A conserved CusR-dependent regulatory pathway couples copper homeostasis with oxidative stress resistance in Klebsiella pneumoniae

Host-derived copper toxicity is a central antimicrobial strategy that restricts bacterial survival during infection, yet how pathogens integrate copper detoxification with oxidative stress resistance remains incompletely understood. Here, we identify the response regulator CusR as a pivotal coordinator of copper homeostasis and oxidative stress defense in Klebsiella pneumoniae . Loss of cusR results in profound defects in copper tolerance, marked intracellular copper accumulation, and hypersensitivity to hydrogen peroxide, accompanied by elevated intracellular reactive oxygen species. Mechanistically, CusR directly activates the cusCFBA copper efflux operon through promoter binding, whereas regulation of the copA transporter occurs independently of CusR. Importantly, a conserved aspartate residue (D56) within CusR is essential for its activity; both plasmid-based and chromosomal substitution of this residue abolish copper efflux regulation, disrupt copper homeostasis, and exacerbate oxidative stress susceptibility. Comparative sequence analysis further demonstrates that this residue is highly conserved across diverse bacterial species, underscoring its evolutionary importance in copper-responsive signaling. Collectively, our findings reveal a conserved Asp-dependent CusR-CusCFBA regulatory axis that couples copper detoxification to oxidative stress resistance, enabling bacterial adaptation to host-imposed copper stress. IMPORTANCE Copper intoxication is an important host-mediated antimicrobial strategy that challenges bacterial pathogens during infection. Although copper resistance systems have been extensively studied, how bacteria coordinate copper detoxification with protection against oxidative stress remains poorly understood. In this study, we identify the response regulator CusR as a key factor linking copper homeostasis and oxidative stress resistance in Klebsiella pneumoniae . We demonstrate that CusR directly controls the cusCFBA copper efflux system through promoter binding and that a conserved aspartate residue is essential for this regulatory function. Loss of CusR disrupts copper balance, increases reactive oxygen species accumulation, and reduces bacterial resistance to oxidative stress. These findings reveal a conserved regulatory mechanism that integrates metal stress adaptation with oxidative stress defense, providing new insights into how bacterial pathogens survive under host-imposed environmental pressures.