Administrative Diversity Supplement for Aimee Potter

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PROJECT SUMMARY/ABSTRACT Neisseria gonorrhoeae (Ngo) is the bacterium that causes the sexually transmitted infection gonorrhea. Infection with Ngo triggers a potent inflammatory response at mucosal epithelial surfaces that is characterized by recruitment of neutrophils, which are unable to clear the bacteria. With mounting antibiotic resistance in Ngo, new therapeutics are urgently needed to treat gonorrhea, and one promising approach is to combat its dependence on metals such as iron and zinc that are acquired from its obligate human host. In humans and other mammalian hosts, essential metals are found within high affinity complexes with proteins and are not freely available. Examples of these proteins are the iron-binding transferrin and hemoglobin, found in abundance in serum/erythrocytes, and zinc-binding lactoferrin and calprotectin, which are found in abundance in neutrophils as well as mucosal secretions. To overcome this restriction (“nutritional immunity”), Ngo produces outer-membrane, surface-exposed transporters that bind to human metal-binding proteins and extract the metal from them directly, via energy supplied by the TonB system. There are 8 known TonB- dependent transporters (Tdts) in Ngo. Four Tdts are the focus of the parent application: TdfH and TdfJ, which bind to human calprotectin and psoriasin, respectively, to acquire zinc; and TdfF and TdfG, which are iron- regulated but whose ligands and functions remain undefined. Four other Tdts are involved in metal acquisition in Ngo: TbpA, which binds human transferrin; LbpA, which binds human lactoferrin; HpuB, which binds human hemoglobin; and FetA, which binds ferric enterobactin, a siderophore produced by members of the microbiota. In this diversity supplement application, we propose three Specific Aims that are independent of the parent R01 award: 1) Test the role for TbpAB, FetAB, and HpuAB in survival and outgrowth of Ngo exposed to primary human neutrophils, including the necessity for ligand binding and TonB-dependent transport; 2) Determine the bioavailable metals and their cognate metal-binding proteins that are available to Ngo during infection of neutrophils; and 3) Define the metabolic pathways Ngo differentially uses in metal-depleted and metal-replete growth conditions. The latter Aim will leverage a recent genome-scale metabolic network reconstruction our lab recently created, contextualized with publicly available metal-dependent transcriptional datasets of Ngo. Completion of these Aims will reveal new insights into the response of Ngo to human nutritional immunity and uncover new biology for future mechanistic investigation at this host-pathogen interface. It will provide the diversity supplement postdoctoral trainee with defined training in bacterial genetic manipulation and neutrophil functional assays, while engaging the trainee in the interdisciplinary research taking place under the auspices of the parent R01. At the same time, the trainee will develop career transitional skills and uncover new research foci, which will help her achieve her career goal of becoming an independent tenure-track principal investigator in Ngo biology and pathogenesis.