Microaerobic Copper Stress Redirects Pyruvate Metabolism and Reveals a CopL-linked Nitrogen Response in Staphylococcus aureus.

Copper is both an essential enzyme cofactor and an antimicrobial agent deployed by the host immune system to eradicate micro-organisms. The epidemic community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) lineage USA300 carries mobile genetic elements that encode copX/B and copL, conferring hyper-resistance to copper, but the role of CopL beyond extracellular copper sequestration remains unclear. We have combined RNA sequencing with targeted metabolite assays under microaerobic conditions, more reflective of host environments, to define key copper induced responses in WT and copL mutant strains. Subinhibitory copper exposure in microaerobic conditions triggered a distinctive transcriptional response across multiple biological functions. Unlike previous studies, copper exposure did not induce an oxidative stress response. Instead, classical copper resistance, teichoic acid modification, immune-evasion factors and core metabolic genes were induced, while genes for stress responses, metal homeostasis and virulence were repressed. Gene set enrichment analysis (GSEA) identified regulation by multiple global regulators, e.g. SigB, CodY, CcpA, Agr and Sae. Copper exposure affected metabolism, redirecting pyruvate flux toward acetoin and lactate production rather than acetate, accompanied by coordinated shifts in TCA cycle and amino acid pathways, including glutamate accumulation. Inactivation of copL revealed a distinct adaptive response, with strong induction of nitrogen metabolism genes and nitrite reduction. Together, these data show that copper functions as a regulatory signal, triggering coordinated transcriptional and metabolic remodelling that potentiates S. aureus fitness in the host.