Parallel identification of novel antimicrobial peptide sequences from multiple anuran species by targeted DNA sequencing

Abstract Background Antimicrobial peptides (AMPs) are multifunctional effector molecules that often combine direct antimicrobial activities with signaling or immunomodulatory functions. The skin secretions of anurans contain a variety of such bioactive peptides. The identification of AMPs from frog species often requires sacrificing several specimens to obtain small quantities of crude peptides, followed by activity based fractionation to identify the active principles. Results We report an efficient alternative approach to selectively amplify AMP-coding transcripts from very small amounts of tissue samples, based on RNA extraction and cDNA synthesis, followed by PCR amplification and high-throughput sequencing of size-selected amplicons. This protocol exploits the highly conserved signal peptide region of the AMP precursors from Ranidae, Hylidae and Bombinatoridae for the design of family-specific, forward degenerate primers, coupled with a reverse primer targeting the mRNA poly-A tail. Conclusions Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides, mostly from Ranidae species, including several novel potential AMPs for functional characterization. This (i) confirms the effectiveness of the experimental approach and indicates points for protocol optimization to account for particular cases, and (ii) encourages the application of the same methodology to other multigenic AMP families, also from other genera, sharing common features as in anuran AMPs

The first transcriptomic resource for the Antarctic scallop Adamussium colbecki

Adamussium colbecki is one of the most common bivalve mollusks found in the coastal waters of the Antarctic continent. Its widespread distribution, easiness of collection and sensitivity to slight alterations of water temperature and presence of pollutants make this cold-adapted scallop a potentially interesting sentinel species for the biomonitoring of the impact of anthropic activities on the Antarctic benthic communities. However, while the availability of genetic and molecular data would represent a resource of the utmost importance to enable the use of this species as a model for environmental studies, this data is presently nearly non-existing. Here we report a high quality de novo assembled and annotated transcriptome for A. colbecki, discussing the long-debated phylogenetic position of this species within the order Pectinida through a Bayesian phylogenomics approach, based on the concatenated multiple sequence alignment of 978 universally conserved orthologous genes

The purplish bifurcate mussel Mytilisepta virgata gene expression atlas reveals a remarkable tissue functional specialization

Abstract Background Mytilisepta virgata is a marine mussel commonly found along the coasts of Japan. Although this species has been the subject of occasional studies concerning its ecological role, growth and reproduction, it has been so far almost completely neglected from a genetic and molecular point of view. In the present study we present a high quality de novo assembled transcriptome of the Japanese purplish mussel, which represents the first publicly available collection of expressed sequences for this species. Results The assembled transcriptome comprises almost 50,000 contigs, with a N50 statistics of ~1 kilobase and a high estimated completeness based on the rate of BUSCOs identified, standing as one of the most exhaustive sequence resources available for mytiloid bivalves to date. Overall this data, accompanied by gene expression profiles from gills, digestive gland, mantle rim, foot and posterior adductor muscle, presents an accurate snapshot of the great functional specialization of these five tissues in adult mussels. Conclusions We highlight that one of the most striking features of the M. virgata transcriptome is the high abundance and diversification of lectin-like transcripts, which pertain to different gene families and appear to be expressed in particular in the digestive gland and in the gills. Therefore, these two tissues might be selected as preferential targets for the isolation of molecules with interesting carbohydrate-binding properties. In addition, by molecular phylogenomics, we provide solid evidence in support of the classification of M. virgata within the Brachidontinae subfamily. This result is in agreement with the previously proposed hypothesis that the morphological features traditionally used to group Mytilisepta spp. and Septifer spp. within the same clade are inappropriate due to homoplasy

A GM1b/asialo-GM1 oligosaccharide-binding R-type lectin from purplish bifurcate mussels Mytilisepta\ua0virgata and its effect on MAP kinases

A 15-kDa lectin, termed SeviL, was isolated from Mytilisepta\ua0virgata (purplish bifurcate mussel). SeviL forms a noncovalent dimer that binds strongly to ganglio-series GM1b oligosaccharide (Neu5Ac\u2512-3Gal\u3b21-3GalNAc\u3b21-4Gal\u3b21-4Glc) and its precursor, asialo-GM1 (Gal\u3b21-3GalNAc\u3b21-4Gal\u3b21-4Glc). SeviL also interacts weakly with the glycan moiety of SSEA-4 hexaose (Neu5Ac\u3b12-3Gal\u3b21-3GalNAc\u3b21-3Gal\u3b11-4Gal\u3b21-4Glc). A partial protein sequence of the lectin was determined by mass spectrometry, and the complete sequence was identified from transcriptomic analysis. SeviL, consisting of 129 amino acids, was classified as an R(icin B)-type lectin, based on the presence of the QxW motif characteristic of this fold. SeviL mRNA is highly expressed in gills and, in particular, mantle rim tissues. Orthologue sequences were identified in other species of the family Mytilidae, including Mytilus galloprovincialis, from which lectin MytiLec-1 was isolated and characterized in our previous studies. Thus, mytilid species contain lectins belonging to at least two distinct families (R-type lectins and mytilectins) that have a common \u3b2-trefoil fold structure but differing glycan-binding specificities. SeviL displayed notable cytotoxic (apoptotic) effects against various cultured cell lines (human breast, ovarian, and colonic cancer; dog kidney) that possess asialo-GM1 oligosaccharide at the cell surface. This cytotoxic effect was inhibited by the presence of anti-asialo-GM1 oligosaccharide antibodies. With HeLa ovarian cancer cells, SeviL showed dose- and time-dependent activation of kinase MKK3/6, p38 MAPK, and caspase-3/9. The transduction pathways activated by SeviL via the glycosphingolipid oligosaccharide were triggered apoptosis. DATABASE: Nucleotide sequence data have been deposited in the GenBank database under accession numbers MK434191, MK434192, MK434193, MK434194, MK434195, MK434196, MK434197, MK434198, MK434199, MK434200, and MK434201

Dr Enrique Cabrero Mendoza, Director-General of CONACYT, United Mexican States

Dr Enrique Cabrero Mendoza, Director-General of CONACYT, United Mexican State