About the Project
The Gram-positive bacterium, Staphylococcus aureus, is a major pathogen of humans and animals. In humans it is a leading cause of community and hospital acquired infections and is associated with life-threatening diseases such as pneumonia, meningitis, endocarditis, toxic shock syndrome, bacteraemia, and sepsis. The organism is notorious for its ability to develop resistance to antibiotics, and is one of the seven bacterial species highlighted by the World Health Organisation as being of critical antimicrobial resistance concern.
Bacterial virulence factors that directly interact with the host must be secreted from the bacterial cytoplasm where they are made. Protein secretion systems are therefore critical to the success of all bacterial pathogens. We and others have shown that the Ess (Type VII) protein secretion system plays a critical role in the ability of S. aureus to cause disease1. The Ess machinery comprises six core components1, four of which are membrane proteins including the essential ATPase protein EssC, one is cytoplasmic and one is extracellular2. Little is known about how the core machinery is organized, and how it is regulated, or how substrate proteins interact with the secretion apparatus during export.
One of the main aims of this project will be to investigate the organisation of the machinery and how substrates are recognised. Analysis of 153 S. aureus genome sequences revealed that the ess locus falls into four distinct clusters that each encode different sets of secreted substrate proteins3. These substrates cluster with a specific variant of the secretion ATPase EssC, strongly implicating EssC in substrate recognition. This will be investigated by expressing EssC proteins in different strains of S. aureus and assessing their ability to export substrates. These genetic experiments will be backed up biochemically by crosslinking and co-purification experiments to identify and isolate EssC-substrate complexes. We have recently designed a fusion protein that acts as a suicide substrate of the Type VII secretion system by interacting with the machinery and preventing export of bona fide substrate proteins. This suicide substrate will be used as a tool to investigate the organisation of a locked form of the transport machinery to identify which of the Ess core proteins interact during substrate transport.
The presence of the Ess system in the core genome of S. aureus coupled with published studies and our preliminary data indicate that it represents a very good potential anti-Staphylococcal drug target. The second aim of the project will be to develop a whole cell assay that will report on Ess secretion activity that can ultimately be miniaturised for running in 384 well format. Assays to be tested will include the use of fluorescent or other reporters for example alkaline phosphatase or biotin. Once a suitable assay has been developed and miniaturised, the project will involve screening one or more of the University of Drug Discovery libraries for molecules that interfere with Ess secretion. Hits will be confirmed by Western blotting of supernatant samples to confirm that secretion of substrates is reduced/abolished. Hits would also be tested against clinical isolates and multidrug resistant strains.
Training provided – the student will receive training in microbiology techniques including S. aureus genetics, protein biochemistry and protein interaction studies, and in drug discovery approaches including an understanding of medicinal chemistry.
***Application details and more information on the BBSRC EASTBIO Doctoral Training Partnership can be found at the website below. You must follow the instructions for your application to be considered:
http://www.eastscotbiodtp.ac.uk/how-apply-0
References
References:
1Kneuper, H, Cao, Z.P., Twomey, K.B., Zoltner, M., Jäger, F., Cargill, J.S., Chalmers, J., van der Kooi-Pol, M.M., van Dijl, J.M., Ryan, R.P., Hunter, W.N. and Palmer, T. (2014) Heterogeneity in ess transcriptional organisation and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains. Molecular Microbiology 93, 928-943.
2Jäger, F., Zoltner, M., Kneuper, H., Hunter, W.H. and Palmer, T. (2016) Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus. FEBS Letters 590, 349-357.
3Warne, B., Harkins, C.P., Harris, S.R., Vatsiou, A., Stanley-Wall, N., Parkhill, J., Peacock, S.J., Palmer, T. and Holden, M.T.G. (2016) The Ess/Type VII Secretion System of Staphylococcus aureus shows unexpected 1 genetic diversity. BMC Genomics 17, 222.