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

阐明宿主碱性磷酸酶在定植和维持有益共生过程中对细菌信号修饰的作用。

• 批准号: 9099058
• 负责人: Bethany Rader
• 金额: $ 43.37万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9099058
• 关键词: Address; Adolescent; Adult; Alkaline Phosphatase; Animals; Antibodies; Architecture; Area; Bacteria; Behavior; Biology; Blood Cells; Cell Line; Cells; Chronic; Communities; Complex; Confocal Microscopy; Detection; Disease; Drug Metabolic Detoxication; Electron Microscopy; Embryonic Development; Endotoxins; Environment; Enzymes; Epithelial Cells; Evaluation; Fluorescence Microscopy; Genes; Genetic Transcription; Gram-Negative Bacteria; Hawaiian population; Homeostasis; Hour; Immune; Immune response; Immunologic Monitoring; In Vitro; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Intestines; Irritable Bowel Syndrome; Label; Lead; Levamisole; Life; Light; Lipid A; Lipopolysaccharides; Location; Maintenance; Mediating; Medicine; Methods; Microbe; Modeling; Modification; Molecular; Monitor; Morphogenesis; Names; Nature; Organ; Phosphoric Monoester Hydrolases; Population; Protein Isoforms; Proteins; Regulation; Role; Signal Transduction; Source; Specificity; Squid; Staining method; Stains; Study models; Symbiosis; Syndrome; System; Up-Regulation; Vent; Vesicle; Vibrio; Vibrio fischeri; Work; cellular microvillus; density; disorder prevention; immunocytochemistry; in vitro activity; insight; interest; juvenile animal; luminescence; mature animal; microbial; novel strategies; phosphatase inhibitor; polyclonal antibody; public health relevance

 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.

 描述（由适用提供）：了解真核宿主及其微生物符号如何相互信号，以建立和维持功能和有益共生，并避免异常炎症是医学和生物学的新兴领域。该提案的目的是阐明细菌信号和促炎性分子脂多糖（LPS）的特定修饰的作用，宿主醇糖磷酸酶（AP）酶促进有益共生并预防LPS介导的感染。我们建议利用独特且研究良好的宿主微叶系统来理解这些相互作用，夏威夷bobtail鱿鱼之间的二元共生，euprymna scolopes和生物发光细菌颤音颤音。 E. scolopes已成为研究宿主微叶相互作用已有25年以上的模型。共生的二进制性质允许将功能清晰地分配给宿主AP，而不会并发症，而脊椎动物混凝土宿主 - 伴侣 - 伴侣 - 伴侣 - 由于细菌LPS在共同符号中的复杂组成而引起的。因此，我们假设E. scolopes Aps（ESAP）在光器官，共生器官中特异性修改了V. fischeri LPS，并且这种修饰是为了保护LPS介导的炎症所必需的。提出了以下特定目的：特定目标1：主机AP对V. fischeri LPS的特定修饰的表征。特定目标2：将宿主AP的定位和传递到光器官的管腔中。目标3：主机AP对细菌信号进行修改的要求，以建立和维护功能象征主义。我们将克隆并表达ESAP同工型ESAP1和ESAP2，以表征这些ESAP在体外特异性去磷酸化V. fischeri LPS的能力。我们将通过荧光显微镜和通过免疫胶质TEM生成与ESAP1和ESAP2的多克隆抗体，并将其用于将ESAP蛋白定位在光器官中。使用该抗体，我们还将确定光器官中的ESAP来源以及将它们传递到V. fischeri LPS的方法。 Finally we will inhibit EsAPs to evaluate the necessaryness of these proteins in A) persistence of the symbol in the light organ Through monitoring bacteria 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 immunocell infiltration in the light organ and transcription f immune-related genes.这些研究的结果将提供有关宿主如何通过调节何时何地来控制细菌信号并确定预防过度炎症疾病（例如炎症性肠综合征）的新型策略来控制感染的洞察力。

项目成果

Aquaculture production of hatchling Hawaiian Bobtail Squid (Euprymna scolopes) is negatively impacted by decreasing environmental microbiome diversity.

• DOI: 10.1111/jam.15350
• 发表时间: 2022-03
• 期刊: JOURNAL OF APPLIED MICROBIOLOGY
• 影响因子: 4
• 作者: Murphy, Trevor R.;Xiao, Rui;Brooks, Marjorie L.;Rader, Bethany A.;Hamilton-Brehm, Scott D.
• 通讯作者: Hamilton-Brehm, Scott D.

Alkaline Phosphatase, an Unconventional Immune Protein.

• DOI: 10.3389/fimmu.2017.00897
• 发表时间: 2017
• 期刊: Frontiers in immunology
• 影响因子: 7.3
• 作者: Rader BA