Administrative Diversity Supplement for Aimee Potter

项目总结/摘要 淋病奈瑟氏菌（Ngo）是导致性传播感染淋病的细菌。 Ngo感染在粘膜上皮表面触发强有力的炎症反应，其特征在于 通过募集中性粒细胞，而中性粒细胞不能清除细菌。随着抗生素耐药性的增加， 非政府组织，新的疗法是迫切需要治疗淋病，一个有前途的方法是打击其 依赖于从其专性人类宿主获得的金属，如铁和锌。在人类和 在其他哺乳动物宿主中，必需金属存在于与蛋白质的高亲和力复合物中， 免费提供。这些蛋白质的例子是铁结合转铁蛋白和血红蛋白，发现于 血清/红细胞中的丰度，以及大量存在的锌结合乳铁蛋白和钙卫蛋白 中性粒细胞和粘膜分泌物中。为了克服这种限制（“营养免疫”），Ngo 产生与人金属结合蛋白结合的外膜、表面暴露的转运蛋白， 通过TonB系统提供的能量直接从其中提取金属。已知有8个TonB- 非政府组织中的依赖性转运蛋白（TDTS）。四个Tdt是父应用程序的焦点：TdfH和TdfJ， 分别与人钙卫蛋白和银屑病蛋白结合以获得锌; TdfF和TdfG是铁- 调节，但其配体和功能仍不明确。另外四家TDT参与金属收购 在Ngo中：TbpA，其结合人转铁蛋白; LbpA，其结合人乳铁蛋白; HpuB，其结合人 血红蛋白;和FetA，其结合铁肠杆菌素，由微生物群成员产生的铁载体。 在这个多样性补充申请中，我们提出了三个独立于母公司的具体目标 R 01奖：1）测试TbpAB、FetAB和HpuAB在暴露于以下物质的Ngo的存活和生长中的作用： 原代人中性粒细胞，包括配体结合和TonB依赖性转运的必要性; 2） 确定生物可利用的金属和它们的同源金属结合蛋白，可用于非政府组织在 中性粒细胞的感染;和3）定义Ngo在金属耗尽和 充满金属的生长条件。后一个目标将利用最近的基因组规模的代谢网络 重建我们的实验室最近创建的，与公开可用的金属依赖性转录 Ngo的数据。这些目标的完成将揭示非政府组织对人类的反应的新见解， 营养免疫和发现新的生物学为未来的机制研究在这个主机病原体 接口.它将为多样性补充博士后培训生提供细菌遗传学方面的明确培训 操作和中性粒细胞功能测定，同时让学员参与跨学科研究 在父R 01的主持下进行。同时，培训生将发展职业过渡 技能和发现新的研究重点，这将有助于她实现成为一个独立的职业目标， 非政府组织生物学和发病机制终身首席研究员。

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.