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 β 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

Common Genetic Denominators for Ca++-Based Skeleton in Metazoa: Role of Osteoclast-Stimulating Factor and of Carbonic Anhydrase in a Calcareous Sponge

Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative) enzyme carbonic anhydrase and the (putative) osteoclast-stimulating factor (OSTF), that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl2) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca2+-depletion condition (1 mM CaCl2). Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes) to human (osteoclast)

Model for the roles of the OSTF and the carbonic anhydrase in sclerocytes of the sponge <i>S. raphanus</i>.

<p>Based on the finding that the expression of the two molecules is upregulated during Ca⁺⁺ depletion condition, it is proposed that these proteins are involved in the development of the precursor sclerocytes to the functionally active catabolic sclerocytes. OSTF forms with Cbl and Src a triple complex that stimulates the membrane-associated PI-3K and lipid metabolism. This metabolic chain is initiated by clustering integrins. These processes finally result in an increased mobility/migration of the catabolic sclerocytes and in an increased expression of the <i>carbonic anhydrase</i> gene. The latter enzyme generates protons that dissolve spicular CaCl₂ (ACC), and causes spicule resorption. Presumably occurring pH shifts within the cells are counterbalanced by a vacuolar H⁺-transporting adenosine triphosphatase and the release of Cl⁻ via the chloride/bicarbonate exchanger (see: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#pone.0034617-Pfaffl1" target="_blank">[58]</a>). In analogy to the differentiation pathway of mammalian osteoclasts it is proposed that also in sponges the differentiation of sclerocytes is under control of, hitherto unknown, differentiation factors and their receptors acting similar like OPG, RANKL and RANK in mammals.</p

Surface architecture of spicules from specimens grown in normal ambient CaCl₂ concentrations (10 mM) and cultivated in a CaCl₂-depleted environment.

<p>The isolation of the spicules has been performed after a short exposure (1 h; sheath-spicules) or after an extended exposure (5 h; purified-spicules) to NaOCl. (<b>A</b>) Sheath-spicules from specimens grown in sea water, supplemented with 10 mM CaCl₂. The surfaces of the abundantly occurring triactines (ts) are covered by organic sheaths (os). (<b>B</b> and <b>C</b>) At a higher magnification those layers, organic sheaths (os), can be resolved as circular netlike ropes whereby the individual fibrils apparently do not fuse to each other; they are tightly attached to the calcite surfaces (><). (<b>D</b> to <b>F</b>) Purified-spicules, diactines (ds) and triactines (ts), devoid of any visible sheath show a smooth surface. (<b>G</b>) Sheath-spicule from a specimen kept for 5 d in CaCl₂-depleted aqueous environment, showing likewise an organic sheath. (<b>H</b> and <b>I</b>) Purified-spicules from similarly cultivated animals; the rough surface architecture is obvious (<>). (<b>J</b>) At higher magnification the rough surface architecture can be resolved as palisade bricks, sticking out about 100 nm radially from the spicules (double-headed arrow). (<b>K</b>) The fissuring of the spicules with an almost identical depth (double-headed arrow) suggests that the calcitic material of the spicules is not homogenous with respect to their density or content in organic material. (<b>L</b>) In comparison, the smooth surface of a purified-spicule, from an animal kept at 10 mM CaCl₂, is shown.</p

The <i>S. raphanus</i> putative carbonic anhydrase.

