<b>The secreted proteases Aur, ScpA, SspA and SspB suppress the virulence of</b><b><i> Staphylococcus aureus</i></b><b> USA300 by shaping the extracellular proteome</b>. Supplemental Figures S1-S4 and Supplemental Tables S1 and S2

Surface-located and secreted virulence factors of the Gram-positive bacterial pathogen <i>Staphylococcus aureus</i> are key drivers for infection of the human host. Proteolytic enzymes may contribute to virulence by breaking primary barriers and host immune defenses. Therefore, the objective of our present study was to chart the contributions of different proteases to <i>S. aureus </i>virulence, and to assess their roles in shaping the staphylococcal surface proteome (the ‘surfacome’) and extracellular proteome (the ‘secretome’). To this end, we applied 12 protease mutants of the <i>S. aureus</i> USA300 lineage. Four mutants lacking the metalloprotease aureolysin (Aur), the cysteine proteases staphopain A (ScpA) and SspB, or the serine protease SspA displayed enhanced cytotoxicity towards human lung epithelial cells, showing that they serve to suppress virulence. Profiling of the surfacomes and secretomes of the four mutants allowed correlation of their increased cytotoxicity to altered virulence factor profiles. Furthermore, enhanced levels of virulence factors were detected in the mutants’ surfacomes, which was shown to be relevant as all four mutants displayed enhanced lung epithelial cell invasion. Enhanced levels of cytoplasmic and membrane proteins in the mutant’s surfacomes showed that Aur, ScpA, SspA and SspB set limits to autolysis by reducing the levels of peptidoglycan hydrolases. We conclude that Aur, ScpA, SspA and SspB have key roles in shaping the surfacome and secretome of <i>S. aureus</i>, thereby controlling the virulence of this major pathogen. This implies that novel antimicrobial agents or vaccines should not target these proteases.