Cellular Effects of Bacterial N-3-Oxo-Dodecanoyl-L-Homoserine Lactone on the Sponge Suberites domuncula (Olivi, 1792): Insights into an Intimate Inter-Kingdom Dialogue.

International audienceSponges and bacteria have lived together in complex consortia for 700 million years. As filter feeders, sponges prey on bacteria. Nevertheless, some bacteria are associated with sponges in symbiotic relationships. To enable this association, sponges and bacteria are likely to have developed molecular communication systems. These may include molecules such as N-acyl-L-homoserine lactones, produced by Gram-negative bacteria also within sponges. In this study, we examined the role of N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) on the expression of immune and apoptotic genes of the host sponge Suberites domuncula. This molecule seemed to inhibit the sponge innate immune system through a decrease of the expression of genes coding for proteins sensing the bacterial membrane: a Toll-Like Receptor and a Toll-like Receptor Associated Factor 6 and for an anti-bacterial perforin-like molecule. The expression of the pro-apoptotic caspase-like 3/7 gene decreased as well, whereas the level of mRNA of anti-apoptotic genes Bcl-2 Homolog Proteins did not change. Then, we demonstrated the differential expression of proteins in presence of this 3-oxo-C12-HSL using 3D sponge cell cultures. Proteins involved in the first steps of the endocytosis process were highlighted using the 2D electrophoresis protein separation and the MALDI-TOF/TOF protein characterization: α and β subunits of the lysosomal ATPase, a cognin, cofilins-related proteins and cytoskeleton proteins actin, α tubulin and α actinin. The genetic expression of some of these proteins was subsequently followed. We propose that the 3-oxo-C12-HSL may participate in the tolerance of the sponge apoptotic and immune systems towards the presence of bacteria. Besides, the sponge may sense the 3-oxo-C12-HSL as a molecular evidence of the bacterial presence and/or density in order to regulate the populations of symbiotic bacteria in the sponge. This study is the first report of a bacterial secreted molecule acting on sponge cells and regulating the symbiotic relationship

Contribution d'une culture axenique de spongiaire à l'étude du dialogue moléculaire entre procaryotes et eucaryotes

