Molecular Characterization of Immunoglobulin M (IgM) and Polymeric Immunoglobulin Receptor (pIgR) and Expression Response to Vibrio harveyi Challenge in Leopard Coral Grouper ( Plectropomus leopardus )

Immunoglobulin M (IgM) is the primary antibody in fsh, which is transported from epithelial cells into the external secretion system by polymeric immunoglobulin receptor (pIgR). In this study, the full length of IgM and pIgR complementary DNA sequences from leopard coral grouper ( Plectropomus leopardus ) was characterized


Introduction
Leopard coral grouper (Plectropomus leopardus) is an important fsh for the marine aquaculture industry [1].Artifcial breeding technology has made a breakthrough in recent years.Te breeding and cultivation of leopard coral grouper were observed in Hainan, Guangdong, and Fujian Provinces, and northern areas.Skin ulceration is a severe disease in leopard coral grouper.Te major pathogen is Vibrio harveyi based on morphological, physiological, and biochemical characteristics, leading to surface ulceration and visceral lesion during the growth period [2,3].A highquality chromosome-level genome of leopard coral grouper has been assembled using 10 × Genomics, high-throughput chromosome conformation capture (Hi-C), and PacBio long-read sequencing technologies.Te genome assembly has a length of 881.55 Mb with a scafold N50 of 34.15 Mb [4][5][6].Te gene families enriched in immune response were immunoglobulin heavy chain (IGH), β-2 microglobulin, PYRIN-containing Apaf1-like (PYPAF1), and nucleotidebinding oligomerization domain (NLRP) [6].
Immunoglobulin heavy chain (IGH) included IgM, IgD, and IgT in teleost.Immunoglobulin M (IgM) is the main compound of antibodies and found in the mucosal secretions on the surface of the intestines, skin, and gills of teleost.IgM has been discovered in many fsh species, including common carp (Cyprinus carpio L.) [7], rainbow trout (Oncorhynchus mykiss) [8], turbot (Scophthalmus maximus) [9], sea bream (Sparus aurata) [10], and olive founder (Paralichthys olivaceus) [11].IgM is tetrameric in most teleost and the monomer has two heavy (H) chains of molecular weight 70 kDa and two light (L) chains of 25 kDa.IgM protects the host against pathogens and its concentration in lymphoid organs was signifcantly upregulated under bacterial infection [12,13], whereas the research of IgM function in leopard coral grouper requires further investigation.
In this study, the IgM and pIgR gene sequences were characterized in leopard coral grouper.Te immune responses of pl-IgM and pl-pIgR at the transcription level were investigated under V. harveyi infection to elucidate protective mechanism of fsh IgM and pIgR.In addition, we found the antimicrobial function of recombinant pl-pIgR protein in Oxford cup assay in vitro, which will provide novel insights into the immune defense mechanism of leopard coral grouper and contribute to the disease control in aquaculture.

V. harveyi Infection and Sample Collection.
A total of 75 juvenile fsh (Average body weight of 15.0 ± 3.3 g) were obtained from Mingbo Aquatic Company (Laizhou, China).Fish were maintained in tanks with recirculation water for a week, continuous aeration, and regular feed in the disconnecting test area.Eight tissues (liver, spleen, kidney, intestine, gill, skin, brain, and muscle) were collected from 5 healthy fsh, transferred into liquid nitrogen, and stored at −80 °C.V. harveyi was isolated from skin ulcer diseased leopard coral grouper and was shaking-grown at 28 °C for 16 h [33].Tirty-fve fsh were randomly selected and intraperitoneally injected with V. harveyi at 1 × 10 5 colony forming unit (CFU)/ml at 0.1 ml/100 g fsh weight as the experiment group.Te remaining 35 fsh were intraperitoneally injected with phosphate-bufered saline (PBS) at 0.1 ml/100 g fsh weight as the control group.Tissue samples, including the spleen, kidney, liver, and intestine, were collected from 5 fsh at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after injection from the experiment group and control group after anesthesia in 5 mg/L MS-222 (tricaine methane sulfonate).