<p>(<b>A</b>) The sponge putative carbonic anhydrase (CA_SYCON) is aligned with the highly related sequences from the demosponge <i>S. domuncula</i>, the silicase (SIA_SUBDO; DD298191), and the carbonic anhydrases from the scleractinian <i>Acropora millepora</i> (CAr1_ACRMIL; ACJ64662.1), and the stony coral <i>Stylophora pistillata</i> (CAa_STYPI; ACA53457.1, EU159467.1), as well as with the human carbonic anhydrase 2 (CA II) (CAHB_HUMAN; O75493). The indicative sites/regions within the <i>Sycon</i> polypeptide are marked, the carbonic anhydrase alpha (vertebrate-like) group stretch (−CA−), including the His residues, functioning as Zn-binding sites, the hydrophobic parts (+hydb+), as well as the signal peptide (:signal:). Residues conserved (identical or similar) in all sequences are shown in white on black; those which share similarity to at least four residues are in black on grey. (<b>B</b>) Radial phylogenetic tree, including the mentioned sequences, together with human carbonic anhydrases of the following isoforms: I (CA-I) (CAH1_HUMAN; P00915); II (CA-II) (CAH2_HUMAN; P00918); III (CA-III) (CAH3_HUMAN; P07451); IV (CAIV_HUMAN; AAA35625.1); IV (CA-IV) (CAH4_HUMAN; P22748); VA (CAH5_HUMAN; P35218); VB (CA5B_HUMAN; CA5B_HUMAN); VI (CA-VI) (CAH6_HUMAN; P23280); VII (CA-VII) (CAH7_HUMAN; P43166); VIII (CA-VIII) (CAH8_HUMAN; P35219); IX (CA-IX) (CAH9_HUMAN; Q16790); 10 (CA-RP X) (CAHA_HUMAN; Q9NS85); XII (CA-XII) (CAHC_HUMAN; O43570); XIV (CA-XIV) (CAHE_HUMAN; Q9ULX7). In addition, the coral sequence from <i>Acropora millepora</i> (CAr2_ACRMIL; ACJ64663.1), as well as the ones from the sea anemone <i>Nematostella vectensis</i> (CAr_NEMVE; XP_001627923.1), the tunicate <i>Ciona intestinalis</i> (CA14_CIONA; XP_002123314.1); the lancelet <i>Branchiostoma floridae</i> (CAr_BRANFLO; XP_002601262.1), the shark <i>Squalus acanthias</i> (CA4_SQUAAC; AAZ03744.1); the fish <i>Oreochromis niloticus</i> (CA4_ORENI; XP_003456174.1), together with the insect enzyme from <i>D. melanogaster</i> (CAr_DROME; NP_572407.3) are included.</p

Production of recombinant <i>S. raphanus</i> carbonic anhydrase (r-CA) and OSTF (r-OSTF).

<p>(<b>A</b>) Preparation of the recombinant <i>S. raphanus</i> putative carbonic anhydrase (r-CA) and analysis by NaDodSO4-PAGE and Western blotting. NaDodSO4-PAGE: (<b>M</b>) Size markers. (<b>Lane a</b>) Proteins in the bacterial pellet, obtained from induced bacteria; (<b>lane b</b>) pattern after lysis with BugBuster; (<b>lane c</b>) affinity purified r-CA. Western blotting; (<b>lane d</b>) the antiserum raised against the r-CA (PoAb-aCA) recognizes the 29-kDa recombinant protein, while (<b>lane e</b>) a pre-immune serum (p.s.) did not react. (<b>B</b>) The recombinant OSTF protein (r-OSTF). (<b>Lane a</b>) NaDodSO4-PAGE analysis of the purified protein. Western blot analysis: (<b>lane b</b>) Reactivity of the antibodies raised against OSTF (PoAb-aOSTFr) to the 25 kDa r-OSTF; while (<b>lane b</b>) the pre-immune serum (p.s.) does not react.</p

The calcareous sponge <i>Sycon raphanus</i> (Schmidt, 1862).

<p>(<b>A</b>) The species <i>S. raphanus</i> had been grouped by Haeckel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#pone.0034617-Haeckel1" target="_blank">[28]</a> to the taxon <i>Sycandra</i>. Here a scheme of the morphology and the skeletal structure of <i>Sycandra hystrix</i> is given <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#pone.0034617-Haeckel1" target="_blank">[28]</a>. (<b>B</b>) <i>S. raphanus</i> specimens, growing on the mussel (m) <i>Mytilus galloprovincialis</i>. The oscule of the specimens is surrounded by a pronounced corona (co), formed of spicules. On the basis of the specimens stolons/buds (st) are seen. They develop after release from the parent sponge asexually to a descendent. (<b>C</b> and <b>D</b>) Cross section through <i>S. raphanus</i> specimens, displaying the external and internal surface layer. In the center the atrium (a) is shown into which the water canals flow in. Radial aquiferous canals traverse the body that originate at the surface of the animal, via the inhalant openings (io), and end at the internal surface via exhalant pores (eo). Between the canals the mesenchyme (m) compartments is radially arranged. The slices were stained with ASTRIN. (<b>E</b>) Non-stained section through the outer part of the sponge showing the location of the two major types of spicules; (i) the diactines spicules (ds), protruding from the distal cones of the outer surface of the specimens, and (ii) the triactines (ts) that are localized within the mesohyl. The mesohyl compartment is filled with eggs/embryos (e).</p

Expression levels of the OSTF (<i>SROSTFr</i>) in <i>S. raphanus</i> in dependence on the level of Ca²⁺ in the culture medium.

<p>Expression levels of the OSTF (<i>SROSTFr</i>) in <i>S. raphanus</i> in dependence on the level of Ca²⁺ in the culture medium.</p