Elucidation of the role of bacterial signal modification by host alkaline phosphatases during colonization and maintenance of beneficial symbiosis.

 描述（由申请人提供）：了解真核宿主及其微生物共生体如何相互发出信号，以建立和维持功能性和有益的共生关系并避免异常炎症，是医学和生物学中一个新兴的感兴趣领域。本提案的目的是阐明宿主碱性磷酸酶（AP）酶对细菌信号和促炎分子脂多糖（LPS）的特异性修饰的作用，以促进有益的共生关系并防止LPS介导的炎症。我们建议利用一种独特且经过充分研究的宿主-微生物系统来了解这些相互作用，即夏威夷短尾鱿鱼、Euprymna scolopes和生物发光细菌费氏弧菌之间的二元共生关系。E. 25年多来，拟南芥一直是研究宿主-微生物相互作用的模型。共生的二元性质允许宿主AP的功能的明确分配，而不会出现脊椎动物聚生宿主-共生体相互作用中由于细菌LPS在共生体中的复杂组成而出现的并发症。因此我们假设E.表位AP（EsAP）特异性修饰费氏弧菌（V. fischeri）光器官（共生器官）中的LPS，并且这种修饰是针对LPS介导的炎症的保护所需的。具体目标1：宿主AP对费氏弧菌LPS的特异性修饰的表征。具体目标2：宿主AP的定位和递送至发光器官的管腔。目的3：宿主AP对细菌信号的修饰是建立和维持功能性共生的必要条件。我们将克隆和表达EsAP同种型EsAP 1和EsAP 2，以表征这些EsAP在体外特异性地使费氏弧菌LPS去磷酸化的能力。我们将产生EsAP 1和EsAP 2的多克隆抗体，我们将使用它们通过荧光显微镜和免疫金TEM在光器官中定位EsAP蛋白。利用该抗体，我们还将鉴定发光器官中EsAP的来源以及它们被递送至费氏弧菌LPS的方法。最后，我们将抑制EsAP以评估这些蛋白在以下中的必要性：A）通过监测细菌数量来使共生体在发光器官中持续存在，B）使用荧光显微镜和TEM来使发光器官内衬的上皮细胞重排，以及C）通过监测发光器官中的免疫细胞浸润和免疫相关基因的转录来调节发光器官中对LPS的炎症反应。这些研究的结果将提供关于宿主如何通过调节何时何地解毒细菌信号来控制炎症的见解，并确定预防过度炎症疾病（如炎症性肠综合征）的新策略。

 DESCRIPTION (provided by applicant): Understanding how eukaryotic hosts and their microbial symbionts signal to each other in order to establish and maintain a functional and beneficial symbiosis and avoid aberrant inflammation is an emerging area of interest in medicine and biology. The objective of this proposal is to elucidate the role of specific modification of bacterial signal and pro-inflammatory molecule lipopolysaccharide (LPS) by host alkaline phosphatase (AP) enzymes to promote beneficial symbiosis and protect against LPS mediated inflammation. We propose to utilize a unique and well-studied host-microbe system for understanding these interactions, the binary symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri. E. scolopes has served as a model for studying host-microbe interactions for more than 25 years. The binary nature of the symbiosis allows for clear assignment of function to host APs without complications that arise in vertebrate consortial host-symbiont interactions due to the complex composition of bacterial LPS in commensal symbioses. We therefore hypothesize that E. scolopes APs (EsAPs) specifically modify V. fischeri LPS in the light organ, the symbiotic organ, and that this modification is required for protection against LPS mediated inflammation. The following specific aims are proposed: Specific Aim 1: Characterization of specific modification of V. fischeri LPS by host APs. Specific Aim 2: Localization and delivery of host APs to the lumen of the light organ. Aim 3: Requirement of modification of bacterial signals by the host APs for establishment and maintenance of functional symbiosis. We will clone and express EsAP isoform EsAP1 and EsAP2 to characterize the ability of these EsAPs to specifically dephosphorylate V. fischeri LPS in vitro. We will produce polyclonal antibodies to EsAP1 and EsAP2 which we will use to localize EsAP proteins in the light organ, through fluorescence microscopy and through immunogold TEM. With this antibody we will also identify the source of EsAPs in the light organ and the method through which they are delivered to the V. fischeri LPS. Finally we will inhibit EsAPs to evaluate the necessity of these proteins in A) persistence of the symbiont in the light organ through monitoring bacterial numbers, B) rearrangement of epithelial cells lining the light organ using fluorescence microscopy and TEM, and C) regulation of the inflammatory response to LPS in the light organ by monitoring immune cell infiltration in the light organ and transcription f immune-related genes. The result of these studies will provide insight on how hosts control inflammation by regulating when and where they detoxify bacterial signals and identify novel strategies for prevention of diseases of excessive inflammation such as inflammatory bowel syndrome.