PROJECT SUMMARY Metal dysbiosis is detrimental to any living system as approximately 40% of proteins use metals as a cofactor or structural component. Therefore, when pathogenic bacteria invade a host, there is a battle for metal micronutrients such as iron, calcium, manganese, and zinc that benefit each organism. While human hosts acquire metals through their diet, bacteria must acquire metals from within the host. However, for bacteria that exist at the host/pathogen interface, some host-utilized metals can be toxic to bacteria. For example, compared to iron, calcium, and manganese concentrations needed for survival, zinc and especially copper are toxic to bacteria even at lower concentrations. As such, bacteria have evolved import and export systems to maintain homeostasis. Complicating metal acquisition is mismetallation, when the unintended metal binds to the protein to diminish function (e.g., low enzymatic turnover or decreased substrate binding), specifically because the stability of complex formation with divalent metal is as follows: Cu >Zn > Fe > Mn > Ca. The human host sequesters beneficial metals (iron, calcium, and manganese) to restrict infection while also bombarding the bacteria with zinc and copper. How bacteria respond to copper + zinc stress and the different concentrations of these metals they encounter in the host are largely unknown. While metal toxicity has been the subject of other studies, most of these have focused on single concentrations of one metal, often in complex media. These media are more lavish than the host environment and may mask portions of the metal response. To address fundamental gaps in knowledge regarding how bacteria respond to metal dysbiosis, we used a multi-omics approach (transcriptomics, metabolomics, and suggest a proteomic arm) to investigate the pathways affected during bacterial disruption via copper and zinc at varying concentrations in a host-adjacent, minimal, and defined media in Streptococcus pneumoniae as