Abstract Our group previously identified a C-terminally processing protease (SaCtpA, ctpASA), as being required for full virulence in Staphylococcus aureus. We demonstrated that SaCtpA is membrane associated and constitutive produced under standard growth, but is elevated upon exposure to conditions encountered during infection (serum, immune cells). When we exposed ctpASA mutants to components of the immune system (human serum, blood and macrophages), we observed increased sensitivity in comparison to the WT. Moreover, using a murine model of sepsis and dissemination, we observed that ctpASA mutant infected mice survived significantly better than those inoculated with the WT, and had multiple log decreases in dissemination to the spleen, heart, brain and kidneys. Recently, a study implicated SaCtpA as inactivating an SOS-induced inhibitor of cell division, in a manner akin to that seen for CtpA in B. subtilis, which cleaves the SOS induced cell division inhibitor, YneA (no homology to SosA). Despite these published works, myriad questions remain regarding how SaCtpA functions in the S. aureus cell, or mediates its key role in virulence. Firstly, SaCtpA is constitutively expressed at high levels (and thus is not part of the SOS response), therefore having a single substrate is very unlikely. Indeed, counterparts from CTP1 and CTP-3 families have a wide array of substrates, from myriad cellular processes. Secondly, the S. aureus enzyme has unique structural features compared to other CTP-enzymes, including a large N-terminal extension in the cytoplasm, an atypical PDZ domain, and a conserved PG binding domain - the relevance of which has yet to be determined. Finally, despite a clear link between SaCtpA and SosA to reboot cell division following DNA damage, the mechanism behind SosA inactivation is completely unexplored. Therefore, we suggest that this array of novel features, alongside myriad unanswered questions regarding how SaCtpA functions in the S. aureus cell, or mediates its role in virulence, make this an important topic for study. Accordingly, we will: 1. Dissect the Influence of SaCtpA: We will begin by performing a structure function study of the SaCtpA enzyme, to explore its many unique features compared to other CTP enzymes. We will then globally capture SaCtpA proteolytic cleavage events using cutting edge mass-spectrometric techniques, developed by ourselves and others. 2. Explore the Impact of CtpA on SosA and Cell Division: There are many questions that remain regarding engagement of SaCtpA with SosA: How do these two proteins interact? Where on SosA does SaCtpA bind? What is the motif(s) recognized by SaCtpA? How does SaCtpA cleave SosA (once, twice, compete degradation)? Are other proteases involved? Thus, it is the focus of this aim to explore these questions, and shine light on the mechanism of SaCtpA-SosA interaction. Similarly, although LexA- controlled cell division inhibitors are widespread in Gram-positive organisms, how they mediate their function is still unknown. Therefore, we will also identify the divisome protein(s) targeted by SosA to inhibit cell division.

Staphylococcus aureus is a highly virulent and widely successful pathogen that is speculated to be the most common cause of human infection. With the continued emergence of multi-drug resistant isolates of S. aureus (such as MRSA), there is an urgent need to understand the mechanisms by which this deadly pathogen causes disease. This proposal seeks to explore the contribution of a membrane associated, C-terminal processing protease to S. aureus virulence.