Molecular Cloning of Complementary DNA (cDNA)
Sequences of pl-IgM and pl-pIgR.Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and stored at −80 °C.Te RNA quality and integrity were assayed at 1% agarose gel electrophoresis and NanoDrop spectrophotometer.cDNA was synthesized from the total RNA with the FastQuant RT Kit (Tiangen, Beijing, China), according to the manufacturer's instructions.Te predicted pl-IgM and pl-pIgR sequences were obtained from the genome data of the leopard coral grouper (Bioproject: PRJNA622646) [4].Open reading frame (ORF) sequences of pl-IgM and pl-pIgR were obtained using polymerase chain reaction (PCR) with specifc primers (IgM-ORF-F/IgM-ORF-R and pIgR-ORF-F/pIgR-ORF-R).All primers used in this study are listed in Table 1.Te PCR products were purifed using a Gel Extraction Kit (CWBio, Beijing, China) after 1% agarose gel electrophoresis and subcloned into the pEASY-T1 simple cloning vector (Transgen Biotech, Beijing, China) for sequencing.

Sequence Analysis, Homology, and Phylogenetic Analysis.
Te predicted protein was analyzed by the Basic Local Alignment Search Tool (BLAST) program of the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/blast).Te putative immunoglobulin domain was identifed using conserved domains search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) by comparing it with IgM and pIgR of other species as listed in Table 2. Te putative amino acid sequence alignment was performed using ClustalX and ESPript 3.0 (https:// espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).Using the neighbor-joining method, the phylogenetic trees of IgM and pIgR were constructed with the sequences listed in Table 2 using Molecular Evolutionary Genetic Analysis (MEGA) 7.0 software.Te reliability of the branching was tested through bootstrap resampling with 1000 replications.

Expression Profle Assay by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).
Te qRT-PCR was performed to examine the expression levels of pl-IgM and pl-pIgR in healthy fsh and bacteria infected fsh.Total RNA and tissue cDNA were synthesized according to the manufacturer's instructions.Te qRT-PCR conditions were as follows: 95 °C for 5 min, followed by 40 cycles at 95 °C for 5 s and 60 °C for 34 s.Relative fold changes of genes were calculated by the methods of 2 −ΔΔCt .β-actin was used as a control for the normalization of expression.

Expression, Purifcation, and Antibacterial Function of
Recombinant pl-pIgR.Te secretory component region of the pl-pIgR cDNA was amplifed by PCR using the primers listed in Table 1, which is inserted into the pET-his expression vector and transformed into Escherichia coli BL21 (DE3) cells (Transgen, Beijing, China).Te positive clones were cultured in 200 ml LB medium with 50 μg/ml ampicillin.When the optical density at 600 nm (OD600) reached 0.5-0.6, a fnal concentration of 0.1 mM isopropyl-bDthiogalactopyranoside (IPTG) was added, and the protein was induced for another 6 h.Te bacteria were centrifuged at 6000 rpm for 5 min at 4 °C and resuspended in lysis bufer (50 mM Tris-Cl pH 8.0, 150 mM NaCl) with 1 mM phenylmethanesulfonyl fuoride (PMSF).After sonicating for 6 min, the sediment was dissolved in precooled solution.Te recombinant pl-pIgR protein was purifed using the HisTrap FF crude purifcation system for six-His-tagged proteins (Invitrogen, Shanghai, China) and renatured by gradient urea dialysis.Te supernatant was collected and stored at −80 °C.In this assay, 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the recombinant protein.Te extracellular antibacterial function of the recombinant pl-pIgR protein was determined with the Oxford cup method.Tree trypticase soy agar plates (TSA) were inoculated with 10 5 CFU/ml of V. harveyi, Vibrio alginolyticus, and Edwardsiella tarda.Ten, 100 μl aliquots of protein (200 ng/μl), ampicillin (5 ng/ μl), and PBS bufer were added to each Oxford cups.Te plates were incubated at 28 °C overnight, and the inhibition zones around the cup were observed and measured.

Statistical Analysis.
Te statistic P values were calculated by one-way analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) 19.0 software, and P value <0.05 was considered statistically signifcant.

Sequence Analysis and Phylogenetic Relationship of pl-IgM
and pl-pIgR.Te pl-IgM shared higher amino acid identities with the grouper IgM.Tere were conserved cysteine residues in each CH domain (CH1-CH2-CH3-CH4), and interand intrachain disulfde bridges were conserved in the teleosts.Comparative analysis showed that ILD1 and ILD2 of pl-pIgR shared homology with other fsh ILD1 and ILD2 (Figure 2).Te distance tree showing the relationship between the amino acid sequences of the constant regions of IgM heavy chain for diferent fsh species was constructed.Te pl-IgM and pl-pIgR were clustered into one branch with other groupers (Figure 3).

