Cryptotanshinone inhibits Staphylococcus aureus NdhC via uncompetitive inhibition: Collateral sensitivity in...

Staphylococcus aureus small colony variants (SCVs) pose a clinical challenge due to antibiotic tolerance and recurrent infections. Targeting the bacterial respiratory chain offers a potential strategy to overcome SCV-mediated resistance. Here we show that cryptotanshinone (CT) selectively inhibits S. aureus's NDH-2 isoform NdhC. Antimicrobial assays showed that ndhC overexpression doubled bacterial sensitivity to CT, while ndhF overexpression had no effect. In vitro enzymatic analysis indicated that CT inhibits NdhC, with noncompetitive inhibition against NADH and uncompetitive inhibition against menadione (MD). Molecular docking and site-directed mutagenesis identified threonine T352 as critical for CT binding to the NdhC-MD complex. Using membrane vesicle assays and cellular thermal shift assays, we obtained evidence that CT acts at NdhC in isolated membrane preparations in a substrate-dependent manner. Notably, gentamicin-induced SCVs carrying fusA and perR mutations exhibited collateral sensitivity to NDH-2 inhibitors. Further characterization of menaquinone (MK) auxotrophic SCVs, which harbor a frameshift mutation in menC and show reduced but detectable NADH dehydrogenase activity, revealed that these variants also display heightened susceptibility to CT, consistent with the notion that residual NdhC activity in respiratory-compromised cells remains targetable. Together, these findings provide insights into the molecular targeting of NdhC by CT and support the concept that the metabolic vulnerability of SCVs may be exploitable for the treatment of refractory S. aureus infections.