Abstract It has been known for centuries that copper is toxic to bacteria. Within the host innate immune system, engulfed bacteria are exposed to high levels of copper within the phagolysosome of macrophages. As a response to counteract this tool used by the innate immune system, bacteria have evolved export systems to pump out copper that enter the bacterial cell. Studying how pathogens respond to this copper stress will help us devise novel antimicrobial strategies. Our model organism, Streptococcus pneumoniae, is a leading cause of meningitis, otitis media, pneumonia, and sepsis worldwide. In response to macrophage-derived copper stress, S. pneumoniae upregulates the cop operon locus, which serves to export copper out from the bacterial cell. Enhancing copper stress above a bacterium’s export capacity can be a mechanism for a novel antimicrobial. Copper chelating compounds that direct copper to macrophages for uptake and use within the phagolysosome will enhance copper stress. A recently developed antifungal, 8-hydroxychloroquine (8-HQ), utilizes copper chelation and uptake into macrophages to enhance killing efficiency. Following screening of several known copper chelators and compounds with similar structural elements, our lab has identified several candidate chelating compounds to test for antimicrobial efficacy. I hypothesize that similar copper chelating compounds enhance kill bacteria and enhance host macrophage killing of engulfed pathogens by increasing the intra-macrophage copper concentration. Current gaps in our knowledge include how pathogens respond to the copper stress induced by these chelating compounds and whether compounds work in vitro and in vivo. To test this hypothesis, I will first define the copper affinity of chelating compounds and the subsequent change in intra-bacterial Cu2+ concentration. Findings from this aim assess whether chelating compounds increase bacterial Cu2+ concentration or increase its intracellular availability. Additionally, I will determine how S. pneumoniae respond to copper stress induced by copper chelating compounds. Findings from this aim will characterize how copper stress is induced by these copper chelating compounds. Lastly, I will determine the role of macrophages in antimicrobial efficacy of our identified synergistic copper chelating compounds. Findings from this aim will show antimicrobial efficacy to be macrophage-dependent or macrophage-independent. While most commercially available antimicrobials target bacterial DNA transcription or mRNA translation, our research in this proposed study will employ the long-known principle of copper toxicity for an under-utilized purpose.