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

伴生放线菌(Aggregatibacter Actinmycetcomitans，AA)与局部侵袭性密切相关 牙周炎(1~3)。在无氧环境下，牙周袋AA暴露于 动态平衡，然而，人们对AA用于获得必要营养的过程知之甚少，例如： 铁，才能适应变化而坚持。研究AA用来感知和响应的机制 为了开发牙周病的治疗方法，环境压力是必不可少的 进一步了解微生物内分泌学领域的知识。两组分系统(TCS)，称为 QseBC由AA表达，在机体的适应和生存过程中发挥重要作用。 厌氧环境。传感器分子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)。YgiW蛋白功能未知，与QseBC共表达 TCS。研究发现，肠动蛋白操纵子基因的调节在存在的情况下保持不变。 CAT-Fe(Weigel 2015)，当参与铁摄取的其他基因下调时，表明它必须 在厌氧环境中对AA的持久性起着重要作用。在编码的四个基因中， 肠动蛋白操纵子有一个外膜肠动蛋白受体，FepA(4)，但AA不产生 铁载体(5)。局限性侵袭性牙周炎促进中性粒细胞浸润增加 (6)中性粒细胞(PMN)。PMN已被证明能释放儿茶酚胺(CAT)，这是一种 铁载体，提示炎症的龈下袋可能是一个富含CAT-Fe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和通过 QseBC，在厌氧的牙龈下环境中启动新陈代谢生长，然后获得 通过肠瘤蛋白复合体摄取CAT-Fe的铁。这一假设将通过以下方式进行检验 解决以下目标：1)表征中性粒细胞合成、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，以及3)确定 YgiW对QSec激活的贡献。研究设计将包括将人中性粒细胞直接暴露于 AA野生型和毒力因子突变体及用ELISA法测定CAT水平以证实其能力 释放CAT(目标1a)并确定释放它们所需的信号(目标1b)。中性粒细胞颗粒将 使用CAT抗体进行分离和筛选，以确定CAT的储存位置(目标1c)。下一步，投标动力 将使用分析来表征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确定YgiW对QSec激活的贡献(目标2b)。最后，有约束力。 将使用动力学分析来确定CAT-Fe与Fepa(Aim 3a)的结合作用，以及 慢性牙周炎动物模型将被用来评估肠杆菌素操纵子在AA毒力中的作用(AIM 3B)。

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).