Structural and Functional Characterization of a Copper Efflux Protein: PcoB from Caulobacter crescentus

Promoter

Prof. Catherine MICHAUX, UNamur, Department of chemistry, Unit of theoretical and structural physico-chemistry (UCPTS), Laboratory of Physical Chemistry of Biomolecules (CPB)

Jury

• Johan WOUTERS (UNamur), Président
• Dr Catherine MICHAUX (UNamur), Secrétaire
• Jean-Yves MATROULE (UNamur)
• Dr Guillaume ROUSSEL (UCLouvain)
• Francesca CECCHET (UNamur)
• Prof. Hennie VALKENIER (ULB)

Summary

Nosocomial infections represent a major public health issue, further exacerbated by the global spread of bacterial resistance to antibiotics. In response to this growing threat, the exploration of alternative or complementary strategies to conventional treatments has become crucial. Among these, the use of copper has attracted renewed attention. This metal has long been recognized for its natural antibacterial properties, known since antiquity. Copper exerts its toxic effects through multiple mechanisms, including membrane disruption, generation of reactive oxygen species, and denaturation of proteins and nucleic acids, ultimately leading to rapid cell death. These characteristics make it a highly effective biocidal agent, particularly in hospital environments.

However, the selective pressure resulting from increased copper exposure in the environment has led to the emergence of specific bacterial resistance systems. These systems enable strict control of copper homeostasis by limiting intracellular accumulation through efflux, sequestration, or oxidation mechanisms. In Caulobacter crescentus, an environmental bacterial model, copper resistance is mediated by the Pco system, which includes the outer membrane protein PcoB. Although its structure has been partially described in E. coli, its precise function remains uncertain. Preliminary observations suggest that PcoB may be involved in the export of copper from the periplasm to the extracellular space, acting as a potential release pathway.

To explore the role of PcoB in bacterial copper resistance, this study focused on the structural and functional characterization of the protein. After extraction and purification, PcoB was incorporated into artificial liposomes to establish an in vitro transport assay aimed at evaluating its ability to mediate copper efflux across a lipid bilayer. In parallel, a truncated mutant lacking the intrinsically disordered N-terminal region was produced and subjected to the same analyses. Comparing the full-length and truncated forms allowed investigation of the contribution of this flexible region to the transport mechanism and protein stability. Overall, these approaches laid the first experimental foundations for studying the transport mechanism of PcoB and represent a first step toward a deeper understanding of the Pco system’s function. They also open perspectives for the development of novel antibacterial strategies targeting bacterial copper homeostasis systems.