Antibacterial and antiviral roles of a fish β-defensin expressed both in pituitary and testis

Abstract

Defensins are a group of cationic peptides that exhibit broad-spectrum antimicrobial activity. In this study, we cloned and characterized a β-defensin from pituitary cDNA library of a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides). Interestingly, the β-defensin was shown to be dominantly expressed in pituitary and testis by RT-PCR and Western blot analysis, and its transcript level is significantly upregulated in reproduction organs from intersexual gonad to testis during the natural and artificial sex reversal. Promoter sequence and the responsible activity region analyses revealed the pituitary-specific POU1F1a transcription binding site and testis-specific SRY responsible site, and demonstrated that the pituitary-specific POU1F1a transcription binding site that locates between -180 and -208 bp is the major responsible region of grouper β-defensin promoter activity. Immunofluorescence localization observed its pituicyte expression in pituitary and spermatogonic cell expression in testis. Moreover, both in vitro antibacterial activity assay of the recombinant β-defensin and in vivo embryo microinjection of the β-defensin mRNA were shown to be effective in killing gram-negative bacteria. And, its antiviral role was also demonstrated in EPC cells transfected with the β-defensin construct. Additionally, the antibacterial activity was sensitive to concentrations of Na(+), K(+), Ca(2+) and Mg(2+). The above intriguing findings strongly suggest that the fish β-defensin might play significant roles in both innate immunity defense and reproduction endocrine regulation.

Figure 1. Molecular characterization of grouper β-defensin.

(A) Nucleotide and deduced amino acid sequences of grouper β-defensin. The signal peptide is underlined. (B) Multiple alignment of grouper β-defensin amino acid sequences with other β-defensin proteins from fish. They are green puffer β-defensin-1 (BN000873) and green puffer β-defensin-2 (BN000874), medaka β-defensin (EU676010), zebrafish β-defensin-1 (AM181358), zebrafish β-defensin-2 (AM181359) and zebrafish β-defensin-3(AM181360), rainbow trout β-defensin-1(AM282655), rainbow trout β-defensin-2 (FM212656), rainbow trout β-defensin-3 (FM212657) and rainbow trout β-defensin-4 (FM212658), tiger puffer β-defensin-1(BN000875), olive flounder β-defensin (GQ414989). The identity values are on the right. The six conservative cystine residues are marked by black box. (C) Phylogenetic tree of grouper β-defensin peptide with other fish β-defensins. The bootstrap values were generated by testing the tree 1000 times. (D) Schematic representation of genomic structure of grouper β-defensin and other seven fish β-defensins.

Figure 2. Adult tissue distribution profiles of grouper β-defensin and the specificity detection of anti-grouper β-defensin antiserum.

(A) Expression pattern analyzed by RT-PCR and Western blot (B). L: liver; K: kidney; S: spleen; F: fat; H: heart; Mu: muscle; P: pituitary; Hy: hypothalamus; Tn: telencephalon; C: cerebellum; MB: midbrain; MOB: medulla oblongata; O: ovary; T: testis. α-tubulin was employed as a positive control. (C) The specificity of anti-grouper β-defensin serum detected by Western blot and immunofluorescence (D). The sections of grouper sexual reversal gonad were immunostained by the anti-grouper β-defensin serum (a), the pre-adsorbed anti-grouper β-defensin serum with extra recombinant grouper β-defensin protein (b), and the pre-immuned rabbit serum (c) respectively. Red fluorescence stained by PI indicates the cellular nucleus.

Figure 3. The expression of grouper β-defensin is upregulated during the period of sex transformation.

(A) The expression of grouper β-defensin in the pituitaries of grouper with gonads in different stages. The total RNAs of pituitaries were isolated from grouper with undevelopmental, mature ovary and mature testis respectively. (B) Differential expression of grouper β-defensin in different stage gonads of red-spotted grouper. The total RNAs of gonads were respectively isolated from the different gonad stages with different sizes: O1: immature ovary from 150 g body weight; O2: ovary with previtellogenic oocytes from 450 g body weight; O3: ovary with vitellogenic oocytes from 700 g body weight; O4: ovary with vitellogenic oocytes from 950 g body weight; T1: testis from 1600 g body weight. α-tubulin was used as control. (C) Expression of grouper β-defensin in different stage gonads of red-spotted grouper during artificial sex inversion. (a) RT-PCR analysis of grouper β-defensin expression. α-tubulin was amplified at the same conditions as a positive control in each sample. (b) The grouper β-defensin mRNA intensities as shown in (a) were analyzed by Band Leader Applification Software Ver. 3.0. Values represent the means ± S.D. of three separate experiments. C01 and C02: the gonads from two individuals before sex inversion experiment, W11, W12, W21, W22, W31, W32, W41, W42, W51, W52, W61 and W62: the gonads from two individuals after feeding with MT for week 1–6, respectively.

