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.

Establishment of Transgenesis in the Demosponge Suberites domuncula.

Sponges (Porifera) represent one of the most basally branching animal clades with key relevance for evolutionary studies, stem cell biology, and development. Despite a long history of sponges as experimental model systems, however, functional molecular studies are still very difficult to perform in these animals. Here, we report the establishment of transgenic technology as a basic and versatile experimental tool for sponge research. We demonstrate that slice explants of the demosponge Suberites domuncula regenerate functional sponge tissue and can be cultured for extended periods of time, providing easy experimental access under controlled conditions. We further show that an engineered expression construct driving the enhanced green fluorescence protein (egfp) gene under control of the Suberites domuncula ß-actin locus can be transfected into such tissue cultures, and that faithfully spliced transcripts are produced from such transfected DNA. Finally, by combining fluorescence-activated cell sorting (FACS) with quantitative PCR, we validate that transfected cells can be specifically reisolated from tissue based on their fluorescence. Although the number of detected enhanced green fluorescent protein (EGFP)-expressing cells is still limited, our approach represents the first successful introduction and expression of exogenous DNA in a sponge. These results represent a significant advance for the use of transgenic technology in a cornerstone phylum, for instance for the use in lineage tracing experiments.

Functional and Structural Characterization of FAU Gene/Protein from Marine Sponge Suberites domuncula.

Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FAU) gene is down-regulated in human prostate, breast and ovarian cancers. Moreover, its dysregulation is associated with poor prognosis in breast cancer. Sponges (Porifera) are animals without tissues which branched off first from the common ancestor of all metazoans. A large majority of genes implicated in human cancers have their homologues in the sponge genome. Our study suggests that FAU gene from the sponge Suberites domuncula reflects characteristics of the FAU gene from the metazoan ancestor, which have changed only slightly during the course of animal evolution. We found pro-apoptotic activity of sponge FAU protein. The same as its human homologue, sponge FAU increases apoptosis in human HEK293T cells. This indicates that the biological functions of FAU, usually associated with "higher" metazoans, particularly in cancer etiology, possess a biochemical background established early in metazoan evolution. The ancestor of all animals possibly possessed FAU protein with the structure and function similar to evolutionarily more recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. It provides an opportunity to use pre-bilaterian animals as a simpler model for studying complex interactions in human cancerogenesis.

Potential biological role of laccase from the sponge Suberites domuncula as an antibacterial defense component.

BACKGROUND: Laccases are copper-containing enzymes that catalyze the oxidation of a wide variety of phenolic substrates. METHODS: We describe the first poriferan laccase from the marine demosponge Suberites domuncula. RESULTS: This enzyme comprises three characteristic multicopper oxidase homologous domains. Immunohistological studies revealed that the highest expression of the laccase is in the surface zone of the animals. The expression level of the laccase gene is strongly upregulated after exposure of the animals to the bacterial endotoxin lipopolysaccharide. To allow the binding of the recombinant enzyme to ferromagnetic nanoparticles, a recombinant laccase was prepared which contained in addition to the His-tag, a Glu-tag at the N-terminus of the enzyme. The recombinant laccase was enzymatically active. The apparent Michaelis constant of the enzyme is 114 µM, using syringaldazine as substrate. Exposure of E. coli to the nanoparticles, coated with Glu-tagged laccase, and to the mediator 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in the presence of lignin, as the oxidizable substrate, resulted in an almost complete inhibition of colony formation. Quantitative studies of the effect of the laccase-coated iron oxide nanoparticles were performed using E. coli grown in suspension in reaction tubes within a magnetic nanoparticle separator. CONCLUSIONS: This newly designed magnetic nanoparticle separator allowed a removal of the nanoparticles after terminating the reaction. Using this system, a strong dose-dependent inhibition of the growth of E. coli by the laccase iron oxide nanoparticles was determined. GENERAL SIGNIFICANCE: From our data we conclude that the sponge laccase is involved in the anti-bacterial defense of the sponge organism.

Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S-transferase (GST).

