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

• 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
• 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

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).

伴放线菌聚集杆菌（Aggregatibacteractinomycetemcomitans，Aa）与局部侵袭性 牙周炎（1-3）。在龈下袋的厌氧环境下，Aa暴露于以下变化： 内稳态，但很少有人知道Aa用于获得必要的营养素的过程，如： 铁，以适应变化和坚持。研究Aa感知和响应 环境压力对于开发牙周病的治疗方法是必不可少的， 促进微生物内分泌学领域的知识。一种双组分系统（TCS），称为 QseBC由Aa表达，在植物的适应和生存过程中起着重要作用。 厌氧环境发现传感分子QseC在Aa（1）的生长和毒力中起作用。 此外，负责激活该TCS的信号被鉴定为儿茶酚胺和铁 （CAT-Fe）（Weigel 2015）。蛋白YgiW没有已知的功能，并且它与QseBC共表达。 TCS。发现肠杆菌素操纵子基因的调节在存在下保持不变。 CAT-Fe（Weigel 2015），当参与铁摄取的其他基因下调时，表明它必须与铁代谢相关。 Aa在厌氧环境中的持久性起重要作用。在编码的四个基因中， 肠杆菌素操纵子存在外膜肠杆菌素受体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 （中性粒细胞）白细胞或中性粒细胞（6）。已显示PMNs释放儿茶酚胺（CAT），一种 铁载体，这表明发炎的龈下袋可作为CAT-Fe丰富的环境。 因此，提出的假设是，Aa利用来自宿主环境的儿茶酚胺，并通过 QseBC，在厌氧龈下环境中启动生长代谢，然后获得 通过肠杆菌素复合物摄取CAT-Fe来吸收铁。该假设将通过以下方式进行检验： 解决以下目标：1）表征由PMNs合成，储存和释放CAT 用Aa刺激，2）确定CAT-Fe与QseC和/或YigiW的相互作用，和3）确定CAT-Fe与QseC和/或YigiW的相互作用。 Yg 1 W对QseC激活的贡献。研究设计将包括将人类PMNs直接暴露于 Aa的野生型和毒力因子突变体，并通过ELISA测量CAT水平，以确认它们的能力， 释放CAT（目标1a），并确定释放CAT所需的信号（目标1b）。中性粒细胞的颗粒 使用CAT抗体进行分离和筛选，以确定CAT储存的位置（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（Aim 2a）和表面的相互作用 等离子体共振将用于定义Yg 1 W对QseC激活的贡献（目标2b）。最后，约束力 将采用动力学分析来确定CAT-Fe与FepA（Aim 3a）的结合相互作用以及 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 3 b）。