<b>Cryptotanshinone Targets NdhC via Selective Uncompetitive Inhibition: Mechanistic Insights into Collateral Sensitivity in Drug-Tolerant </b><b><i>Staphylococcus aureus</i></b><b> </b><b>SCVs</b>

Staphylococcus aureus (SA) small colony variants (SCVs) are a major clinical challenge due to antibiotic tolerance and recurrent infections. Targeting the bacterial respiratory chain is a potential strategy to overcome SCV-mediated resistance. We previously found that natural monomer cryptotanshinone (CT) inhibits SA’s respiratory chain; this study further confirms its selective inhibitory effect on the two NDH-2 isoforms of SA (NdhC and NdhF). Antimicrobial assays showed ndhC overexpression doubled bacterial sensitivity to CT, while ndhF overexpression had no effect. In vitro enzymatic analysis confirmed CT specifically inhibits NdhC, with noncompetitive inhibition against NADH and uncompetitive inhibition against menadione (MK). Molecular docking and site-directed mutagenesis revealed threonine T352 mediates CT binding to the NdhC-MK complex, and T352 mutation abolished CT’s inhibition. Notably, gentamicin-induced SA-SCVs (with perR nonsense and fusA missense mutations) had reduced susceptibility to conventional antibiotics but ≥ 2-fold enhanced collateral sensitivity to NDH-2 inhibitors (CT, HQNO, thioridazine). This study uncovers CT’s molecular targeting of NdhC and supports targeting SCV metabolic vulnerability for refractory SA infections.金黄色葡萄球菌（Staphylococcus aureus, SA）小菌落变体（small colony variants, SCVs）因抗生素耐受与反复感染成为临床重大挑战。靶向细菌呼吸链是克服SCV介导耐药性的潜在策略。我们此前发现天然单体隐丹参酮（cryptotanshinone, CT）可抑制SA的呼吸链；本研究进一步证实其对SA的两种NDH-2同工酶（NdhC与NdhF）具有选择性抑制作用。抗菌实验显示，ndhC过表达可使细菌对CT的敏感性提升一倍，而ndhF过表达则无此效果。体外酶学分析证实，CT可特异性抑制NdhC：其对烟酰胺腺嘌呤二核苷酸（NADH）呈现非竞争性抑制，对甲萘醌（menadione, MK）则呈现反竞争性抑制。分子对接与定点诱变实验揭示，苏氨酸残基T352介导CT与NdhC-MK复合物的结合，而T352突变可完全消除CT的抑制作用。值得注意的是，庆大霉素诱导形成的SA-SCVs（携带perR无义突变与fusA错义突变）对常规抗生素的敏感性降低，但对NDH-2抑制剂（CT、HQNO、硫利达嗪）的侧敏感性提升至少2倍。本研究阐明了CT对NdhC的分子靶向机制，并为针对难治性SA感染靶向SCV的代谢脆弱性提供了理论支持。