A <i>Staphylococcus aureus</i> Virulence Inhibitor Identified by SaeRS Refactoring and Screening in <i>Bacillus subtilis</i>

Bacteria utilize two-component system (TCS) signal transduction pathways to sense environmental and physiological stimuli and mount appropriate responses. In opportunistic pathogens such as <i>Staphylococcus aureus</i>, TCSs activate virulence programs in response to host defense systems. Due to their critical role in pathogenesis, TCSs are important targets for antivirulence drug discovery campaigns. However, challenges associated with screening TCSs in pathogens and <i>in vitro</i> have limited the output of such efforts to a small number of characterized drug candidates. Here, we functionally express the <i>S. aureus</i> virulence-regulating TCS SaeRS from synthetic gene regulatory elements in the model bacterium <i>Bacillus subtilis</i> to reliably screen this system against a small molecule library under simple culturing conditions. Our approach reveals the compound NSC97920 as a strong inhibitor of SaeRS signaling. We combine <i>in situ</i>, <i>in vivo</i>, <i>in silico,</i> and <i>in vitro</i> characterization to demonstrate that NSC97920 suppresses the critical step of autophosphorylation in the SaeS histidine kinase, resulting in strong antivirulence activity. Our work shows that heterologous expression and screening of TCSs in model bacteria could accelerate the development of therapeutics against antibiotic-resistant pathogens.