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

 描述(申请人提供)：了解真核宿主和它们的微生物共生体如何相互传递信号，以建立和维持功能和有益的共生，并避免异常炎症是医学和生物学中的一个新兴领域。本研究的目的是阐明宿主碱性磷酸酶(AP)对细菌信号和促炎分子脂多糖(LPS)的特异性修饰在促进有益共生和预防LPS介导的炎症反应中的作用。我们建议利用一个独特的、研究得很好的宿主-微生物系统来理解这些相互作用，即夏威夷短尾鱿鱼、Euprymna scolope和生物发光细菌费氏弧菌之间的二元共生。25年来，E.Scolope一直是研究宿主-微生物相互作用的模型。共生的二元性允许明确地将功能分配给宿主AP，而不会由于共生环境中细菌内毒素的复杂组成而导致脊椎动物联合宿主-共生体相互作用的并发症。因此，我们假设E.scolope APs(EsAP)特异性地修饰共生器官--光器官中的内毒素，并且这种修饰是保护内毒素介导的炎症所必需的。提出了以下具体目标：具体目标1：宿主AP对裂叶弧菌内毒素的特异性修饰的特征。具体目标2：宿主AP的定位和递送到光器官的管腔。目的3：宿主AP需要对细菌信号进行修饰，以建立和维持功能共生。我们将克隆和表达EsAP异构体EsAP1和EsAP2，以研究这些EsAP在体外特异性地去磷酸化裂殖吸虫内毒素的能力。我们将生产针对EsAP1和EsAP2的多克隆抗体，我们将使用这些抗体通过荧光显微镜和免疫金电子显微镜定位EsAP蛋白在光器官中的位置。利用这一抗体，我们还将确定光器官中EsAP的来源以及它们被传递到V.facheri LPS的方法。最后，我们将通过抑制EsAPs来评估这些蛋白质在A)通过监测细菌数量在光器官中持续共生的必要性，B)利用荧光显微镜和电子显微镜观察光器官衬里上皮细胞的重排，以及C)通过监测光器官中的免疫细胞渗透和免疫相关基因的转录来调节对内毒素的炎症反应。这些研究的结果将为宿主如何通过调节它们何时何地解毒细菌信号来控制炎症提供洞察力，并确定预防炎症性肠道综合征等过度炎症疾病的新策略。

 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.