The Prevalence and Genetic Characterization of Strains of Borrelia Isolated from Ixodes Tick Vectors in Belarus (2012-2019).

Borrelia burgdorferi sensu lato (s.l.) is the most common pathogen of medical significance transmitted by ticks of the family Ixodidae in Belarus. Human infection with B. burgdorferi causes Lyme borreliosis, most commonly referred to as Lyme disease. Currently, 20 species of Lyme disease-associated Borrelia and more than 20 relapsing fever-associated Borrelia species have been identified. These etiologic agents belong to the genus Borrelia in the family Spirochaetaceae. Genetically characterized isolates with specific sequences have proven that these pathogens are endemically transmitted in many European and Asian countries. In addition, joinpoint regression analysis is often applied to characterize infection trends over time and to identify the time point(s) at which the trend significantly changes. In this epidemiological investigation, joinpoint analysis was applied to investigate the temporal trend of B. burgdorferi s.l. infections in 4070 ticks collected between April and October 2012-2019. Detection of Borrelia species in ticks is an important tool to determine temporal and geographic distribution and abundance, and to predict the risk of Lyme disease to people in different regions. Our data provide a basis for further studies to determine the distribution and abundance of B. burgdorferi s.l. species in Belarus.

The Prevalence of Different Human Pathogenic Microorganisms Transmitted by Ixodes Tick Vectors in Belarus.

Pathogens transmitted by ticks cause several important diseases in humans, including Lyme disease, the incidence of which has been increasing in Belarus. Between April and October 2017, a total of 504 questing Ixodid ticks (77% Ixodes ricinus and 23% Dermacentor reticulatus) were collected from six regions and city of Minsk, in Belarus. All ticks were analyzed by RT-PCR amplification for the presence of Borrelia burgdorferi sensu lato, tick-borne encephalitis virus (TBEV), Anaplasma phagocytophillum, Ehrlihia muris, and Borrelia miyamotoi. B. burgdorferi s.l. and Rickettsia spp. were the most commonly detected tick-borne pathogens, with prevalence rates of 31.08% and 33.7%, respectively. A. phagocytophillum was found in 104 (20.63%), and 107 (21.2%) ticks were positive for E. muris. TBEV was detected in 83 (16.47%). Circulation of Borrelia miyamotoi spirochete in I. ricinus ticks in Brest, Gomel, and Minsk region was detected for the first time. Our data provide a basis for further studies to determine the distribution and abundance of different tick species in Belarus and therefore a capacity to predict where cases of important tick-borne diseases may occur.

Bio-sintering processes in hexactinellid sponges: fusion of bio-silica in giant basal spicules from Monorhaphis chuni.

The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processes. Here, we study the different stages of a process that we termed bio-sintering, within the giant basal spicule (GBS) of Monorhaphis chuni. During this study, a major GBS protein component (27 kDa) was isolated and analyzed by MALDI-TOF-MS. The sequences were used to isolate and clone the encoding cDNA via degenerate primer PCR. Bioinformatic analyses revealed a significant sequence homology to silicatein. In addition, the native GBS protein was able to mediate bio-silica synthesis in vitro. We conclude that the syntheses of bio-silica in M. chuni, and the subsequent fusion of nanoparticles to lamellae, and finally to spicules, are enzymatically-driven by a silicatein-like protein. In addition, evidence is now presented that in hexactinellids those fusions involve sintering-like processes.

Symbiotic interaction between dinoflagellates and the demosponge Lubomirskia baicalensis: aquaporin-mediated glycerol transport.