Expression Profles of pl-IgM and pl-pIgR.
Te pl-IgM messenger RNA (mRNA) was detected in immune-related tissues, with higher expression levels in the intestine, kidney, and spleen and middle expression levels in the gill, liver, skin, and brain of fsh (Figure 4(a)).Te pl-pIgR mRNA was highly expressed in the intestine and spleen and weakly expressed in the skin, gill, and kidney, which was similar to pl-IgM mRNA (Figure 4(b)).

Expression Levels of pl-IgM and pl-pIgR in Immune-
Related Tissues after V. harveyi Infection 3.4.1.pl-IgM.Te expression levels of pl-IgM increased signifcantly in immune-related tissues after V. harveyi infection.pl-IgM mRNA expression pattern in 4 tissues of infected fsh showed a signifcant increase compared with uninfected fsh.Te pl-IgM expression fuctuated in the intestine, head kidney, and spleen from 6 h.Te highest expression of pl-IgM was observed in the intestine at 6 h (∼4 fold) and 24 h (∼7 fold) after bacterial challenge (Figure 5(a)).In the kidney and spleen, the expression levels were upregulated at 2 peak times (6/12 h and 72 h).Te expression levels were 5∼6 fold compared to 0 h (Figures 5(b) and 5(c)).In the liver, the expression level of pl-IgM was up to the peak time at 72 h, and the expression level was up to 20-fold postinfection (Figure 5(d)).

pl-pIgR.
Experimental infection with V. harveyi caused signifcant induction of pl-pIgR expression level in infected fshes compared with uninfected fshes.Te peak time point of pl-pIgR expression level was 12 h after infection in the intestine and head kidney (Figures 6(a) and 6(b)).Te pink time point of pl-pIgR expression levels in the spleen and liver were 24 h and 48 h, respectively (Figures 6(c) and 6(d)).Te peak expression levels of pl-pIgR in the intestine, kidney, spleen, and liver were 8, 5, 3, and 5 times higher than those in 0 h, respectively (Figure 6).

Recombinant Expression and Antibacterial Function of the pl-pIgR Protein.
Te recombinant pl-pIgR protein was obtained using the pET-His vector.After sonicating, the supernatant and sediment of uninduced and IPTG-induced E. coli DE3 (BL21) were assessed separately.As shown in Figure 7(a), a specifc band of approximately 42 kDa was identifed in the lane of sediment of IPTG-induced E. coli DE3 (BL21).Tis protein was consistent with the predicted molecular mass of the recombinant protein of pl-pIgR with His-tag.Te recombinant protein was purifed and dissolved in a 2 mg/ml concentration with neutral Tris bufer.On the Oxford cup assay, each plate contained 200 ng/μl pl-pIgR protein as experiment group, 5 ng/μl ampicillin as a positive control, and PBS was used as negative controls.Te recombinant pl-pIgR protein displayed antibacterial activity against V. harveyi and V. alginolyticus according to the

