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

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 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.