AbstractIt has been known for centuries that copper is toxic to bacteria. Within the host innate immune systemengulfed bacteria are exposed to high levels of copper within the phagolysosome of macrophages. As aresponse to counteract this tool used by the innate immune system bacteria have evolved export systems topump out copper that enter the bacterial cell. Studying how pathogens respond to this copper stress will helpus devise novel antimicrobial strategies. Our model organism Streptococcus pneumoniae is a leading causeof meningitis otitis media pneumonia and sepsis worldwide. In response to macrophage-derived copperstress S. pneumoniae upregulates the cop operon locus which serves to export copper out from the bacterialcell. Enhancing copper stress above a bacteriums export capacity can be a mechanism for a novelantimicrobial. Copper chelating compounds that direct copper to macrophages for uptake and use within thephagolysosome 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 ofseveral known copper chelators and compounds with similar structural elements our lab has identified severalcandidate chelating compounds to test for antimicrobial efficacy. I hypothesize that similar copper chelatingcompounds enhance kill bacteria and enhance host macrophage killing of engulfed pathogens byincreasing the intra-macrophage copper concentration. Current gaps in our knowledge include howpathogens respond to the copper stress induced by these chelating compounds and whether compounds workin vitro and in vivo. To test this hypothesis I will first define the copper affinity of chelating compounds and thesubsequent change in intra-bacterial Cu2+ concentration. Findings from this aim assess whether chelatingcompounds increase bacterial Cu2+ concentration or increase its intracellular availability. Additionally I willdetermine how S. pneumoniae respond to copper stress induced by copper chelating compounds. Findingsfrom this aim will characterize how copper stress is induced by these copper chelating compounds. Lastly I willdetermine the role of macrophages in antimicrobial efficacy of our identified synergistic copper chelatingcompounds. Findings from this aim will show antimicrobial efficacy to be macrophage-dependent ormacrophage-independent. While most commercially available antimicrobials target bacterial DNA transcriptionor mRNA translation our research in this proposed study will employ the long-known principle of coppertoxicity for an under-utilized purpose.