Silicatein from Suberites domuncula was known to catalyze silica deposition in vitro under near neutral pH and ambient temperature conditions. In this study, we employed GST-glutathione (GSH) interaction system to increase the production of silicatein and develop an efficient protein immobilization method. Recombinant silicatein fused with GST (GST-SIL) was produced in E. coli and the GST-SIL protein was employed on GSH-coated glass plate. GST-SIL bound surface or matrix can catalyze the formation of silica layer in the presence of tetraethyl orthosilicate as a substrate at an ambient temperature and neutral pH. During silicatein-mediated silicification, green fluorescent protein (GFP) or horseradish peroxidase (HRP) can be efficiently immobilized on the silica surface. Immobilized GFP or HRP retained their activity and were released gradually. This biocompatible silica coating technique can be employed to prepare biomolecule-immobilized surfaces or matrixes, which are useful for the development of biocatalytic, diagnostic and biosensing system, or tissue culture scaffolds.

Nocturnin in the demosponge Suberites domuncula: a potential circadian clock protein controlling glycogenin synthesis in sponges.

Sponges are filter feeders that consume a large amount of energy to allow a controlled filtration of water through their aquiferous canal systems. It has been shown that primmorphs, three-dimensional cell aggregates prepared from the demosponge Suberites domuncula and cultured in vitro, change their morphology depending on the light supply. Upon exposure to light, primmorphs show a faster and stronger increase in DNA, protein and glycogen content compared with primmorphs that remain in the dark. The sponge genome contains nocturnin, a light/dark-controlled clock gene, the protein of which shares a high sequence similarity with the related molecule of higher metazoans. The sponge nocturnin protein was found showing a poly(A)-specific 3'-exoribonuclease activity. In addition, the cDNA of the glycogenin gene was identified for subsequent expression studies. Antibodies against nocturnin were raised and used in parallel with the cDNA to determine the regional expression of nocturnin in intact sponge specimens; the highest expression of nocturnin was seen in the epithelial layer around the aquiferous canals. Quantitative PCR analyses revealed that primmorphs after transfer from light to dark show a 10-fold increased expression in the nocturnin gene. In contrast, the expression level of glycogenin decreases in the dark by 3-4-fold. Exposure of primmorphs to light causes a decrease in nocturnin transcripts and a concurrent increase in glycogenin transcripts. It was concluded that sponges are provided with the molecular circadian clock protein nocturnin that is highly expressed in the dark where it controls the stability of a key metabolic enzyme, glycogenin.

Inducible ASABF-type antimicrobial peptide from the sponge Suberites domuncula: microbicidal and hemolytic activity in vitro and toxic effect on molluscs in vivo.

Since sponges, as typical filter-feeders, are exposed to a high load of attacking prokaryotic and eukaryotic organisms, they are armed with a wide arsenal of antimicrobial/cytostatic low-molecular-weight, non-proteinaceous bioactive compounds. Here we present the first sponge agent belonging to the group of ASABF-type antimicrobial peptides. The ASABF gene was identified and cloned from the demosponge Suberites domuncula. The mature peptide, with a length of 64 aa residues has a predicted pI of 9.24, and comprises the characteristic CSα ß structural motif. Consequently, the S. domuncula ASABF shares high similarity with the nematode ASABFs; it is distantly related to the defensins. The recombinant peptide was found to display besides microbicidal activity, anti-fungal activity. In addition, the peptide lyses human erythrocytes. The expression of ASABF is upregulated after exposure to the apoptosis-inducing agent 2,2'-dipyridyl. During the process of apoptosis of surface tissue of S. domuncula, grazing gastropods (Bittium sp.) are attracted by quinolinic acid which is synthesized through the kynurenine pathway by the enzyme 3-hydroxyanthranilate 3,4-dioxygenase (HAD). Finally, the gastropods are repelled from the sponge tissue by the ASABF. It is shown that the effector peptide ASABF is sequentially expressed after the induction of the HAD gene and a caspase, as a central enzyme executing apoptosis.

Sponge biosilica formation involves syneresis following polycondensation in vivo.

