Abstract A number of studies performed by ourselves and others have investigated the contribution of S. aureus extracellular proteases to disease causation. Until recently, these data proved inconclusive, however work by our group has definitively shown that secreted protease are key mediators of S. aureus disease. Their role appears to be biphasic as: i) Secreted protease deletion leads hypervirulence in infected animals; whilst ii) A complete protease-null strain has impaired survival in human blood, decreased resistance to phagocytosis, increased sensitivity to AMPs and impaired ability for dissemination and/or survival during infection. An explanation for these findings stems from their differing roles, and substrates, during infection. Specifically, the enhanced mortality is driven by an increased abundance of virulence factors, which exist unchecked upon secreted protease deletion. Conversely, the virulence attenuation is mediated by these enzymes attacking the host, cleaving proteins that facilitate nutrition, immune evasion, and dissemination. However, despite this, much remains unknown about how these enzymes are regulated, how they themselves regulate infection, and how they enhance the fitness of S. aureus in vivo. To fill in these gaps we will explore: 1. Regulation by Secreted Proteases During S. aureus Infection. We currently do not know which proteases cleave which virulence factors, or how this influences the progression of infection. As such, in this aim we will connect in vitro virulence factor proteolysis to the pathogenic potential of S. aureus in vivo. 2. The Regulation of Secreted Proteases During S. aureus Infection. Although secreted proteases are produced alongside virulence factors, their synthesis must be (and indeed is) tightly controlled, so as to tailor virulence factor abundance during disease causation. Accordingly, in this aim we will fill in major knowledge gaps regarding secreted protease regulation in vitro, and connect this to what happens in vivo. 3. The Role of Secreted Proteases During S. aureus Infection. The next frontier of protease biology in the context of pathogenic bacteria is an understanding of the host degradome (the complete set of host proteins cleaved by bacterial proteases). Therefore, in this aim we will use cutting edge proteomic techniques to gain insight into the infectious process, and interaction of host with pathogen. Through these studies we will define specific pathways that directly govern S. aureus disease, providing a unique and detailed insight into the molecular events that occur during infection. This will produce key findings regarding S. aureus pathogenesis that has potential to be used for the future generation of novel anti-virulence based therapeutics.