"Failure To Launch": Development of a Reproductive Organ Linked to Symbiotic Bacteria.

Developmental processes in animals are influenced by colonization and/or signaling from microbial symbionts. Here, we show that bacteria from the environment are linked to development of a symbiotic organ that houses a bacterial consortium in female Hawaiian bobtail squid, Euprymna scolopes. In addition to the well-characterized light organ association with the bioluminescent bacterium Vibrio fischeri, female E. scolopes house a simple bacterial community in a reproductive organ, the accessory nidamental gland (ANG). In order to understand the influences of bacteria on ANG development, squid were raised in the laboratory under conditions where exposure to environmental microorganisms was experimentally manipulated. Under conditions where hosts were exposed to depleted environmental bacteria, ANGs were completely absent or stunted, a result independent of the presence of the light organ symbiont V. fischeri. When squid were raised in the laboratory with substrate from the host's natural environment containing the native microbiota, normal ANG development was observed, and the bacterial communities were similar to wild-caught animals. Analysis of the bacterial communities from ANGs and substrates of wild-caught and laboratory-raised animals suggests that certain bacterial groups, namely, the Verrucomicrobia, are linked to ANG development. The ANG community composition was also experimentally manipulated. Squid raised with natural substrate supplemented with a specific ANG bacterial strain, Leisingera sp. JC1, had high proportions of this strain in the ANG, suggesting that once ANG development is initiated, specific strains can be introduced and subsequently colonize the organ. Overall, these data suggest that environmental bacteria are required for development of the ANG in E. scolopes. IMPORTANCE Microbiota have profound effects on animal and plant development. Hosts raised axenically or without symbionts often suffer negative outcomes resulting in developmental defects or reduced organ function. Using defined experimental conditions, we demonstrate that environmental bacteria are required for the formation of a female-specific symbiotic organ in the Hawaiian bobtail squid, Euprymna scolopes. Although nascent tissues from this organ that are involved with bacterial recruitment formed initially, the mature organ failed to develop and was absent or severely reduced in sexually mature animals that were not exposed to microbiota from the host's natural environment. This is the first example of complete organ development relying on exposure to symbiotic bacteria in an animal host. This study broadens the use of E. scolopes as a model organism for studying the influence of beneficial bacteria on animal development.

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

AIMS: The Hawaiian Bobtail Squid (Euprymna scolopes) is a model organism for investigating host-symbiont relationships. The current scientific focus is on the microbiome within E. scolopes, while very little is known about the microbiome of the tanks housing E. scolopes. We examined the hypothesis that bacterial communities and geochemistry within the squid tank environment correlate with the production of viable paralarval squid. METHODS AND RESULTS: Total DNA was extracted from sediment and filtered water samples from 'productive' squid cohorts with high embryonic survival and paralarval hatching, 'unproductive' cohorts with low embryonic survival and paralarval hatching. As a control total DNA was extracted from environmental marine locations where E. scolopes is indigenous. Comparative analysis of the bacterial communities by the 16S rRNA gene was performed using next generation sequencing. Thirty-eight differentially abundant genera were identified in the adult tank waters. The majority of the sequences represented unclassified, candidate or novel genera. The characterized genera included Aquicella, Woeseia and Ferruginibacter, with Hyphomicrobium and Rhizohapis were found to be more abundant in productive adult tank water. In addition, nitrate and pH covaried with productive cohorts, explaining 67% of the bacterial populations. The lower abundance of nitrate-reducing bacteria in unproductive adult tank water could explain detected elevated nitrate levels. CONCLUSIONS: We conclude that microbiome composition and water geochemistry can negatively affect E. scolopes reproductive physiology in closed tank systems, ultimately impacting host-microbe research using these animals. SIGNIFICANCE AND IMPACT OF STUDY: These results identify the tight relationship between the microbiome and geochemistry to E. scolopes. From this study, it may be possible to design probiotic counter-measures to improve aquaculture conditions for E. scolopes.

Alkaline Phosphatase, an Unconventional Immune Protein.

