Taming the symbiont for coexistence: a host PGRP neutralizes a bacterial symbiont toxin

In horizontally-transmitted mutualisms between marine animals and their bacterial partners, the host environment promotes the initial colonization by specific symbionts that it harvests from the surrounding bacterioplankton. Subsequently, the host must develop long-term tolerance to immunogenic bacterial molecules, such as peptidoglycan and lipopolysaccaride derivatives. We describe the characterization of the activity of a host peptidoglycan-recognition protein (EsPGRP2) during establishment of the symbiosis between the squid Euprymna scolopes and its luminous bacterial symbiont Vibrio fischeri. Using confocal immunocytochemistry, we localized EsPGRP2 to all epithelial surfaces of the animal, and determined that it is exported in association with mucus shedding. Most notably, EsPGRP2 was released by the crypt epithelia into the extracellular spaces housing the symbionts. This translocation occurred only after the symbionts had triggered host morphogenesis, a process that is induced by exposure to the peptidoglycan monomer (TCT), a bacterial ‘toxin’ that is constitutively exported by V. fischeri. Enzymatic analyses demonstrated that, like many described PGRPs, EsPGRP2 has a TCT-degrading amidase activity. The timing of EsPGRP2 export into the crypts provides evidence that the host does not export this protein until after TCT induces morphogenesis, and thereafter EsPGRP2 is constantly present in the crypts ameliorating the effects of V. fischeri TCT

Peptidoglycan Induces Loss of a Nuclear PGRP During Host Tissue Development in a Beneficial Animal–Bacterial Symbiosis

Peptidoglycan recognition proteins (PGRPs) are mediators of innate immunity and recently have been implicated in developmental regulation. To explore the interplay between these two roles, we characterized a PGRP in the host squid Euprymna scolopes (EsPGRP1) during colonization by the mutualistic bacterium Vibrio fischeri. Previous research on the squid-vibrio symbiosis had shown that, upon colonization of deep epithelium-lined crypts of the host light organ, symbiont-derived peptidoglycan monomers induce apoptosis-mediated regression of remote epithelial fields involved in the inoculation process. In this study, immunofluorescence microscopy revealed that EsPGRP1 localizes to the nuclei of epithelial cells, and symbiont colonization induces the loss of EsPGRP1 from apoptotic nuclei. The loss of nuclear EsPGRP1 occurred prior to DNA cleavage and breakdown of the nuclear membrane, but followed chromatin condensation, suggesting that it occurs during late stage apoptosis. Experiments with purified peptidoglycan monomers and with V. fischeri mutants defective in peptidoglycan-monomer release provided evidence that these molecules trigger nuclear loss of EsPGRP1 and apoptosis. The demonstration of a nuclear PGRP is unprecedented, and the dynamics of EsPGRP1 during apoptosis provide a striking example of a connection between microbial recognition and developmental responses in the establishment of symbiosis

Structural and Functional Features of a Developmentally Regulated Lipopolysaccharide-Binding Protein

ABSTRACT Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses. IMPORTANCE Mammalian lipopolysaccharide (LPS)-binding protein (LBP) is implicated in conveying LPS to host cells and potentiating its signaling activity. In certain disease states, such as obesity, the overproduction of this protein has been a reliable biomarker of chronic inflammation. Here, we describe a symbiosis-induced invertebrate LBP whose tertiary structure and LPS-binding characteristics are similar to those of mammalian LBPs; however, the primary structure of this distantly related squid protein (EsLBP1) differs in key residues previously believed to be essential for LPS binding, suggesting that an alternative strategy exists. Surprisingly, symbiotic expression of eslbp1 is induced by peptidoglycan derivatives, not LPS, a pattern converse to that of RegIIIγ, an important mammalian immunity protein that binds peptidoglycan but whose gene expression is induced by LPS. Finally, EsLBP1 occurs along the apical surfaces of all the host’s epithelia, suggesting that it was recruited from a general defensive role to one that mediates specific interactions with its symbiont

