The C-type lectin family with the signature C-type lectin–like domain promotes antibacterial host defense within the animal kingdom. We examined the role of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards) (Decapoda: Grapsidae) Ig domain–containing C-type lectin (EsIgLectin), a novel and poorly understood member of the C-type lectin family. EsIgLectin was expressed primarily by both hemocytes (E. sinensis) and intestines, with significantly induced mRNA expression on intestinal or hemolymph bacterial infections. As a soluble protein, both its C-type lectin–like domain and the Ig domain were required for bacterial binding, bacterial agglutination, bacterial growth inhibition, and in vivo bacterial clearance. Polymeric EsIgLectin could be constructed via the disulfide bond in the Ig domain, significantly enhancing EsIgLectin antibacterial activity. EsIgLectin promoted bacterial phagocytosis in an Ig domain–dependent manner in hemocytes, while it controlled microbial homeostasis and protected against bacteria-induced inflammation in the intestine. Protein interaction studies revealed that the EsIgLectin Ig domain bound to the first Ig domain of the polymeric Ig receptor, which was essential for EsIgLectin-induced bacterial phagocytosis. The temporal sequence of cell interactions during intestinal inflammation is only beginning to be understood. In this article, we show that hemocyte-derived EsIgLectin entered the intestinal wall at the later phase of intestinal inflammation. Moreover, EsIgLectin protected the host against intestinal and hemolymph infections in a polymeric Ig receptor–dependent manner. Therefore, the EsIgLectin promoted bacterial clearance and protected against inflammatory disease through an independent or synergistic effect of hemocytes and intestines in invertebrates.

Visual Abstract

C-type lectins are a superfamily of >1000 proteins that are defined by having one or more characteristic C-type lectin–like domains and are subdivided into 17 subgroups based on their phylogeny and domain organizations, which confer specificity for mannose- and galactose-type carbohydrates (1). In mammals, C-type lectins are present as soluble or transmembrane proteins implicated in a diverse range of physiological functions, resulting from their ability to recognize endogenous and exogenous ligands (2). However, much less is known about invertebrate lectins, especially in nonmodel organisms. Most invertebrate C-type lectins contain a single carbohydrate-recognition domain, and C-type lectins with other functional domains have been identified in insects (36) and shrimp (7, 8). In the C-type lectins with other functional domains group, in addition to the carbohydrate-recognition domain, other functional domains, such as complement control protein modules, the epidermal growth factor-like domain, the extracellular domain, the discoidin domain family, the chitin-binding domain, and the Ig domain, have been identified (39). These domains, other than the classical C-type lectin–like domain, may confer additional immune functions on C-type lectins, allowing them to participate more widely in different host immune responses.

Adaptive immunity, which has long been thought to be restricted to vertebrates, is largely dependent on Igs of the Ig superfamily. Generally, Ig superfamily proteins are encountered more frequently in vertebrates than invertebrates (10). However, some invertebrate proteins with Ig-like domains are essential in host defense (11). Several studies have described Ig-containing C-type lectins, which are members of the C-type lectin superfamily. Lecticans, which are lectins with an Ig domain and one or more epidermal growth factor domains, are involved in extracellular matrix development and organization by binding to hyaluronan or chondroitin sulfate (12, 13). Another C-type lectin with several Ig and fibronectin type 3 domains has been identified in Drosophila. The BSCLT protein in Botryllus schlosseri has a C-type lectin–like domain and an Ig domain at its N and C termini, respectively (14). In contrast, the BgSel protein and C-type lectin–related proteins from Biomphalaria glabrata have an N-terminal Ig domain and a C-terminal C-type lectin–like domain (15). However, the role of Ig domain immune functions in invertebrates remains largely unclear.

Invertebrates are the most diverse species of animal on the planet and live in adverse environments with many pathogenic microorganisms (16). During their long-term coevolution with pathogens, invertebrates have developed an effective innate immune system to compensate for the lack of a lymphocyte-mediated adaptive immune system, to defend against microorganisms (17), in which large numbers of C-type lectins play a crucial role (18). Aquatic animals in direct and continual contact with the water environment are usually stressed by the changes in multiple factors of the rearing water, particularly that of bacterioplankton. In an environment with frequent pathogen stress, efficient hemocyte immunity and healthy intestinal microbiota are especially important for aquatic animals to prevent disease resulting from the interplay among the host, environment, and microbes. For hemocyte immunity, C-type lectins have multiple functions as pathogen receptors, which include regulating melanization, respiratory burst, agglutination, phagocytosis, antimicrobial activity, and antiviral responses (9, 19, 20). For intestinal immunity, C-type lectins are especially important for regulating commensal microbiota. For example, the mammalian intestinal C-type lectin, RegIIIγ, prevents infection by maintaining the spatial segregation of bacteria and the intestinal epithelial surface under homeostatic conditions (21). In mosquitoes, C-type lectins coat and help intestinal microbiota to evade antimicrobial peptide deposition and antimicrobial peptide–mediated elimination (22); in crustaceans, they also mediate biofilm formation by intestinal bacteria (23). These studies demonstrated the important and pleiotropic roles of C-type lectins in regulating vertebrate and invertebrate immunity.

In this study, we found that in Chinese mitten crab an Eriocheir sinensis (H. Milne-Edwards) (Decapoda: Grapsidae) Ig domain–containing C-type lectin, designated EsIgLectin, expressed by both hemocytes and intestine, was required for bacterial binding, bacterial agglutination, bacterial growth inhibition, and in vivo bacterial clearance and was essential for bacterial phagocytosis by hemocytes, controlled microbial homeostasis, and protection against Vibrio parahaemolyticus–induced inflammation in the intestine. Moreover, the polymeric Ig receptor (EspIgR) was essential for EsIgLectin-promoted hemocyte phagocytosis through protein–protein interactions. Surprisingly, hemocyte-derived EsIgLectin could execute transcytosis into the intestine at the later phase of intestinal inflammation, thus establishing the linkage between the intestine and hemocytes. The EsIgLectin therefore regulated hemocyte immunity and intestinal homeostasis, to promote bacterial clearance and protect against inflammatory disease.

Healthy Chinese mitten crabs (mean body weight, 26–30 g each) were obtained from a local aquatic farm (Shanghai, China). After quick transfer to our laboratory, the crabs were maintained in filtered, aerated freshwater and fed daily with a commercially formulated antibiotic-free diet. To assess the healthy status of crabs that were used in this study, we conducted both behavioral observation and bacterial detection with previously described methods (24) with slight modifications. For behavioral observation, the healthy crabs showed an aggressive manner and high activity. For bacterial detection, 5% of the total experimental crabs were randomly checked by PCR using 16S rDNA Bacterial Identification PCR Kit (Takara, Japan) to ensure that the crabs were free of Staphylococcus aureus and V. parahaemolyticus that were used for stimulation in this study.

E. sinensis hemolymph was collected from the nonsclerotized membrane of the posterior walking leg using a 1-ml sterile syringe preloaded with 0.5 ml precooled sterile anticoagulant (0.14 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM EDTA [pH 4.6]) at a ratio of 1:1. The collected hemolymph was immediately centrifuged at 300 × g for 10 min at 4°C. Then the serum was removed and hemocytes were washed with PBS.

We searched the EsIgLectin, EspIgR, and other related cDNA and amino acid sequences of E. sinensis and other species using GenBank (https://www.ncbi.nlm.nih.gov/genbank/), and aligned them using the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The potential functional domains of the amino acids were predicted using the Simple Modular Architecture Research Tool (http://smart.embl.de/), and the organization of domains was displayed using Adobe Illustrator (version 2018; https://www.adobe.com/products/illustrator.html). Multiple sequence alignment was performed with ClustalW Multiple Alignment (http://www.clustal.org/). A phylogenetic tree was constructed using the neighbor-joining method with Molecular Evolutionary Genetics Analysis (MEGA, version 6; https://www.megasoftware.net/).

