Characterisation of the Ess protein secretion system of Staphylococcus aureus, a key virulence factor.

Almost all bacteria cause disease by producing toxins that they secrete into the host. Many of these toxins are protein molecules, and the bacterium has specialised machines in the cell membrane that allows the controlled passage of these toxins to the outside. We are working on one of these machines - the recently discovered Ess system - seeking to understand a central aspect of microbiology, namely how protein secretion machines work. This system has been shown to be essential for the virulence of Staphylococcus aureus.

S. aureus is a commensal Gram positive bacterium responsible for a number of illnesses in humans ranging from minor skin infections, such as pimples and abscesses, Toxic shock syndrome, through to septicaemia and pneumonia. It is most widely known as a major cause of hospital acquired infections, and is a frequent cause of post-surgical wound infections. This situation is exacerbated by the fact that some strains of S. aureus are resistant to many antibiotics (e.g. Methicillin Resistant Staphylococcus aureus; MRSA), making it a severe and difficult to treat problem. An understanding, at the molecular level, of S. aureus biology and pathogenesis is essential if we are to design new treatments to prevent or cure infection.

Our preliminary work and results from others has suggested that the Ess transport system drives the secretion of at least five different proteins from S. aureus, and it is known that when this system is inactivated S. aureus shows a dramatic reduction in its ability to cause infection. We seek knowledge of the architecture and function of this distinctive bacterial secretion machine and to determine the function of one of the secreted proteins.

In order to secrete proteins, the machine needs to be energised. It is very likely that the EssC protein is responsible for providing the energy for this process, by breaking down an energy-rich compound in the cell called ATP. We want to understand the molecular basis for how the machine works. We have significant progress towards this by purifying and crystallising a large part of the EssC protein. We now wish to exploit single crystal X-ray diffraction methods to derive an accurate molecular structure for EssC. We want to build on this by understanding how EssC binds and hydrolyses ATP, and which other proteins in the Ess machine it interacts with.

We want to confirm our preliminary results to determine which proteins are secreted by this machine. We would like to determine molecular structures for one of these proteins to understand how they work. Finally we want to understand how these proteins are secreted by the machinery in particular to see if they interact with EssC during their export.

Knowing the structures of the protein components and how they interact with each other is important because this might, in the longer term, lay the foundation for studies directed to the design or discovery of compounds that will prevent these proteins from working with each other or prevent the motive force from being used to secrete out the proteins that establish and prolong infection. Information on proteins that are secreted and of the structures found on the surface of the bacterium may also provide opportunities for vaccine design.

The Type VII/Ess protein secretion system is a general protein export pathway found in many Gram positive bacteria including the serious pathogens Mycobacterium tuberculosis and Staphylococcus aureus. Previous studies have shown that this secretion pathway is essential for the virulence of both of these organisms. Indeed the loss of one of these systems from a strain of M. bovis is the basis for the attenuated BCG vaccine. Although the Type VII/Ess export pathway shares a number of common components between bacterial species there are clear differences in the composition of the machinery between Actinobacteria (including M. tuberculosis) and firmicutes (S. aureus).

We have been working on the characterisation of the Ess secretion system from S. aureus. We have shown that EssC is essential for Ess secretion system function in S. aureus. We have obtained diffracting crystals of the large C-terminal domain of EssC and have shown that it binds ATP. Proteomic analysis of our essC mutant strain has led to the identification of several novel candidate substrates of the Ess machinery. Finally we have shown that The Ess secretion system of S. aureus RN3690 is essential for virulence in the mouse pneumonia model of infection.
We now seek to build on our preliminary data to obtain high-resolution crystal structures for EssC and for one of the secreted substrate proteins. In addition we will probe the organisation of the membrane-bound secretion complex in S. aureus using cross-linking and native level protein purification coupled with yeast two-hybrid studies and co-purification approaches. Finally we will determine whether substrate proteins interact with EssC during export by the machinery.

Beneficiaries of this research include:

i) Biotech companies interested in protein secretion, bacterial virulence, surface display, anti-infectives. Such companies will benefit from the proposed research programme since it will generate new knowledge on novel pathways for surface display and protein secretion in Gram positive bacteria, and on the virulence of a key human pathogen, Staphylococcus aureus. We will act to protect any intellectual property and to maximise opportunities for collaborative research or licensing. The Dundee research and innovation team have a wealth of industrial contacts and close links to Scottish Enterprise, and will help maximise the impact of all findings of commercial value. As and when appropriate, results will be peer-reviewed and published.

ii) Members of the wider academic community. The primary mechanism for communication of this research will be through publication in peer review international journals. Open access publishing options will be used. We will liaise at the time of publication with the University of Dundee and MRC Press offices to ensure publicity of results of interest to the general public. Our results will also be made available on our regularly updated web site. Strains and other resources will be made available as appropriate.

iii) The staff employed on this project. The University of Dundee takes training of early career researchers seriously, thereby ensuring a successful contribution to the knowledge-led economy of UK Plc. The appointed PDs will be encouraged to be innovative in their work. There will be opportunities for them to train undergraduate, postgraduate and visiting scientists. They will be given multiple opportunities to present their findings at major research conferences, facilitating their career development through the acquisition and refining of key presentational and networking skills. Furthermore, the appointed PDs will have access to training in transferable/generic skills through the professional development schemes. In line with the Concordat 2009, the PDs will be actively encouraged to undertake at least 5 days training in personal professional development per annum. In addition, the University of Dundee has an annual appraisal scheme to actively facilitate the career development of staff, including PDs and PIs. The PDs will also be encouraged to design and supervise undergraduate projects and to become involved in science communication.

iv) The general public. It is important that members of the general public are aware and supportive of how tax payers' money is spent on scientific research. Therefore as part of our work on this project, we will engage with local communities, through face-to-face discussion of our work and family focussed scientific event days. The PIs are experienced science communicators. For example, the Division of Molecular Microbiology has teamed up with the Dundee Science Centre to organize a 2-day event in May 2010, March 2012 and March 2014 entitled "Magnificent Microbes", in which the primary applicant fully participated. This event attracted teachers, children, the general public and the local media to learn about the research going on in the Division of Molecular Microbiology and educated, inspired and entertained the public about microbiology research. We will be running new versions of this event in 2016 and 2018 which would be during the course of the project.