New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides

Abstract

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

Keywords: NK-lysin; Salmo salar; antimicrobial peptide; cytokines; immune response.

Figure 1

Multiple alignment of salmonid NK-lysin sequences. Multiple alignment of NK-lysin sequences from S. salar and O. mykiss was performed with the Clustal Omega tool. The sequences corresponding to the signal peptide predicted with SignalP 5.0 are framed. The physicochemical properties of the amino acid residues are represented by different colors, blue: acidic; red: small ]small+ hydrophobic (incl.aromatic -Y)]; magenta: basic and green: hydroxyl + sulfhydryl + amine + G. In addition, “*” indicates positions with a single fully conserved residue, “:” indicates conservation between residues with highly similar properties, and “.” indicates conservation between residues with weakly similar properties. Cysteine residues are highlighted in bold.

Figure 2

Phylogenetic analysis of NK-lysin amino acid sequences. A phylogenetic tree was constructed using the Neighbor-joining method. Numbers above the branches indicate frequencies per 1000 Bootstrap analysis. NK-lysin protein sequences without signal peptides from mammals, birds, and teleosts were used for alignment and phylogenetic tree construction. NCBI GenBank accession numbers of sequences used are listed in Table 1 .

Figure 3

Modeled structure of Salmo salar NK-lysin peptides (SWISS‐MODEL server). The region corresponding to the NK-lysin-derived peptides is indicated in magenta. The cysteines involved in the disulfide bonds are also indicated.

Figure 4

Hemolytic activity of the three Salmo salar NK-lysin-derived peptides against fish (A) and human (B) erythrocytes. The percentage of hemolysis was defined relative to the hemolysis obtained with the erythrocyte suspension treated with 0.1% SDS (100% hemolysis).

Figure 5

Relative SsNK-lysin 1, 2, and 4 mRNA expression profiles in nine uninfected Atlantic salmon (S. salar) tissues. The EF-1α reference gene was used as a normalizer, and the stomach tissue was used as a control or calibrator. The relative expression was determined according to Paff’s mathematical model (2001) (55). The data was shown as mean ± SD (n=5).

Figure 6

Effect of NK-lysin-derived peptides (NK1, NK2, and NK4) on cell viability (MTT assay) in head kidney leucocytes from Salmo salar after 24 and 48 hours of treatment with peptides at a concentration of 50 μM. Values are expressed as means ± S.D. (n=6).

Figure 7

Relative expression of immune-related genes induced by SsNK-lysin-derived peptides in S. salar head kidney leucocytes. Cells were stimulated with 50 μM of the synthetic NK-lysin-derived peptides for 6, 12, and 48 h. Expression levels were analyzed by Real-time PCR. The expression of the mRNA was analyzed as 2^−ΔΔCT relative quantification. The comparative threshold cycle values were normalized for EF-1α. The comparative threshold cycle values were normalized for EF-1α. Data were expressed as the means ± S.D. of three independent experiments, each in triplicate. Data were analyzed by ANOVA followed by Dunnett’s multiple comparisons test (* p<0.05; ** p<0.01; ***p < 0.001; **** p<0.0001). The Dunnett post hoc test was used to compare means from experimental groups against a control group mean.

Figure 8

Activation of the MAPK signaling pathway by SsNK-lysin-derived peptides in S. salar HKLs. HKLs were pretreated for 2 hours with 10 μM of SB202190 inhibitor (p38 inhibitor), U0126 inhibitor (mitogen-activated protein kinase 1/2 (MEK1/MEK2) inhibitor) or SP600125 inhibitor (JNK inhibitor). As a negative control, cells were incubated with 0.1% DMSO as a vehicle for 2 hours. Cells were then incubated with 50 μM of NK1, NK2, NK4, or medium for 12 hours. The IL-1β relative expression was analyzed by qRT-PCR. The expression of the mRNA was analyzed as 2^−ΔΔCT relative quantification. The comparative threshold cycle values were normalized for EF-1α. Data were expressed as the means ± S.D. of three independent experiments, each in triplicate. Data were analyzed by ANOVA followed by Šidák’s multiple comparisons test (* p<0.05; ** p<0.01; ***p < 0.001; **** p<0.0001). Šídák method performs simultaneous joint pairwise comparisons for all possible pairwise combinations of means.

Figure 9

Modulation of LPS- or poly(I:C)-induced immune responses by co-administration of SsNK-lysin-derived peptides. SHK-1 cells were co-stimulated with 50 μM of SsNK-lysin-derived peptides (NK1, NK2, and NK4) and 1 µg/mL lipopolysaccharide or 1 µg/mL poly(I:C) for 6 and 12 hours. In addition, cells were treated with only LPS, poly(I:C), or peptides. Cells with culture medium alone were included as a negative control. The relative expressions of TNF-α, IL-1β, and IL-8 as LPS-induced responses and of IFN-1α and Mx involved in the antiviral response induced by poly(I:C) were determined by qRT-PCR. The expression of the mRNA was analyzed as 2^−ΔΔCT relative quantification. The comparative threshold cycle values were normalized for EF-1α. Data were expressed by the means ± S.D. of three independent experiments, each analyzed in triplicate, and by ANOVA followed by Šidák’s multiple comparisons test (* p<0.05; ** p<0.01; ***p < 0.001; **** p<0.0001). Šídák method performs simultaneous joint pairwise comparisons for all possible pairwise combinations of means.

Figure 10

Effects on phagocytic activity of S. salar head kidney leukocytes (HKL). HKLs were incubated with pHrodo Green-conjugated E. coli bioparticles in the absence or presence of SsNK-lysin-derived peptides (NK1, NK2, and NK4) at 0, 10, and 50 μM. Phagocytosis in the absence of peptides was set at 100%. Data were expressed as percentages relative to cells incubated with bioparticles alone. Data represent means ± S.D. of three independent experiments performed in triplicate. Data were analyzed by ANOVA followed by Šidák’s multiple comparisons test. Asterisks indicate statistically significant differences compared to phagocytosis in the absence of stimuli (** p<0.01; ***p < 0.001; **** p<0.0001). Šídák method performs simultaneous joint pairwise comparisons for all possible pairwise combinations of means.

Figure 11

Relative transcriptional level of the immune-related gene in fish injected with Salmo salar NK-lysin-derived peptides at 1, 3-, 7-, 14- and 21 days post-injection. mRNA expression in Atlantic salmon’s head kidney after intraperitoneal injection of NK-lysin-derived peptides. The graph shows the cytokine fold induction compared to the control group, i.e., fish injected with phosphate-buffered saline. Data were expressed the means ± S.E. and analyzed by ANOVA followed by Dunnett’s multiple comparisons test (* p<0.05; ** p<0.01; ***p < 0.001; **** p<0.0001) (n = 5). The Dunnett post hoc test compared means from experimental groups against a control group mean.