S. aureus and V. parahaemolyticus were obtained from the National Pathogen Collection Center for Aquatic Animals (Shanghai Ocean University, Shanghai, China) and cultured in Luria–Bertani (LB) medium (1% NaCl, 1% tryptone, 0.5% yeast extract) overnight at 37°C, collected by centrifugation at 6000 × g, and resuspended in sterile PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 [pH 7.4]). Bacterial counts were determined using agar plating.

Abdominal injection was performed in accordance with previously described methods (25) with slight modifications. Briefly, 120 crabs were randomly divided into three groups, and 100 μl of S. aureus and V. parahaemolyticus solution (1 × 106 CFUs per crab) was injected into the hemolymph from the nonsclerotized membrane of the posterior walking leg but at a different site from that used for hemolymph collection; sterile PBS (100 μl) was used in the control. Afterward, the crabs were maintained in freshwater for the study. Six crabs were used to prepare each sample from each group at specific time points (0, 2, 6, 12, and 24 h) poststimulation. The data are representative of three independent experiments. As for the oral injection experiment, 120 crabs were randomly divided into three groups. The experiment was performed by delivering 200 µl of S. aureus and V. parahaemolyticus solution (1 × 108 CFUs per crab) into the crab oral cavity using a sterile flexible microsyringe. The control animals were treated orally with an equal volume of sterile PBS. Afterward, the crabs were maintained in freshwater for the study. Six crabs were used to prepare each sample from each group at specific time points (0, 2, 6, 12, and 24 h) poststimulation. The data are representative of three independent experiments. Total RNA was extracted using TRIzol Reagent (catalog number: 15596018; Invitrogen, Carlsbad, CA) and used to synthesize first-strand cDNA using a PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (catalog number: RR047A; Takara, Japan) according to the manufacturer’s instructions.

The relative gene expression levels of EsIgLectin/EspIgR/AMPs in the hemocytes and intestine of crab that received bacterial oral injection and EsIgLectin/EspIgR in the hemocytes and intestine of crab that received bacterial abdominal injection were determined by quantitative real-time PCR using the CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA) and ChamQ Universal SYBR qPCR Master Mix (catalog number: Q711-02; Vazyme, Nanjing, China) with gene-specific primers (Table I) and the following reaction conditions: 94°C for 3 min, followed by 40 cycles at 94°C for 10 s and 60°C for 1 min, and then melting from 65°C to 95°C. β-Actin was used as the internal reference. The obtained data were normalized to the control samples using the comparative threshold cycle (2−ΔΔCT) method. The results are expressed as the mean ± SD of three independent experiments.

Table I.

Primers used in this study

PrimerSequence (5′–3′)
qRT-PCR  
EsIgLectin-RT-F 5′-GCGGCGTATGGTGAGAATGA-3′ 
EsIgLectin-RT-R 5′-TGAAGTGCATCTCTTTGGTGCA-3′ 
EspIgR-RT-F 5′-GCAAAGTTTTCGTGGTCGG-3′ 
EspIgR-RT-R 5′-GAGGCTGTGATGTCGGTGTC-3′ 
Esβ-actin-RT-F 5′-GCATCCACGAGACCACTTACA-3′ 
Esβ-actin-RT-R 5′-CTCCTGCTTGCTGATCCACATC-3′ 
RNAi  
dsEsIgLectin-T7-F 5′-GGATCCTAATACGACTCACTATAGGCT CCGCTGCACCTCGACTG-3′ 
dsEsIgLectin-R 5′-CTCACCATACGCCGCCAAG-3′ 
dsEsIgLectin-F 5′-CTCCGCTGCACCTCGACTG-3′ 
dsEsIgLectin-T7-R 5′-GGATCCTAATACGACTCACTATAGGCT CACCATACGCCGCCAAG-3′ 
dsEspIgR-T7-F 5′-GGATCCTAATACGACTCACTATAGGCA CCCGCTAATACCTCCG-3′ 
dsEspIgR-R 5′-GTCATGGTTGCTTGTTTCAGA-3′ 
dsEspIgR-F 5′-CACCCGCTAATACCTCCG-3′ 
dsEspIgR-T7-R 5′-GGATCCTAATACGACTCACTATAGGGTC ATGGTTGCTTGTTTCAGA-3′ 
dsGFP-T7-F 5′-GGATCCTAATACGACTCACTATAGGCGA CGTAAACGGCCACAAGTT-3′ 
dsGFP-R 5′-ATGGGGGTGTTCTGCTGGTAG-3′ 
dsGFP-F 5′-CGACGTAAACGGCCACAAGTT-3′ 
dsGFP-T7-R 5′-GGATCCTAATACGACTCACTATAGGATG GGGGTGTTCTGCTGGTAG-3′ 
Recombinant expression  
EspIgR-SC-ex-F 5′-CCGGAATTCCATAACCAGATAGTCGTG-3′ 
EspIgR-SC-ex-R 5′-CCGCTCGAGCTTGACTCTGTACCGAGT-3′ 
EspIgR-Ig1-ex-F 5′-CCGGAATTCCATAACCAGATAGTCGTG-3′ 
EspIgR-Ig1-ex-R 5′-CCGCTCGAGTTTGATTGTGACTACCAC-3′ 
EspIgR-Ig2-ex-F 5′-CCGGAATTCGAGCGGCCCGTGGTGTGC-3′ 
EspIgR-Ig2-ex-R 5′-CCGCTCGAGGGGTGCGGAAAGTGTCTG-3′ 
EspIgR-Ig3-ex-F 5′-CCGGAATTCACAACATTCTACCAAATC-3′ 
EspIgR-Ig3-ex-F 5′-CCGCTCGAGCTTGACTCTGTACCGAGT-3′ 
FISH  
EsIgLectin probe 5′-FITC-AGUGCAUCUCUUUGGUGCAGU-3′ 
PrimerSequence (5′–3′)
qRT-PCR  
EsIgLectin-RT-F 5′-GCGGCGTATGGTGAGAATGA-3′ 
EsIgLectin-RT-R 5′-TGAAGTGCATCTCTTTGGTGCA-3′ 
EspIgR-RT-F 5′-GCAAAGTTTTCGTGGTCGG-3′ 
EspIgR-RT-R 5′-GAGGCTGTGATGTCGGTGTC-3′ 
Esβ-actin-RT-F 5′-GCATCCACGAGACCACTTACA-3′ 
Esβ-actin-RT-R 5′-CTCCTGCTTGCTGATCCACATC-3′ 
RNAi  
dsEsIgLectin-T7-F 5′-GGATCCTAATACGACTCACTATAGGCT CCGCTGCACCTCGACTG-3′ 
dsEsIgLectin-R 5′-CTCACCATACGCCGCCAAG-3′ 
dsEsIgLectin-F 5′-CTCCGCTGCACCTCGACTG-3′ 
dsEsIgLectin-T7-R 5′-GGATCCTAATACGACTCACTATAGGCT CACCATACGCCGCCAAG-3′ 
dsEspIgR-T7-F 5′-GGATCCTAATACGACTCACTATAGGCA CCCGCTAATACCTCCG-3′ 
dsEspIgR-R 5′-GTCATGGTTGCTTGTTTCAGA-3′ 
dsEspIgR-F 5′-CACCCGCTAATACCTCCG-3′ 
dsEspIgR-T7-R 5′-GGATCCTAATACGACTCACTATAGGGTC ATGGTTGCTTGTTTCAGA-3′ 
dsGFP-T7-F 5′-GGATCCTAATACGACTCACTATAGGCGA CGTAAACGGCCACAAGTT-3′ 
dsGFP-R 5′-ATGGGGGTGTTCTGCTGGTAG-3′ 
dsGFP-F 5′-CGACGTAAACGGCCACAAGTT-3′ 
dsGFP-T7-R 5′-GGATCCTAATACGACTCACTATAGGATG GGGGTGTTCTGCTGGTAG-3′ 
Recombinant expression  
EspIgR-SC-ex-F 5′-CCGGAATTCCATAACCAGATAGTCGTG-3′ 
EspIgR-SC-ex-R 5′-CCGCTCGAGCTTGACTCTGTACCGAGT-3′ 
EspIgR-Ig1-ex-F 5′-CCGGAATTCCATAACCAGATAGTCGTG-3′ 
EspIgR-Ig1-ex-R 5′-CCGCTCGAGTTTGATTGTGACTACCAC-3′ 
EspIgR-Ig2-ex-F 5′-CCGGAATTCGAGCGGCCCGTGGTGTGC-3′ 
EspIgR-Ig2-ex-R 5′-CCGCTCGAGGGGTGCGGAAAGTGTCTG-3′ 
EspIgR-Ig3-ex-F 5′-CCGGAATTCACAACATTCTACCAAATC-3′ 
EspIgR-Ig3-ex-F 5′-CCGCTCGAGCTTGACTCTGTACCGAGT-3′ 
FISH  
EsIgLectin probe 5′-FITC-AGUGCAUCUCUUUGGUGCAGU-3′ 

