ABSTRACT
BACKGROUND: The liver-expressed antimicrobial peptide 2 (LEAP2) plays a pivotal role in the host's immune response against pathogenic microorganisms. Numerous such antimicrobial peptides have recently been shown to mitigate infection risk in fish, and studying those harboured by the economically important fish Acrossocheilus fasciatus is imperative for enhancing its immune responses against pathogenic microorganisms. In this study, we cloned and sequenced LEAP2 cDNA from A. fasciatus to examine its expression in immune tissues and investigate the structure-activity relationships of its intramolecular disulphide bonds. RESULTS: The predicted amino acid sequence of A. fasciatus LEAP2 was found to include a signal peptide, pro-domain, and mature peptide. Sequence analysis indicated that A. fasciatus LEAP2 is a member of the fish LEAP2A cluster and is closely related to Cyprinus carpio LEAP2A. A. fasciatus LEAP2 transcripts were expressed in various tissues, with the head kidney exhibiting the highest mRNA levels. Upon exposure to Aeromonas hydrophila infection, LEAP2 expression was significantly upregulated in the liver, head kidney, and spleen. A mature peptide of A. fasciatus LEAP2, consisting of two disulphide bonds (Af-LEAP2-cys), and a linear form of the LEAP2 mature peptide (Af-LEAP2) were chemically synthesised. The circular dichroism spectroscopy result shows differences between the secondary structures of Af-LEAP2 and Af-LEAP2-cys, with a lower proportion of alpha helix and a higher proportion of random coil in Af-LEAP2. Af-LEAP2 exhibited potent antimicrobial activity against most tested bacteria, including Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Staphylococcus warneri. In contrast, Af-LEAP2-cys demonstrated weak or no antibacterial activity against the tested bacteria. Af-LEAP2 had a disruptive effect on bacterial cell membrane integrity, whereas Af-LEAP2-cys did not exhibit this effect. Additionally, neither Af-LEAP2 nor Af-LEAP2-cys displayed any observable ability to hydrolyse the genomic DNA of P. aeruginosa. CONCLUSIONS: Our study provides clear evidence that linear LEAP2 exhibits better antibacterial activity than oxidised LEAP2, thereby confirming, for the first time, this phenomenon in fish.
Subject(s)
Amino Acid Sequence , Animals , Structure-Activity Relationship , Fish Diseases/microbiology , Antimicrobial Peptides/chemistry , Antimicrobial Peptides/pharmacology , Antimicrobial Peptides/genetics , Fish Proteins/genetics , Fish Proteins/chemistry , Disulfides/chemistry , Phylogeny , Aeromonas hydrophila/drug effects , Base SequenceABSTRACT
The safety of feed derived from genetically modified (GM) crops is one of the focuses of attention. To evaluate the ecotoxicological effects of transgenic mCry1Ac maize (BT799) on fish, zebrafish (Danio rerio) were fed extruded feeds containing either 20% GM maize (GMF) or its parental control maize (PF), GM maize meal (GMM) or its parental control maize meal (PMM), and a control commercial feed (CF), respectively. The growth performance, histopathology, reproduction, antioxidant enzyme activity and mRNA expression levels of sensitive protein in the liver were investigated over the course of a 98-day feeding trial. The results showed that transgenic mCry1Ac maize had no significant effect on growth, histopathology of the liver, brain and intestinal tract, fecundity, hatching rate of fertilized eggs, superoxide dismutase (SOD), catalase (CAT) activity, mRNA expression levels of SOD and CAT, or heat shock protein 70 (HSP70) and vitellogenin (VTG) in the liver. However, zebrafish fed the commercial feed exhibited significantly greater weight, longer length, and higher specific growth rate than those fed feeds (GMF and PF) and maize meals (GMM and PMM). The hatching rate of zebrafish in the feed groups was significantly lower than that of the maize meal groups and the commercial feed group. The mRNA transcriptional levels of VTG were significantly higher in the liver for the feed groups (3.85±0.76) than that for the maize meal groups (1.60±0.56). These results suggest that transgenic mCry1Ac maize has no ecotoxicological effects on zebrafish. However, the differences in nutrient composition and palatability between the extruded experimental feeds and the commercial feed would lead to significant diffe-rences in some parameters.
