Neisseria gonorrhoeae metal transporters that subvert nutritional immunity

淋病奈瑟菌金属转运蛋白破坏营养免疫

• 批准号: 9218801
• 负责人: CYNTHIA N CORNELISSEN
• 金额: $ 66.27万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-09 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9218801
• 关键词: Affect; Affinity; Bacterial Genes; Binding; Binding Proteins; Bioinformatics; Biological Assay; Blocking Antibodies; Cell secretion; Cells; Characteristics; Chelating Agents; Coenzymes; Complex; Crystallography; Data; Disease; Drug Metabolic Detoxication; Employee Strikes; Environment; Exposure to; Gene Expression; Gonorrhea; Growth; Human; Imagery; Imaging Techniques; Immune; Immunity; Infection; Inflammation; Iron; Lactoferrin; Leukocyte L1 Antigen Complex; Ligands; Mass Spectrum Analysis; Measures; Membrane; Membrane Transport Proteins; Metabolism; Metals; Multi-Drug Resistance; Mutagenesis; Natural Immunity; Neisseria gonorrhoeae; Nutrient; Nutritional; Oxygen; Pharmaceutical Preparations; Proteins; S100 Proteins; S100A8 gene; S100A9 gene; Seminal Plasma; Seminal fluid; Sexually Transmitted Diseases; Site; Source; Species Specificity; Specimen; Structure; Superbug; System; Testing; Time; Transferrin; Transition Elements; Vaccines; Vagina; Variant; Zinc; biophysical techniques; combat; extracellular; in vivo; inhibitor/antagonist; innovation; insight; macrophage; microbial; mutant; neutrophil; novel; novel therapeutics; nucleotide metabolism; pathogen; prevent; protein expression; protein protein interaction; receptor; small molecule inhibitor; uptake; zinc-binding protein

The human-specific bacterial pathogen Neisseria gonorrhoeae (Ngo) causes the sexually-transmitted infection gonorrhea. Ngo expresses outer membrane transporters to acquire iron and other metals in a TonB-dependent manner. In order to prevent microbial growth and infectivity, humans use high affinity metal-binding proteins to sequester essential metals, a phenomenon known as “nutritional immunity.” Ngo subverts nutritional immunity by using dedicated transporters that bind to and remove the metals from human metal-binding proteins such as transferrin and lactoferrin. Our preliminary data indicates that two poorly-characterized Ngo outer membrane transporters, TdfH and TdfJ, enable Ngo to internalize zinc (Zn). Furthermore, we show that TdfH affords Ngo the ability to overcome Zn sequestration imposed by the innate immunity protein, calprotectin (CP), which is produced in abundance by neutrophils and macrophages and accumulates to high concentrations at sites of inflammation. We hypothesize that TdfH and TdfJ are Zn transporters that enable Ngo to overcome the growth inhibitory effects of the innate immunity proteins produced during inflammation. These studies are significant because knowing how Ngo acquires essential metals like Zn from the human host opens new opportunities for developing new therapeutics against `superbug' strains, whether they be targets for a protective vaccine or small molecule inhibitors of crucial transporters. The overarching hypothesis to be tested in the proposed study is that Ngo subverts nutritional immunity imposed by S100 proteins produced by neutrophils and macrophages by the deployment of outer-membrane transporters that bind to and relieve these proteins of their sequestered transition metal cargo. The specific aims are as follows: Aim 1 will define the characteristics of ligand interactions with TdfH and TdfJ using crystallography, biophysical approaches to protein-protein interactions and mutagenesis to confirm interactions. We will also ascertain if ligand interactions are specific to human proteins, given that humans are the only natural host for Ngo. Aim 2 will define the nutritional and metal environment sensed by Ngo upon exposure to human immune cells and secretions, employing mass spectrometry and imaging techniques alongside bacterial gene expression. Aim 3 will explore how Zn uptake systems impact survival of Ngo after exposure to human immune cells and secretions that contain S100 proteins. These studies will utilize Ngo survival assays with macrophages and neutrophils, combined with real-time confocal visualization and S100 inhibition studies. Overall these innovative studies will provide an in-depth analysis of two new outer membrane transport proteins involved in Zn acquisition and will provide novel insights into the human environmental niches in which Ngo normally resides.

人类特异性细菌病原体淋病（NGO）引起性传播感染 淋病。非政府组织表达外膜转运蛋白以获取铁和其他金属的依赖性 方式。为了防止微生物生长和感染，人类使用高亲和力金属结合蛋白 隔离金属，一种被称为“营养免疫原”的现象。非政府组织颠覆营养免疫。 通过使用与人类金属结合蛋白（例如 转铁蛋白和乳铁蛋白。我们的初步数据表明两个特征较差的非政府组织外膜 TDFH和TDFJ的转运蛋白使NGO能够内化锌（Zn）。此外，我们证明TDFH提供了非政府组织 克服先天免疫蛋白钙蛋白钙蛋白酶（CP）施加的Zn固存的能力， 由中性粒细胞和巨噬细胞以丰富的丰富产生，并在 炎。我们假设TDFH和TDFJ是Zn转运蛋白，使NGO能够克服增长 炎症过程中产生的先天免疫蛋白的抑制作用。这些研究很重要 因为知道非政府组织如何从人类主机那里获取诸如Zn之类的基本金属为新的机会打开了新的机会 开发针对“超级咬合”菌株的新疗法，无论它们是受保护疫苗还是小的靶标 关键转运蛋白的分子抑制剂。在拟议的研究中要检验的总体假设 是非政府组织颠覆由中性粒细胞和巨噬细胞产生的S100蛋白施加的营养免疫力 通过部署与其隔离的这些蛋白质结合和拯救的外膜转运蛋白 过渡金属货物。具体目的如下：AIM 1将定义配体相互作用的特征 使用TDFH和TDFJ使用晶体学，蛋白质 - 蛋白质相互作用的生物物理方法和 诱变以确认相互作用。我们还将确定配体相互作用是否特定于人类蛋白质， 鉴于人类是非政府组织的唯一自然寄主。 AIM 2将定义营养和金属环境 通过质谱法和分泌物的暴露于人类免疫核管和分泌物时，通过非政府组织感应 成像技术与细菌基因表达一起。 AIM 3将探讨Zn吸收系统如何影响 暴露于人类免疫细胞和包含S100蛋白的分泌物后的非政府组织的存活。这些研究 将使用巨噬细胞和中性粒细胞利用非政府组织的生存评估，并结合实时共聚焦可视化 和S100抑制研究。总体而言，这些创新研究将对两个新的外部进行深入分析 涉及锌获取的膜运输蛋白，并将为人类提供新颖的见解 非政府组织通常居住的环境利基市场。