In Vitro Culture of the Insect Endosymbiont Spiroplasma poulsonii Highlights Bacterial Genes Involved in Host- Symbiont Interaction

Endosymbiotic bacteria associated with eukaryotic hosts are omnipresent in nature, particularly in insects. Studying the bacterial side of host-symbiont interactions is, however, often limited by the unculturability and genetic intractability of the symbionts. <i>Spiroplasma poulsonii</i> is a maternally transmitted bacterial endosymbiont that is naturally associated with several <i>Drosophila</i> species. <i>S. poulsonii</i> strongly affects its host's physiology, for example by causing male killing or by protecting it against various parasites. Despite intense work on this model since the 1950s, attempts to cultivate endosymbiotic <i>Spiroplasma in vitro</i> have failed so far. Here, we developed a method to sustain the <i>in vitro</i> culture of <i>S. poulsonii</i> by optimizing a commercially accessible medium. We also provide a complete genome assembly, including the first sequence of a natural plasmid of an endosymbiotic <i>Spiroplasma</i> species. Last, by comparing the transcriptome of the <i>in vitro</i> culture to the transcriptome of bacteria extracted from the host, we identified genes putatively involved in host-symbiont interactions. This work provides new opportunities to study the physiology of endosymbiotic <i>Spiroplasma</i> and paves the way to dissect insect-endosymbiont interactions with two genetically tractable partners. <b>IMPORTANCE</b> The discovery of insect bacterial endosymbionts (maternally transmitted bacteria) has revolutionized the study of insects, suggesting novel strategies for their control. Most endosymbionts are strongly dependent on their host to survive, making them uncultivable in artificial systems and genetically intractable. <i>Spiroplasma poulsonii</i> is an endosymbiont of <i>Drosophila</i> that affects host metabolism, reproduction, and defense against parasites. By providing the first reliable culture medium that allows a long-lasting <i>in vitro</i> culture of <i>Spiroplasma</i> and by elucidating its complete genome, this work lays the foundation for the development of genetic engineering tools to dissect endosymbiosis with two partners amenable to molecular study. Furthermore, the optimization method that we describe can be used on other yet uncultivable symbionts, opening new technical opportunities in the field of host-microbes interactions

The Drosophila melanogaster gut microbiota provisions thiamine to its host

The microbiota of Drosophila melanogaster has a substantial impact on host physiology and nutrition. Some effects may involve vitamin provisioning, but the relationships between microbe-derived vitamins, diet, and host health remain to be established systematically. We explored the contribution of microbiota in supplying sufficient dietary thiamine (vitamin B1) to support D. melanogaster at different stages of its life cycle. Using chemically defined diets with different levels of available thiamine, we found that the interaction of thiamine concentration and microbiota did not affect the longevity of adult D. melanogaster Likewise, this interplay did not have an impact on egg production. However, we determined that thiamine availability has a large impact on offspring development, as axenic offspring were unable to develop on a thiamine-free diet. Offspring survived on the diet only when the microbiota was present or added back, demonstrating that the microbiota was able to provide enough thiamine to support host development. Through gnotobiotic studies, we determined that Acetobacter pomorum, a common member of the microbiota, was able to rescue development of larvae raised on the no-thiamine diet. Further, it was the only microbiota member that produced measurable amounts of thiamine when grown on the thiamine-free fly medium. Its close relative Acetobacter pasteurianus also rescued larvae; however, a thiamine auxotrophic mutant strain was unable to support larval growth and development. The results demonstrate that the D. melanogaster microbiota functions to provision thiamine to its host in a low-thiamine environment. Importance: There has been a long-standing assumption that the microbiota of animals provides their hosts with essential B vitamins; however, there is not a wealth of empirical evidence supporting this idea, especially for vitamin B1 (thiamine). To determine whether this assumption is true, we used Drosophila melanogaster and chemically defined diets with different thiamine concentrations as a model. We found that the microbiota does provide thiamine to its host, enough to allow the development of flies on a thiamine-free diet. The power of the Drosophila-microbiota system allowed us to determine that one microbiota member in particular, Acetobacter pomorum, is responsible for the thiamine provisioning. Thereby, our study verifies this long-standing hypothesis. Finally, the methods used in this work are applicable for interrogating the underpinnings of other aspects of the tripartite interaction between diet, host, and microbiota

Attenuation of Host NO Production by MAMPs Potentiates Development of the Host in the Squid-Vibrio Symbiosis

The presence of bacterial pathogens typically upregulates the host’s production of nitric oxide synthase (NOS) and nitric oxide (NO) as antimicrobial agents. This dramatic response is often mediated by microbe-associated molecular patterns (MAMPs) of the pathogen. In contrast, previous studies of the beneficial Euprymna scolopes/Vibrio fischeri symbiosis demonstrated that symbiont colonization results in an attenuation of host NOS/NO, which occur in high levels in the hatchling light organ. In the present study, we sought to determine whether V. fischeri MAMPs, specifically lipopolysaccharide (LPS) and the peptidoglycan derivative tracheal cytotoxin (TCT), attenuate NOS/NO, and whether this activity mediates the MAMPs-induced light organ morphogenesis. Using confocal microscopy, we visualized and quantified the levels of NOS with immunocytochemistry and NO with a NO-specfic fluorochrome. When added exogenously to seawater containing hatchling animals, V. fischeri LPS and TCT together, but not individually, induced normal NOS/NO attenuation. Further, V. fischeri mutants defective in TCT release did not. Experiments with NOS inhibitors and NO donors provided evidence that NO mediates the apoptosis and morphogenesis associated with symbiont colonization. Attenuation of NOS/NO by LPS and TCT in the squid-vibrio symbiosis provides another example of how the host’s response to MAMPs depends on the context (i.e., beneficial or pathogenic bacteria). These data also provide a mechanism by which symbiont MAMPs regulate host development through NO attenuation

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

Shaping the microenvironment: Evidence for the influence of a host galaxin on symbiont acquisition and maintenance in the squid-vibrio symbiosis

Most bacterial species make transitions between habitats, such as switching from free-living to symbiotic niches. We provide evidence that a galaxin protein, EsGal1, of the squid Euprymna scolopes participates in both: (i) selection of the specific partner Vibrio fischeri from the bacterioplankton during symbiosis onset and, (ii) modulation of V. fischeri growth in symbiotic maintenance. We identified two galaxins in transcriptomic databases and showed by qRT-PCR that one (esgal1) was dominant in the light organ. Further, esgal1 expression was upregulated by symbiosis, a response that was partially achieved with exposure to symbiont cell-envelope molecules. Confocal immunocytochemistry of juvenile animals localized EsGal1 to the apical surfaces of light-organ epithelia and surrounding mucus, the environment in which V. fischeri cells aggregate before migration into the organ. Growth assays revealed that one repeat of EsGal1 arrested growth of Gram-positive bacterial cells, which represent the cell type first ‘winnowed’ during initial selection of the symbiont. The EsGal1-derived peptide also significantly decreased the growth rate of V. fischeri in culture. Further, when animals were exposed to an anti-EsGal1 antibody, symbiont population growth was significantly increased. These data provide a window into how hosts select symbionts from a rich environment and govern their growth in symbiosis

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

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host