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

Dual Inhibitor of Staphylococcus aureus Virulence and Biofilm Attenuates Expression of Major Toxins and Adhesins

Staphylococcus aureus is a major bacterial pathogen that invades and damages host tissue by the expression of devastating toxins. We here performed a phenotypic screen of 35 molecules that were structurally inspired by previous hydroxyamide-based S. aureus virulence inhibitors compiled from commercial sources or designed and synthesized de novo. One of the most potent compounds, <b>AV73</b>, not only reduced hemolytic alpha-hemolysin production in S. aureus but also impeded in vitro biofilm formation. The effect of <b>AV73</b> on bacterial proteomes and extracellular protein levels was analyzed by quantitative proteomics and revealed a significant down-regulation of major virulence and biofilm promoting proteins. To elucidate the mode of action of <b>AV73</b>, target identification was performed using affinity-based protein profiling (AfBPP), where among others YidC was identified as a target

Profiling withanolide A for therapeutic targets in neurodegenerative diseases

To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects