Abstract

The escalating threat of antimicrobial resistance in Klebsiella pneumoniae necessitates novel therapeutic vulnerabilities. Here, we identify the redox-sensing regulator SoxR as a specific target of zinc toxicity and reveal that SoxR regulatory networks differ fundamentally even among closely related Enterobacteriaceae. Integrated in vitro and in vivo analyses show that zinc competitively displaces the essential SoxR [2Fe–2S] cluster, causing protein destabilization and functional inactivation. Consequently, bacteria fail to activate the canonical soxS–sodA defense axis, rendering them hypersensitive to vitamin K3-induced superoxide stress. Building on this mechanism, we established a synergistic strategy combining zinc with vitamin K3. This combination elicited potent bactericidal activity against K. pneumoniae (including ciprofloxacin-resistant strains) and Salmonella Typhimurium, with a remarkably low propensity for resistance development. Furthermore, treatment significantly improved survival in a Galleria mellonella acute infection model without detectable host toxicity. Collectively, highlighting SoxR [2Fe–2S] cluster integrity as a critical vulnerability, this work extends metal–redox crosstalk-based antibacterial intervention from Escherichia coli to a clinically important pathogen and provides a conceptual framework for developing redox-targeting therapies against drug-resistant gram-negative bacteria.