Lake Baikal is rich in endemic sponge species, among them the arborescently growing species Lubomirskia baicalensis. During winter when the lake is covered by ice, this species reproduces sexually, reflecting a high metabolic activity. Throughout the year, L. baicalensis lives in association with dinoflagellates, which - according to the data presented herein - are symbiotic. The dinoflagellates have been determined on the basis of their rDNA/ITS characteristics and were found to display high sequence similarity to Gymnodinium sanguineum. The dinoflagellates give the sponge its characteristic green color, reflecting the high chlorophyll content (chlorophyll-a content in March and September of 3.2 +/- 0.6 microg/g and 1.9 +/- 0.5 microg/g of protein, respectively). With the in vitro cell culture system for sponges, the primmorphs, it could be demonstrated that [(14)C] glycerol is readily taken up by sponge cells; this process can be inhibited by phloretin, an aquaporin channel blocker. In order to prove the effect of cholesterol on the intermediate metabolism of the sponge cells, molecule probes, cDNAs for key enzymes in gluconeogenesis, glycolysis, and citric acid, have been applied in Northern blot studies. The data revealed that the genes coding for the enzymes citrate synthase and fructose-1,6-bisphosphatase are strongly upregulated after exposure of primmorphs to glycerol. This effect is abolished by phloretin. The genes encoding the phosphoglucose isomerase and pyruvate dehydrogenase do not respond to glycerol supply, suggesting that their expression is not under genetic control in L. baicalensis. To prove the assumption that the aquaporin channel is involved in the influx of glycerol in sponge cells, this cDNA was cloned and applied for in situ hybridization studies. The results obtained show that cells surrounding the dinoflagellates become brightly stained after hybridization with the aquaporin this probe. This demonstrates that L. baicalensis cells respond to glycerol, a metabolite which might be supplied by the dinoflagellates and imported via the aquaporin channel into the sponge cells.

Bioencapsulation of living bacteria (Escherichia coli) with poly(silicate) after transformation with silicatein-alpha gene.

Bioencapsulation is an intriguing way to immobilize biological materials, including cells, in silica, metal-oxides or hybrid sol-gel polymers. Until now only the sol-gel precursor technology was utilized to immobilize bacteria or yeast cells in silica. With the discovery of silicatein, an enzyme from demosponges that catalyzes the formation of poly(silicate), it became possible to synthesize poly(silicate) under physiological (ambient) conditions. Here we show that Escherichia coli can be transformed with the silicatein gene, its expression level in the presence of isopropyl beta-D-thiogalactopyranoside (IPTG) can be efficiently intensified by co-incubation with silicic acid. This effect could be demonstrated on the level of recombinant protein synthesis as well as by immunostaining analysis. The heterologously produced silicatein is enzymatically active, as confirmed by staining with Rhodamine 123 (formation for poly[silicate] from silicic acid) and by reacting free silicic acid with the beta-silicomolybdato color system. Electron microscopic analysis revealed that the bacteria that express silicatein form a viscous cover around them when growing in the presence of silicic acid. Finally, we demonstrate that the growth kinetics of E. coli remains unaffected whether or not the bacteria had been transformed with silicatein or grown in medium, supplemented with silicic acid. It is concluded that silicatein-mediated encapsulation of bacteria with silica might improve, extend and optimize the range of application of bacteria for the production of recombinant protein.

The 2'-5'-oligoadenylate synthetase in the lowest metazoa: isolation, cloning, expression and functional activity in the sponge Lubomirskia baicalensis.

Aquatic animals, especially filter feeders such as sponges [phylum Porifera], are exposed to a higher viral load than terrestrial species. Until now, the antiviral defense system in the evolutionary oldest multicellular organisms, sponges, is not understood. One powerful protection of vertebrates against virus infection is mediated by the interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase [(2-5)A synthetase] system. In the present study we cloned from the freshwater sponge Lubomirskia baicalensis a cDNA encoding a 314 aa long ORF with a calculated size of 35748Da, a putative (2-5)A synthetase, and raised antibodies against the recombinant protein. The native enzyme was identified in a crude extract from L. baicalensis by application of a novel separation procedure based on polymer coated ferromagnetic nanoparticles. The particles were derivatized with a synthetic double-stranded RNA [dsRNA], synthetic poly(I:C), a known allosteric activator of the latent (2-5)A synthetase. These particles were used to separate a single 35kDa protein from a crude extract of L. baicalensis, which cross-reacted with antibodies raised against the sponge enzyme. In situ hybridization studies revealed that highest expression of the gene is seen in cells surrounding the aquiferous canals. Finally primmorphs, an in vitro cell culture system, from L. baicalensis were exposed to poly(I:C); they responded to this dsRNA with an increased expression of the (2-5)A synthetase gene already after a 1-day incubation period. We conclude that sponges contain the (2-5)A synthetase antiviral protection system.