Les spongiaires vivent en symbiose avec une grande communauté de bactéries. Ces animaux filtreurs se nourrissent de bactéries. Cependant, une partie de cette population bactérienne vit en association étroite avec les cellules d éponges. Malgré la présence d un système immunitaire innée chez les spongiaires, ces individus semblent tolérer certaines bactéries. A travers cette association, ces deux organismes ont dû développer des mécanismes afin de communiquer au sein de cette association étroite. Nous avons émis l hypothèse que cette communication pouvait s effectuer, entre autre, par l émission de molécules de communication inter-règne. Les éponges et les bactéries sont des organismes qui sont naturellement capables d excréter des molécules de communication pour réguler des phénomènes physiologiques. Nous avons suspecté leur intervention au sein du modèle bactéries/éponge Suberites domuncula. La présence in vivo de molécules de communication inter-règne au sein de l éponge a été recherchée par LC-ESI-MS/MS, produites potentiellement par les cellules d éponges (bioamines dérivées de la tyrosine) et par les bactéries résidentes (homosérine lactone (HSL), quinolones, rhamnolipides). Nous avons identifié plusieurs HSL au sein d extraits bruts d éponge S. domuncula ainsi qu au sein de surnageants de culture de bactéries isolées préalablement de l éponge, suggérant l origine bactérienne de ces molécules. Nous avons ensuite étudié le rôle d une de ces HSL, la 3-oxo-C12-HSL, dans le dialogue potentiel avec l hôte. Dans un premier temps, nous avons analysé l effet de cette molécule sur l expression de gènes des systèmes immunitaires et apoptotiques de l éponge S. domuncula. Les résultats démontrent que la 3-oxo-C12-HSL semble inhiber la réponse immunitaire et apoptotique de l éponge. Afin de déterminer la capacité d une bactérie isolée de l éponge Hex 311, productrice de 3-oxo-C12-HSL, l effet des LPS de cette bactérie a été comparé à celui des LPS d E. coli sur l expression de certains gènes du système immunitaire et apoptotique de l éponge. L expression de Traf 6-like est stimulée en présence des LPS de E. coli mais pas en présence de LPS de Hex 311, suggérant la nature symbiotique de cette bactérie. Dans un second temps, nous avons évalué les profils électrophorétiques de primmorphs axéniques d éponge (culture cellulaire 3D) en présence vs en absence de 3-oxo-C12-HSL. Plusieurs protéines sont surexprimées ou bien recrutées aux membranes cellulaires en présence de cette molécule, suggérant une réponse spécifique. La caractérisation de ces protéines nous a fourni des informations sur l implication potentielle de la 3-oxo-C12-HSL dans les premières étapes de l endocytose. Des expériences complémentaires nous permettrons de confirmer l implication de la 3-oxo-C12-HSL dans ce processus. La 3-oxo-C12-HSL semblerait masquer la présence physique des bactéries aux systèmes immunitaires et apoptotiques de l éponge. En contrepartie, l éponge pourrait se servir de ce signal comme témoin moléculaire de la présence/densité bactérienne dans le but de réguler la population symbiotique dans l éponge.Porifera live in close association with numerous bacterial partners. Indeed, as filter feeders, sponges prey on bacteria. Nevertheless, a part of these bacteria lives a peculiar relationship with sponge cells. The sponge tolerates their presence, despite it possesses an innate immune system fighting against microbial pathogens. Through this association, sponges and bacteria must have developed molecular means tightly interacting with their respective physiologic states. We hypothesized that both partners may interact through inter-kingdom communication molecules. Bacteria and sponge cells are naturally able to produce molecules to communicate and regulate physiological events. We suspect their involvement within the sponge/bacteria model: Suberites domuncula. We searched the in vivo presence within the sponge of inter-kingdom communication molecules, potentially produced by the sponge (tyrosine-derivate bioamines) and by inhabiting bacteria (homoserine lactones (HSL), quinolones, rhamnolipids). Using LC-ESI-MS/MS, we identified several HSL in sponge crude extracts and furthermore in the supernatant of cultured bacteria initially isolated from sponge, suggesting the bacterial origin of the molecules. We then investigated the role of these molecules in a potential dialogue with the host. We studied the expression of specific immune and apoptotic genes in presence of 3-oxo-C12-HSL. This molecule seems to inhibit the immune and apoptotic response of the sponge. In order to check the ability of a sponge-isolated bacterium Hex 311 to elude the sponge immune system, the effect of membrane lipopolysaccharides (LPS) was compared in parallele to this of LPS from E. coli on sponge immune and apoptotic genes. The expression of TRAF 6-like was stimulated in presence of LSP from E. coli but not in presence of LPS from Hex 311, suggesting that this bacterium may be symbiotic. Moreover, we analyzed differential 2D-electophoretic patterns of axenic sponge primmorphs (3D cell culture) in presence vs absence of the 3-oxo-C12-HSL. Several proteins were overexpressed in presence of this molecule or were recruited to the cell membranes, suggested a specific dedicated response. Characterization of these proteins led us to suspect an implication of the 3-oxo-C12-HSL in the early phagocytosis process. Complementary experiments may confirm the involvement of the phagocytosis pathway in response to this quorum - sensing molecule. 3-oxo-C12-HSL may hide the presence of bacteria to the sponge apoptotic and immune systems. Nethertheless, sponge may sense the 3-oxo-C12-HSL as a molecular witness of the bacterial presence and density in order to regulate the population of symbiotic bacteria in the sponge.LORIENT-BU (561212106) / SudocSudocFranceF

The Challenge of the Sponge Suberites domuncula (Olivi, 1792) in the Presence of a Symbiotic Bacterium and a Pathogen Bacterium

