Harnessing iron acquisition to hinder enterobacterial pathogenesis

PROJECT SUMMARY The primary objective of this application is to investigate siderophore-based immunization and antibiotic delivery strategies designed to inhibit the growth of Escherichia coli and non-typhoidal Salmonella (NTS). These Gram-negative facultative anaerobic bacteria are major causes of infections in diverse patient populations. E. coli includes commensal organisms, pathogens, and pathobionts (organisms that are usually harmless but are pathogenic in some settings) and cause infections that include urinary tract infections (UTI), bacteremia, meningitis, and sepsis. Moreover, a pathovar known as adherent-invasive E. coli (AIEC) is commonly isolated from patients with Crohn's disease, a form of inflammatory bowel disease. NTS, including Salmonella enterica serovar Typhimurium (STm), are major causes of inflammatory diarrhea. The primary site of E. coli and NTS colonization is the gastrointestinal tract, where these organisms thrive during colitis and disseminate to other body sites. Recent studies, including work from our laboratories, demonstrate that iron (Fe) availability is a key factor for the progression of E. coli and NTS colonization in the gut, motivating the research proposed in this grant application. Our central hypothesis is that targeting siderophores and their uptake machineries can limit enteric pathogen growth in vitro and in vivo. Both E. coli and NTS deploy the catecholate siderophores enterobactin (Ent) and salmochelin (DGE, diglucosylated enterobactin) in the gut to scavenge Fe3+ from the host. We propose that blocking Ent&DGE-mediated Fe3+ acquisition by bacterial pathogens or targeting Ent&DGE transport systems to deliver antibiotics will provide a means to inhibit the growth of STm and AIEC in the inflamed gut. In support of this notion, we developed a siderophore-based immunization based on Ent that inhibits STm and AIEC growth in the murine gut, and we synthesized and evaluated siderophore-antibiotic conjugates (SACs) based on the Ent&DGE scaffold that target E. coli and STm. In Aim 1, we will use mutants in Fe acquisition genes in STm and AIEC to test whether CTB-Ent immunization results in specific inhibition of pathogen growth and association with the gut mucosa when the pathogen produces Ent&DGE; determine the effect of CTB-Ent immunization on the mucosal-associated versus luminal microbiota; and ascertain whether neutralizing anti- Ent&DGE Ig mediate protection by limiting pathogen association with the mucosa. In Aim 2, we will evaluate the antimicrobial activity of three Ent&DGE-based SACs. Studies in vitro will largely focus on how key environmental variables that characterize diverse host environments affect the antimicrobial activity of SACs, whereas studies in vivo will evaluate the consequences of SAC administration on the gut microbiome composition as well as on inhibiting mucosal expansion of STm and AIEC during colitis. This work may lead to future development of siderophore-binding antibodies and SACs as therapeutics to limit colonization of enteric pathogens and pathobionts in the inflamed gut.