An annotated cDNA library of juvenile Euprymna scolopes with and without colonization by the symbiont Vibrio fischeri

BACKGROUND: Biologists are becoming increasingly aware that the interaction of animals, including humans, with their coevolved bacterial partners is essential for health. This growing awareness has been a driving force for the development of models for the study of beneficial animal-bacterial interactions. In the squid-vibrio model, symbiotic Vibrio fischeri induce dramatic developmental changes in the light organ of host Euprymna scolopes over the first hours to days of their partnership. We report here the creation of a juvenile light-organ specific EST database. RESULTS: We generated eleven cDNA libraries from the light organ of E. scolopes at developmentally significant time points with and without colonization by V. fischeri. Single pass 3' sequencing efforts generated 42,564 expressed sequence tags (ESTs) of which 35,421 passed our quality criteria and were then clustered via the UIcluster program into 13,962 nonredundant sequences. The cDNA clones representing these nonredundant sequences were sequenced from the 5' end of the vector and 58% of these resulting sequences overlapped significantly with the associated 3' sequence to generate 8,067 contigs with an average sequence length of 1,065 bp. All sequences were annotated with BLASTX (E-value < -03) and Gene Ontology (GO). CONCLUSION: Both the number of ESTs generated from each library and GO categorizations are reflective of the activity state of the light organ during these early stages of symbiosis. Future analyses of the sequences identified in these libraries promise to provide valuable information not only about pathways involved in colonization and early development of the squid light organ, but also about pathways conserved in response to bacterial colonization across the animal kingdom

Identifying Components of the NF-κB Pathway in the Beneficial Euprymna scolopes-Vibrio fischeri Light Organ Symbiosis

The Toll/NF-κB pathway is a common, evolutionarily conserved innate immune pathway that modulates the responses of animal cells to microbe-associated molecular patterns (MAMPs). Because MAMPs have been implicated as critical elements in the signaling of symbiont-induced development, an expressed sequence tag library from the juvenile light organ of Euprymna scolopes was used to identify members of the Toll/NF-κB pathway. Full-length transcripts were identified by using 5′ and 3′ RACE PCR. Seven transcripts critical for MAMP-induced triggering of the Toll/NF-κB phosphorylation cascade have been identified, including receptors, signal transducers, and a transcription factor. Further investigations should elucidate the role of the Toll/NF-κB pathway in the initiation of the beneficial symbiosis between E. scolopes and Vibrio fischeri

Taming the symbiont for coexistence: a host PGRP neutralizes a bacterial symbiont toxin

In horizontally transmitted mutualisms between marine animals and their bacterial partners, the host environment promotes the initial colonization by specific symbionts that it harvests from the surrounding bacterioplankton. Subsequently, the host must develop long-term tolerance to immunogenic bacterial molecules, such as peptidoglycan and lipopolysaccaride derivatives. We describe the characterization of the activity of a host peptidoglycan recognition protein (EsPGRP2) during establishment of the symbiosis between the squid Euprymna scolopes and its luminous bacterial symbiont Vibrio fischeri. Using confocal immunocytochemistry, we localized EsPGRP2 to all epithelial surfaces of the animal, and determined that it is exported in association with mucus shedding. Most notably, EsPGRP2 was released by the crypt epithelia into the extracellular spaces housing the symbionts. This translocation occurred only after the symbionts had triggered host morphogenesis, a process that is induced by exposure to the peptidoglycan monomer tracheal cytotoxin (TCT), a bacterial \u27toxin\u27 that is constitutively exported by V. fischeri. Enzymatic analyses demonstrated that, like many described PGRPs, EsPGRP2 has a TCT-degrading amidase activity. The timing of EsPGRP2 export into the crypts provides evidence that the host does not export this protein until after TCT induces morphogenesis, and thereafter EsPGRP2 is constantly present in the crypts ameliorating the effects of V. fischeri TCT