Big defensins and mytimacins, new AMP families of the Mediterranean mussel Mytilus galloprovincialis

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Abstract

Antimicrobial peptides (AMPs) play a fundamental role in the innate immunity of invertebrates, preventing the invasion of potential pathogens. Mussels can express a surprising abundance of cysteine-rich AMPs pertaining to the defensin, myticin, mytilin and mytimycin families, particularly in the circulating hemocytes.

Based on deep RNA sequencing of Mytilus galloprovincialis, we describe the identification, molecular diversity and constitutive expression in different tissues of five novel transcripts pertaining to the macin family (named mytimacins) and eight novel transcripts pertaining to the big defensins family (named MgBDs). The predicted antimicrobial peptides exhibit a N-terminal signal peptide, a positive net charge and a high content in cysteines, allegedly organized in intra-molecular disulfide bridges. Mytimacins and big defensins therefore represent two novel AMP families of M. galloprovincialis which extend the repertoire of cysteine-rich AMPs in this bivalve mollusk.

Highlights

► Identification of macin and big defensin in Mytilus galloprovincialis. ► They exhibit a signal peptide, a positive net charge and a high content in cysteines. ► Macins are widespread in Metazoans. ► Big defensins are limited to Bivalves, Merostomata and amphioxus.

Introduction

Antimicrobial peptides (AMPs) are humoral components of the innate immunity, present in all metazoans and essential to the immediate defense reactions of invertebrate organisms lacking adaptive immunity. Antibacterial activity was first reported in mollusks in the ‘80s (Kubota et al., 1985) whereas the isolation and characterization of true AMPs from the mussels Mytilus galloprovincialis (Hubert, 1996) and Mytilus edulis (Charlet et al., 1996) date back to 1996.

In the Mediterranean mussel M. galloprovincialis, cysteine-rich antimicrobial peptides are produced as precursor molecules and processed into mature peptides within the hemocyte granules (Mitta et al., 2000c). All the four AMP classes described so far in mussels, namely defensins (Hubert, 1996, Mitta et al., 2000a, Mitta et al., 1999b), myticins (Mitta et al., 1999a, Pallavicini et al., 2008), mytilins (Mitta et al., 2000a, Mitta et al., 2000b, Roch et al., 2008) and the strictly antifungal mytimycins (Charlet et al., 1996, Sonthi et al., 2011), retain a cysteine array essential to stabilize the mature peptide in a highly compact, cationic and amphipatic structure (Mitta et al., 2000c, Yeaman and Yount, 2007). More in detail, eight cysteine residues defining four intra-molecular disulfide bridges are present in defensins, myticins and mytilins, whereas 12 cysteines and two additional disulfide bridge characterize mytimycins. The structures of mussel defensin (Yang et al., 2000) and mytilin (Roch et al., 2008) have been determined by NMR, confirming the expected pattern of intra-molecular disulfide bonds.

Each of the above mentioned AMP classes comprises several members and the recent identification of 12 additional sequence transcripts sensibly extended the number of mussel AMPs in M. galloprovincialis (Venier et al., 2011). New massive sequencing of the M. galloprovincialis transcriptome allowed us to prepare a high-coverage transcript collection and to study identity and molecular variability of two classes of previously uncharacterized cysteine-rich mussel AMPs, namely big defensins (MgBDs) and mytimacins.

Big defensins have been originally identified in the horseshoe crab Tachypleus tridentatus (Saito et al., 1995), specifically stored in granules within hemocytes (Kawabata and Iwanaga, 1997) likewise many molluscan AMPs (Mitta et al., 2000c). The structure of big defensins typically includes one N-terminal highly hydrophobic region, one C-terminal cysteine-rich and positively charged region, and six cysteine residues arranged to form 1–5, 2–4, 3–6 disulfide bonds in the mature peptide (Saito et al., 1995), in a similar fashion to mammalian β-defensins (Kouno et al., 2008, Selsted et al., 1993, Zhao et al., 2010). The disulfide array is therefore different from the classic 1–4, 2–5, 3–6 cysteines arrangement of arthropod defensins (Dimarcq et al., 1998). Furthermore the cysteine-stabilized a-helix and b-sheet (CSαβ) motif characterizing many plant and invertebrate defensins (including those of mussel) (Cornet et al., 1995) cannot be observed in big defensins.

The two terminal regions of the molecule display remarkable differences in antimicrobial properties, with the N-terminal fragment being more active towards Gram− bacteria and the C-terminal fragment being more effective against Gram+ bacteria (Saito et al., 1995). NMR-based studies indicated that a globular N-terminal hydrophobic domain plays a fundamental role in the dynamic interaction with target membranes (Kouno et al., 2009). To date only two other big defensins have been extensively studied: AiBD of the bay scallop Argopecten irradians and VpBD of the clam Venerupis philippinarum were significantly up-regulated in the bivalve hemocytes in response to bacterial challenges and both displayed a broad spectrum of antimicrobial activity (Zhao et al., 2010, Zhao et al., 2007). Transcripts encoding big defensins have been also identified in the mollusks Crassostrea gigas, Mytilus chilensis and Bathymodiolus azoricus and in the lancelets Branchiostoma belcheri tsingtauense and Branchiostoma floridae, suggesting a broader taxonomic distribution of this AMP class.

