Neisseria gonorrhoeae metal transporters that subvert nutritional immunity

人类特有的细菌病原体淋病奈瑟菌（Ngo）导致性传播感染 淋病Ngo表达外膜转运蛋白，以TonB依赖的方式获得铁和其他金属。 方式为了防止微生物生长和传染性，人类使用高亲和力金属结合蛋白来 螯合必需金属，这种现象被称为"营养免疫"。ngo颠覆营养免疫 通过使用专用的转运蛋白，其结合并从人金属结合蛋白中去除金属， 转铁蛋白和乳铁蛋白。我们的初步数据表明，两个特征不佳的Ngo外膜 转运蛋白TdfH和TdfJ使Ngo能够内化锌（Zn）。此外，我们表明，TdfH提供Ngo 克服由先天免疫蛋白钙卫蛋白（CP）施加的锌螯合的能力， 由嗜中性粒细胞和巨噬细胞大量产生，并在 炎症我们假设TdfH和TdfJ是锌转运蛋白，使Ngo克服生长 炎症过程中产生的先天免疫蛋白的抑制作用。这些研究意义重大 因为了解Ngo如何从人类宿主中获得锌等必需金属， 开发针对"超级细菌"菌株的新疗法，无论它们是保护性疫苗的目标还是小的 关键转运蛋白的分子抑制剂。拟议研究中要检验的总体假设 是Ngo破坏了由中性粒细胞和巨噬细胞产生的S100蛋白所施加的营养免疫力， 通过部署外膜转运蛋白，结合并释放这些蛋白质的螯合， 过渡金属货物。具体目标如下：目标1将定义配体相互作用的特征 与TdfH和TdfJ使用晶体学，生物物理方法蛋白质-蛋白质相互作用， 诱变以确认相互作用。我们还将确定配体相互作用是否对人类蛋白质具有特异性， 因为人类是Ngo的唯一自然宿主。目标2将定义营养和金属环境 Ngo在暴露于人类免疫细胞和分泌物后，采用质谱法检测， 成像技术与细菌基因表达。目标3将探讨锌吸收系统如何影响 Ngo在暴露于含有S100蛋白的人类免疫细胞和分泌物后的存活。这些研究 将利用巨噬细胞和中性粒细胞的Ngo存活测定，结合实时共聚焦可视化， 和S100抑制研究。总的来说，这些创新研究将为两个新的外部环境提供深入分析。 膜转运蛋白参与锌的收购，并将提供新的见解，人类 非政府组织通常居住的环境小生境。

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.