*4 Year MRC PhD Programme* The role the Type VII protein secretion system in Staphylococcus aureus virulence

he 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. Protein secretion is key to bacterial virulence, and we and others have shown that the Type VII protein secretion system plays a critical role in the ability of S. aureus to cause disease1.



The major aim of this project will be to identify substrates of the Type VII secretion system and determine their roles in disease. Analysis of S. aureus genome sequences has revealed that genes encoding the S. aureus Type VII secretion system cluster into four lineages, each having different suites of secreted substrates2. The Type VII-secretion proteome will be determined for one of these specific lineages, and candidate substrates will subsequently be verified by secretion assays. Deletion mutants will be constructed that lack genes for individual substrate proteins. The virulence of these strains will be determined using a murine pneumonia infection model and in vitro using cell-based models of infection. For substrates that have roles in infection, functional analysis will be undertaken. This will involve overproduction and purification of the protein/s of interest for structural analysis and also for assays to determine for example if the purified protein has pore-forming, protease or nuclease activity. It will also be determined whether purified substrates are internalised into eukaryotic cells e.g. neutrophils, or if it they are toxic when transfected into human cell lines. Tagged variants of the protein internalised or produced in human cells may also be used as bait to co-purify interacting eukaryotic partner proteins.

Training provided – the student will receive training in microbiology techniques including S. aureus genetics, protein biochemistry and virulence models of bacterial infection.