CuiT is a Cu importer required for metal homeostasis in Salmonella enterica

Copper (Cu) is an essential micronutrient that serves as a cofactor for redox enzymes but becomes toxic when unregulated. In bacteria, while Cu efflux systems are well characterized, mechanisms of Cu import remain poorly understood. Here, we characterize the major facilitator superfamily transporter CuiT (STM1486) as a key Cu importer in Salmonella enterica. Comparative genomics revealed that cuiT is evolutionarily conserved across Enterobacteriaceae, and structural modeling predicts a 12-transmembrane-helix architecture with conserved His, Met, and Cys residues suitable for Cu coordination. Functional analyses demonstrated that deletion of cuiT reduces intracellular Cu accumulation, slows Cu uptake kinetics, and diminishes expression of Cu-responsive genes, including copA, cueP, cueO, and golB. Conversely, overexpression of CuiT increases intracellular Cu but sensitizes cells to Cu stress, highlighting the need for tight regulation. Kinetic modeling indicates that CuiT mediates rapid Cu import, supporting larger intracellular Cu pools compared to Pseudomonas influx transporters. These findings position CuiT as a central component of the Salmonella Cu homeostasis network, linking Cu import to transcriptional regulation, redox balance, and stress adaptation. Our work provides mechanistic insights into bacterial Cu acquisition and suggests CuiT and associated pathways as potential targets for antimicrobial strategies. SignificanceCopper (Cu) is essential for bacterial redox enzymes but toxic when dysregulated. While Cu efflux pathways are well studied, mechanisms of Cu import are poorly understood. We identify CuiT, a conserved major facilitator superfamily transporter, as a key Cu importer in Salmonella enterica. CuiT controls intracellular Cu levels, influences Cu-responsive gene expression, and maintains redox balance and stress adaptation. Disruption or overexpression of CuiT perturbs Cu homeostasis, highlighting its regulatory importance. These findings reveal a critical bacterial Cu acquisition pathway and suggest CuiT and its network as potential antimicrobial targets, advancing understanding of metal homeostasis in pathogens.