are made, across a lipid membrane and onto the cell surface. We have discovered that this step requires a special secretion system, called the accessory Sec system. This secretion system, and the proteins it transports, are essential to the life of the organism and its ability to cause disease, making it an ideal target for the future development of new therapies for the treatment of CDAD. However there are many important gaps in our knowledge of how this system operates, including how the system recognises the proteins it secretes and how the system works with other proteins in the cell to enable secretion. It is essential that we develop a detailed understanding of its operation in order to allow the development of drugs that will block secretion and kill the bacteria.We will adopt a multidisciplinary approach to answer four key questions:1. Where in the cell is the accessory Sec system located?We will use advanced microscopy techniques to visualise where on the surface of the cell new protein appears, to determine if the accessory Sec system is localised to a particular part of the cell. Our preliminary data indicates that the there may be a small number of distinct secretion channels in the cell.2. What additional proteins are required for efficient secretion via the accessory Sec system?At the core of the accessory Sec system is a molecular motor, SecA2, which powers the system and three proteins, SecYEG, which form a channel in the membrane through which the secreted protein is transported. To date our work has focussed on these four components. However other similar systems require several more proteins for full efficiency. We will use the motor protein SecA2 to isolate and identify other proteins it interacts with in the cell.3. How does the accessory Sec system recognise the proteins it transports?The most important characteristic of the accessory Sec system is the ability to recognise and transport a few specific proteins with astonishing accuracy. We will use a combination of biochemistry and genetics to determine how the system recognises its targets. This information will be crucial to the development of drugs which inhibit this system specifically.In the longer term we will build on insights gained from this project to develop chemicals that interfere with the accessory Sec system in C. difficile. This could lead to the development of new treatments for CDAD.