Neisseria gonorrhoeae metal transporters that subvert nutritional immunity

人类特有的细菌病原体淋病奈瑟氏菌(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得铁和其他金属的白蛋白依赖 举止。为了防止微生物生长和传染性，人类使用高亲和力的金属结合蛋白来 隔离必需金属，一种被称为“营养免疫”的现象。非政府组织颠覆营养免疫 通过使用专门的转运蛋白与人类金属结合蛋白结合并从中去除金属，例如 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内化锌。此外，我们证明了TdfH为NGO提供了 克服天然免疫蛋白钙保护素(CP)造成的锌隔离的能力，钙保护素是 由中性粒细胞和巨噬细胞大量产生，并在 发炎。我们推测TdfH和TdfJ是锌的转运蛋白，使Ngo能够克服生长 炎症过程中产生的天然免疫蛋白的抑制作用。这些研究具有重要意义 因为了解Ngo是如何从人类宿主那里获得锌等必需金属的为 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型疫苗的目标 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NGO颠覆了由中性粒细胞和巨噬细胞产生的S100蛋白施加的营养免疫 通过外膜转运体的部署，结合并解除这些蛋白的隔离 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 TdfH和TdfJ使用结晶学，蛋白质-蛋白质相互作用的生物物理方法和 突变以确认相互作用。我们还将确定配体之间的相互作用是否专属于人类蛋白质， 鉴于人类是NGO的唯一自然宿主。目标2将定义营养和金属环境 Ngo在接触人类免疫细胞和分泌物时感觉到的，使用质谱仪和 成像技术与细菌基因表达同时进行。目标3将探索锌吸收系统如何影响 NGO暴露于人类免疫细胞和含有S100蛋白的分泌物后存活。这些研究 将利用巨噬细胞和中性粒细胞的NGO存活分析，结合实时共聚焦可视化 和S100抑制研究。总体而言，这些创新研究将深入分析两个新的外部 膜转运蛋白参与锌的获取，将为人类提供新的见解 NGO通常所在的环境利基。

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.