Morphogenetic activity of silica and bio-silica on the expression of genes controlling biomineralization using SaOS-2 cells.

In a previous study (Schröder et al., J Biomed Mater Res B Appl Biomater 75:387-392, 2005) we demonstrated that human SaOS-2 cells, when cultivated on bio-silica matrices, respond with an increased hydroxyapatite deposition. In the present contribution we investigate if silica-based components (Na-silicate, tetraethyl orthosilicate [TEOS], silica-nanoparticles) (1) change the extent of biomineralization in vitro (SaOS-2 cells) and (2) cause an alteration of the expression of the genes amelogenin, ameloblastin, and enamelin, which are characteristic for an early stage of osteogenesis. We demonstrate that the viability of SaOS-2 cells was not affected by the silica-based components. If Na-silicate or TEOS was added together with ss-glycerophosphate, an organic phosphate donor, a significant increase in biomineralization was measured. Finally, expression levels of the amelogenin, ameloblastin, and enamelin genes were determined in SaOS-2 cells during exposure to the silica-based components. After exposure for 2 days, expression levels of amelogenin and enamelin strongly increased in response to the silica-based components, while no significant change was seen for ameloblastin. In contrast, exposure of SaOS-2 cells to ss-glycerophosphate resulted in increased expression of all three genes. We conclude that the levels of the structural molecules of the enamel matrix, amelogenin and enamelin, increase in the presence of silica-based components and substantially contribute to the extent of hydroxyapatite crystallite formation. These results demonstrate that silica-based components augment hydroxyapatite deposition in vitro and suggest that enzymatically synthesized bio-silica (via silicatein) might be a promising route for tooth reconstruction in vivo.

Analysis of the axial filament in spicules of the demosponge Geodia cydonium: different silicatein composition in microscleres (asters) and megascleres (oxeas and triaenes).

The skeleton of the siliceous sponges (Porifera: Hexactinellida and Demospongiae) is supported by spicules composed of bio-silica. In the axial canals of megascleres, harboring the axial filaments, three isoforms of the enzyme silicatein (-alpha, -beta and -gamma) have been identified until now, using the demosponges Tethya aurantium and Suberites domuncula. Here we describe the composition of the proteinaceous components of the axial filament from small spicules, the microscleres, in the demosponge Geodia cydonium that possesses megascleres and microscleres. The morphology of the different spicule types is described. Also in G. cydonium the synthesis of the spicules starts intracellularly and they are subsequently extruded to the extracellular space. In contrast to the composition of the silicateins in the megascleres (isoforms: -alpha, -beta and -gamma), the axial filaments of the microscleres contain only one form of silicatein, termed silicatein-alpha/beta, with a size of 25kDa. Silicatein-alpha/beta undergoes three phosphorylation steps. The gene encoding silicatein-alpha/beta was identified and found to comprise the same characteristic sites, described previously for silicateins-alpha or -beta. It is hypothesized, that the different composition of the axial filaments, with respect to silicateins, contributes to the morphology of the different types of spicules.

Cold stress defense in the freshwater sponge Lubomirskia baicalensis. Role of okadaic acid produced by symbiotic dinoflagellates.

