PepTraq: a toolbox for in silico data mining and fast sequence filtering.

The development of de novo sequencing tools has led to the massive production of genomes and transcriptomes from many unconventional animal models. To tackle this huge flow of data, PepTraq brings together many functionalities generally scattered in multiple tools, so that sequences can be filtered on the basis of multiple criteria. It is particularly suitable for the identification of non-annotated transcripts, re-annotation, extraction of secretomes, neuropeptidomes, targeted search for peptides and proteins, preparing specific proteomics/peptidomics fasta files for mass spectrometry (MS) applications, MS data processing, etc. PepTraq is developed in Java, and is available as a desktop application that can be downloaded from https://peptraq.greyc.fr . It is also available as a web application at the same URL for processing small files (10-20 MB). The source code is open under a CeCILL-B licence.

Marine Transcriptomics Analysis for the Identification of New Antimicrobial Peptides.

Antimicrobial peptides (AMPs) participate in the immune system to avoid infection, are present in all living organisms and can be used as drugs. Fish express numerous AMP families including defensins, cathelicidins, liver-expressed antimicrobial peptides (LEAPs), histone-derived peptides, and piscidins (a fish-specific AMP family). The present study demonstrates for the first time the occurrence of several AMPs in lionfish (Pterois volitans). Using the lionfish transcriptome, we identified four transcript sequences encoding cysteine-rich AMPs and two new transcripts encoding piscidin-like peptides. These AMPs are described for the first time in a species of the Scorpaenidae family. A functional approach on new pteroicidins was carried out to determine antimicrobial sequences and potential uses, with a view to using some of these AMPs for human health or in aquaculture.

Involvement of RFamide neuropeptides in polyp contraction of the adult scleractinian corals Euphyllia ancora and Stylophora pistillata.

The distribution and functions of neurons in scleractinian corals remain largely unknown. This study focused on the Arg-Phe amide family of neuropeptides (RFamides), which have been shown to be involved in a variety of biological processes in animals, and performed molecular identification and characterization in the adult scleractinian coral Euphyllia ancora. The deduced amino acid sequence of the identified RFamide preprohormone was predicted to contain 20 potential neuropeptides, including 1 Pro-Gly-Arg-Phe (PGRF) amide and 15 Gln-Gly-Arg-Phe (QGRF) amide peptides. Tissue distribution analysis showed that the level of transcripts in the tentacles was significantly higher than that in other polyp tissues. Immunohistochemical analysis with the FMRFamide antibody showed that RFamide neurons were mainly distributed in the epidermis of the tentacles and mouth with pharynx. Treatment of E. ancora polyps with synthetic QGRFamide peptides induced polyp contraction. The induction of polyp contraction by QGRFamide peptide treatment was also observed in another scleractinian coral, Stylophora pistillata. These results strongly suggested that RFamides play a role in the regulation of polyp contraction in adult scleractinians.

Involvement of GLWamide neuropeptides in polyp contraction of the adult stony coral Euphyllia ancora.

The existence and function of neurons remain largely unexplored in scleractinian corals. To gain a better understanding of neuronal functions in coral physiology, this study focused on Glycine-Leucine-Tryptophan-amide family neuropeptides (GLWamides), which have been shown to induce muscle contraction and larval metamorphosis in other cnidarians. Molecular identification and functional characterization of GLWamides in the adult stony coral Euphyllia ancora were performed. We successfully elucidated the full-length cDNA of GLWamide preprohormone in E. ancora (named EaGLW preprohormone). The deduced amino acid sequence was predicted to contain six potential GLWamide peptides. Tissue distribution analysis demonstrated that transcripts of EaGLW preprohormone were mainly expressed in the mouth (including the pharynx) and tentacles of the polyps. Immunodetection with an anti-GLWamide monoclonal antibody revealed that GLWamide neurons were mainly distributed in the epidermis of the mouth region and tentacle, in agreement with the distribution patterns of the transcripts. Treatment of the isolated mouth and tentacles with synthetic GLWamide peptides induced the contraction of these isolated tissues. Treatment of polyps with synthetic GLWamide peptides induced the contraction of polyps. These results suggest that GLWamides are involved in polyp contraction (myoactivity) in adult scleractinians. Our data provide new information on the physiological function of neuropeptides in scleractinians.

Identification and structural characterization of the factors involved in vitellogenesis and its regulation in the African Osteoglossiforme of aquacultural interest Heterotis niloticus (Cuvier, 1829).