The amino acid sequence of EsIgLectin mature peptide, EsIg, EsCTLD and mutated EsIgLectin mature peptide, EsIg, EsCTLD were optimized by the newest codon optimization software MaxCodon Optimization Program (V13) from Merrybio (Nanjing, China). All genes were synthesized and inserted into expression vector pET30a (+) through restriction enzyme digestion sites NdeI and HindIII. The accuracy of the final expression vector was confirmed by enzyme digestion and sequencing. Finally, the vector was transformed into Escherichia coli BL21(DE3) Chemically Competent Cell [E. coli BL21(DE3)] (catalog number: CD601-03; TransGen Biotech, Beijing, China). Proteins with His-tag were induced by 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 6 h, and then proteins were purified by High Affinity Ni-NTA Resin (catalog number: L00250-50; GenScript, Nanjing, China) according to the manufacturer’s instructions. To remove most of the endotoxin contamination, we used 10 column volumes of cold 0.1% Triton X-114 (Sangon Biotech, Shanghai, China) to fully wash the column before final elution of proteins, and the endotoxin level in the protein was <3 endotoxin units/mg; thus, the endotoxin levels did not have an impact on the experiments (26). To prepare the anti-EsIgLectin Ab, we thoroughly mixed 1 ml of purified rEsIgLectin (2 mg/ml) with an equal volume of CFA (catalog number: F5881; Sigma-Aldrich, St. Louis, MO) to immunize the New Zealand white rabbit. Immunization was repeated 21 d later with incomplete adjuvant (catalog number: F5506; Sigma-Aldrich), and this process was repeated three times. After testing the titer of the antiserum at day 7 after last immunization, the rabbit was sacrificed to obtain the antiserum. Subsequently, the Ab was purified by using protein A resin column (L00210; GenScript, Nanjing, China). To evaluate the specificity of the Ab, we used Western blotting to detect the EsIgLectin protein in the crab hemocytes through anti-EsIgLectin, and only a single band at the known mass (26 kDa) for the target was found.

The specific primers EspIgR-ex-F and EspIgR-ex-R (Table I) were used to amplify the extracellular fragment of EspIgR (SC). The PCR procedure was as follows: 94°C for 3 min; 35 cycles at 94°C for 15 s, 60°C for 15 s, and 72°C for 1 min; and one cycle at 72°C for 5 min. The PCR fragments were digested with restriction enzymes EcoRI and XhoI and then ligated into the pGEX-4T-1 vector (GE Healthcare, Piscataway, NJ). The recombinant plasmid was transformed into E. coli BL21(DE3). GST-tagged EspIgR-SC recombinant expression was induced with 0.5 mM IPTG at 37°C for 6 h. The recombinant protein was expressed in the inclusion bodies and purified using an affinity chromatography with Glutathione Resin (catalog number: L00206-50; GenScript, Nanjing, China) according to the manufacturer’s instructions. Most of the endotoxin was removed according to the method described earlier. The three Ig domains (i.e., Ig1 [first Ig domain], Ig2, Ig3) of EspIgR were also expressed and purified in the same way.

A total of 300 μg of purified anti-EsIgLectin Ab was coupled with 150 mg of CNBr-activated Sepharose 4B (catalog number: 170430; Cytiva) with gentle rotation at 25°C for 1 h. The resin was washed five times with 1 ml of coupling buffer (0.1 M NaHCO3, 0.5 M NaCl [pH 8.3]), then equilibrated in 0.1 M Tris–HCl (pH 8.0) at 25°C for 2 h. After four times of alternate washing with acetic acid buffer (0.1 M sodium acetate, 0.5 M NaCl [pH 4.0]) and Tris buffer (0.1 M Tris–HCl, 0.5 M NaCl [pH 8.0]), the resin was equilibrated in 0.1 M Tris–HCl (pH 8.0). The plasma (3 ml) from crabs that received abdominal injection with PBS, S. aureus, or V. parahaemolyticus at specific time points (0, 2, 6, 12, and 24 h) was then mixed with the resin and incubated at 4°C overnight with gentle rotation. The resin was washed five times with 0.1 M Tris–HCl (pH 8.0) and eluted with 0.1 M glycine (pH 2.5). The elution was immediately neutralized with 1 M Tris–HCl (pH 8.5). The purified nature of EsIgLectin was detected by Western blotting.

Protein samples from crab hemolymph, hemocytes, and intestine were quantified with Pierce Coomassie Plus (Bradford) Assay Reagent (Thermo Fisher Scientific). The cells were lysed using radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Roche Applied Science, Penzburg, Germany). Protein concentrations were quantified using a Pierce BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific). The proteins were then separated by 10% SDS-PAGE and transferred to an Immobilon-FL polyvinylidene difluoride membrane (catalog number: IPFL00005; Merck Millipore, Burlington, MA), which was blocked for 1–2 h with BSA Blocking Buffer (catalog number: CW0054; Cwbio, China); the membrane was then incubated overnight with the rabbit anti-EsIgLectin (1:15,000) in TBST at 4°C. After three washes with TBST, the membrane was incubated with goat anti-rabbit IgG (H+L) (DyLight 800 4 × PEG Conjugate) (catalog number: 5151S; 1:30,000; Cell Signaling Technology, Danvers, MA) and analyzed on an Odyssey CLx Imaging System (LI-COR Biosciences, Lincoln, NE) according to the manufacturer’s instructions.

The spatial distribution of intestinal EsIgLectin mRNA was evaluated using fluorescence in situ hybridization (FISH). EsIgLectin-specific primers (Table I) labeled with FITC were synthesized by Sangon Biotech (Shanghai, China) and used for hybridization. Briefly, removed and washed intestine tissues were immediately put into the fixative (diethyl pyrocarbonate water preparation) for 6 h, dehydrated with gradient alcohol, and then waxed and embedded. Paraffin sections were prepared using a microtome (RM2235; Leica, Germany) and dried at 62°C for 2 h; paraffin sections were dewaxed to water with the following procedures: xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and diethyl pyrocarbonate washing. The slices were then boiled in the repair solution for 12 min and cooled naturally, digested with protease K (20 μg/ml) at 37°C for 30 min, and washed with pure water and PBS three times. Prehybrid solution was added and incubated at 37°C for 1 h; then the prehybridization solution was dumped, and the probe was dropped into the hybridization solution (8 ng/μl) and incubated at 37°C for overnight hybridization. After that, slides were washed with saline sodium citrate, and DAPI dye solution was dropwise added to sections, followed by incubation in the dark for 8 min and mounted by a drop of antifluorescent quenching sealer. The sections were examined, and images were captured using laser confocal microscopy (Leica TCS SP8).

