<b>Cryptotanshinone Inhibits </b><b><i>Staphylococcus aureus</i></b><b> NdhC via Uncompetitive Inhibition: Collateral Sensitivity in Respiratory-Deficient SCVs</b>

Targeting the bacterial respiratory chain offers a potential strategy to overcome SCV-mediated resistance. Here we show that cryptotanshinone (CT) selectively inhibits <i>S. aureus</i>’s NDH-2 isoform NdhC.

Antimicrobial assays showed that ndhC overexpression doubled bacterial sensitivity to CT, while ndhF overexpression had no effect. <i>In vitro</i> 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 <i>fusA</i> and <i>perR </i>mutations exhibited collateral sensitivity to NDH-2 inhibitors.