Abstract

Adherent-invasive Escherichia coli (AIEC) LF82 is closely linked to Crohn’s disease and can persist within macrophages in a quiescent, growth-arrested state. Here, we show that cupric gluconate (Cu Glu) promoted cell death in E. coli MG1655, yet drove E. coli LF82 into a viable-but-non-culturable (VBNC) state. VBNC induction was time dependent and modulated by temperature. VBNC cells remained susceptible to ampicillin and tetracycline but were tolerant to ciprofloxacin. To probe whether capsule-associated factors contribute to the strain-dependent outcome, we heterologously expressed kpsM , kpsT , or kpsMT in MG1655. This increased MG1655 tolerance to Cu Glu but did not induce a VBNC phenotype. Cu Glu increased intracellular Cu + and ROS in LF82 without detectable lipid peroxidation or DNA damage. Copper or ferrous-iron chelation prevented VBNC entry and rescued cells otherwise destined to die, whereas redox modulators shifted VBNC outcomes. Glutathione and catalase resuscitated a small subset, implicating H 2 O 2 -driven oxidative stress in VBNC fate. Proteomics revealed repression of energy metabolism together with enhanced outer-membrane maintenance and Fe-S cluster repair. Notably, ascorbic acid (Vc) abolished resuscitation and rapidly killed VBNC cells by promoting labile Fe 2+ release and, together with Cu + , amplifying Fenton chemistry to damage membranes without a lipid-peroxidation signature. These findings define a copper-dependent VBNC program in LF82 and provide mechanistic insight into how metal redox imbalance and oxidative stress shape VBNC maintenance and clearance.