Studies of microorganisms binding activity detection were performed as previously described with slight modifications (27). Briefly, Gram-positive bacteria (Bacillus subtilis, Streptococcus agalactiae, S. aureus) and Gram-negative bacteria (Vibrio harveyi, Vibrio anguillarum, V. parahaemolyticus, Vibrio vulnificus, and Aeromonas hydrophila) were cultured overnight at 37°C with rotation in LB medium. The microorganisms were collected by centrifugation at 6000 × g for 3 min, washed by 1 × TBS (Beyotime Institute of Biotechnology, Shanghai, China) thrice, and resuspended in 500 μl of 1 × TBS. Then the microorganisms (1 × 107 CFUs) were incubated with 10 μg recombinant proteins (i.e., rEsIgLectin, rEsIg, rEsCTLD, or rEsCTLDΔQPD) for 1 h with gentle shaking at 28°C. After 5 min at 6000 × g centrifugation, the microorganisms were collected and washed five times with TBS, and 100 μl of 7% SDS was used to elute the binding protein for 10 min. After centrifugation at 6000 × g for 5 min, the elution solution and the bacterial precipitate were collected and analyzed using 12% SDS-PAGE. The proteins in the gel were transferred to an Immobilon-FL polyvinylidene difluoride membrane for Western blot analysis using an anti–His-tag mouse mAb (catalog number: CW0286; Cwbio) and a goat anti-mouse IgG (H+L) (DyLight 680 Conjugate) (catalog number: 5470S; 1:15,000; Cell Signaling Technology, Danvers, MA) as the primary and secondary Abs. The recombinant proteins (i.e., rEsIgLectin, rEsIg, rEsCTLD, or rEsCTLDΔQPD) were also subjected to Western blot analysis as a positive control.

S. aureus and V. parahaemolyticus were cultured in LB medium to the logarithmic phase and harvested by centrifugation at 6000 × g for 5 min. The collected bacteria were heat inactivated at 72°C for 30 min, washed with 0.1 M NaHCO3 (pH 9.0), and labeled with FITC (Sigma) at a final concentration of 1 mg/ml at 28°C for 1 h. The FITC-labeled bacteria were washed with PBS three times to eliminate free FITC and then resuspended in PBS to a final OD600 of 0.5. A 50-μl microbe suspension was mixed with 50 μl recombinant proteins (i.e., rEsIgLectin, rEsIg, rEsCTLD, or rEsCTLDΔQPD) (1 mg/ml) in the presence or absence of 10 mM CaCl2. The mixtures were incubated at 28°C for 1 h. BSA instead of recombinant proteins (i.e., rEsIgLectin, rEsIg, rEsCTLD) (1 mg/ml) was used as a negative control. After incubation, agglutinating reactions were observed under an Echo Revolve Hybrid Microscope (Echo Laboratories, San Diego, CA).

The binding activity of recombinant proteins (i.e., rEsIgLectin, rEsIg, rEsCTLD) to LPS from E. coli and peptidoglycan from S. aureus or B. subtilis was analyzed using an ELISA. Briefly, polysaccharides (80 μg/ml; 50 μl) were added to wells of a 96-well plate and incubated at 37°C overnight, followed by incubation at 60°C for 30 min. After blocking with 200 μl of BSA (1 mg/ml dissolved in TBS) at 37°C for 2 h, each recombinant protein was then added into the wells with serial doses ranging from 0.1 to 100 μM, and the mixture was maintained at 28°C for 3 h. After TBS washing, 100 μl of the anti-His Ab (1:2000 diluted in 0.1 mg/ml BSA) was added to the wells and incubated at 4°C overnight. After washing three times with TBST, the wells were incubated with 1:5000 diluted HRP-conjugated goat anti-rabbit Abs for 1 h at 37°C, and 0.01% 3,3′,5,5′-tetramethylbenzidine (Sigma) was added to determine bound HRP activity after washing. The reaction was terminated by adding 2 M H2SO4, and the absorbance at 450 nm was read by a microplate reader (Bio-Rad).

The antimicrobial activity of the recombinant proteins (i.e., rEsIgLectin, rEsIg, rEsCTLD, rEsIgΔCys42, 102 [EsIgLectin position 42 and 102 cysteine residue–deleted Ig domain recombinant protein], rEsCTLDΔQPD) against S. aureus and V. parahaemolyticus was examined using LB agar plates. The different bacteria strains were cultured in LB medium, collected by 10-min centrifugation (6000 × g), and suspended in PBS. The prepared microbes were smeared on 100 mm × 20 mm LB agar plates. Then sterile filter paper was attached to the plates by using tweezers. Equal concentrations of rEsIgLectin, rEsIg, or rEsCTLD protein (1 µg/μl, 20 μl) were added to the sterile filter paper. Then the plates were cultured at 37°C until a bacteriostatic ring appeared. The controls were ampicillin (2 μg/μl, 20 μl), kanamycin sulfate (2 μg/μl, 20 μl), and 20 μl PBS.

Bacterial clearance assays were performed according to a previously reported method with slight modifications (24). Bacterial suspensions (S. aureus and V. parahaemolyticus) were cultured overnight in LB medium at 37°C and adjusted to OD600 = 0.4. The recombinant proteins (i.e., rEsIgLectin, rEsIg, rEsCTLD, rEsIgLectinΔCys42, 102 [full-length EsIgLectin mutated protein], rEsIgΔCys42, 102, and rEsCTLDΔQPD) (50 µg) were mixed with 500 µl of the suspensions (5 × 107 CFUs) and incubated under gentle rotation at 28°C for 1 h. After thorough washing with PBS, the bacteria were collected by centrifugation at 6000 × g for 5 min and resuspended in 50 µl PBS, followed by abdominal injection into each crab as described earlier, and the hemolymph was collected at 30 min postinjection. After serial dilution, the number of residual bacteria was determined by plating the samples onto LB agar plates. The controls were incubated with bacteria with PBS or BSA. For the clearance test, three crabs were used for each group, and the data are representative of three independent experiments.

A cross-linking assay was performed to detect oligomerization in vitro by the chemical cross-linker, suberic acid bis (3-sulfo-N-hydroxysuccinimide ester) sodium salt (BS3, catalog number: S5799; Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, BS3 was added to the recombinant proteins (i.e., rEsIgLectin, rEsIg, rEsCTLD) (1 mg/ml) or native EsIgLectin that was purified from the plasma (3 ml) from abdominal injection with V. parahaemolyticus (12 h) with a final concentration of 5 mM, and the mixture was incubated at room temperature for 1 h. After incubation, SDS-PAGE sample loading buffer was added for reaction termination and treated in a boiling water bath for 5 min followed by SDS-PAGE or Western blotting.

