Elucidating the Orchestrated Bacterial Response to Copper Toxicity

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 the human host 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 the concentrations of iron, calcium, and manganese 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 decrease substrate binding), specifically because the stability of complex formation with divalent metal is as follows, Cu >Zn > Fe > Mn > Ca. To restrict infection, the human host sequesters beneficial metals (iron, calcium, and manganese) 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 and 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 and metabolomics) 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 the bacterial system. Using these data and leveraging our technical expertise in metallobiology and microbiology, we will determine how metal influx, efflux, and internal metal concentrations (the “metallome”) respective to copper and zinc affect the transcriptome and metabolome. These data will not only be used to determine which systems are affected, but also when these systems are being turned on respective to concentration dependence. We will determine how some of these systems are being regulated based on mismetallation of proteins such as DNA transcription factors. We will determine how metal dysbiosis affects other metal transport. Lastly, we will determine if an affected system is poisoned by copper and/or zinc or being used to overcome that stress based on examining the metabolite level respective to the direction of the system’s transcriptomic profile.