The Functions of β-Defensin in Flounder (Paralichthys olivaceus): Antibiosis, Chemotaxis and Modulation of Phagocytosis

Abstract

Most defensins are cationic antimicrobial peptides with broad-spectrum killing activity against bacteria, fungi and enveloped viruses. However, it should be recognized that there are some non-cationic β-defensins in organisms, which need to be further studied. In this study, a new spliced isoform of anionic β-defensin from flounder (Paralichthys olivaceus, fBD) was identified, and its antibiosis, chemotaxis and modulation of phagocytosis were examined. In addition, the contributions of fBD to the antimicrobial activity of extracellular traps (ETs) were also analyzed. The recombinant fBD (rfBD) could effectively inhibit the growth of Gram-positive bacteria (S. aureus, Micrococcus luteus) and Gram-negative bacteria (E. coli, V. alginolyticus, V. anguillarum). An indirect immunofluorescence assay showed that the fBD was co-localized in the extracellular traps released by the leukocytes. When the ETs were blocked with antibodies against rfBD, the proliferation of S. aureus and E. coli incubated with ETs tended to increase compared with that in the control group. In addition, the results obtained by flow cytometry showed that the rfBD could significantly chemoattract leukocytes and increase phagocytic activity in vitro. In conclusion, this study provides new insights into the biological function of anionic defensins, which can serve as one of the important effectors in extracellular traps and as a bridge between innate and adaptive immunity in teleosts.

Keywords: antibiosis; chemotaxis; extracellular traps; phagocytosis; β-defensin.

Figure 1

Sequence structure and phylogenetic analysis of fBD. (A) Nucleotide and amino acid sequences of fBD. The initiation and termination codons are marked with bold letters. The signal peptide is marked with a highlight, the propiece region is shown in red, and fixed cysteines are framed by boxes. (B) Phylogenetic tree analysis of rfBD from flounder and other species. The tree was structured according to the “maximum-likelihood” method using MEGAX. The GenBank accession numbers of α-, β- and θ-defensins are listed as follows: Homo sapiens: AF529417.1; Ctenopharyngodon idella: MG652749.1; Oncorhynchus mykiss: NM_001195183.2; Anser cygnoides: EU606039.1; Mus musculus: AY591385.1; Oreochromis niloticus: MH674361.1; Ictalurus punctatus: KX211992.1; Cercopithecus preussi: EU126877.1; Arvicanthis niloticus: XM_034520497.1; Arvicola amphibius: XM_038325539.1; Cricetulus griseus: XM_027435861.1; Grammomys surdaster: XM_028784038.1; Mesocricetus auratus: XM_040733512.1; Microtus oregoni: XM_041673564.1; Cercocebus atys: XM_012078007.1; Chlorocebus sabaeus: XM_037984733.1; Macaca fascicularis: XM_005562541.2; Macaca mulatta: AF191100.1; Macaca nemestrina: XM_011711827.2; Papio anubis: FJ030939.1; Paralichthys olivaceus: OL631146.

Figure 2

The preparation of recombinant fBD. (A) The agarose gel electrophoresis of fBD gene amplified from head kidney by PCR. Lane M, DNA marker; lane 1, PCR products of fBD. (B) SDS-PAGE analysis of rfBD peptide. Lane M, protein marker; lane 1, uninduced whole-bacteria lysate; lane 2, induced whole-bacteria lysate; lane 3, purification of rfBD peptide with Trx tag. (C) SDS-PAGE analysis of rfBD peptide after digestion. Lane M, protein marker; lane 1, purification of rfBD peptide with Trx tag; lane 2, purification of rfBD peptide without Trx tag. Full figure of PAGEs can be found in Supplementary Materials.

Figure 3

The localization of fBD in peritoneal cells. After incubation with anti-rfBD or anti-Trx antibody, the peritoneal cells were detected using Alexa Flour 488–labeled secondary antibody. The nucleus was visualized using DAPI. In both cases, the right panels were merges of the left and middle panels. The arrows indicate some representative fBD-positive cells. Bar, 20 μm.

Figure 4

Antibacterial activities of rfBD. The broth dilution method was used to study the inhibitory effects of different concentrations of rfBD on two Gram-positive bacteria (A) and three Gram-negative bacteria (B). The results were expressed as the absorbance value of the culture solution at 600 nm (The data was summarized in Table S1). The values in the graph are expressed as mean ± SD (n = 3).

Figure 5

The localization and antibacterial activities of fBD in extracellular traps. (A) PMA-stimulated ET-producing cells were treated with antibodies against Trx or rfBD followed by Alexa Fluor–labeled secondary antibody. Arrows without tail indicate ETs and arrows with tails indicate fBD in ETs. Bar, 20 μm. (B) Effect of rfBD on bacteriostatic capabilities of ETs. ET-producing cells were infected by E. coli, Ed. tarda and S. aureus with or without (control) of anti-rfBD, and bacterial survival was counted at different times. Data are the means of three independent assays and are presented as means ± SEM. ** p < 0.01.

Figure 6

Chemotactic effects of rfBD on leukocytes from head kidney and spleen in vitro. Migrated leukocytes to the lower chamber of the transwell were counted by flow cytometry. Bars represent the mean number of migrated leukocytes ± SEM (n = 3). The level of significance compared with the Trx treatment group is represented by ** p < 0.01.

Figure 7

The phagocytosis of leukocytes from head kidney and spleen with or without rfBD stimulation detected by flow cytometric analysis. FITC fluorescence histograms showed the ratio of leukocyte with (B,E) or without (A,D) rfBD stimulation. The phagocytosis rate of leukocytes from the head kidney (C) and the spleen (F), respectively, with rfBD stimulation was analyzed using SPSS. All the error bars represent the standard deviation of three biological replicates. ** p < 0.01.