Quantitative Map of β-Lactone-Induced Virulence Regulation

β-Lactones
have recently been introduced as the first selective
ClpP inhibitors that attenuate virulence of both sensitive Staphylococcus aureus and multiresistant strains
(MRSA). Although previous knockout studies showed that ClpP is essential
for S. aureus alpha-toxin production,
a link between β-lactone inhibition and molecular virulence
mechanisms has been lacking so far. We here perform a chemical–proteomic
approach to elucidate antivirulence pathways. First, we demonstrate
by gel-free activity-based protein profiling that ClpP is the predominant
target of β-lactones. Only a few off-targets were discovered,
which, unlike ClpP, were not involved in the reduction of alpha-toxin
expression. Second, in-depth mechanistic insight was provided by a
full proteomic comparison between lactone treated and untreated S. aureus cells. Quantitative mass-spectrometric
analysis revealed increased repressor of toxin (Rot) levels and a
corresponding down-regulation of α-toxin, providing the first
direct connection between the lactone-dependent phenotype and a corresponding
cellular mechanism. By building up a quantitative virulence regulation
network, we visualize the impact of ClpP inhibition in a systems biology
context. Interestingly, a lack of in vitro Rot degradation by either
ClpXP or ClpCP calls either for a proteolysis mechanism with yet unknown
adaptor proteins or for an indirect mode of action that may involve
ClpX-mediated RNA signaling and feedback circuits.