The African bonytongue (Heterotis niloticus) is an excellent candidate for fish farming because it has outstanding biological characteristics and zootechnical performances. However, the absence of sexual dimorphism does not favor its reproduction in captivity or the understanding of its reproductive behavior. Moreover, no molecular data related to its reproduction is yet available. This study therefore focuses on the structural identification of the different molecular actors of vitellogenesis expressed in the pituitary gland, the liver and the ovary of H. niloticus. A transcriptomic approach based on de novo RNA sequencing of the pituitary gland, ovary and liver of females in vitellogenesis led to the creation of three transcriptomes. In silico analysis of these transcriptomes identified the sequences of pituitary hormones such as prolactin (PRL), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their ovarian receptors (PRLR, FSHR, LHR). In the liver and ovary, estrogen receptors (ER) beta and gamma, liver vitellogenins (VtgB and VtgC) and their ovarian receptors (VLDLR) were identified. Finally, the partial transcript of an ovarian Vtg weakly expressed compared to hepatic Vtg was identified based on structural criteria. Moreover, a proteomic approach carried out from mucus revealed the presence of one Vtg exclusively in females in vitellogenesis. In this teleost fish that does not exhibit sexual dimorphism, mucus Vtg could be used as a sexing biomarker based on a non-invasive technique compatible with the implementation of experimental protocols in vivo.

Design of antimicrobial peptides from a cuttlefish database.

No antimicrobial peptide has been identified in cephalopods to date. Annotation of transcriptomes or genomes using basic local alignment Search Tool failed to yield any from sequence identities. Therefore, we searched for antimicrobial sequences in the cuttlefish (Sepia officinalis) database by in silico analysis of a transcriptomic database. Using an original approach based on the analysis of cysteine-free antimicrobial peptides selected from our Antimicrobial Peptide Database (APD3), the online prediction tool of the Collection of Anti-Microbial Peptides (CAMPR3), and a homemade software program, we identified potential antibacterial sequences. Nine peptides less than 25 amino acids long were synthesized. The hydrophobic content of all nine of them ranged from 30 to 70%, and they could form alpha-helices. Three peptides possessed similarities with piscidins, one with BMAP-27, and five were totally new. Their antibacterial activity was evaluated on eight bacteria including the aquatic pathogens Vibrio alginolyticus, Aeromonas salmonicida, or human pathogens such as Salmonella typhimurium, Listeria monocytogenes, or Staphylococcus aureus. Despite the prediction of an antimicrobial potential for eight of the peptides, only two-GR21 and KT19-inhibited more than one bacterial strain with minimal inhibitory concentrations below 25 µM. Some sequences like VA20 and FK19 were hemolytic, while GR21 induced less than 10% of hemolysis on human blood cells at a concentration of 200 µM. GR21 was the only peptide derived from a precursor with a signal peptide, suggesting a real role in cuttlefish immune defense.

How Egg Case Proteins Can Protect Cuttlefish Offspring?

Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection.

Transcriptomic and peptidomic analysis of protein hydrolysates from the white shrimp (L. vannamei).

An RNAseq approach associated to mass spectrometry was conducted to assess the composition, molecular mass distribution and primary sequence of hydrolytic peptides issued from hydrolysates of white shrimp (Litopenaeus vannamei) by-products. High performance size exclusion chromatography (HPSEC) analyses indicated that 69.2% of the 214-nm-absorbing components had apparent molecular masses below 1000 Da, and 88.3% below 2000 Da. OFFGEL-nLC-MALDI-TOF/TOF and nLC-ESI-MS/MS analyses led to the identification of 808 peptides based on the NCBI EST databank (161,397 entries) completed by the new L. vannamei databank (58,508 entries) that we created from the RNAs of tissues used for hydrolysate production. Whereas most of hydrolytic peptides have a MW below 2000 Da, preliminary investigations of antimicrobial properties revealed three antibacterial fractions that demonstrate functional activities. The abundance of small peptides as well as the biological activities detected could imply very interesting applications for shrimp hydrolysate in the field of aquaculture feeding.

Dual role of the cuttlefish salivary proteome in defense and predation.

We characterized the proteome of the posterior salivary glands of the cephalopod S. officinalis by combining de novo RNA sequencing and mass spectrometry. In silico analysis of the transcriptome revealed the occurrence of three main categories of proteins: enzymes, immune factors and toxins. Protein identification by SDS-PAGE and MALDI-TOF/TOF confirmed the occurrence of proteins essential to venom-like enzymes: peptidase S1 under four isoforms, phospholipase A2 and two toxins. The first toxin is a cystein rich secreted protein (CRISP), a common toxin found in all venomous animals. The second one is cephalotoxin, which is specific to decabrachia cephalopods. Secretions of the posterior salivary glands are transported to the cephalopodium; they are involved in prey catching but also in gamete storage, fertilization and egg-laying. The paralyzing activity and the antimicrobial effect of saliva suggest a dual role in predation and in immune defense in cuttlefish. BIOLOGICAL SIGNIFICANCE: The originality of this study lies in the use of a transcriptomic approach (de novo RNA sequencing) coupled to a proteomic approach to get an overview of posterior salivary glands in S. officinalis. In cephalopods, these glands are involved in predation, more precisely in paralyzing preys and digesting them. Our in silico analysis equally reveals a role in immune defense as observed in mammals' saliva. Our study also shows the specificity of cuttlefish venom, with the identification of cephalotoxins, proteins that are not found in octopuses. Finally, we show that cuttlefish saliva is a complex mixture that has antibacterial and crippling properties, but no lethal effect.