The binding capacity of recombinant proteins (i.e., rEsIgLectin, rEsIg, rEsCTLD) (1 mg/ml) to hemocytes was investigated in vivo by injecting recombinant proteins into crabs (10 µg/g). Hemolymph was collected at 30 min postinjection, and the hemocytes were isolated according to the method described earlier. The hemocytes were fixed with 4% paraformaldehyde in PBS for 10 min. The fixed hemocytes (50 µl) were dripped onto poly-l-lysine–coated glass slides and allowed to settle for 30 min at room temperature. After blocking with 5% BSA in PBS for 30 min, an anti–His-tag mouse mAb (catalog number: CW0286; 1:1000; Cwbio) was added and incubated for 8 h at 4°C, and the slides were washed six times with PBS. After blocking with 5% BSA in PBS for 5 min, FITC-conjugated goat anti-mouse IgG (H+L) (catalog number: F0257; 1:1000; Sigma-Aldrich) was added to the glass slide, incubated for 1 h at 37°C in the dark, and washed six times with PBS for 10 min each. Then hemocytes were incubated with DAPI (catalog number: C1005; Beyotime Institute of Biotechnology) for 10 min and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (catalog number: C1036; Beyotime Institute of Biotechnology) (1:500 dilution in PBS) for 20 min to stain nuclei and membranes, washed again six times with PBS for 10 min, and images were obtained by laser confocal microscopy (Leica TCS SP8). Three crabs were used for each group. Immunocytochemical images are representative of three independent experiments.

Overnight-cultured S. aureus and V. parahaemolyticus were heat killed and conjugated to FITC (Sigma-Aldrich). The bacterial suspension (1 × 108 microbes/ml, 1 ml) in PBS was mixed and incubated with 500 μg of recombinant proteins by gentle rotation for 1 h at 28°C to ensure full coating. The bacteria were pelleted and washed three times with PBS by centrifugation. Then the bacterial suspension (100 μl) was injected into the crab hemolymph at one of the posterior walking legs. After 30 min, the hemocytes (6 × 105 cells) were isolated from the other posterior walking legs and centrifuged at 300 × g for 10 min at 4°C and washed with PBS three times. Extracellular fluorescence was quenched by adding 1 μl of 0.5% trypan blue to the cell suspension (28). Next, the phagocytosis rate in 1 ml of each sample was determined by flow cytometry using a CytoFLEX apparatus (Beckman Coulter, Indianapolis, IN), and the data were analyzed using CytExpert software. A total of 10,000 hemocytes from three crabs was counted for each sample. The experiments were repeated three times.

V. parahaemolyticus was used to establish an intestinal inflammation model through oral injection. A bacterial suspension (200 μl) (1 × 108 CFUs per crab) was delivered into the crab oral cavity using a sterile flexible microsyringe, daily over the course of 12 d in total. Every day at 12 h after bacterial injection, the crab intestines were collected, washed with PBS, and then used for morphological detection and histological analysis by H&E staining under standard procedures. Briefly, the intestine was fixed in Bouin’s solution for 24 h, dehydrated in a graded series of ethyl alcohol, and then waxed and embedded. Paraffin sections were prepared using a microtome (RM2235; Leica). The thickness of the paraffin section was 4 μm. Then the paraffin sections were stained with H&E and observed with a light microscope (model BX51; Olympus, Tokyo, Japan). In addition, the antimicrobial peptide expression was analyzed by quantitative RT-PCR (qRT-PCR). For studying EsIgLectin rescue of bacteria-induced intestinal inflammation, crabs were orally infected with 200 μl bacterial suspension (1 × 108 CFUs per crab) daily for a total of 12 d, while rEsIgLectin, rEsIg, and rEsCTLD were orally injected into the crabs daily from days 6 to 12. Every day at 12 h after recombinant protein injection, the crab intestines were collected, washed with PBS, and then used for morphological detection and histological analysis by H&E staining as described earlier and analysis of antimicrobial peptide expression on day 7 of the intestinal inflammation model with or without rEsIgLectin injection on days 6–12. The midgut of the intestine was used for morphological detection and histological analysis.

Total microbial DNA was extracted from crab intestine using a QIAamp PowerFecal Pro DNA Kit (catalog number: 51804; QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. At least 24 animals were used to prepare each sample. The DNA quality and quantity were verified using agarose gel electrophoresis and a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA). The hypervariable region of the 16S rRNA gene was amplified using the universal primers 343F and 798R. The sequencing libraries were built using a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA) and sequenced using the Illumina HiSeq 2500 platform by Oebiotech (Shanghai, China). Raw sequences were demultiplexed, quality-filtered by Trimmomatic, and merged by Fast Length Adjustment of Short Reads (http://ccb.jhu.edu/software/FLASH/). Sequences were considered the same operational taxonomic unit if their similarities were >97%. Taxonomy was determined using the Ribosomal Database Project Classifier algorithm (http://rdp.cme.msu.edu/) with a confidence threshold of 80% and the GreenGene database (http://greengenes.lbl.gov/download) as the reference database. The α-diversity to show the complexity of species in a sample was determined using Quantitative Insights into Microbial Ecology and displayed using R software (https://www.r-project.org/).

Panning was performed according to our published method with slight modifications (29). Briefly, 40 μg purified EsIg protein was added to the wells of a 96-well plate and incubated at 4°C overnight. After the unbound protein had been removed by PBS washing, 10 μl T7 phage display library derived from E. sinensis hemocytes was added to the wells for incubation. After washing three times with TBST, the rEsIgLectin-bound phages were eluted using 200 μl 1% SDS and then centrifuged at 6000 × g for 5 min; then the supernatant (10 μl) was inoculated into 1 ml of BLT5403 cells and cultured at 37°C for 2 h. After centrifugation, the supernatant was plated on an agarose plate containing BLT5403 cells and cultured at 37°C for 3 h. Extraction buffer (100 mM NaCl, 6 mM MgSO4, 20 mM Tris–HCl [pH 8.0]) was added to the wells, incubated at 4°C overnight, and then collected for the next panning. After three repeats, single plaques appeared, and the 15 longest fragments were sequenced.

Pull-down assays were performed to determine the interaction between EsIgLectin and EspIgR proteins. Briefly, purified GST-tagged proteins (EspIgR-SC, EspIgR-Ig1, EspIgR-Ig2, EspIgR-Ig3) (200 μg) were incubated with His-tagged proteins (EsIgLectin, EsIg, EsCTLD) (1:1) separately at 4°C overnight. Next, 200 μl of glutathione resin was incubated with the protein mixture to bind GST-tagged proteins for 1 h at 4°C. After washing six times with PBS. Elution buffer (10 mM reduced glutathione, 50 mM Tris–HCl [pH 8.0]) was added to wash out the bound proteins and then was analyzed by 12% SDS-PAGE.

For the in vivo RNA interference (RNAi) experiments, T7 RiboMAX Express RNAi System (catalog number: P1700; Promega, Madison, WI) was used to generate dsRNA against EsIgLectin (dsEsIgLectin), dsRNA against EspIgR (dsEspIgR), and dsGFP (control) with primers containing a 5′ T7 RNA polymerase binding site (Table I). The dsRNA quality was checked after annealing by gel electrophoresis. The dsRNA was injected into crabs at 5 μg of dsRNA per gram of body weight with a microliter syringe through the arthrodial membrane of the fifth walking leg, which in this study was designated on the ipsilateral side of the crab. The volume of the dsRNA inoculum was adjusted to 50 μl. The RNAi efficiency was determined using qRT-PCR at 24 h after dsRNA injection. Three crabs were used to prepare each sample, and the data are representative of three independent experiments.

The crabs were randomly divided into two groups, each containing 20 animals. The crabs were injected with the target gene dsRNA or dsGFP control, respectively. After EsIgLectin or EspIgR was knocked down by dsRNA injection, all crabs were injected with S. aureus or V. parahaemolyticus (1 × 106 CFUs per crab, 100 μl) with or without 5 or 10 μg of recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD) either abdominally or orally. Dead crabs were counted daily in each group, and survival was tabulated for 6 d.