Recent years have seen an increase in the number of studies focusing on alkaline phosphatases (APs), revealing an expanding complexity of function of these enzymes. Of the four human AP (hAP) proteins, most is known about tissue non-specific AP (TNAP) and intestinal AP (IAP). This review highlights current understanding of TNAP and IAP in relation to human health and disease. TNAP plays a role in multiple processes, including bone mineralization, vitamin B6 metabolism, and neurogenesis, is the genetic cause of hypophosphatasia, influences inflammation through regulation of purinergic signaling, and has been implicated in Alzheimer's disease. IAP regulates fatty acid absorption and has been implicated in the regulation of diet-induced obesity and metabolic syndrome. IAP and TNAP can dephosphorylate bacterial-derived lipopolysaccharide, and IAP has been identified as a potential regulator of the composition of the intestinal microbiome, an evolutionarily conserved function. Endogenous and recombinant bovine APs and recombinant hAPs are currently being explored for their potential as pharmacological agents to treat AP-associated diseases and mitigate multiple sources of inflammation. Continued research on these versatile proteins will undoubtedly provide insight into human pathophysiology, biochemistry, and the human holobiont.

Regulation of Bioluminescence in Photobacterium leiognathi Strain KNH6.

UNLABELLED: Bacterial bioluminescence is taxonomically restricted to certain proteobacteria, many of which belong to the Vibrionaceae. In the most well-studied cases, pheromone signaling plays a key role in regulation of light production. However, previous reports have indicated that certain Photobacterium strains do not use this regulatory method for controlling luminescence. In this study, we combined genome sequencing with genetic approaches to characterize the regulation of luminescence in Photobacterium leiognathi strain KNH6, an extremely bright isolate. Using transposon mutagenesis and screening for decreased luminescence, we identified insertions in genes encoding components necessary for the luciferase reaction (lux, lum, and rib operons) as well as in nine other loci. These additional loci encode gene products predicted to be involved in the tricarboxylic acid (TCA) cycle, DNA and RNA metabolism, transcriptional regulation, and the synthesis of cytochrome c, peptidoglycan, and fatty acids. The mutagenesis screen did not identify any mutants with disruptions of predicted pheromone-related loci. Using targeted gene insertional disruptions, we demonstrate that under the growth conditions tested, luminescence levels do not appear to be controlled through canonical pheromone signaling systems in this strain. IMPORTANCE: Despite the long-standing interest in luminous bacteria, outside a few model organisms, little is known about the regulation and function of luminescence. Light-producing marine bacteria are widely distributed and have diverse lifestyles, suggesting that the control and significance of luminescence may be similarly diverse. In this study, we apply genetic tools to the study of regulation of light production in the extremely bright isolate Photobacterium leiognathi KNH6. Our results suggest an unusual lack of canonical pheromone-mediated control of luminescence and contribute to a better understanding of alternative strategies for regulation of a key bacterial behavior. These experiments lay the groundwork for further study of the regulation and role of bioluminescence in P. leiognathi.

Initial symbiont contact orchestrates host-organ-wide transcriptional changes that prime tissue colonization.

Upon transit to colonization sites, bacteria often experience critical priming that prepares them for subsequent, specific interactions with the host; however, the underlying mechanisms are poorly described. During initiation of the symbiosis between the bacterium Vibrio fischeri and its squid host, which can be observed directly and in real time, approximately five V. fischeri cells aggregate along the mucociliary membranes of a superficial epithelium prior to entering host tissues. Here, we show that these few early host-associated symbionts specifically induce robust changes in host gene expression that are critical to subsequent colonization steps. This exquisitely sensitive response to the host's specific symbiotic partner includes the upregulation of a host endochitinase, whose activity hydrolyzes polymeric chitin in the mucus into chitobiose, thereby priming the symbiont and also producing a chemoattractant gradient that promotes V. fischeri migration into host tissues. Thus, the host responds transcriptionally upon initial symbiont contact, which facilitates subsequent colonization.

Host/microbe interactions revealed through "omics" in the symbiosis between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri.

The association between Euprymna scolopes, the Hawaiian bobtail squid, and Vibrio fischeri, a bioluminescent bacterium, has served as a model for beneficial symbioses for over 25 years. The experimental tractability of this association has helped researchers characterize many of the colonization events necessary for symbiosis. Recent technological advances, such as the sequenced genome of V. fischeri, DNA microarrays, and high-throughput transcriptomics and proteomics, have allowed for the identification of host and symbiont factors that are important in establishing and maintaining specificity in the association. We highlight some of these findings pertaining to quorum sensing, luminescence, responses to reactive oxygen and nitrogen species, recognition of microbe-associated molecular patterns by the innate immune system of the host, and a diel rhythm that helps regulate the symbiont population. We also discuss how comparative genomics has allowed the identification of symbiont factors important for specificity and why sequencing the host's genome should be a priority for the research community.

