Tropolone and hinokitiol act against Staphylococcus aureus by concentration-dependent iron restriction or toxic iron overload

ABSTRACT Transition metal homeostasis is a critical component of bacterial physiology and thus represents a promising opportunity for antimicrobial intervention. Here, we demonstrate that the naturally occurring chelators tropolone (TRO) and hinokitiol (HNK) inhibit Staphylococcus aureus growth through concentration-dependent shifts in metal stress. Integrated metallomic and transcriptomic profiling revealed that low doses of both compounds promote toxic iron loading and a PerR-regulated oxidative stress response, consistent with iron-induced redox toxicity. However, at higher concentrations, both chelators trigger iron and manganese starvation responses, copper and zinc accumulation, and repression of respiratory and fermentative pathways, consistent with widespread disruption of metal homeostasis and failed metabolic adaptation. Despite these broadly similar responses, TRO, but not HNK, produced an unexpected biphasic growth phenotype. Experiments confirmed an analogous response in the distantly related gram-positive species Bacillus subtilis . These findings highlight how small molecule chelators can interfere with multiple metal systems to disrupt bacterial metabolism and how TRO can be exploited in a dose-dependent fashion to disrupt S. aureus proliferation. IMPORTANCE There is an urgent need to develop alternative therapies to combat the drug-resistant pathogen Staphylococcus aureus . One solution is to disrupt the intricate balance of metal availability using small-molecule chelators. We show that tropolone and hinokitiol arrest bacterial growth at low and high doses by radically different mechanisms. At low concentrations, both chelators induce iron overload, whereas at higher concentrations, access to iron and manganese is blocked, and instead, toxic levels of intracellular copper accumulate. Despite the similarity in their cellular effects, only tropolone confers bacterial growth restriction at low and high doses, while permitting recovery at intermediate levels. Such contrasting concentration-dependent antibacterial activities could be harnessed, by iterative switching of doses, to radically perturb metal metabolism as a unique means to control S. aureus infections.