Most data in this study were analyzed using Student t test, and significance was accepted at p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). For the tissue distribution assay, the results were analyzed using one-way ANOVA, followed by Turkey multiple comparison test using GraphPad Prism software (GraphPad Software, La Jolla, CA). For the survival assay, data were analyzed using the log rank (Mantel-Cox) test using GraphPad Prism.

The full-length EsIgLectin open reading frame cDNA was cloned from E. sinensis hemocytes (GenBank accession number: OK020090, https://www.ncbi.nlm.nih.gov/nuccore/OK020090). The EsIgLectin protein contained a conserved C-type lectin–like domain at the C terminus, but also a novel Ig domain at the N terminus, with the signature QPD and FND motifs located in the C-type lectin–like domain (Supplemental Fig. 1). EsIgLectin was expressed in all tested crab tissues, including hemocytes and the intestine (Fig. 1A), which are important immune-related organs in invertebrates. To verify this result, we constructed three recombinant proteins, i.e., the recombinant Ig domain (rEsIg), C-type lectin–like domain (rEsCTLD), and full-length EsIgLectin (rEsIgLectin) (Fig. 1B), and synthesized pAb against EsIgLectin (Fig. 1C). Western blotting showed that the selected organs expressed EsIgLectin protein (Fig. 1D), which agreed with the results at the mRNA level (Fig. 1A).

FIGURE 1.

EsIgLectin transcription profile on bacterial infection. (A) qRT-PCR detection of the tissue distribution of EsIgLectin mRNA with β-actin as the reference gene. Six crabs were used to prepare each sample, and the data are representative of three independent experiments. (B) Recombinant protein construction of EsIgLectin, Ig, and C-type lectin–like domain. Constructs pET30a (+)-EsIg, -EsCTLD, or -EsIgLectin were transformed into E. coli, and the expression of proteins was induced by IPTG and analyzed by SDS-PAGE. Lane M, protein markers; lane 1, proteins from E. coli with pET30a (+)-EsIg, -EsCTLD, or -EsIgLectin without IPTG induction; lane 2, proteins from E. coli with pET30a (+)-EsIg, -EsCTLD, or -EsIgLectin with 0.5 mM IPTG induction for 6 h at 37°C; lane 3, the supernatant of the E. coli with treatment by ultrasound; lane 4, the precipitation of the E. coli with treatment by ultrasound; lane 5, purified recombinant protein pET30a (+)-EsIg, -EsCTLD, or -EsIgLectin. (C) Synthesized polyclonal Ab against EsIgLectin. (D) Western blot detection of the tissue distribution of EsIgLectin with β-actin detected as the reference. Total protein samples were extracted from the tissues of healthy E. sinensis. Six crabs were pooled together as one sample, and the data are representative of three independent repeats. (E and F) EsIgLectin expression patterns in hemocytes (F) of crabs that received abdominal injections of bacteria (E) or PBS; the control was untreated crab hemocytes. The expression level was normalized to that in untreated crab. Six crabs were used to prepare each sample from each group at specific time points (0, 2, 6, 12, and 24 h) poststimulation. The data are representative of three independent experiments. (G and H) Illustration of procedure for native EsIgLectin purification. EsIgLectin expression patterns in the hemolymphs of crabs that received EsIgLectin abdominal injections with bacteria (G) as detected by Western blotting with EsIgLectin polyclonal Abs. The data are representative of three independent experiments (H). (I) FISH detection of the expressional localization of EsIgLectin in crab intestines. EsIgLectin probe was used to hybridize with the EsIgLectin, and DAPI was used to visualize cell nuclei. Data shown are representative of two independent experiments. Scale bars, 300 μm (left), 150 μm (middle), or 115 μm (right). (J) Schematic model of the bacterial oral injection method and detection of the bacterial FITC signal in the intestine. (K) EsIgLectin expression patterns in the intestine of crabs that received oral injection of bacteria or PBS; the control was untreated crab intestine. The expression level was normalized to that in untreated crab. Six crabs were used to prepare each sample from each group at specific time points (0, 2, 6, 12, and 24 h) poststimulation. The data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student t test (F and K) or lowercase letters indicating the significance by one-way ANOVA (A).

FIGURE 1.

EsIgLectin transcription profile on bacterial infection. (A) qRT-PCR detection of the tissue distribution of EsIgLectin mRNA with β-actin as the reference gene. Six crabs were used to prepare each sample, and the data are representative of three independent experiments. (B) Recombinant protein construction of EsIgLectin, Ig, and C-type lectin–like domain. Constructs pET30a (+)-EsIg, -EsCTLD, or -EsIgLectin were transformed into E. coli, and the expression of proteins was induced by IPTG and analyzed by SDS-PAGE. Lane M, protein markers; lane 1, proteins from E. coli with pET30a (+)-EsIg, -EsCTLD, or -EsIgLectin without IPTG induction; lane 2, proteins from E. coli with pET30a (+)-EsIg, -EsCTLD, or -EsIgLectin with 0.5 mM IPTG induction for 6 h at 37°C; lane 3, the supernatant of the E. coli with treatment by ultrasound; lane 4, the precipitation of the E. coli with treatment by ultrasound; lane 5, purified recombinant protein pET30a (+)-EsIg, -EsCTLD, or -EsIgLectin. (C) Synthesized polyclonal Ab against EsIgLectin. (D) Western blot detection of the tissue distribution of EsIgLectin with β-actin detected as the reference. Total protein samples were extracted from the tissues of healthy E. sinensis. Six crabs were pooled together as one sample, and the data are representative of three independent repeats. (E and F) EsIgLectin expression patterns in hemocytes (F) of crabs that received abdominal injections of bacteria (E) or PBS; the control was untreated crab hemocytes. The expression level was normalized to that in untreated crab. Six crabs were used to prepare each sample from each group at specific time points (0, 2, 6, 12, and 24 h) poststimulation. The data are representative of three independent experiments. (G and H) Illustration of procedure for native EsIgLectin purification. EsIgLectin expression patterns in the hemolymphs of crabs that received EsIgLectin abdominal injections with bacteria (G) as detected by Western blotting with EsIgLectin polyclonal Abs. The data are representative of three independent experiments (H). (I) FISH detection of the expressional localization of EsIgLectin in crab intestines. EsIgLectin probe was used to hybridize with the EsIgLectin, and DAPI was used to visualize cell nuclei. Data shown are representative of two independent experiments. Scale bars, 300 μm (left), 150 μm (middle), or 115 μm (right). (J) Schematic model of the bacterial oral injection method and detection of the bacterial FITC signal in the intestine. (K) EsIgLectin expression patterns in the intestine of crabs that received oral injection of bacteria or PBS; the control was untreated crab intestine. The expression level was normalized to that in untreated crab. Six crabs were used to prepare each sample from each group at specific time points (0, 2, 6, 12, and 24 h) poststimulation. The data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student t test (F and K) or lowercase letters indicating the significance by one-way ANOVA (A).

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To test the EsIgLectin expression pattern during bacterial infection, we established two models, i.e., abdominal injection delivering bacteria to the hemolymph (Fig. 1E) and oral injection delivering bacteria to the intestine (Fig. 1J), to emulate injury and oral infections in nature, respectively.

EsIgLectin expression was significantly induced after abdominal injection by S. aureus or V. parahaemolyticus (Fig. 1F). As a soluble protein, hemocyte-expressed EsIgLectin may secrete into the hemolymph, thereby functioning in different organs because of its open circulatory system. EsIgLectin-specific Ab-based technology (Fig. 1G) and Western blotting showed significantly induced EsIgLectin protein levels in the hemolymph after abdominal injection with S. aureus or V. parahaemolyticus (Fig. 1H), with its expression pattern almost the same as that of hemocytes (Fig. 1F), suggesting that hemocytes mainly contributed to hemolymph EsIgLectin protein levels during hemolymph infection.