Figure 4. Map and deletion analysis of grouper β-defensin promoter.

(A) Sequence of grouper β-defensin promoter region. Consensus nucleotide sequences corresponding to potential transcription binding sites are underlined and labeled. (B) Schematic diagrams and comparison of potential transcription factor binding sites between grouper β-defensin promoter and medaka β-defensin promoter. (C) The progressive 5′ deletion series and their luciferase activities in CO (grey bars) and EPC (black bars). pRL-TK was used as internal control. The promoter activity is presented as relative light units (RLU) normalized to Renilla luciferase activity. The data shown are derived from a representative experiment reported as the mean (n = 3) ± SD.

Figure 5. Immunofluorescence detection of grouper β-defensin protein (green) in grouper pituitary.

(A) TSH signals in the anterior pituitary. (B) Grouper β-defensin signals in the posterior pituitary of transversal section of pituitary, and (C-D) are higher magnifications of positive signal area in B. (E) Grouper β-defensin signals in the posterior pituitary of sagittal section of pituitary. (F) is higher magnification of positive signal area in E. RPD:rostral pars distalis; PPD: proximal pars distalis; PN: pars noversa. Red fluresecence was stained by Propidium Iodide for cellular nucleus.

Figure 6. Spermatogonium-specific immunofluorescence localization of grouper β-defensin protein (green) in the grouper gonads.

(A) Ovary section; (B) Gonad section at the early sex reversal stage; (C) Gonad section at intersex stage; (D) Testis section. PO: previtellogenic oocyte; SG: spermatogonia; PSP: primary spermatocytes; SSP: secondary spermatocytes; SPZ: spermatozoa. Red fluresecence was stained by Propidium Iodide for the nuclei.

Figure 7. Antimicrobial activity of grouper β-defensin.

(A) Antimicrobial activities of grouper β-defensin in vitro to eight different strains (shown in respective chart). X-axis indicates the protein gradient concentration (µg/ml) and the Y-axis shows the survival rate (means ± SD). At least three independent experiments were conducted. (B) Effects of NaCl, KCl, CaCl₂ and MgCl₂ concentration on the antimicrobial activity of grouper β-defensin. E.coli were incubated for 12 h with 64 µg/ml grouper β-defensin in the culture medium containing various concentration ions. Values shown are mean ± SD. (C) Antimicrobial activity of grouper β-defensin in vivo. (a) Western blot was employed to detect the expression of grouper β-defensin by embryos. (b) At 24hpf, the control embryos injected with pure water and embryos injected grouper β-defensin mRNA were challenged with the Gram-negative bacterium Vibrio flurialis and Gram-positive bacterium Micrococcus luteus, respectively. The survival rates were recorded at the indicated times after infection. The experiments were repeated three times with 70 embryos per group.

Figure 8. Antiviral activity of grouper β-defensin.

(A) EPC cells seeded in 24-well plates were transfected for 24 h with 0.5 µg of pcDNA3.1-grouper β-defensin or pcDNA3.1 as control. Grouper β-defensin mRNA and the protein was detected by RT-PCR and Western blot respectively, and (B) the other group of transfected was challenged with RGV with different dose (10⁵ TCID₅₀/ml, 10⁴ TCID₅₀/ml, 10³ TCID₅₀/ml and 10² TCID₅₀/ml), respectively. 48 h later, cells were then stained with crystal violet for detection of CPE, and (C) the culture supernatants were collected to detect the virus titers. (D) EPC cells seeded in 24-well plates were transfected for 24 h with pcDNA3.1-grouper β-defensin or pcDNA3.1 at dose of 1 µg, 0.5 µg, 0.25 µg and 0.1 µg, respectively. Grouper β-defensin mRNA and the protein was detected by RT-PCR and Western blot respectively, and (E) the other of transfected cells was challenged with RGV at multiplicity of infection 10³ TCID₅₀/ml. 48 h later, cells were then stained with crystal violet for detection of CPE, and (F) the culture supernatants were collected to detect the virus titers. The data shown is a representative of three independent experiments. Differences between control cells and grouper β-defensin transfected cells are significant *p<0.05.