Syneresis is a process observed during the maturation/aging of silica gels obtained by sol-gel synthesis that results in shrinkage and expulsion of water due to a rearrangement and increase in the number of bridging siloxane bonds. Here we describe how the process of biosilica deposition during spicule ("biosilica" skeleton of the siliceous sponges) formation involves a phase of syneresis that occurs after the enzyme-mediated polycondensation reaction. Primmorphs from the demosponge Suberites domuncula were used to study syneresis and the inhibition of this mechanism. We showed by scanning electron microscopy that spicules added to primmorphs that have been incubated with manganese sulfate fuse together through the deposition of silica spheres and bridges. Energy-dispersive X-ray mapping of the newly formed deposits showed high silicon and oxygen content. These biosilica deposits contain a comparably higher percentage of water than mature/aged spicules. Quantitative real-time polymerase chain reaction analyses revealed that the addition of silicate to primmorph cultures resulted in a marked upregulation of the expression of the aquaporin gene and of the genes encoding the silica anabolic enzyme silicatein-α and the silica catabolic enzyme silicase. On the other hand, addition of manganese sulfate, either alone or together with silicate, caused a strong reduction in the level of aquaporin transcripts, although this metal ion did not essentially affect the silicate-induced increase in silicatein-α and silicase gene expression. We conclude that the secondary silica deposits formed on spicules under physiological conditions in the presence of silicate fuse together and subsequently undergo syneresis, which is facilitated by the removal of water through aquaporin channels. In growing spicules, these processes of biosilica formation and syneresis in the lamellar monolithic structures precede the final step of "biosintering" during which the massive biosilica rods of the spicules are formed.

MAP kinase cell signaling pathway as biomarker of environmental pollution in the sponge Suberites domuncula.

In the present study, we analyzed the effects of two major pollutants of the environment, tributyltin (TBT) and water-accommodated fraction (WAF) of diesel oil, on MAP kinase activation, apoptosis induction and DNA damage, in the marine sponge Suberites domuncula. Our results clearly demonstrated a differential activation of the MAPKs depending on the chemicals tested. TBT induced the activation of p38 and JNK while diesel oil enhanced activation of both ERK and p38. The activation of MAPKs was observed after 1 h exposure and 6 and 24 h of recovery in seawater. In addition, DNA fragmentation, assessed by two techniques, the Fast micromethod(®) and the TUNEL assay, was detected after sponges were treated with both chemicals. Moreover, the study of caspase 3/7 activity showed that apoptosis was induced and triggered with all concentrations of TBT but only at high diesel oil concentrations. After TBT exposure, a correlation was observed between JNK activation, caspase 3 activity and DNA damage while p38 activation followed the two latter parameters at high concentrations of diesel oil, suggesting that sponges enhanced a specific apoptotic pathway depending on the xenobiotic tested. This study demonstrated a high signal response by the sponge Suberites domuncula to the tested chemicals. Cell signaling pathway studies may thus be of use in water quality biomonitoring programs.

Characterization of Nme6-like gene/protein from marine sponge Suberites domuncula.

Nucleoside diphosphate kinases (NDPKs) are evolutionarily conserved enzymes involved in many biological processes such as metastasis, proliferation, development, differentiation, ciliary functions, vesicle transport and apoptosis in vertebrates. Biochemical mechanisms of these processes are still largely unknown. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestors' genomic features. The purpose of this study was to address structural and functional properties of group II Nme6 gene/protein ortholog from the marine sponge Suberites domuncula, Nme6, in order to elucidate its evolutionary history. Sponge Nme6 gene and promoter were sequenced and analysed with various bioinformatical tools. Nme6 and Nme6Δ31 proteins were produced in E. coli strain BL21 and NDPK activity was measured using a coupled pyruvate kinase-lactate dehydrogenase assay. Subcellular localization in human tumour cells was examined by confocal scanning microscopy. Our results show that the sponge Nme6 compared to human Nme6 does not possess NDPK activity, does not localize in mitochondria at least in human cells although it has a putative mitochondrial signal sequence, lacks two recent introns that comprise miRNAs and have different transcriptional binding sites in the promoter region. Therefore, we conclude that the structure of Nme6 gene has changed during metazoan evolution possibly in correlation with the function of the protein.