Copper as a central element critical to interkingdom interactions within a polymicrobial wound environment.

Staphylococcus aureus is a global health concern, estimated to cause over one million deaths each year. To colonise and survive in humans, S. aureus must overcome the host-immune system and compete with other native and invading microorganisms. As a frequent coloniser of wounds, it forms inter-kingdom polymicrobial biofilms, which are associated with impaired wound healing and poorer clinical outcomes. However, the microbial processes that contribute to the competitiveness of S. aureus within infection-related inter-kingdom communities are poorly understood. To address this, we established a polymicrobial in vitro wound-like model (WLM) consisting of two common bacterial pathogens Staphylococcus aureus and Pseudomonas aeruginosa, as well as a the fungal pathobiont Candida albicans. Using a collection of clinical S. aureus strains we then applied a genome-wide association study (GWAS) approach to identify bacterial factors that contribute to S. aureus fitness within this environment. Amongst the six S. aureus loci identified was copA, which encodes a copper exporter that prevents the accumulation of copper in the bacterial cytoplasm. Characterising the molecular processes involved, we identified a 4-way interaction, where C. albicans releases copper from the ceruloplasmin found in the human hosts plasma, which in turn causes a build-up of copper within the S. aureus cytoplasm, rendering the bacteria more susceptible to killing by P. aeruginosa. Human infection data validates the clinical relevance of this, where there is an association between mutations in copA and an origin of infection that is wound-based, highlighting the importance of understanding the entire infection ecosystem if improvements therapeutic approaches are to be developed.