Understanding the role of host hemocytes in a squid/vibrio symbiosis using transcriptomics and proteomics.

The symbiosis between the squid, Euprymna scolopes, and the bacterium, Vibrio fischeri, serves as a model for understanding interactions between beneficial bacteria and animal hosts. The establishment and maintenance of the association is highly specific and depends on the selection of V. fischeri and exclusion of non-symbiotic bacteria from the environment. Current evidence suggests that the host's cellular innate immune system, in the form of macrophage-like hemocytes, helps to mediate host tolerance of V. fischeri. To begin to understand the role of hemocytes in this association, we analyzed these cells by high-throughput 454 transcriptomic and liquid chromatography/tandem mass spectrometry (LC-MS/MS) proteomic analyses. 454 high-throughput sequencing produced 650, 686 reads totaling 279.9 Mb while LC-MS/MS analyses of circulating hemocytes putatively identified 702 unique proteins. Several receptors involved with the recognition of microbial-associated molecular patterns were identified. Among these was a complete open reading frame to a putative peptidoglycan recognition protein (EsPGRP5) with conserved residues for amidase activity. Assembly of the hemocyte transcriptome showed EsPGRP5 had high coverage, suggesting it is among the 5% most abundant transcripts in circulating hemocytes. Other transcripts and proteins identified included members of the conserved NF-κB signaling pathway, putative members of the complement pathway, the carbohydrate binding protein galectin, and cephalotoxin. Quantitative Real-Time PCR of complement-like genes, cephalotoxin, EsPGRP5, and a nitric oxide synthase showed differential expression in circulating hemocytes from adult squid with colonized light organs compared to those isolated from hosts where the symbionts were removed. These data suggest that the presence of the symbiont influences gene expression of the cellular innate immune system of E. scolopes.

Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization.

Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and k(cat) and K(m) values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.

Modulation of symbiont lipid A signaling by host alkaline phosphatases in the squid-vibrio symbiosis.

UNLABELLED: The synergistic activity of Vibrio fischeri lipid A and the peptidoglycan monomer (tracheal cytotoxin [TCT]) induces apoptosis in the superficial cells of the juvenile Euprymna scolopes light organ during the onset of the squid-vibrio symbiosis. Once the association is established in the epithelium-lined crypts of the light organ, the host degrades the symbiont's constitutively produced TCT by the amidase activity of a peptidoglycan recognition protein (E. scolopes peptidoglycan recognition protein 2 [EsPGRP2]). In the present study, we explored the role of alkaline phosphatases in transforming the lipid A of the symbiont into a form that changes its signaling properties to host tissues. We obtained full-length open reading frames for two E. scolopes alkaline phosphatase (EsAP) mRNAs (esap1 and esap2); transcript levels suggested that the dominant light organ isoform is EsAP1. Levels of total EsAP activity increased with symbiosis, but only after the lipid A-dependent morphogenetic induction at 12 h, and were regulated over the day-night cycle. Inhibition of total EsAP activity impaired normal colonization and persistence by the symbiont. EsAP activity localized to the internal regions of the symbiotic juvenile light organ, including the lumina of the crypt spaces where the symbiont resides. These data provide evidence that EsAPs work in concert with EsPGRPs to change the signaling properties of bacterial products and thereby promote persistent colonization by the mutualistic symbiont. IMPORTANCE: The potential for microbe-associated molecular patterns (MAMPs) to compromise host-tissue health is reflected in the often-used nomenclature for these molecules: lipopolysaccharide (LPS) is also called "endotoxin" and the peptidoglycan monomer is also called "tracheal cytotoxin" (TCT). With constant presentation of MAMPs by the normal microbiota, mechanisms to tolerate their effects have developed. The results of this contribution provide evidence that host alkaline phosphatases (APs) dephosphorylate and inactivate the symbiont MAMP lipid A. As such, APs work in synergy with a peptidoglycan recognition protein, which inactivates symbiont-exported TCT, to alter the symbiont MAMPs and promote persistence of the partnership. Not only may these activities serve to "tame" the MAMPs, but also the resulting products may themselves be important signals in persistent mutualisms. The finding of lipid A modification by APs in an invertebrate mutualism provides evidence that this specific strategy for dealing with symbiotic partners is conserved across the animal kingdom.

