Figure 1.
Genomic structure and location of Nile tilapia piscidin genes.
a. Comparison of the genomic organization of piscidin genes of Nile tilapia. The five Nile tilapia piscidin genes were located between the ninth and tenth exons of exostosin-1a-like. Both were transcribed in reverse. b. The length of the coding sequence of each exon is indicated by numbers above the boxes, and each intron is indicated by numbers above the line. Red indicates regions of translated amino acid sequences, and gray indicates the 5′ and 3′ untranslated regions (UTRs).
Figure 2.
Sequence alignment between piscidins.
Multiple sequence alignments of the Nile tilapia piscidin peptides with selected piscidin or with other members of the piscidin family. Gaps were inserted to obtain maximum homology. Sequences used for comparison with the references for GenBank accession numbers are described in Supplementary Table 3. All gene sequences of the coding region were input into the dendogram for alignment. The same amino acid is indicated by the same color, such as methionine (M) being indicated by yellow.
Figure 3.
Amphipathic α-helical structures and three-dimensional structures of five different Nile tilapia piscidins.
A Schiffer-Edmunson plot of (a) Nile tilapia piscidin 1 (TP1), (b) -2, (c) -3, (d) -4, and (e) -5 was produced with the PEPWHEEL program. Amino acid residues are successively numbered and connect each amino acid following the sequence with lines that represent its relative position along the helix. Red indicates hydrophilic, green indicates hydrophobic, blue triangles indicate negatively charged, and blue pentagons indicate positively charged residues. Structural models of (f) TP1∼5. Pictures were generated with the Viewerlite program (Accelrys, San Diego, CA, USA).
Figure 4.
Differential expressions of Nile tilapia piscidin genes in tissues and after stimulation with bacteria.
(a) A qRT-PCR analysis of Nile tilapia piscidins (TP2, -3, and -4) and ubiquitin expression in various tissues (gills, liver, intestines, skin, head kidneys (abbreviated as ‘kidneys’), and spleen) of Nile tilapia. (b) qRT-PCR analysis of Nile tilapia piscidin transcripts in various tissues (gills, liver, intestines, skin, kidney, and spleen) at two time points (24 and 48 h) after an intraperitoneal injection of Streptococcus agalactiae (SA47) or Vibrio vulnificus (204). Wild-type (WT) indicates no bacteria were injected, and the fish experienced no injection. Expression data are presented as the mean (± standard error; SE) relative quantity normalized to ubiquitin. Relative quantities were calculated to the individual (WT) with the lowest normalized piscidin expression. Data (mean ± SE) with different letters significantly differ (p<0.05) among tissues for single-gene comparisons.
Figure 5.
Hemolytic activities of Nile tilapia piscidin 1∼5 (TP1, -2, -3, -4, and -5) peptides against fish erythrocytes. Data (mean ± standard error) with different letters significantly differ (p<0.05) among the experimental groups.
Table 1.
Activities of Nile tilapia piscidin 1∼5 (TP1, -2, -3, -4, and -5) peptides against gram-positive and -negative bacteria.
Figure 6.
Effects of piscidins on cell proliferation.
Fish cells (tilapia ovary (TO2) cell line) were treated with different doses of piscidin peptides (TP1∼5) for 24 h, followed by an MTS assay. Each concentration was repeated in eight wells for three independent experiments. Piscidin treatment affected cell viability in a dose-dependent manner. There were significant differences between treatment groups for (a) TP3 (40 µg/ml) and (b) TP4 (12 µg/ml) compared to 0 µg/ml treatment. There were significant differences between treatment groups for the lower concentrations in (c) TP2 (30 µg/ml) and TP5 (10 µg/ml) compared to 0 µg/ml treatment. But survival of cells treated with 100 µg/ml of TP1, -2, and -5 were still >80%. Data are from three separate experiments. Data (mean ± SE) with different letters significantly differ (p<0.05) among the experimental groups.