The endemic freshwater sponge Lubomirskia baicalensis lives in Lake Baikal in winter (samples from March have been studied) under complete ice cover at near 0 degrees C, and in summer in open water at 17 degrees C (September). In March, specimens show high metabolic activity as reflected by the production of gametes. L. baicalensis lives in symbiosis with green dinoflagellates, which are related to Gymnodinium sanguineum. Here we show that these dinoflagellates produce the toxin okadaic acid (OA), which is present as a free molecule as well as in a protein-bound state. In metazoans OA inhibits both protein phosphatase-2A and protein phosphatase-1 (PP1). Only cDNA corresponding to PP1 could be identified in L. baicalensis and subsequently isolated from a L. baicalensis cDNA library. The deduced polypeptide has a molecular mass of 36 802 Da and shares the characteristic domains known from other protein phosphatases. As determined by western blot analysis, the relative amount of PP1 is almost the same in March (under ice) and September (summer). PP1 is not inhibited by low OA concentrations (100 nm); concentrations above 300 nm are required for inhibition. A sponge cell culture system (primmorphs) was used to show that at low temperatures (4 degrees C) expression of hsp70 is strongly induced and hsp70 synthesis is augmented after incubation with 100 nm OA to levels measured at 17 degrees C. In the enriched extract, PP1 activity at 4 degrees C is close to that measured at 17 degrees C. Immunoabsorption experiments revealed that hsp70 contributes to the high protein phosphatase activity at 4 degrees C. From these data we conclude that the toxin OA is required for the expression of hsp70 at low temperature, and therefore contributes to the cold resistance of the sponge.

Novel mechanism for the radiation-induced bystander effect: nitric oxide and ethylene determine the response in sponge cells.

Until now the bystander effect had only been described in vertebrates. In the present study the existence of this effect has been demonstrated for the phylogenetically oldest metazoan phylum, the Porifera. We used the demosponge Suberites domuncula for the experiments in the two-chamber-system. The lower dish contained irradiated "donor" cells (single cells) and the upper dish the primmorphs ("recipient" primmorphs). The "donor" cells were treated with UV-B light (40 mJ/cm2) and 100 microM hydrogen peroxide (H2O2), factors that exist also in the natural marine aquatic environment of sponges; these factors caused a high level of DNA strand breaks followed by a reduced viability of the cells. If these cells were added to the "recipient" primmorphs these 3D-cell cultures started to undergo apoptosis. This effect could be abolished by the NO-specific scavenger PTIO and ethylene. The conclusion that NO is synthesized by the UV-B/H2O2-treated cells was supported analytically. The cDNA encoding the enzyme dimethylarginine dimethylaminohydrolase (DDAH) was isolated from the "donor" cells. High levels of DDAH transcripts were measured in UV-B/H2O2-treated "donor" cells while after ethylene treatment the steady-state level of expression drops drastically. We conclude that in the absence of ethylene the concentration of the physiological inhibitor for the NO synthase ADMA is low, due to the high level of DDAH. In consequence, high amounts of NO are released from "donor" cells which cause apoptosis in "recipient" primmorphs. In contrast, ethylene reduces the DDAH expression with the consequence of higher levels of ADMA which prevent the formation of larger amounts of NO. This study describes the radiation-induced bystander effect also for the most basal metazoans and demonstrates that this effect is controlled by the two gases NO and ethylene.

Molecular control of serial module formation along the apical-basal axis in the sponge Lubomirskia baicalensis: silicateins, mannose-binding lectin and mago nashi.

The freshwater sponge Lubomirskia baicalensis (from Lake Baikal) is characterized by a body plan composed of serial modules which are arranged along an apical-basal axis. In shallow water, the sponge occurs only encrusting, while in deeper environment (>3 m), this species forms branches and grows in an arborescent manner. Each module is stabilized by bundles of spined oxeas (amphioxeae spicules). The spicules are surrounded by an organic matrix. cDNAs for structural proteins (silicatein and mannose-binding lectin (MBL)) as well as for one regulatory protein (mago nashi) were isolated from L. baicalensis. Surprisingly the silicatein alpha molecule exists in several, at least four, isoforms (a1 to a4). Expression studies revealed that the steady-state levels of transcripts for the silicateins, the mannose-binding lectin, and mago nashi are highest at the top of the branches, while only very low levels are found in cells at the base. Based on in situ hybridization studies, evidence is presented that the spicule formation (1) starts and is completed inside of the bundles, and (2) occurs together with the mannose-binding lectin from the surfaces of the bundles. The data suggest that the modules are sequentially formed. It is speculated that the expression of the silicateins and the mannose-binding lectin might be (partially) controlled by mago nashi.