To further confirm the expression of EsIgLectin in the crab intestine, we conducted a FISH assay. The results showed that EsIgLectin was expressed in crab intestinal epithelial cells (Fig. 1I), with significantly induced expression after oral injection with S. aureus or V. parahaemolyticus (Fig. 1K).

In invertebrates, antimicrobial activity is the universal function of C-type lectins through the C-type lectin–like domain (9), but whether the EsIgLectin Ig domain has the same function is unknown. We therefore tested bacterial binding, bacterial agglutination, bacterial growth inhibition, and bacterial clearance capacities of the Ig domain, C-type lectin–like domain, and full-length EsIgLectin. Both the rEsIg and rEsC-type lectin–like domain bound different bacteria in varying degrees, whereas the rEsIgLectin exhibited comparatively strong abilities to bind all investigated bacteria (Fig. 2A), which were concordant with the results of the polysaccharide binding assay (Fig. 2B). Moreover, rEsIg, rEsCTLD, and rEsIgLectin showed bacterial agglutination activities in a calcium-dependent manner, where rEsCTLD and rEsIgLectin had much stronger activities than rEsIg (Fig. 2C). Furthermore, rEsIg, rEsCTLD, and rEsIgLectin had significant bacterial growth inhibition (Fig. 2D) and in vivo bacterial clearance activity against S. aureus (Fig. 2E) and V. parahaemolyticus (Fig. 2F). Together, these results indicate that EsIgLectin has efficient antimicrobial activities that depend on both the C-type lectin–like domain and Ig domains.

FIGURE 2.

Antimicrobial activity of EsIgLectin and its domains. (A) The bacterial binding properties of EsIgLectin. Western blotting was performed to analyze the incubation of eight bacterial species (B. subtilis, S. agalactiae, S. aureus, V. harveyi, V. anguillarum, V. parahaemolyticus, Vibrio vulnificus, and A. hydrophila) with rEsIgLectin, rEsIg, and rEsCTLD. (B) Quantitative binding of EsIgLectin to pathogen-associated molecular patterns. The binding activities of rEsIgLectin, rEsIg, and rEsCTLD to LPS from E. coli and peptidoglycan from S. aureus or B. subtilis were analyzed with ELISA; PBS was used as the control. The results are presented as the mean ± SD derived from three independent repeats. (C) Microorganism agglutination assays of EsIgLectin in the absence or presence of calcium. S. aureus and V. parahaemolyticus were incubated with rEsIgLectin, rEsIg, or rEsCTLD. PBS and BSA were used as the negative controls. The data shown are representative of three independent repeats. Scale bars, 100 μm. (D) Antibacterial activity of EsIgLectin against S. aureus and V. parahaemolyticus on LB agar plates. S. aureus and V. parahaemolyticus cultured overnight at 37°C and plated onto LB agar plates. The rEsIgLectin, rEsIg, rEsCTLD, ampicillin (Amp), PBS, H2O, BSA, and kanamycin sulfate (Kana) were added to each sterile filter paper on the plate. The plates were cultured at 37°C until a bacteriostatic ring appeared. (E and F) EsIgLectin promoted bacterial clearance in crabs. The rEsIgLectin, rEsIg, or rEsCTLD was incubated with S. aureus (E) or V. parahaemolyticus (F) suspension, abdominally injected into the crabs, and the hemolymph was collected. The number of residual bacteria in the hemolymph was determined by plating onto LB agar plates. PBS- and BSA-coated bacteria were used as controls. For the clearance test, three crabs were used for each group. Data are representative of three independent experiments. (G) Amino acid sequence alignment of the C-type lectin–like domain from different invertebrate species. (H) The predicted QPD motif in the C-type lectin–like domain and illustration of the mutated rEsCTLD. (I) Binding properties of rEsCTLD and rEsCTLDΔQPD to bacteria. The eight bacterial species incubated with the rEsCTLD or QPD motif–deleted rEsCTLD protein were analyzed with Western blotting. (J) Microorganism agglutination assays of rEsCTLD or rEsCTLDΔQPD in the absence or presence of calcium. Scale bars, 100 μm. (K) Antibacterial activities of rEsCTLDΔQPD against S. aureus and V. parahaemolyticus on LB agar plates. (L and M) The rEsCTLDΔQPD regulates S. aureus (L) and V. parahaemolyticus (M) clearance in crabs. For the clearance test, three crabs were used for each group. Data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t test (E, F, L, and M).

FIGURE 2.

Antimicrobial activity of EsIgLectin and its domains. (A) The bacterial binding properties of EsIgLectin. Western blotting was performed to analyze the incubation of eight bacterial species (B. subtilis, S. agalactiae, S. aureus, V. harveyi, V. anguillarum, V. parahaemolyticus, Vibrio vulnificus, and A. hydrophila) with rEsIgLectin, rEsIg, and rEsCTLD. (B) Quantitative binding of EsIgLectin to pathogen-associated molecular patterns. The binding activities of rEsIgLectin, rEsIg, and rEsCTLD to LPS from E. coli and peptidoglycan from S. aureus or B. subtilis were analyzed with ELISA; PBS was used as the control. The results are presented as the mean ± SD derived from three independent repeats. (C) Microorganism agglutination assays of EsIgLectin in the absence or presence of calcium. S. aureus and V. parahaemolyticus were incubated with rEsIgLectin, rEsIg, or rEsCTLD. PBS and BSA were used as the negative controls. The data shown are representative of three independent repeats. Scale bars, 100 μm. (D) Antibacterial activity of EsIgLectin against S. aureus and V. parahaemolyticus on LB agar plates. S. aureus and V. parahaemolyticus cultured overnight at 37°C and plated onto LB agar plates. The rEsIgLectin, rEsIg, rEsCTLD, ampicillin (Amp), PBS, H2O, BSA, and kanamycin sulfate (Kana) were added to each sterile filter paper on the plate. The plates were cultured at 37°C until a bacteriostatic ring appeared. (E and F) EsIgLectin promoted bacterial clearance in crabs. The rEsIgLectin, rEsIg, or rEsCTLD was incubated with S. aureus (E) or V. parahaemolyticus (F) suspension, abdominally injected into the crabs, and the hemolymph was collected. The number of residual bacteria in the hemolymph was determined by plating onto LB agar plates. PBS- and BSA-coated bacteria were used as controls. For the clearance test, three crabs were used for each group. Data are representative of three independent experiments. (G) Amino acid sequence alignment of the C-type lectin–like domain from different invertebrate species. (H) The predicted QPD motif in the C-type lectin–like domain and illustration of the mutated rEsCTLD. (I) Binding properties of rEsCTLD and rEsCTLDΔQPD to bacteria. The eight bacterial species incubated with the rEsCTLD or QPD motif–deleted rEsCTLD protein were analyzed with Western blotting. (J) Microorganism agglutination assays of rEsCTLD or rEsCTLDΔQPD in the absence or presence of calcium. Scale bars, 100 μm. (K) Antibacterial activities of rEsCTLDΔQPD against S. aureus and V. parahaemolyticus on LB agar plates. (L and M) The rEsCTLDΔQPD regulates S. aureus (L) and V. parahaemolyticus (M) clearance in crabs. For the clearance test, three crabs were used for each group. Data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t test (E, F, L, and M).

