The Diverse Piscidin Repertoire of the European Sea Bass (Dicentrarchus labrax): Molecular Characterization and Antimicrobial Activities

Abstract

Fish rely on their innate immune responses to cope with the challenging aquatic environment, with antimicrobial peptides (AMPs) being one of the first line of defenses. Piscidins are a group of fish specific AMPs isolated in several species. However, in the European sea bass (Dicentrarchus labrax), the piscidin family remains poorly understood. We identified six different piscidins in sea bass, performed an in-depth molecular characterization and evaluated their antimicrobial activities against several bacterial and parasitic pathogens. Sea bass piscidins present variable amino acid sequences and antimicrobial activities, and can be divided in different sub groups: group 1, formed by piscidins 1 and 4; group 2, constituted by piscidins 2 and 5, and group 3, formed by piscidins 6 and 7. Additionally, we demonstrate that piscidins 1 to 5 possess a broad effect on multiple microorganisms, including mammalian parasites, while piscidins 6 and 7 have poor antibacterial and antiparasitic activities. These results raise questions on the functions of these peptides, particularly piscidins 6 and 7. Considering their limited antimicrobial activity, these piscidins might have other functional roles, but further studies are necessary to better understand what roles might those be.

Antimicrobial Peptides: Identification of two Beta-Defensins in a Teleost Fish, the European Sea Bass (Dicentrarchus labrax)

“…Beta-defensin genes in salmonids derived from common ancestry and then expanded, after these events of WGDs [12], and diverged from other species, including the European sea bass. While in Salmonid species several beta-defensins can be found, in Perciformes and perhaps other related species, they turned into a more restricted group, with other families of AMPs being more diversified, such as hepcidins or piscidins [50,51].…”

“…Healthy European sea bass (D. labrax), with an average weight of 30 g, were provided by a commercial fish farm (Sonríonansa, Pesués, Cantabria, Spain) [50]. Prior to the experiments, fish were acclimated for 30 days to the fish-holding facilities of the Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Porto.…”

“…Pairs of oligonucleotide PCR primers were designed according to conserved regions of beta-defensins' mRNA sequences from sea bass and other fish species, with sea bass expressed sequence tags (ESTs) and whole-genome shotgun sequences (WGSS) available in the National Center for Biotechnology Information nucleotide database (http: //www.ncbi.nlm.nih.gov, accessed on April 2021) and Ensembl genome browser 103 (http://www.ensembl.org, accessed on April 2021). cDNA preparations from the whole intestine, kidney and gill were used in PCR amplifications [50,69]. PCR products were run on 1% agarose gels, and then relevant fragments were purified with the NZYGelpure kit (NZYtech, Lisbon, Portugal), cloned into pCR 2.1-TOPO vectors, propagated in One Shot Mach1-T1R competent cells (Invitrogen, Life Technologies, Carlsbad, CA) and sent for sequencing (GATC, A Eurofins Genomics Company, Ebersberg, Germany).…”

“…Genomic DNA was isolated from sea bass red blood cells, using the NZY Blood gDNA Isolation kit (NZYtech), as previously described [50,69]. Quantification was performed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and quality was checked by agarose gel electrophoresis.…”

Deuterium Solid State NMR Studies of Intact Bacteria Treated With Antimicrobial Peptides

“…A major challenge in AMP research has been in developing a unified picture of AMP mechanism(s) that is consistent, at least for the particular AMP under scrutiny, with the results from a spectrum of experimental approaches, from simple model systems to whole cells to whole organisms. For example, on the one hand, function is often studied via minimal inhibitory concentration (MIC) assays with bacteria, which indicate the minimum concentration of AMP needed to prevent bacterial growth (9)(10)(11). On the other hand, NMR and other biophysical studies provide details of AMP structure and AMP-induced alterations to the bilayer structure, such as bilayer thinning, formation of toroidal pores, solubilizing the membrane into micellar structures, or lipid clustering (1,(12)(13)(14).…”