Sponges, which are in close contact with numerous bacteria in prey/predator, symbiotic and pathogenic relationships, must provide an appropriate response in such situations. This starts with a discriminating recognition of the partner either by a physical contact or through secreted molecules or both. We investigated the expression of the Toll-like receptor, Caspase 3/7, Tumor Necrosis Factor receptor-associated factor 6, Bcl-2 homology protein-2 and macrophage expressed genes of axenic sponge cells in the presence of a symbiotic bacterium (Endozoicomonas sp. Hex311), a pathogen bacterium (Pseudoalteromonas sp. 1A1), their exoproducts and lipopolysaccharides. The vast majority of answers are in line with what could be observed with the symbiotic bacterium. The pathogenic bacterium seems to profit from the eukaryotic cell: suppression of the production of the antibacterial compound, inhibition of the apoptosis caspase-dependent pathway, deregulation of bacterial recognition. This work contributes new scientific knowledge in the field of immunology and apoptosis in early branching metazoan harboring within its tissue and cells a large number of symbiotic bacteria

lsolement et caractérisation des bactéries de Suberites domuncula (Olivi, 1792) associées : le point sur les potentielles molécules de communication

Cinquante et une bactéries cultivables ont été isolées d partir de l'éponge Suberifes domuncula Récoltee en Bretagne (France). Celles-ci se repartissent entre les Gammaproteobacteria (é3%), les Firmicutes (18%), les Alphaproteobacteria (12o/o) et les Bacteroides (7%). La production d'homosérines lactones (HSL) a été recherchée chez 115 de ces isolats bactériens en utilisant un test, en microplaque, d'induction de la luminescence d'une souche d'Escherichia colipSB40é. Les surnageants de culture a 24, 48,72 et 9éh contenant potentiellement des HSL ont été testés. Des valeurs positives d'induction (des AHL Ont été produites) ainsi que des valeurs négatives ont été obtenues. Des expériences complémentaires ont Montre que pour ces dernières, les inhibitions de la luminescence étaient dues à un effet bactériostatique/bactéricide. Soixante quinze bactéries produisirent des HSL au moins une fois au cours D une phase de leur culture. Dix neuf bactéries isolées ont produit des AHL tout au long de leur culture, i.e.De 24 d 9é heures ; 52 d'entre-elles présentaient des effets mixtes (favorisant et inhibant la production de Luminescence), 30 une inhibition et 4 n'ont présenté aucune activité. La production d'HL estimée au court De la croissance, était d 24, 48,72 et 9é heures respectivement de 22,31o/o, 42,31o/o 3é,47% 35,30%. La production de composés inhibant la luminescence de la souche E. coli pSB40é était quant A elle, d ces mémes temps de culture, de 47,é90/o, 27,é9Yo, 47,01Yo et 55,29o/o. Parmi les producteurs d'HSL (au moins une fois au cours de la culture), 4é des 75 isolats (é8%) étaient des Gammaproteobacteria,9 (13%) des Alphaproteobacteria, T (10%) des Firmicutes et é (9%) des Flavobacteriia. Les producteurs d'HSL au sein de la famille des Gammaproteobacteria se répartissaient entre Pseudoalteromonas (15), Shewanella (7), Endozoicomonas et Coweillia (é), Vibrio (5), Pseudomonas (4) el Microbulbifer el Cobetia (1). Pour les Alphaproteobacteria un genre contenait 9 espdces : celui des Pseudovibrio. Le groupe des Flavobacteriia contenait le genre Tenacibaculum avec cinq espdces et le genre Flavobacterium une seule espdce. Sept inducteurs de luminescence d'E. coli pSB40é étaient des Firmicutes. Dans cette