Expression of silicatein in spicules from the Baikalian sponge Lubomirskia baicalensis.

Lake Baikal harbors the largest diversity of sponge species [phylum Porifera] among all freshwater biotopes. The abundantly occurring species Lubomirskia baicalensis was used to study the seasonal silicatein metabolism; the spicules of this species have an unusually thick axial filament, consisting of silicatein, which remains constant in diameter during their growth. In the course of maturation, the size of the silicic acid shell grows, until the final diameter of the spicules of about 8 microm is reached. The seasonal content of silicatein was assessed by use of antibodies raised against silicatein; they stained specifically the axial filaments. In addition we determined, by application of the enzyme-linked immunosorbent assay system, that the proteinaceous content of the spicules, the silicatein, increases from spring to late summer by 8-fold. As molecular markers to quantify the seasonal changes in expression levels of genes coding for proteins/enzymes, the genes for the calumenin-like protein and the kinesin-related protein, were selected. The expression of calumenin-like gene, involved in the intracellular signaling, is highest during September, whereas the expression of the kinesin-related protein does not change during the annual course. These results suggest that the highest metabolic activity of L. baicalensis occurs in late summer (September), in parallel with the highest accumulation of silicatein, a structural protein/enzyme of the spicules.

Selenium affects biosilica formation in the demosponge Suberites domuncula. Effect on gene expression and spicule formation.

Selenium is a trace element found in freshwater and the marine environment. We show that it plays a major role in spicule formation in the demosponge Suberites domuncula. If added to primmorphs, an in vitro sponge cell culture system, it stimulates the formation of siliceous spicules. Using differential display of transcripts, we demonstrate that, after a 72-h exposure of primmorphs to selenium, two genes are up-regulated; one codes for selenoprotein M and the other for a novel spicule-associated protein. The deduced protein sequence of selenoprotein M (14 kDa) shows characteristic features of metazoan selenoproteins. The spicule-associated protein (26 kDa) comprises six characteristic repeats of 20 amino acids, composed of 10 distinct hydrophobic regions ( approximately 9 amino acids in length). Recombinant proteins were prepared, and antibodies were raised against these two proteins. Both were found to stain the central axial filament, which comprises the silicatein, as well as the surface of the spicules. In the presence of selenium, only the genes for selenoprotein M and spicule-associated protein are up-regulated, whereas the expression of the silicatein gene remains unchanged. Finally we show that, in the presence of selenium, larger silica aggregates are formed. We conclude that selenium has a stimulatory effect on the formation of siliceous spicules in sponges, and it may be involved in the enzymatic synthesis of biosilica components.

Mineralization of SaOS-2 cells on enzymatically (silicatein) modified bioactive osteoblast-stimulating surfaces.

There is a demand for novel bioactive supports in surgery, orthopedics, and tissue engineering. The availability of recombinant silica-synthesizing enzyme (silicatein) opens new possibilities for the synthesis of silica-containing bioactive surfaces under ambient conditions that do not damage biomolecules like proteins. Here it is shown that growth of human osteosarcoma SaOS-2 cells on cluster plates precoated with Type 1 collagen is not affected by additional coating of the plates with the recombinant silicatein and incubation with its enzymatic substrate, tetraethoxysilane (TEOS). However, the enzymatic modification of the plates by biosilica deposition on the protein-coated surface caused a marked increase in calcium phosphate formation of SaOS-2 cells as revealed by alizarin red-S staining to quantify calcium mineral content. The increased occurrence of calcium-phosphate nodules on the modified surface was also observed by scanning electron microscopy. These results suggest that by supporting calcium-phosphate deposition in vitro, biosilica (silicatein)-modified surfaces are potentially bioactive in vivo, by stimulating osteoblast mineralization function.

Innate immune defense of the sponge Suberites domuncula against bacteria involves a MyD88-dependent signaling pathway. Induction of a perforin-like molecule.

