Design of antimicrobial peptides from a cuttlefish database

Abstract

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 (CAMP), 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-GR and KT-inhibited more than one bacterial strain with minimal inhibitory concentrations below 25 µM. Some sequences like VA and FK were hemolytic, while GR induced less than 10% of hemolysis on human blood cells at a concentration of 200 µM. GR was the only peptide derived from a precursor with a signal peptide, suggesting a real role in cuttlefish immune defense.

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

“…Among the differentially expressed transcripts between the control and challenged conditions, we selected sequences of less than 100 amino acids presenting a signal peptide, using our homemade PepTraq software program. We applied some of the specific structural AMP criteria previously defined by Houyvet and collaborators [35] and selected five candidates (Table 2). Among them, three were down-regulated after V. splendidus exposure, with fold changes of 0.39, 0.84 and 0.85.…”

“…AV19 possessed 40% similarity with two frog antibacterial peptides, namely temporin-1Cd from Rana clamitans [66] and temporin-1Ola from R. akaloosae [67]. GK28 possessed 35.48% similarity with marmelittin from the venom of the scorpion Mesobuthus eupeus [68] and a C-terminal end similar to the one of GR21 [35], in addition to the lowest hydrophobic ratio and the highest net charge of the selected peptides. Both NF19 and II19 possessed similarity with AMPs from the bovine rumen microbiome-47.36% with P20 and 41.66% with lynronne-2, respectively [69].…”

“…Considering that these three peptides were identified from a precursor with a signal peptide, it would be interesting to validate their biological function in cuttlefish. The methodology used in this study, based on Houyvet and collaborators [35] as an alternative to classical methods requiring a lot of biological material, was focused on the reduction of animal pain and use, consistent with directive 2010/63/EU on animal care and protection.…”

“…In Cephalopods, no AMP has been identified using classical approaches such as homology search using in silico analysis or immune challenge response. The only two AMPs described were designed in silico using a cuttlefish (Sepia officinalis) transcriptomic database including central nervous system, reproductive tissues and posterior salivary glands extracts, but no hct extract [35].…”

OctoPartenopin: Identification and Preliminary Characterization of a Novel Antimicrobial Peptide from the Suckers of Octopus vulgaris

“…In particular, it is believed that cephalopod antimicrobial peptides (AMPs) are trapped in the egg capsule and mainly expressed by female accessory sex glands, conferring on them efficient protection of organs against microorganisms [24]. In this context, studies reporting the identification of AMPs in cephalopods were conducted on squids and cuttlefish [25,26]. Nevertheless, there is still a scarcity of knowledge regarding the defense mechanisms involved in O. vulgaris immune response, even if more researchers are focusing their attention on this topic [13,[27][28][29][30][31].…”