CueO Possesses Copper Detoxification Activity Linking Bacterial Virulence and Drug Resistance in Brucella

Brucellosis is a zoonotic disease caused by Brucella spp. that poses a significant threat to human health. Copper resistance is a key determinant of the virulence of many intracellular pathogens, including those expressing the multicopper oxidase CueO. However, the role of CueO in Brucella remains poorly understood. In this study, bioinformatics tools were used to perform sequence homology comparison and conserved domain analysis of the CueO protein. Unmarked cueO deletion mutant and complemented strain of Brucella were constructed using homologous recombination technology. Subsequently, multiple experiments were carried out, including in vitro copper tolerance tests, RAW264.7 macrophage intracellular survival assays, in vivo virulence detection in mice, antibiotic susceptibility tests (AST), and so on. We found that CueO contains highly conserved domains across bacteria (motifs 1-10). Notably, the Brucella homolog contains only motifs 2, 3, 5, 6, and 10; it remains highly conserved within the genus. Despite this reduced domain composition, CueO is important for Brucella to resist copper toxicity. Notably, CueO contributes to intracellular survival in RAW264.7 macrophages and to the maintenance of copper homeostasis in Brucella. Deletion of cueO attenuated bacterial virulence in a mouse model. In addition, CueO influences bacterial susceptibility to doxycycline and levofloxacin. Overall, our results indicate that, despite lacking several typical domains, CueO retains critical functionality and links copper detoxification to intracellular survival, virulence, and antibiotic resistance. These findings enhance our understanding of CueO function and provide new insights into the pathogenesis of Brucella.