Role of M3 peptidases in Staphylococcus aureus pathogenesis

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7. Project Summary/Abstract The SaeR/S two component system is a central regulator of virulence in Staphylococcus aureus. It is composed of an intramembrane sensor kinase SaeS and a response regulator DNA binding protein SaeR. During a screen for factors that modulate SaeR/S activity, we discovered insertions in two genes that encode putative oligoendopeptidase F (PepF) enzymes (hereafter referred to as PepF1 and PepF2 due to homology with Bacillus subtilis and Lactococcus lactis PepFs). PepFs are members of the M3 family of zinc metallopeptidases. Unlike eukaryotic M3 peptidases, prokaryotic M3 peptidases are understudied and have been enigmatic since their discovery. B. subtilis PepF is the only functionally characterized M3 peptidase to date, and when overproduced cleaves a short peptide, inhibiting a key sporulation signaling cascade in that organism. S. aureus pepF1 and pepF2 mutants reduced SaeR/S activity individually and additively. While S. aureus pepF null mutants are unaffected for growth in laboratory media, including one rich in peptides, pepF mutants show defects in expression of Sae-dependent genes, and survival in whole human blood. Notably, a pepF1 pepF2 double mutant is attenuated for virulence in a systemic infection model. Altogether, our preliminary data suggest a role in regulation and not nutrition or general protein turnover. Despite the characterization of eukaryotic M3 peptidases to date and only a small number of studies with prokaryotic M3 peptidases – all with test substrates – there is a major knowledge gap for these important peptidases. In this proposal, we will unravel the role of PepFs in promoting pathogenesis in this important human pathogen and, more broadly, explore PepF biology and M3 peptidase activity. We will do this using two specific aims. First, we will use an unbiased and cutting-edge mass spectrometry-based peptidomics approach to identify substrates of PepF1 and PepF2 and perform functional studies on confirmed substrates. Second, we will reveal the regulatory cascade leading to gene regulation by PepFs. To do so, we will use RNA-Sequencing to delineate the set of S. aureus genes affected by S. aureus PepFs, and then use mutagenesis to identify factors that control pepF gene expression. In the end, by collaborating with an expert in the field of mass spectrometry-based proteomics, we will generate data that, for the first time, will identify cleavage preference and substrate specificity determinants for the M3 family. At the same time, we will gain a deeper understanding how one of the most important virulence regulators in S. aureus is controlled and reveal how environmental and physiological signals are integrated into the virulence regulatory network to precisely upregulate pathogenic potential of this human pathogen.