Antimicrobial and Immunoregulatory Activities of TS40, a Derived Peptide of a TFPI-2 Homologue from Black Rockfish (Sebastes schlegelii)

Abstract

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor. Previous reports have shown that TFPI-2 plays an important role in innate immunity, and the C-terminal region of TFPI-2 proved to be active against a broad-spectrum of microorganisms. In this study, the TFPI-2 homologue (SsTFPI-2) of black rockfish (Sebastods schegelii) was analyzed and characterized, and the biological functions of its C-terminal derived peptide TS40 (FVSRQSCMDVCAKGAKQHTSRGNVRRARRNRKNRITYLQA, corresponding to the amino acid sequence of 187-226) was investigated. The qRT-PCR (quantitative real-time reverse transcription-PCR) analysis showed that the expression of SsTFPI-2 was higher in the spleen and liver. The expression of SsTFPI-2 increased significantly under the stimulation of Listonella anguillarum. TS40 had a strong bactericidal effect on L. anguillarum and Staphylococcus aureus. Further studies found that TS40 can destroy the cell structure and enter the cytoplasm to interact with nucleic acids to exert its antibacterial activity. The in vivo study showed that TS40 treatment could significantly reduce the transmission of L. anguillarum and the viral evasion in fish. Finally, TS40 enhanced the respiratory burst ability, reactive oxygen species production and the expression of immune-related genes in macrophages, as well as promoted the proliferation of peripheral blood leukocytes. These results provide new insights into the role of teleost TFPI-2.

Keywords: Sebastods schegelii; TFPI-2; antibacterial; antiviral; immunomodulatory.

Figure A1

Structural domains and spatial structure of SsTFPI-2. (A) The predicted structural domain of protein motifs of SsTFPI-2 predicted by SMART. Signal peptides, KU domains and regions of compositional complexity are represented in red, green and pink, respectively. (B) The secondary structure of protein motifs of SsTFPI-2 predicted by PBIL. The alpha helix region is shown in blue, the extended strand region is shown in red, and the random coil region is shown in yellow. (C) The 3D structure of SsTFPI-2 predicted. Model dimensions (Å): X:40.642 Y:62.841 Z:54.951.

Figure A2

Multiple sequence alignments of SsTFPI-2. Dots denote gaps introduced for maximum matching. Black indicates consensus residues, red indicates the ≥75% identical residues in the aligned sequences, and blue indicates the ≥50% identical residues.

Figure A3

Phylogenetic analysis of SsTFPI-2. The numbers at the forks indicate the bootstrap values. The species and the GenBank accession numbers are as follows: Sebastes umbrosus (XP_037649587.1), Perca fluviatilis (XP_039675145.1), Sciaenops ocellatus (ADM64310.1), Etheostoma cragini (XP_034747376.1), Collichthys lucidus (TKS88549.1), Etheostoma spectabile (XP_032390827.1), Sander lucioperca (XP_031179144.1), Chelmon rostratus (XP_041811714.1), Perca flavescens (XP_028453602.1), Morone saxatilis (XP_035536691.1), Solea senegalensis (KAG7499865.1), Oreochromis aureus (XP_031612754.2), Micropterus salmoides (XP_038580246.1), Pundamilia nyererei (XP_005728783.1), Melanotaenia boesemani (XP_041867209.1), Scophthalmus maximus (XP_035476204.1), Danio rerio (ABD52009.1), Mus musculus (NP_001368836.1) and Homo sapiens (AAH05330.1).

Figure 1

The nucleotide and amino acid sequences of SsTFPI-2. In the cDNA sequence, the translation start and stop codons are in bold. In the amino acid sequence, the signal peptide is shaded in red, the three Kunitz domains are shown in shades of grey and the low complexity sequence is shaded blue. * Represents the stop codon.

Figure 2

SsTFPI-2 expression in black rockfish tissues. SsTFPI-2 expressions in various tissues were determined by qRT-PCR. Each result is the average of three independent experiments, shown as means ± SEM. * 0.01< p < 0.05, ** p < 0.01.

Figure 3

SsTFPI-2 expression in response to Listonella anguillarum challenge. L. anguillarum was used to infect black rokerfish, and PBS was used as the control. After infection, the liver (A), spleen (B) and head kidney (C) were taken for aseptic treatment at 4 h, 8 h, 12 h, 24 h, 48 h and 72 h. The SsTFPI-2 expression in each tissue was determined by qRT-PCR at various time points. Each result is the average of three independent experiments, shown as means ± SEM. ** p < 0.01.

Figure 4

Bacterial killing kinetics of TS40 against (A) Staphylococcus aureus and (B) Listonella anguillarum. Each result is the average of three independent experiments, shown as means ± SEM. ** p < 0.01.

Figure 5

Effect of TS40 on the cell structure of Listonella anguillarum. L. anguillarum was treated with TS40 for 0 h (A), 2 h (B) or 4 h (C) and then observed by transmission electron microscopy (TEM). Magnification: 60,000× (A), 20,000× (B), 50,000× (C). Scale bar: 500 nm (A), 1 µm (B), 500 nm (C).

Figure 6

Fluorescent localization of TS40 in target bacteria. Listonella anguillarum interacted with FITC-labeled TS40 (D,E,G,H) or FITC-labeled P86P15 (A,B) for 2 h under the fluorescence channel (B,E,H) and white light channel (A,D,G) and was observed after quenching the external fluorescence. (C), (F), (I) are the merged images of (A and B), (D and E), (G and H), respectively. Magnification, 400×. The scale bar is 20 µm.

Figure 7

Effect of TS40 on the genomic DNA of Listonella anguillarum in vitro. (A) Electrophoresis images of L. anguillarum genomic DNA incubated with different concentrations of TS40, P86P15 or PBS; (B) Electrophoresis images of proteinase K degradation of TS40 at a high temperature.

Figure 8

Effect of TS40 on total RNA of Listonella anguillarum in vitro. The RNA identification diagram of L. anguillarum total RNA treated with TS40, P86P15 or PBS.

Figure 9

In vivo effect of TS40 on bacterial infection. The bacterial amounts in the liver (A), spleen (B) and kidney (C) of different treatment groups were determined after infection. Each result is the average of three independent experiments, shown as means ± SEM. ** p < 0.01.

Figure 10

Effect of TS40 on virus infection. Virus copies at each time point in the spleen of fish after treatment with RBIV-C1 under different conditions. Each result is the average of three independent experiments, shown as means ± SEM. ** p < 0.01.

Figure 11

Effect of TS40 on the activity of macrophages. The effects of different concentrations of TS40 on (A) respiratory burst, (B) reactive oxygen species (ROS) and (C) immune-related gene expression. Each result is the average of three independent experiments, shown as means ± SEM. * 0.01 < p < 0.05, ** p < 0.01.

Figure 12

The proliferation effect of TS40 on the peripheral blood leukocytes. The proliferation activity of cells treated with different concentrations of TS40 or P86P15 was measured by an MTT assay. Each result is the average of three independent experiments, shown as means ± SEM. ** p < 0.01.