Helicobacter pylori perceives the quorum-sensing molecule AI-2 as a chemorepellent via the chemoreceptor TlpB.

Helicobacter pylori moves in response to environmental chemical cues using a chemotaxis two-component signal-transduction system. Autoinducer-2 (AI-2) is a quorum-sensing signal produced by the LuxS protein that accumulates in the bacterial environment in a density-dependent manner. We showed previously that a H. pylori luxS mutant was defective in motility on soft agar plates. Here we report that deletion of the luxS gene resulted in swimming behaviour with a reduced frequency of stops as compared to the wild-type strain. Stopping frequency was restored to wild-type levels by genetic complementation of the luxS mutation or by addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Synthetic DPD also increased the frequency of stops in wild-type H. pylori, similar to the behaviour induced by the known chemorepellent HCl. We found that whereas mutants lacking the chemoreceptor genes tlpA, tlpC or tlpD responded to an exogenous source of synthetic DPD, the chemoreceptor mutant tlpB was non-responsive to a gradient or uniform distribution of the chemical. Furthermore, a double mutant lacking both tlpB and luxS exhibited chemotactic behaviour similar to the tlpB single mutant, whereas a double mutant lacking both tlpB and the chemotransduction gene cheA behaved like a nonchemotactic cheA single mutant, supporting the model that tlpB functions in a signalling pathway downstream of luxS and upstream of cheA. We conclude that H. pylori perceives LuxS-produced AI-2 as a chemorepellent via the chemoreceptor TlpB.

The quorum-sensing molecule autoinducer 2 regulates motility and flagellar morphogenesis in Helicobacter pylori.

The genome of the gastric pathogen Helicobacter pylori contains a homologue of the gene luxS, which has been shown to be responsible for production of the quorum-sensing signal autoinducer 2 (AI-2). We report here that deletion of the luxS gene in strain G27 resulted in decreased motility on soft agar plates, a defect that was complemented by a wild-type copy of the luxS gene and by the addition of cell-free supernatant containing AI-2. The flagella of the luxS mutant appeared normal; however, in genetic backgrounds lacking any of three flagellar regulators--the two-component sensor kinase flgS, the sigma factor sigma28 (also called fliA), and the anti-sigma factor flgM--loss of luxS altered flagellar morphology. In all cases, the double mutant phenotypes were restored to the luxS+ phenotype by the addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Furthermore, in all mutant backgrounds loss of luxS caused a decrease in transcript levels of the flagellar regulator flhA. Addition of DPD to luxS cells induced flhA transcription in a dose-dependent manner. Deletion of flhA in a wild-type or luxS mutant background resulted in identical loss of motility, flagella, and flagellar gene expression. These data demonstrate that AI-2 functions as a secreted signaling molecule upstream of FlhA and plays a critical role in global regulation of flagellar gene transcription in H. pylori.

The stringent response is required for Helicobacter pylori survival of stationary phase, exposure to acid, and aerobic shock.

The gastric pathogen Helicobacter pylori must adapt to fluctuating conditions in the harsh environment of the human stomach with the use of a minimal number of transcriptional regulators. We investigated whether H. pylori utilizes the stringent response, involving signaling through the alarmone (p)ppGpp, as a survival strategy during environmental stresses. We show that the H. pylori homologue of the bifunctional (p)ppGpp synthetase and hydrolase SpoT is responsible for all cellular (p)ppGpp production in response to starvation conditions. Furthermore, the H. pylori spoT gene complements the growth defect of Escherichia coli mutants lacking (p)ppGpp. An H. pylori spoT deletion mutant is impaired for stationary-phase survival and undergoes a premature transformation to a coccoid morphology. In addition, the spoT deletion mutant is unable to survive specific environmental stresses, including aerobic shock and acid exposure, which are likely to be encountered by this bacterium during infection and transmission.