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

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.

C. difficile causes intestinal infections varying in severity from a mild, self-limiting diarrhea to severe, often life threatening, inflammatory complications such as toxic megacolon and pseudomembranous colitis. Infection commonly follows antibiotic-induced intestinal dysbiois that gives C. difficile a competitive advantage. There is an urgent need for novel therapies that treat C. difficile infection without causing further dysbiosis.

Our research focuses on the biogenesis of the C. difficile cell surface and, in particular, the translocation of cell wall associated proteins across the membrane. The C. difficile surface is completely covered with a paracrystalline S-layer comprised of the S-layer protein, SlpA, and an additional 28 cell wall proteins which functionalise the surface. SlpA and the dominant cell wall protein, CwpV, are translocated by the accessory SecA2 system, a second parallel Sec machinery found in some Gram positive bacteria. The ATPase at the core of the accessory Sec system in C. difficile, SecA2, is an essential protein, making this an attractive target for therapeutic intervention. SlpA and CwpV are critical virulence factors contributing to colonisation of the host and interactions with the immune system. We aim to fully characterise the C. difficile accessory Sec system, localising protein secretion within the cell, identifying all of the proteins required for efficient secretion and elucidating the molecular basis of substrate recognition. We will adopt a highly multidisciplinary approach, complementing our expertise in molecular microbiology and protein biochemistry with key collaborations adding world-class mass spectrometry and advanced super-resolution microscopy. The insights gained from our analysis of S-layer secretion will contribute to the development of targeted inhibitors that block the accessory SecA2 system and thereby kill C. difficile.

The proposed project deals with the characterisation of a potential new antimicrobial target in the nosocomial pathogen Clostridium difficile. This project will provide a number of benefits to industry and the general public in the UK and farther afield.

Industry:
In the context of the current antibiotic resistance crisis the development of novel antimicrobials is an absolute priority. Biotech and pharmaceutical companies with an interest in the development of antimicrobials will benefit both directly and indirectly from this project. We will provide crucial fundamental mechanistic information about the C. difficile accessory SecA2 system that will enable its exploitation as an antimicrobial target. We will protect the IP we generate in the course of this project and will seek to partner with appropriate companies to fully exploit the outputs of our research. We are developing a number of genetic tools, including our innovative transposon mutagenesis system, that will be broadly applicable to all aspects of research into the life and pathogenesis of C. difficile. We will also employ a range of innovative methodologies, many of which we are pioneering in the study of C. difficile pathogenesis. These approaches will be broadly applicable to the study of many facets of C. difficile biology and will be of benefit to any company with an interest in C. difficile. The Sheffield Science Gateway (SSG) fosters partnerships between academics and the public and private sectors. We will engage with SSG from the outset of the project to maximise our exploitable output.

National Health Service:
Management of C. difficile infection is a current NHS priority and is a major financial drain on the health service. Development of novel therapies to treat C. difficile associated disease will have a major impact on the NHS.

The local community in Sheffield:
The local community will benefit from our outreach activities, gaining a broader knowledge of this important scientific area and an appreciation of the research happening within their city.

Society:
The development of a new treatment for C. difficile will have a major impact on society in general. There were 1600 deaths involving C. difficile in England and Wales in 2012 in addition to considerable morbidity. Treatment of C. difficile infections and the resulting extended hospital stays also place a huge burden on the NHS budget.

Students and Staff:
Undergraduate and postgraduate students at the University of Sheffield will benefit from their involvement in the project, developing knowledge and skills that will contribute to successful future careers. The postdoctoral research associate employed on the project will also develop crucial research and career development skills, with opportunities for involvement in outreach, science communication and industry collaborations.