PROJECT SUMMARY The primary objective of this research initiative is to use siderophore-antibiotic conjugates based on native siderophore platforms to deliver antibacterial cargo to Gram-negative bacterial pathogens and pathobionts on the basis of siderophore receptor expression and the molecular recognition that occurs between a native siderophore and its receptor(s). Iron is an essential nutrient for almost all human pathogens, and siderophores are small-molecule iron chelators and virulence factors produced by bacteria for acquiring this essential nutrient in the vertebrate host. We hypothesize that the exquisite interactions between native siderophores and dedicated siderophore receptors allow siderophore-appended antibiotics to be directed to select groups of bacterial pathogens, resulting in targeted antibiotic delivery. This approach allows for species- and strain- specific targeting of antibiotics to select pathogens and pathobionts on the basis of siderophore receptor expression. We address this hypothesis by investigating enterobactin and salmochelins, siderophores produced by enteric Gram-negative bacteria, including Escherichia coli and non-typhoidal Salmonella. In Aim 1, we will expand upon our preliminary results on enterobactin- and salmochelin-mediated delivery of the ?- lactam antibiotics ampicillin (Amp) and amoxicillin (Amx) to the periplasm of Escherichia coli and perform in vitro studies with E. coli and Salmonella to address the uptake and antibacterial mechanisms of these compounds. In Aim 2, we will perform animal model studies and determine whether administration of the siderophore-?-lactam conjugates affects the composition of the commensal microbiota. Moreover, we will employ animal models of E. coli and Salmonella colonization and infection to test our hypothesis that administration of our siderophore-?-lactam conjugates will more selectively target E. coli and Salmonella and be effective at lower doses than the parent ?-lactam antibiotics. Taken together, the results from these investigations will provide the foundation for the future development of siderophore-based tools to study and manipulate the microbiota, as well as therapeutic approaches to treat bacterial infections caused by Gram- negative pathogens that harness the iron acquisition systems employed by these deleterious organisms when colonizing the mammalian host.