étude, dix espdces bactériennes ont été repérées comme probablement endemiques de l'éponge S. domuncula relative d la zone de l'étude. Les plus proches des bactéries apparentées d ce groupe serait une bactérie non cultivable, le clone Past-O03, provenant d'un corail (Porites asfreiodes) avec lequel elle présente 93-9é% d'identite. Le représentant cultivable le plus proche serait I'espdce Endozoicomonas elysicola MKT110 isolee dr partir d'un nudibranche, Elysia ornata (91 -93%). L'analyse phylogénétique Complémentaire des séquences disponibles d partir de NCBI a révélé que la bactérie provenant de S. domuncula etait plus liée au groupe de la bactérie non cultivable avec une valeur de bootstrap de 85% qu'd celui du groupe des Endozoicomonas (valeur de bootstrap de é0%). Ce travail est le premier d signaler que la famille Endozoicomonas serait en mesure de produire des HSL. En effet, parmi les cinq isolats testés, I'un d'entre eux, Endozoicomonas sp. Cdtt2, a démontré une production d'HSL pendant toute la phase de croissance, soit pendant les 9é heures de culture. L'espdce Endozoicomonas sp. Hex311 produirait quant d elle un (des) compose(s) inhibiteur(s) tout au long de sa culture. Les trois autres isolats démontrent une inhibition de la luminescence de la souche E. coli raportrice tout au long de leur croissance. Néanmoins la possibilite que des HSL soient également produites ne peut étre negligee. Par ailleurs, dans cette étude, deux bactéries ont été sélectionnées pour isoler des composés actifs présents dans les surnageants de culture: Pseudoalteromonas sp. Hex322 el Pseudomonas sp. H312. Pseudoalteromonas sp Hex322 montrait un effet inhibiteur de la luminescence d'E. coli pSB40é. L'etude des fractions de son surnageant a montré que le(s) compose(s) inhibiteur (s) etait(ent) présents dans les fractions non-polaires alors que les fractions polaires présentaient un effet anti-biofilm. Malheureusement, en raison des quantités infimes récoltées de fractions non-polaires et polaires avec une caractéristique trds polaire, aucun composé actif n'a eté isolé. Quant d Pseudomonas sp. H312, trois composés purs ont été isolées : I'acide phénazine-1-carboxylique, un di-rhamnolipide C10-C10 et des glycolipides. Les expériences que nous avons menées en mettant en contact ces deux bactéries ont montré qu'elles seraient compétitrices I'une de l'autre. Mais, des expériences complémentaires seront nécessaires pour conclure sur ce point.One hundred and fifty one cultivable bacteria were isolated from the sponge Suberifes domuncula collected in Brittany (France). This bacterial population consisted of Gammaproteobacteria (é20/0), Firmicutes (18Yo), Alphaproteobacteria (13%) and Bacteroides (7%). One hundred and fifteen were subjected to a microplate luminescence assay using E. coli pSB40é as a reporter strain in order to determine a production of AHLs, using the supernatants at 24, 48,72 and 9é hours. These molecules are a possible mean to communicate between resident bacteria and/or the host. Both positive luminescence values as well as negative ones were obtained. After further experimentations, the negative values were the result of bacteriostatic/bactericidal effects. Among the AHLs producers 75 did not produce these molecules constantly but, at least once, during the time of the culture. Only 19 of them produced AHLs throughouttheir growth (from24 to 9é hours). Fifty two bacteria exhibited both effects (enhancement and inhibition of the luminescence), 3é showed a inhibition effect and 4 had no activity. ln term of AHLs the productions at specific growth's time, at 24 hours of growth only 22.31o/o of them were able to produced AHL, while at the 48 hours growth, the number of isolates that produced AHL were 42.31o/o, then at 72 and 9é hours, the percentage of the AHL producers were 3é.47o/o and 35.30%, respectively. As for the production of E. coll inhibitor compound(s) at specific growth time was 47.