A complex set of sex pheromones identified in the cuttlefish Sepia officinalis.

BACKGROUND: The cephalopod mollusk Sepia officinalis can be considered as a relevant model for studying reproduction strategies associated to seasonal migrations. Using transcriptomic and peptidomic approaches, we aim to identify peptide sex pheromones that are thought to induce the aggregation of mature cuttlefish in their egg-laying areas. RESULTS: To facilitate the identification of sex pheromones, 576 5'-expressed sequence tags (ESTs) were sequenced from a single cDNA library generated from accessory sex glands of female cuttlefish. Our analysis yielded 223 unique sequences composed of 186 singletons and 37 contigs. Three major redundant ESTs called SPα, SPα' and SPß were identified as good candidates for putative sex pheromone transcripts and are part of the 87 unique sequences classified as unknown. The alignment of translated SPα and SPα' revealed a high level of conservation, with 98.4% identity. Translation led to a 248-amino acid precursor containing six peptides with multiple putative disulfide bonds. The alignment of SPα-α' with SPß revealed a partial structural conservation, with 37.3% identity. Translation of SPß led to a 252-amino acid precursor containing five peptides. The occurrence of a signal peptide on SPα, SPα' and SPß showed that the peptides were secreted. RT-PCR and mass spectrometry analyses revealed a co-localization of transcripts and expression products in the oviduct gland. Preliminary in vitro experiments performed on gills and penises revealed target organs involved in mating and ventilation. CONCLUSIONS: The analysis of the accessory sex gland transcriptome of Sepia officinalis led to the identification of peptidic sex pheromones. Although preliminary functional tests suggested the involvement of the α3 and ß2 peptides in ventilation and mating stimulation, further functional investigations will make it possible to identify the complete set of biological activities expected from waterborne pheromones.

Characterization of GnRH-related peptides from the Pacific oyster Crassostrea gigas.

Gonadotropin-releasing hormone (GnRH), a key neuropeptide regulating reproduction in vertebrates has now been characterized in a number of non-vertebrate species. Despite the demonstration of its ancestral origin, the structure and the function of this family of peptides remain poorly known in species as distant as lophotrochozoans. In this study, two GnRH-related peptides (Cg-GnRH-a and CgGnRH-G) were characterized by mass spectrometry from extracts of the visceral ganglia of the Pacific oyster Crassostrea gigas. These peptides showed a high degree of sequence identity with GnRHs of other mollusks and annelids and to a lesser extent with those of vertebrates or with AKH and corazonins of insects. Both the mature peptides and the transcript encoding the precursor protein were exclusively expressed in the visceral ganglia. Significant differences in transcriptional activity of Cg-GnRH encoding gene were recorded in the ganglia along the reproductive cycle and according to trophic conditions with a higher level in fed animals compared to starved animals. This suggests the involvement of Cg-GnRHs as synchronizers of nutritional status with energy requirements during reproduction in oyster. Evidence for a role of Cg-GnRHs as neuroregulators and as neuroendocrine factors in bivalve is discussed.

Structure and mechanism of action of a de novo antimicrobial detergent-like peptide.

The K4 peptide (KKKKPLFGLFFGLF) was recently demonstrated to display good antimicrobial activities against various bacterial strains and thus represents a candidate for the treatment of multiple-drug resistant infections. In this study, we use various techniques to study K4 behaviour in different media: water, solutions of detergent micelles, phospholipid monolayers and suspension of phospholipid vesicles. First, self-assembly of the peptide in water is observed, leading to the formation of spherical objects around 10nm in diameter. The addition of micelles induces partial peptide folding to an extent depending on the charge of the detergent headgroups. The NMR structure of the peptide in the presence of SDS displays a helical character of the hydrophobic moiety, whereas only partial folding is observed in DPC micelles. This peptide is able to destabilize the organization of monolayer membranes or bilayer liposomes composed of anionic lipids. When added on small unilamellar vesicles it generates larger objects attributed to mixed lipid-peptide vesicles and aggregated vesicles. The absence of calcein leakage from liposomes, when adding K4, underlines the original mechanism of this linear amphipathic peptide. Our results emphasize the importance of the electrostatic effect for K4 folding and lipid destabilization leading to the microorganisms' death with a high selectivity for the eukaryotic cells at the MIC. Interestingly, the micrographs obtained by electronic microscopy after addition of peptide on bacteria are also consistent with the formation of mixed lipid-peptide objects. Overall, this work supports a detergent-like mechanism for the antimicrobial activity of this peptide.