Discussion
In this study, the pl-IgM and pl-pIgR genes were characterized in leopard coral grouper, and the transcriptome profles of these two genes were analyzed in healthy fsh and after V. harveyi infection.Te SC part of pl-pIgR was expressed in the E. coli system.Te recombinant protein had an antibacterial function against two tested Vibrio (V.harveyi and V. alginolyticus).
Several conserved amino acid residues of the pl-IgM and pl-pIgR were found in the multiple sequence alignment, such as the conserved cysteine in the Ig-like domains, which suggests the stability of protein structure.Te pIgR homologs in teleost have 2 ILDs, which are homologous with the ILD1 and ILD5 of the mammalian pIgR.Tese 2 ILDs are sufcient for interaction with tetrameric IgM, and there should be further investigation on the binding sites.pIgR mediates IgM across the intestinal epithelium into gut mucus and the epithelia in Japanese founder [18,34].Te protein structure and phylogenetic analysis showed that the evolution of leopard coral grouper IgM and pIgR genes had the nearest neighbor-joining with other groupers, such as Epinephelus coioides and Epinephelus lanceolatus, due to the same origin of the grouper family of teleost.
IgM is the principal immunoglobulin in humoral and mucosal immunity in teleost.IgM activates the complement system and can cause pathogen lysis [35].Te tissue distribution of pl-IgM and pl-pIgR demonstrated the vital function in immune-related tissues such as intestine, spleen, and kidney.Te highest expression levels of pl-IgM and pl-pIgR were observed both in the mucosal-associated tissues and systemic immune tissues, including the intestine, spleen, kidney, gill, liver, and skin.Te present result is consistent with the previous studies on Japanese founder, turbot, and half-smooth tongue sole [13,24,30].Te expression levels of IgM and pIgR were afected in fsh under infection with bacteria, parasites, and viruses.In the orange-spotted grouper, the transcript level of IgM was upregulated in the head, kidney, and spleen, followed by V. alginolyticus infection [36].After exposure to Flavobacterium columnare, grass carp IgM and pIgR mRNA levels were signifcantly upregulated and downregulated to control levels [37].We focused on the changes in the expression levels of pl-IgM and pl-pIgR in   pIgR is a core gene in bridging innate and adaptive immune responses, and its extracellular ligand-binding region (SC part) has intrinsic antimicrobial properties [14].In this study, an antibacterial activity analysis based on the inhibition zone proved that the recombinant SC part protein of pl-pIgR exhibited antibacterial function against two tested Vibrio species in the observed inhibition zones, which indicates its potential usage in anti-vibriosis.

Conclusion
Our study characterized the IgM and pIgR genes in leopard coral grouper and analyzed mRNA expression profles of pl-IgM and pl-pIgR in healthy fshes and in response to V. harveyi infection.Te protein sequences and pl-IgM and pl-pIgR structures had the highest similarity with other groupers.Under V. harveyi infection, pl-IgM and pl-pIgR were upregulated in immune-related tissues with similar expression trends.Te recombinant pl-pIgR protein has the antibacterial function against two tested vibrio species, indicating the potential value in replacing antibiotics.

Figure 1 :Figure 2
Figure 1: Te nucleotide and deduced amino acid sequences of the heavy chain of pl-IgM (a) and pl-pIgR (b).Te number on the left showed the positions of nucleotide and amino acid.Te stop codon is represented with an asterisk ( * ).

Figure 2 :
Figure 2: Multiple sequence alignment of pl-IgM (a,b) and pl-pIgR (c) deduced amino acid sequences with the corresponding sequences of other species.Sequences corresponding to signal peptide, immunoglobulin-like domain (ILD), transmembrane region, and extracellular region are noted in the brackets at the top of the sequence matrix.

Figure 3 :Figure 4 :Figure 5 :
Figure 3: Phylogenetic trees were constructed with the neighbor-joining method based on the amino acid sequences of pl-IgM (a) and pl-pIgR (b) from leopard coral grouper and other species.Te reliability of each node is estimated by bootstrapping with 1000 replications implemented in MEGA 7.0.Te scale bar (0.1) represents the genetic distance.Te numbers marked near the nodes indicate the bootstrap test scores.
Tis study was supported by Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang) (ZJW-2019-06), Key Research and Development Project of China

Figure 6 :
Figure 6: Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of expression of pl-pIgR messenger RNA (mRNA) after being infected with V. harveyi.Te asterisk on the bars indicates the statistical signifcance of pl-pIgR compared to the 0 h group ( * P < 0.05, * * P < 0.01).Te mRNA levels of pl-pIgR were determined by normalized relative to β-actin.

Figure 7 :
Figure 7: Recombinant expression and antibacterial activity analysis of recombinant pl-pIgR protein.(a) Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis of recombinant pl-pIgR protein.M protein marker; lane 1: uninduced E. coli total bacteria; lane 2: supernatant of uninduced E. coli; 3: induced E. coli total bacteria; 4: supernatant of induced E. coli; 5: sediment of induced E. coli.Te arrow indicates the recombinant pl-pIgR protein.(b) Antibacterial activity of the recombinant pl-pIgR protein in Oxford cup assay.
2.1.Ethical Statement.Experiments were performed according to the guidelines and ethical standards of Regulations for the Administration of Afairs Concerning Experimental Animals of the State Science and Technology Commission of Shandong Province, China.Tis study was approved by the Ethics Committee of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.

Table 2 :
Te protein ID of IgM and pIgR in fsh and other species.