Form and function in the ESX-1 secretion system; elucidation of mechanism and structural determinants of bacterial virulence.

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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 ESX-1 system seeking to understand a central aspect of microbiology, namely how protein secretion machines work. This ESX-1 system has been shown to be essential for the virulence of important pathogens (M. tuberculosis, B. anthracis and S. aureus) and so there is a biomedical and agricultural aspect to this basic research. We have identified that the Staphylococcus aureus ESX-1 appears to be the most tractable for structure-function studies. S. aureus is a commensal Gram positive bacterium responsible for a number of illnesses in humans and animals that range from minor skin infections, such as pimples and abscesses, Toxic shock syndrome (TSS) through to septicaemia and mastitis in dairy herds and pigs. However, 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), making it a severe and difficult to treat problem. The number of deaths attributed to S. aureus infection is comparable to that attributed to acquired immune deficiency syndrome. 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. The ESX-1 transport system drives the secretion of at least three 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 identification of its cargo. The ESX-1 system consists of 5-8 different protein components. We will investigate how these proteins interact to assemble the secretion machine, and the molecular basis for how the machine works. To attain this goal we must derive the structure and function of each component, elucidate how the components interact and quantify the association, determine the molecular basis for cargo recognition, and the generation of the motive force. We have already made significant progress with six of these proteins cloned, expressed and purified and two crystallised. We will exploit single crystal X-ray diffraction methods to derive accurate molecular structures and a battery of biophysical techniques to characterise the protein-protein interactions. 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.