Characterization of myeloperoxidase and its contribution to antimicrobial effect on extracellular traps in flounder (Paralichthys olivaceus)

Abstract

Myeloperoxidase (MPO) is a cationic leukocyte haloperoxidase and together with other proteins, they possess activities against various microorganisms and are involved in extracellular trap (ET) formation. The present work describes the gene and deduced protein sequences, and functions of MPO in flounder (PoMPO). The PoMPO possesses a 2313 bp open reading frame (ORF) that encodes a protein of 770 amino acids. The highest PoMPO mRNA expression levels were found in the head kidney, followed by peritoneal cells, gill, spleen, skin, muscle, and liver. PoMPO was expressed in MHCII⁺ and GCSFR⁺ cells which indicated that PoMPO mainly is expressed in flounder macrophages and granulocytes. Bacterial lipopolysaccharide-stimulated peritoneal leukocytes showed an increased protein level of PoMPO while it seemed that LPS also promoted the migration of MPO⁺ cells from the head kidney into the peripheral blood and peritoneal cavity. After phorbol 12-myristate 13-acetate (PMA) or bacterial stimulation, flounder leukocytes produced typical ET structures containing DNA with decoration by MPO. The ETs containing DNA and PoMPO effectively inhibited the proliferation of ET-trapped bacteria. Blocking PoMPO with antibodies decreased the enzymatic activity, which attenuated the antibacterial activity of ETs. This study pinpoints the involvement of ETs in flounder innate responses to pathogens.

Keywords: antibiosis; extracellular traps; fish; immune response; myeloperoxidase.

Figure 1

Phylogenetic tree showed the relationship between flounder MPO/EPO and other vertebrate amino acid MPO/EPO sequences. Numbers in each branch indicate the percentage bootstrap values from 1000 replicates. The accession numbers of MPO and EPO amino acid sequences are as follows, MPO: Paralichthys olivaceus, XP_019938121.1; Siniperca chuatsi, ABC72122.1; Danio rerio, AAK83239.1; Pogona vitticeps, XP_020659555.1; Gallus gallus, XP_015151399.1; Epinephelus coioides, APM83155.1; Channa argus, QCY41338.1; Pygoscelis adeliae, KFW66803.1 (partial); Anabas testudineus, XP_026195736.1; Larimichthys crocea, KAE8292175.1; Latimeria chalumnae, XP_005992326.1; Chelonoidis abingdonii, XP_032625834.1; Ictalurus punctatus, ACV69995.1; Scophthalmus maximus, XP_035464009.1. EPO: Gallus gallus, XP_015151415.1; Pogona vitticeps, XP_020659588.1; Homo sapiens, AAA58458.1; Xenopus laevis, NP_001081848.1; Bufo bufo, XP_040279578.1; Mus caroli, XP_021033325.1.

Figure 2

Comparison of the location of MPO genes in different fish species. The chromosomes and scaffold were identified in NCBI. Boxes represent the deduced genes. Arrows indicate the deduced orientation of gene transcription. Dashed lines connecting boxes suggest a homologous relationship.

Figure 3

The expression of PoMPO was determined by Western blotting (A) and quantitative real-time PCR (B) in different tissues. (C) Relative gene expression of PoMPO in the head kidney at different time points after V. anguillarum, S. aureus, or HIRRV infection. The results were calculated using relative expression method with 18S as the housekeeping gene. Different letters above the bar represent the statistical significance (p < 0.05) compared to each other at the same time point, and vertical bars represented the mean ± SD, n = 5.

Figure 4

The expression of PoMPO in leukocytes at different time points upon LPS stimulation. (A) Cell lysates were harvested and subjected to Western blotting analysis using anti-rPoMPO and anti-GAPDH Abs and the semi‐quantification of PoMPO was shown (B). (C) Flow cytometric analysis of MPO⁺ peritoneal leukocytes in the gate of FSC area (FSC-A)/SSC area (SSC-A) of flounder. Data are representative from three independent experiments, and vertical bars represent the mean ± SD. Different letters represent the statistical significance (p < 0.05) compared to each other at the same tissue.

Figure 5

Identification of MPO⁺ cells in flounder. (A) Peritoneal cells were double-stained using anti-rPoMPO Abs (red) and anti-Zap-70, anti-MHCII, or anti-GCSFR Abs (green), respectively. The blue color showed the DAPI dye nuclei. Arrows indicated double-positive cells. Bar = 10 µm. (B) Peritoneal cells were double-stained and analyzed by flow cytometry. Each figure is representative from three analysis (mean ± SD, n = 5).

Figure 6

PoMPO positive cells (continuous arrows or red double arrow) and MPO protein on ETs (yellow double arrow) in peritoneal cells (A, B) and head kidney leukocytes (C, D). (A, C) show the control cells without stimulation; (B, D) show PMA-induced ET formation. Blue indicates the nuclei and DNA fibers stained by DAPI. Bar = 10 µm.

Figure 7

SEM analysis of the entrapment of bacteria by ETs and the proliferation of the entrapped bacteria. (A) PMA-induced production of ETs. (B) The quantitative analysis of the formation of ETs by leukocytes in response to PMA, E. coli, E. tarda, and S. aureus for 3 hours. (C, E, G) show E. coli, E. tarda, and S. aureus induced the ET formation (arrows with tail) and entrapped bacteria (arrows without tail), respectively. (D, F, H) show the effect of PoMPO on the antimicrobial activity of ETs. ETs-producing cells were incubated with E. coli, E. tarda, or S. aureus in the presence or absence of anti-PoMPO Abs. Bacterial survival was determined at various time points. Different letters above the bar represent the statistical significance compared to each other at the same time point, and vertical bars represented the mean ± SD, n = 5 (P< 0.05).