é9% at 24 hours of growth, 27.é9Yo at 48 hours, 47.01% at 72 hours and 55.29% at 9é hours. Among the AHL producers (at least once throughout the culture time), 4é out of 75 isolates (é8%) were Gammaproteobacteria, 9 (13Yo) were Alphaproteobacteria,7 (10o/o) were Firmicufes and é (9%) were Flavobacteriia. The AHLs producers within the Gammaproteobacteria were those from the genera Pseudoalteromonas (15 bacteria), Shewanella (7), Endozoicomonas (é) and Coweillia (é), Vlbrio (5), Pseudomonas (4), Microbulbifer (1) and Cobetia (1). The Alphaproteobacteria contained only one genus: Pseudovibrio with nine isolates. fhe Flavobacteriia group contained the genus Tenacibaculum with five isolates and one from the genus F/avobacterium. Seven inducing the luminescence were from the Phylum Firmicutes. ln this study ten isolates probably belonged to a sponge-specialist bacterial symbiont group endemic to S. domuncula collected from the area of the study. lndeed, the closest related bacterium for this group was an Uncultured bacterium, clone Past-O03, coming from a coral (Porites asfreiodes) (93-9é% of identity) while the closest cultivable representative was Endozoicomonas elysicola MKT110 isolated from the sea slug, Elysia ornata (91-93%). Further phylogenetic analysis of the available sequences from NCBI revealed that this group was more related to the group of the uncultured bacterium with a bootstrap value of 85% rather than to lhe Endozoicomonas group (bootstrap value: é0%). Besides, we reported for the first time, the capability of lhe Endozoicomonas family to induce luminescence relative to the production of AHLs. Among five sponge- Endozoicomonas tested, one of lhem, Endozoicomonas sp. Cdtt2, showed a possibility of AHLs production throughoutthe 9éh of culture. An other Endozoicomonas, Hex311 as for it, produced an inhibitor compound(s) al24 hours; then, from 48 to 9é hours AHLs were produced. The other three Endozoicononas displayed an inhibition of the E. coli luminescence reporter strain throughout their growth. Nevertheless the possibility of an AHLs production cannot be neglected this one being hidden. For other studies, we selected two sponge-associated bacteria to proceed to the isolation of active compounds from their culture supernatants: Pseudoalteromonas sp. Hex322 and Pseudomonas sp. H312. The Pseudoalteromonas sp. Hex322 was choosen because it inhibited the luminescence of the E. coli reporter strain so, produced a bacteriostatic/bactericidal compound(s). Further investigations of the fractions showed that the inhibitor compound(s) was present in the non-polar fractions while the polar fractions exhibited an anti-biofilm effect. Unfortunately, due to the minute quantity of the non-polar and polar fractions with a very polar characteristic, none of the active compounds were successfully isolated. Concerning Pseudomonas sp. H312, three pure compounds were isolated: the phenazine-1-carboxylic acid, a dirhamnolipid Cro-Cro and a glycolipid. The experiments setting in contact both bacteria showed that they seemed to be in competition, but to gain conclusive insights more experiments are neededLORIENT-BU (561212106) / SudocSudocFranceF

