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.

In-Depth In Silico Search for Cuttlefish (Sepia officinalis) Antimicrobial Peptides Following Bacterial Challenge of Haemocytes.

Cuttlefish (Sepia officinalis) haemocytes are potential sources of antimicrobial peptides (AMPs). To study the immune response to Vibrio splendidus and identify new AMPs, an original approach was developed based on a differential transcriptomic study and an in-depth in silico analysis using multiple tools. Two de novo transcriptomes were retrieved from cuttlefish haemocytes following challenge by V. splendidus or not. A first analysis of the annotated transcripts revealed the presence of Toll/NF-κB pathway members, including newly identified factors such as So-TLR-h, So-IKK-h and So-Rel/NF-κB-h. Out of the eight Toll/NF-κB pathway members, seven were found up-regulated following V. splendidus challenge. Besides, immune factors involved in the immune response were also identified and up-regulated. However, no AMP was identified based on annotation or conserved pattern searches. We therefore performed an in-depth in silico analysis of unannotated transcripts based on differential expression and sequence characteristics, using several tools available like PepTraq, a homemade software program. Finally, five AMP candidates were synthesized. Among them, NF19, AV19 and GK28 displayed antibacterial activity against Gram-negative bacteria. Each peptide had a different spectrum of activity, notably against Vibrio species. GK28-the most active peptide-was not haemolytic, whereas NF19 and AV19 were haemolytic at concentrations between 50 and 100 µM, 5 to 10 times higher than their minimum inhibitory concentration.

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.

Neuropeptidome of the Cephalopod Sepia officinalis: Identification, Tissue Mapping, and Expression Pattern of Neuropeptides and Neurohormones during Egg Laying.

Cephalopods exhibit a wide variety of behaviors such as prey capture, communication, camouflage, and reproduction thanks to a complex central nervous system (CNS) divided into several functional lobes that express a wide range of neuropeptides involved in the modulation of behaviors and physiological mechanisms associated with the main stages of their life cycle. This work focuses on the neuropeptidome expressed during egg-laying through de novo construction of the CNS transcriptome using an RNAseq approach (Illumina sequencing). Then, we completed the in silico analysis of the transcriptome by characterizing and tissue-mapping neuropeptides by mass spectrometry. To identify neuropeptides involved in the egg-laying process, we determined (1) the neuropeptide contents of the neurohemal area, hemolymph (blood), and nerve endings in mature females and (2) the expression levels of these peptides. Among the 38 neuropeptide families identified from 55 transcripts, 30 were described for the first time in Sepia officinalis, 5 were described for the first time in the animal kingdom, and 14 were strongly overexpressed in egg-laying females as compared with mature males. Mass spectrometry screening of hemolymph and nerve ending contents allowed us to clarify the status of many neuropeptides, that is, to determine whether they were neuromodulators or neurohormones.

Identification of a New Set of Polypeptidic Sex Pheromones from Cuttlefish (Sepia officinalis).

The common English Channel cuttlefish (Sepia officinalis) reproduces every year on very localized coastal spawning areas after a west-east horizontal migration of several tens of kilometers (80-200 km). The massive arrival of spawners on the coasts of west Cotentin and the Bay of Seine is suspected to be driven by the action of sex pheromones expressed and secreted by the genitals of sexually mature females. The present study aims to verify the existence of polypeptide pheromones, of a higher molecular weight than those described above. Their size could confer them a wider range of action than that of the previously identified peptide pheromones. The implementation of an experimental strategy combining transcriptomics and proteomics with functional tests and an in silico study led to the identification of a cocktail of pheromones with molecular weights ranging between 22 and 26 kDa. Proteomic analyses combined to functional tests revealed partial pheromone release in the environment, and their accumulation in the outer capsule of the egg, suggesting the eggs as pheromone diffusers, also able to induce stimulation by contact when the eggs are handled by females.

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.

Characterization of a novel LFRFamide neuropeptide in the cephalopod Sepia officinalis.

From a single LC-MS/MS analysis, a new C-terminally extended RFamide neuropeptide was characterized in Sepia officinalis. The experimental strategy was based on the specific neutral loss associated with RFamide breakdown. Mass losses of 17 Da (C-terminally amide) and 320 Da (RFamide) have been observed for three known peaks of m/z 581.7 (FLRFamide), 599.8 (FMRFamide), 1096.3 (ALSGDAFLRFamide) and one unknown of m/z 752.8. The primary sequence of the peptide of m/z 752.8 was GNLFRFamide. MS/MS analyses revealed that this novel neuropeptide, called sepFRF1, is largely distributed in the central nervous system of cuttlefish of both sexes. Probably transported in the visceral nerve from the subesophageal mass (the peptide was not detected in the hemolymph), this neuropeptide targeted the rectum in agreement with its peripheral distribution. From concentrations as low as 10(-9)M, sepFRF1 increased the frequency, tonus and amplitude of rectal contractions. SepFRF1 is the first RFamide peptide identified in Sepia officinalis that is not derived from the FaRPs precursor. SepFRF1 could belong to a RFamide subfamily identified in gastropods and may be involved in feeding behavior.

Ovarian and sperm regulatory peptides regulate ovulation in the oyster Crassostrea gigas.

For more than six decades, several studies have shown that genital products to entering the mantle cavity via the incurrent siphon, initiate in oyster, strong and rhythmic contractions of the adductor muscle (AM). In order to characterize the regulatory peptides capable of triggering AM contractions, we focused on the identification of putative myotropic peptides from genital products. Two experimental approaches were developed. The first one, based on a mass spectrometry screening of the male genital products, led to the identification of the tetrapeptide APGWamide. This neuropeptide was also detected in the seminal secretions of the cephalopod Sepia officinalis. In this species, APGWamide is directly involved in the oocyte transport. In Crassostrea, in vitro bioassay demonstrated that APGWamide modulates the AM contractions that insure the release of oocytes in the external medium. Exposure of oysters to a physiological concentration of APGWamide triggered repetitive shell closures. The second experimental approach was based on the monitoring of HPLC purification by a myotropic bioassay using the cuttlefish oviduct contractions as a target. The successive purification steps of the acidic extraction of ovaries from mature female oysters, led to the characterization of the hexapeptide PIESVD. When applied to mature female oysters, this peptide triggered the increase of shell closure frequency. The activity of these two regulatory peptides is the first experimental evidence of a peptidergic control of egg-laying in oyster. APGWamide and PIESVD could be used, in commercial and experimental hatcheries, for the identification of mature females to be selected for in vitro fertilization.

Identification of SepCRP analogues in the cuttlefish Sepia officinalis: a novel family of ovarian regulatory peptides.

In the cuttlefish, Sepia officinalis, the ovary appears to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying. Following the identification of the SepCRP-1, which is a peptide extracted from ovary and involved in egg capsule secretion, investigations were focused on the identification of related peptides. Seven related-Sepia Capsule Releasing Peptides (R-SepCRPs) were identified by means of mass spectrometry and characterized using MS/MS spectra and/or Edman degradation. Finally, primary structures were verified by the comparison of MS/MS spectra from endogenic and synthetic peptides. This new ovarian peptide family exhibits a conserved SLXKD tag involved in the biological activity. LC-MS/MS screening clearly demonstrates that R-SepCRPs are restricted to the female genital tract. Expressed during vitellogenesis, they are released by vitellogenic follicles and full-grown oocytes (FGO) in the genital coelom. Biological activities suggest that R-SepCRPs would be responsible for the storage of FGO before mating and would take part in the mechanical secretion of egg capsule products, as previously described for SepCRP-1.