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

Abstract

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.

Combined Proteotranscriptomic-Based Strategy to Discover Novel Antimicrobial Peptides from Cone Snails

“…For example, the investigation of scorpion venoms led to the discovery of Hadrurin, Scorpine, Vejovine, StCT2, Pandinin 1, and Pandinin 2, which are effective antimicrobial peptides [ 19 , 20 , 21 , 22 , 23 ]. Similar studies also found antimicrobial peptides in snakes, spiders, bees, ants, centipedes, lugworms, wasps, and cuttlefish [ 15 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ].…”

Marine Transcriptomics Analysis for the Identification of New Antimicrobial Peptides

“…To complete the analysis and search for peptides with low sequence homology such as piscidins, we used the homemade software program PepTraq [ 65 , 87 , 88 ]. The strategy developed to search for new piscidins was to use the sequence homologies observed at the signal peptide level of the precursors encoding piscidins.…”

Helminthic host defense peptides: using the parasite to defend the host

“…Unlike BLAST, such methods do not require the detection of sequence homology for the identification of candidate HDP, relying on the identification of primary sequence features shared by a training set of peptides with known antimicrobial activity, but uncommon in peptides with other biological functions. While an in-depth overview of these methods goes beyond the scope of this review (for a comprehensive description, see [58][59][60]), several recent publications showcase the potential of machine learning approaches in the discovery of novel HDP in non-model invertebrates [61][62][63]. It is however important to remark that no matter the level of sophistication that the current computational approaches utilize, laboratory functional validation remains a fundamental confirmatory step for any candidate HDP.…”