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleukin-1 receptor domain (found in MyD88 and in Toll-like receptors) and a death domain (present in MyD88 and interleukin-1 receptor-associated kinase). Northern blot experiments and in situ hybridization studies showed that after LPS treatment, the level of the LPS-interacting protein remains unchanged, whereas MyD88 is strongly up-regulated. A perforin-like molecule (Mr 74,171), the macrophage-expressed protein, was identified as an executing molecule of this pathway. This gene is highly expressed after LPS treatment, especially at the surfaces of the animals. The recombinant protein possesses biological activity and eliminates gram-negative bacteria; it is inactive against gram-positive bacteria. These data indicate that S. domuncula is provided with an innate immune system against gram-negative bacteria; the ligand LPS (a pathogen-associated molecular pattern) is recognized by the pattern recognition receptor (LPS-interacting protein), which interacts with MyD88. A signal transduction is established, which results in an elevated expression of MyD88 as well as of the macrophage-expressed protein as an executing protein.

Arginine kinase in the demosponge Suberites domuncula: regulation of its expression and catalytic activity by silicic acid.

In Demospongiae (phylum Porifera) the formation of the siliceous skeleton, composed of spicules, is an energetically expensive reaction. The present study demonstrates that primmorphs from the demosponge Suberites domuncula express the gene for arginine kinase after exposure to exogenous silicic acid. The deduced sponge arginine kinase sequence displays the two characteristic domains of the ATP:guanido phosphotransferases; it can be grouped to the 'usual' mono-domain 40 kDa guanidino kinases (arginine kinases). Phylogenetic studies indicate that the metazoan guanidino kinases evolved from this ancestral sponge enzyme; among them are also the 'unusual' two-domain 80 kDa guanidino kinases. The high expression level of the arginine kinase gene was already measurable 1 day after addition of silicic acid by northern blot, as well as by in situ hybridization analysis. Parallel determinations of enzyme activity confirmed that high levels of arginine kinase are present in primmorphs that had been exposed for 1-5 days to silicic acid. Finally, transmission electron-microscopical studies showed that primmorphs containing high levels of arginine kinase also produce siliceous spicules. These data highlight that silicic acid is an inorganic morphogenetic factor that induces the expression of the arginine kinase, which in turn probably catalyzes the reversible transfer of high-energy phosphoryl groups.

Bronchial hyperreactivity and other inhalation lung injuries in rescue/recovery workers after the World Trade Center collapse.

BACKGROUND: The collapse of the World Trade Center (WTC) on September 11, 2001 created a large-scale disaster site in a dense urban environment. In the days and months thereafter, thousands of rescue/recovery workers, volunteers, and residents were exposed to a complex mixture of airborne pollutants. METHODS: We review current knowledge of aerodigestive inhalation lung injuries resulting from this complex exposure and present new data on the persistence of nonspecific bronchial hyperreactivity (methacholine PC20 < or =8 mg/mL) in a representative sample of 179 Fire Department of the City of New York (FDNY) rescue workers stratified by exposure intensity (according to arrival time) who underwent challenge testing at 1, 3, 6, and 12 months post-collapse. RESULTS: Aerodigestive tract inflammatory injuries, such as declines in pulmonary function, reactive airways dysfunction syndrome (RADS), asthma, reactive upper airways dysfunction syndrome (RUDS), gastroesophageal reflux disease (GERD), and rare cases of inflammatory pulmonary parenchymal diseases, have been documented in WTC rescue/recovery workers and volunteers. In FDNY rescue workers, we found persistent hyperreactivity associated with exposure intensity, independent of airflow obstruction. One year post-collapse, 23% of highly exposed subjects were hyperreactive as compared with only 11% of moderately exposed and 4% of controls. At 1 yr, 16% met the criteria for RADS. CONCLUSIONS: While it is too early to ascertain all of the long-term effects of WTC exposures, continued medical monitoring and treatment is needed to help those exposed and to improve our prevention, diagnosis, and treatment protocols for future disasters.

Dynamics of skeleton formation in the Lake Baikal sponge Lubomirskia baicalensis. Part II. Molecular biological studies.