Macins are positively charged secreted peptides which have been first described in the annelids Theromyzon tessulatum (Tasiemski et al., 2004) and Hirudo medicinalis (Schikorski et al., 2008) and have been later identified in the cnidarian Hydra magnipapillata (Jung et al., 2009) and in the mollusk Hyriopsis cumingii (Xu et al., 2010). Macins are characterized by a disulfide array of eight cysteines, with the optional presence of a fifth intra-molecular disulfide bridge involving a C-terminal sequence extension in theromacin. The structure of hydramacin has been determined by NMR, revealing a compact organization with an uneven distribution of positively charged residues which divide the molecular surface into two large hydrophobic hemispheres, characterized by the arrangement of cysteine bonds in a knottin fold, found in all the proteins pertaining the scorpion-toxin-like superfamily members, including mussel defensins (Jung et al., 2009).

Contrary to the majority of cysteine-rich AMPs, macins are not specifically expressed in the circulating cells, being instead localized in the endodermal epithelium (Bosch et al., 2009) or peripheral Large Fat Cells (LFCs) functionally resembling the insect fat body and often in close contact with the coelomic cavity (Tasiemski et al., 2004) or, in the case of neuromacin, in the central nervous system (Schikorski et al., 2008). The only reported exception is represented by the freshwater pearl oyster H. cumingii theromacin-like protein, which was found to be preferentially expressed in hemocytes (Xu et al., 2010). Macin expression is induced after exposure to bacteria (Tasiemski et al., 2004, Xu et al., 2010), and neuromacin localizes especially at the site of tissue injury (Schikorski et al., 2008). Increased expression of a theromacin-like transcript was also observed in response to both infection and tissue injury in the snail Biomphalaria glabrata (Ittiprasert et al., 2010).

Macins display membrane aggregating and permeabilizing activity, effective against Gram+ bacteria in theromacin and neuromacin (Schikorski et al., 2008, Tasiemski et al., 2004) and against Gram− bacteria in hydramacin (Jung et al., 2009). On the basis of the tertiary structure of hydramacin determined by NMR, a mechanistic model postulates its interaction with the bacterial membranes, with cell aggregation and microbe morphology changes preceding full permeabilization and their effective killing (Jung et al., 2009).

Although both macins and big defensins have already been reported in mollusks, the knowledge of these two AMP families is still extremely limited and their occurrence and evolutionary relationship in the animal kingdom have not been adequately studied. Here we report the identification in M. galloprovincialis, thanks to a whole-transcriptome sequencing approach, of novel transcripts pertaining to the macin family (named mytimacins) and to the big defensins family (named MgBDs) and discuss their molecular diversity and constitutive expression in different tissues.

Section snippets

Identification of transcripts encoding macins and big defensins from M. galloprovincialis

Using second generation sequencing systems (454 Life Sciences and Illumina platforms) we sequenced the transcriptome of Mediterranean mussels (M. galloprovincialis) from tissues (hemocytes, gills and digestive gland) of different individuals. Following accurate processing, we could locally assemble a transcript collection which updates and enrich the pre-existing Mytibase (http://mussel.cribi.unipd.it) (Venier et al., 2009). The predicted peptides originated from the assembly process were

Computational identification and sequence features of MgBDs

The mussel transcriptomic collection, assembled starting from a total of 24901 Sanger, 150857 454 Life Sciences and 108620377 Illumina sequencing reads, conprises110259 contigs (with an average length of 590 nucleotides; the N50 parameter of the assembly was 658). In this transcriptomic mussel collection we could identify eight different sequences encoding big defensins, named MgBD 1–6 and deposited at EMBL under the accession IDs FR873266–FR873273. Three sequences showing remarkable similarity

Conclusions

The advent of next generation sequencing technologies provided a valuable resource for the bioinformatic identification of previously uncharacterized protein families in non-model organisms on a transcriptomic or on a genomic scale (Patrzykat and Douglas, 2003). Such methodologies have also been successfully used also in the identification of potential AMPs in plants (Belarmino and Benko-Iseppon, 2010, Graham et al., 2008) and more recently also in invertebrate genomes (Tian et al., 2010, Wang

Acknowledgements

This work was supported by the project FP7-KBBE-2010-4-266157 (Bivalife) and by Regione Friuli Venezia Giulia, Direzione Centrale Risorse Agricole, Naturali, Forestali e Montagna, L.R. 26/2005 prot. RAF/9/7.15/47174.

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