Exploitation of the host immune response by Aggregatibacter actinomycetemcomitans mediated by QseBC

QseBC 介导的放线聚集菌利用宿主免疫反应

基本信息

批准号：
10080721
负责人：
Hazel Ozuna
金额：
$ 3.33万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10080721
关键词：
Actinobacillus actinomycetemcomitans Address Anaerobic Bacteria Animal Model Antibodies Binding Catecholamines Complex Cytoplasmic Granules Development Endocrinology Enterobactin Environment Enzyme-Linked Immunosorbent Assay Exposure to Foundations Future Gene Expression Genes Growth Homeostasis Hormones Human Immune response Infection Infiltration Inflammatory Iron Kinetics Knowledge Ligands Location Measures Mediating Membrane Metabolism Microbial Biofilms Nutrient Operon Organism Outcome Periodontal Diseases Periodontitis Phagocytes Play Process Proteins Regulation Research Design Respiration Role Siderophores Signal Transduction Stress Surface Plasmon Resonance System Testing Therapeutic Transferrin Virulence Virulence Factors Work design enteric pathogen environmental change exposed human population macrophage microbial mutant neutrophil periplasm receptor response sensor therapeutic target uptake

项目摘要

Aggregatibacter actinomycetemcomitans (Aa) has been strongly associated with localized aggressive periodontitis (1-3). At the anaerobic environment of the sub-gingival pocket Aa is exposed to changes in homeostasis, yet little is known about the processes used by Aa to acquire the necessary nutrients such as: iron, in order to adjust to changes and persist. The study the mechanism used by Aa to sense and respond to environmental stress is essential in order to to develop therapeutic treatments for periodontal disease and further the knowledge in the field of microbial endocrinology. A two component system (TCS), known as QseBC, is expressed by Aa and it plays an important role in the process of adaptation and survival in the anaerobic environment. The sensor molecule QseC was found to play a role in growth and virulence of Aa (1). Further, the signals responsible for the activation of this TCS were identified as being catecholamines and iron (CAT-Fe) (Weigel 2015). The protein YgiW, has no known function and it is co-expressed with the QseBC TCS. The regulation of the enterobactin operon genes were found to remain unchanged in the presence of CAT-Fe (Weigel 2015), when other genes involved in iron uptake were downregulated, suggesting that it must play an important role for Aa persistence in the anaerobic environment. Among the four genes encoded in the enterobactin operon there is an outer membrane enterobactin receptor, FepA (4), yet Aa does not produce siderophores (5). Localized aggressive periodontitis promotes increased infiltration of polymorphonuclear (PMNs) leukocytes or neutrophils (6). PMNs have been shown to release catecholamines (CAT), a type of siderophore, suggesting that the inflamed sub-gingival pocket may serve as a CAT-Fe rich environment. Therefore, the proposed hypothesis is that Aa utilizes catecholamines from the host environment and via QseBC, priming metabolism for growth in the anaerobic sub-gingival environment, and then acquiring iron through the uptake of CAT-Fe via the enterobactin complex. The hypothesis will be tested by addressing the following aims: 1) to characterize the synthesis, storage and release of CAT by PMNs stimulated with Aa, 2) to define the interaction of CAT-Fe with QseC and/or YigiW, and 3) determine the contribution of YgiW on QseC activation. The research design will consist of exposing human PMNs directly to wild type and virulence factors mutants of Aa and measuring CAT levels by ELISA to confirm their ability to release CAT (Aim 1a) and identify the signals required for their release (Aim 1b). The granules of PMNs will be isolated and screened using CAT antibodies, to identify where CAT are stored (Aim 1c). Next, biding kinetic analysis will be used to characterize the interaction of CAT-Fe to QseC and/or YgiW (Aim 2a) and surface plasmon resonance will be used to define YgiW contribution to QseC activation (Aim 2b). Lastly, binding kinetic analysis will be employed to determine the binding interaction of CAT-Fe to FepA (Aim 3a) and the chronic periodontitis animal model will be use to asses the role of the enterobactin operon in Aa virulence (Aim 3b).

放线菌聚集杆菌 (Aa) 与局部侵袭性密切相关牙周炎 (1-3)。在龈下袋 Aa 的厌氧环境下，Aa 会发生变化体内平衡，但人们对 Aa 获取必要营养物质的过程知之甚少，例如：铁，才能适应变化并坚持下去。研究 Aa 用于感知和响应的机制环境压力对于开发牙周病的治疗方法至关重要进一步了解微生物内分泌学领域的知识。二元系统 (TCS)，称为 QseBC，由Aa表达，在生物体的适应和生存过程中发挥着重要作用。厌氧环境。研究发现传感器分子 QseC 在 Aa 的生长和毒力中发挥作用 (1)。此外，负责激活该 TCS 的信号被确定为儿茶酚胺和铁。（CAT-Fe）（Weigel 2015）。蛋白质 YgiW 没有已知的功能，它与 QseBC 共表达 TCS。发现肠杆菌素操纵子基因的调节在存在以下物质的情况下保持不变 CAT-Fe (Weigel 2015)，当其他参与铁吸收的基因被下调时，这表明它必须 Aa 在厌氧环境中的持久性发挥着重要作用。编码的四个基因中肠杆菌素操纵子有一个外膜肠杆菌素受体 FepA (4)，但 Aa 不产生铁载体 (5)。局部侵袭性牙周炎促进多形核细胞浸润增加 (PMN) 白细胞或中性粒细胞 (6)。 PMN 已被证明可以释放儿茶酚胺 (CAT)，这是一种铁载体，表明发炎的龈下袋可能是富含 CAT-Fe 的环境。因此，提出的假设是 Aa 利用宿主环境中的儿茶酚胺并通过 QseBC，在厌氧的龈下环境中启动新陈代谢以促进生长，然后获得铁通过肠杆菌素复合物吸收 CAT-Fe。该假设将通过以下方式进行检验实现以下目标：1）表征 PMN 合成、储存和释放 CAT 的过程用 Aa 刺激，2) 定义 CAT-Fe 与 QseC 和/或 YigiW 的相互作用，以及 3) 确定 YgiW 对 QseC 激活的贡献。研究设计将包括将人类中性粒细胞直接暴露于野生型和 Aa 毒力因子突变体并通过 ELISA 测量 CAT 水平以确认其能力释放 CAT（目标 1a）并识别释放所需的信号（目标 1b）。 PMN 颗粒将使用 CAT 抗体进行分离和筛选，以确定 CAT 的储存位置（目标 1c）。接下来，投标动力学分析将用于表征 CAT-Fe 与 QseC 和/或 YgiW（目标 2a）以及表面的相互作用等离子共振将用于定义 YgiW 对 QseC 激活的贡献（目标 2b）。最后，绑定将采用动力学分析来确定 CAT-Fe 与 FepA 的结合相互作用（目标 3a）以及慢性牙周炎动物模型将用于评估肠杆菌素操纵子在 Aa 毒力中的作用（目的 3b).