Role of M3 peptidases in Staphylococcus aureus pathogenesis

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.

Grant Number: 5R21AI173882-02
NIH Institute/Center: NIH
Principal Investigator: Shaun Brinsmade