In a preceding study it has been reported that the freshwater sponge Lubomirskia baicalensis, living in Lake Baikal (East Siberia), is composed of spicules forming a characteristic pattern which follows radiate accretive growth. Here we report that the spicules are synthesized by the enzyme silicatein, a protein which is related to cathepsin L. The cDNAs for silicatein and the related cathepsin L were isolated and used as probes to show that the mRNA levels of silicatein in the bases of the spicule skeleton of the animals are low, while the mRNA level of cathepsin L in this region exceeds that of the growing zone. This is the first comprehensive study on the importance of the axial filament/silicatein as an essential structural and functional component determining the growth and stability of demosponge spicules.

Matrix-mediated canal formation in primmorphs from the sponge Suberites domuncula involves the expression of a CD36 receptor-ligand system.

Sponges (Porifera), represent the phylogenetically oldest metazoan phylum still extant today. Recently, molecular biological studies provided compelling evidence that these animals share basic receptor/ligand systems, especially those involved in bodyplan formation and in immune recognition, with the higher metazoan phyla. An in vitro cell/organ-like culture system, the primmorphs, has been established that consists of proliferating and differentiating cells, but no canals of the aquiferous system. We show that after the transfer of primmorphs from the demosponge Suberites domuncula to a homologous matrix (galectin), canal-like structures are formed in these 3D-cell aggregates. In parallel with the formation of these structures a gene is expressed whose deduced protein falls into the CD36/LIMPII receptor family. The receptor was cloned and found to be strongly expressed after adhesion to the galectin matrix. This process was suppressed if primmorphs were co-incubated with a homologous polypeptide containing the CSVTCG domain, as found in thrombospondin-1 (and related) molecules of vertebrates. In situ hybridization studies revealed that the S. domuncula CD36/LIMPII receptor is localized in the pinacocytes that surround the canals of the sponge. Furthermore, a secondary metabolite from a sponge-associated bacterium was isolated and characterized, the 2-methylthio-1,4-naphthoquinone (MTN). MTN causes inhibition of cell proliferation of vertebrate tumor cells at concentrations of >80 ng/ml. However, doses of only 2 ng are required to potently inhibit angiogenesis in the chick chorio-allantoic membrane assay. At concentrations of 10 ng/ml this compound was also found to suppress the expression of the S. domuncula CD36/LIMPII; this result is a first indication that this secondary metabolite has a conserved functional activity: the suppression of the formation of the circulation system, from sponges to vertebrates.

Oxygen-controlled bacterial growth in the sponge Suberites domuncula: toward a molecular understanding of the symbiotic relationships between sponge and bacteria.

Sponges (phylum Porifera), known to be the richest producers among the metazoans of bioactive secondary metabolites, are assumed to live in a symbiotic relationship with microorganisms, especially bacteria. Until now, the molecular basis of the mutual symbiosis, the exchange of metabolites for the benefit of the other partner, has not been understood. We show with the demosponge Suberites domuncula as a model that the sponge expresses under optimal aeration conditions the enzyme tyrosinase, which synthesizes diphenols from monophenolic compounds. The cDNA isolated was used as a probe to determine the steady-state level of gene expression. The gene expression level parallels the level of specific activity in sponge tissue, indicating that without aeration the tyrosinase level drops drastically; this effect is reversible. The SB2 bacterium isolated from the sponge surface grew well in M9 minimal salt medium supplemented with the dihydroxylated aromatic compound protocatechuate; this carbon source supported growth more than did glucose. From the SB2 bacterium the protocatechuate gene cluster was cloned and sequenced. This cluster comprises all genes coding for enzymes involved in the conversion of protocatechuate to acetyl coenzyme A. Expression is strongly induced if the bacteria are cultivated on M9-protocatechuate medium; the genes pcaQ (encoding the putative transcriptional activator of the pca operon) and pcaDC were used for quantitative PCR analyses. We conclude that metabolites, in this case diphenols, which might be produced by the sponge S. domuncula are utilized by the sponge surface-associated bacterium for energy generation. This rationale will help to further uncover the symbiotic pathways between sponges and their associated "nonculturable" microorganisms; our approach is flanked by the establishment of an EST (expressed sequence tags) database in our laboratory.