Écobiologie du bivalve invasif Fulvia fragilis (Forskal in Niehbur, 1775) des cotes tunisiennes

Ce travail traite de l éco biologie de F. fragilis (Forskal in Niehbur, 1775), bivalve lessepsien, signalé en Tunisie depuis 1996. La biologie de la reproduction a été étudiée et un suivi la croissance relative et absolue a été effectué, à deux biotopes différents (la baie de Tunis et la lagune de Bizerte). Le statut de F. fragilis en Tunisie a été établi. Les études histologiques ont révélé que c est une espèce hermaphrodite simultanée. L échelle de maturation sexuelle a été dressée. La fécondation interne et l auto fécondation sont possibles chez cette espèce. La ponte est continue sur toute l année. Ce mode de reproduction optimise le succès de l installation de l espèce mais est coûteux en énergie. D ailleurs, des mortalités estivales ont été notées et une déficience du cycle de reproduction chez F. fragilis de la lagune de Bizerte, site fortement pollué, a été observée. Le suivi de la cinétique du diamètre ovocytaire a permis la validation de l échelle de maturation sexuelle. L indice de condition constitue un bon indicateur de l état physiologique de F. fragilis. L étude de la croissance relative de F. fragilis, aux deux sites de collecte, révèle des différences majeures. La coque présente une forme plus allongée à la baie de Tunis. La croissance pondérale de ce bivalve invasif est généralement défavorisée par rapport à celle de la longueur coquillière, à Tunis, inversement à la population de Bizerte. Au second site, la croissance coquillière pourrait être affectée par la pollution. Le suivi de la croissance absolue dans les deux sites atteste que le recrutement de ce bivalve serait étalé dans le temps à Tunis et très faible à Bizerte.This work deals with the biology of Eco F. fragilis (Forskal in Niebuhr, 1775), bivalve lessepsian, reported in Tunisia since 1996. The reproductive biology has been studied and monitored the relative and absolute growth was performed at two different biotopes (the Bay of Tunis and Bizerte Lagoon). The status of F. fragilis was established in Tunisia. Histological studies have revealed that it is a simultaneous hermaphrodite. The scale of sexual maturation has been established. Internal fertilization and self fertilization are possible in this species. Spawning is continuous throughout the year. This mode optimizes reproductive success of the installation of the species but is energetically costly. Moreover, summer mortality were noted and an impaired reproductive cycle in F. fragilis from the lagoon of Bizerte, heavily polluted site, was observed. Monitor the kinetics of oocyte diameter allowed the validation of the scale of sexual maturation. The condition index is a good indicator of the physiological state of F. fragilis. The study of the relative growth of F. fragilis, the two collection sites, reveals major differences. The shell has a more elongated in the Bay of Tunis. Weight gains of this invasive bivalve is generally disadvantaged relative to the length of shellfish in Tunis, inversely to the population of Bizerte. At the second site, shell growth could be affected by pollution. Monitoring of the absolute growth in both sites confirm that the recruitment of this bivalve is spread over time in Tunis and Bizerte to very low.LORIENT-BU (561212106) / SudocSudocFranceF

Lipopolysaccharides from Commensal and Opportunistic Bacteria: Characterization and Response of the Immune System of the Host Sponge Suberites domuncula

Marine sponges harbor a rich bacterioflora with which they maintain close relationships. However, the way these animals make the distinction between bacteria which are consumed to meet their metabolic needs and opportunistic and commensal bacteria which are hosted is not elucidated. Among the elements participating in this discrimination, bacterial cell wall components such as lipopolysaccharides (LPS) could play a role. In the present study, we investigated the LPS chemical structure of two bacteria associated with the sponge Suberites domuncula: a commensal Endozoicomonas sp. and an opportunistic Pseudoalteromonas sp. Electrophoretic patterns indicated different LPS structures for these bacteria. The immunomodulatory lipid A was isolated after mild acetic acid hydrolysis. The electrospray ionization ion-trap mass spectra revealed monophosphorylated molecules corresponding to tetra- and pentaacylated structures with common structural features between the two strains. Despite peculiar structural characteristics, none of these two LPS influenced the expression of the macrophage-expressed gene S. domuncula unlike the Escherichia coli ones. Further research will have to include a larger number of genes to understand how this animal can distinguish between LPS with resembling structures and discriminate between bacteria associated with it

Insights into bioaccumulation and bioconcentration of potentially toxic elements in marine sponges from the Northwestern Mediterranean coast of Morocco

International audienceThe present research aimed to investigate the concentrations and patterns of six potentially toxic elements (PTEs) in three common sponge species collected along the Moroccan Mediterranean coast, as well as their levels in ambient seawater and sediments. Distinct inter-species variability in PTEs bioaccumulation was observed among the three species, suggesting that sponges have distinct selectivity for assimilating PTEs from the surrounding environment. C. crambe had a higher enrichment capacity for Cu, As, Cr and Ni, while P. ficiformis and C. reniformis exhibited the highest concentration of Cd and Pb, respectively. Interestingly, a similar spatial distribution patterns of PTEs was observed in the three media, with high values occurring in Tangier and Al-Hoceima locations. Overall, our results confirm that sponges reliably reflect the bioavailability of PTEs in their immediate environment, especially C. crambe, whose PTE tissue contents were highly and positively correlated with the contents of all PTEs in the sediment