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Alignment of the amino acid sequences of the C-type lectin–like domains from different invertebrate animals suggested a conserved QPD motif within the EsIgLectin (Fig. 2G), which may have a critical role in C-type lectin–like domain antimicrobial activity (Table I). To test that possibility, we constructed the QPD motif–deleted C-type lectin–like domain (Fig. 2H) recombinant proteins (Supplemental Fig. 2), and the following assays showed the essential role of the QPD motif in bacterial binding (Fig. 2I), bacterial agglutination (Fig. 2J), bacterial growth inhibition (Fig. 2K), and in vivo S. aureus (Fig. 2L) or V. parahaemolyticus (Fig. 2M) clearance activity, through comparison of QPD motif–deleted C-type lectin–like domain and wild-type C-type lectin–like domain recombinant protein.

The conventional model of vertebrate polymeric IgM involves a unique structure in which the heavy chains and J chain are joined by well-defined disulfide bonds (30, 31). After we analyzed the amino acid sequences of the EsIgLectin Ig domain and C-type lectin–like domain, disulfide bridges within both domains (Fig. 3A) were predicted, which suggested that EsIgLectin may generate a polymeric protein structure. To test this hypothesis, we purified native EsIgLectin from the plasma that was detected by Western blotting, which showed clear monomeric and polymeric EsIgLectin structures in the presence of the cross-linker BS3 [suberic acid bis (3-sulfo-N-hydroxysuccinimide ester) sodium salt] or under nonreducing (NR) conditions without DTT in the sample buffer (Fig. 3B). To analyze whether the EsIgLectin polymeric structures constructed by the Ig domain or C-type lectin–like domain, Coomassie brilliant blue (CBB)-stained SDS-PAGE and Western blotting (Fig. 3C, 3D) were conducted using His-tag Abs for rEsIgLectin, rEsIg, or rEsCTLD. The results showed that monomer, dimer, trimer, and even tetramer structures of EsIgLectin existed in the presence of the BS3, which was dependent on both the Ig domain and C-type lectin–like domain (Fig. 3C). Because the BS3 method may cause false-positive results, we performed CBB and Western blotting under reducing (R) and NR conditions with or without DTT in the sample buffer, respectively. The results showed that the full-length EsIgLectin, Ig domain, or C-type lectin–like domain resolved as a single species under R conditions, but that EsIgLectin and the Ig domain appeared to assemble into large disulfide-linked polymers, whereas C-type lectin–like domain appeared monomeric during both R and NR conditions (Fig. 3D), indicating that only the disulfide bond within the Ig domain contributed to the polymeric structure of EsIgLectin.

FIGURE 3.

EsIgLectin generates polymeric and monomeric structures and differences in antimicrobial activity. (A) Predicted disulfide bridges in the EsIgLectin Ig domain and C-type lectin–like domain. (B) Oligomerization of native EsIgLectin. BS3 was added to the native EsIgLectin that was purified from the plasma (3 ml) after abdominal injection of crabs with V. parahaemolyticus (12 h) with a final concentration of 5 mM, and the mixture was incubated at room temperature for 1 h. After incubation, SDS-PAGE sample loading buffer was added for reaction termination and treated in a boiling water bath for 5 min followed by Western blotting or the native EsIgLectin under R and NR conditions with or without DTT in sample buffer, respectively, and then detected by Western blotting. Anti-EsIgLectin Ab was used. (C) Polymeric EsIgLectin was detected in vitro using CBB-stained SDS-PAGE (left) and Western blotting (right) after BS3 treatment of the recombinant protein. His-Tag Abs against rEsIgLectin, rEsIg, and rEsCTLD were used. (D) Polymeric EsIgLectin was detected in vitro using CBB (left) and Western blotting (right) under R and NR conditions with or without DTT in sample buffer, respectively. (E and F) Predicted disulfide bridge in the Ig domain with Cys42 and Cys102 and illustration of the mutated rEsIg (E) and the recombinant protein (F). (G) The rEsIgΔCys42, 102 lost the ability to generate polymeric protein in vitro, as shown with CBB staining (left) and Western blotting (right) under R and NR conditions. (H and I) Predicted disulfide bridge in the Ig domain with Cys42 and Cys102 and illustration of the mutated rEsIgLectin (H) and the recombinant protein (I). (J) The rEsIgLectinΔCys42, 102 lost the ability to generate polymeric protein in vitro, as shown with CBB staining (left) and Western blotting (right) under R and NR conditions. (K) Antibacterial activity of rEsIgΔCys42, 102 against S. aureus and V. parahaemolyticus on LB agar plates. (L and M) rEsIgΔCys42, 102 and rEsIgLectinΔCys42, 102 regulated S. aureus (L) and V. parahaemolyticus (M) clearance in crab. For the clearance test, three crabs were used for each group, and the data are representative of three independent experiments. Results shown are the mean ± SD. **p < 0.01, ***p < 0.001 by Student t test (L and M).

FIGURE 3.

EsIgLectin generates polymeric and monomeric structures and differences in antimicrobial activity. (A) Predicted disulfide bridges in the EsIgLectin Ig domain and C-type lectin–like domain. (B) Oligomerization of native EsIgLectin. BS3 was added to the native EsIgLectin that was purified from the plasma (3 ml) after abdominal injection of crabs with V. parahaemolyticus (12 h) with a final concentration of 5 mM, and the mixture was incubated at room temperature for 1 h. After incubation, SDS-PAGE sample loading buffer was added for reaction termination and treated in a boiling water bath for 5 min followed by Western blotting or the native EsIgLectin under R and NR conditions with or without DTT in sample buffer, respectively, and then detected by Western blotting. Anti-EsIgLectin Ab was used. (C) Polymeric EsIgLectin was detected in vitro using CBB-stained SDS-PAGE (left) and Western blotting (right) after BS3 treatment of the recombinant protein. His-Tag Abs against rEsIgLectin, rEsIg, and rEsCTLD were used. (D) Polymeric EsIgLectin was detected in vitro using CBB (left) and Western blotting (right) under R and NR conditions with or without DTT in sample buffer, respectively. (E and F) Predicted disulfide bridge in the Ig domain with Cys42 and Cys102 and illustration of the mutated rEsIg (E) and the recombinant protein (F). (G) The rEsIgΔCys42, 102 lost the ability to generate polymeric protein in vitro, as shown with CBB staining (left) and Western blotting (right) under R and NR conditions. (H and I) Predicted disulfide bridge in the Ig domain with Cys42 and Cys102 and illustration of the mutated rEsIgLectin (H) and the recombinant protein (I). (J) The rEsIgLectinΔCys42, 102 lost the ability to generate polymeric protein in vitro, as shown with CBB staining (left) and Western blotting (right) under R and NR conditions. (K) Antibacterial activity of rEsIgΔCys42, 102 against S. aureus and V. parahaemolyticus on LB agar plates. (L and M) rEsIgΔCys42, 102 and rEsIgLectinΔCys42, 102 regulated S. aureus (L) and V. parahaemolyticus (M) clearance in crab. For the clearance test, three crabs were used for each group, and the data are representative of three independent experiments. Results shown are the mean ± SD. **p < 0.01, ***p < 0.001 by Student t test (L and M).

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To test the role of the disulfide bridge in the Ig domain in generating the polymeric EsIgLectin structure, we constructed rEsIgΔCys42, 102 (Fig. 3F) and rEsIgLectinΔCys42, 102 (Fig. 3I). The results showed that rEsIgΔCys42, 102 and rEsIgLectinΔCys42, 102 could not generate any polymeric structure in both the R and NR conditions (Fig. 3G, 3J). Furthermore, rEsIgΔCys42, 102 and rEsIgLectinΔCys42, 102 showed lower bacterial growth inhibition (Fig. 3K) and reduced in vivo S. aureus (Fig. 3L) or V. parahaemolyticus (Fig. 3M) clearance activity compared with the wild-type Ig domain and full-length EsIgLectin, respectively. Together, these results indicate the essential role of disulfide bonds in the polymeric EsIgLectin structure and further show that polymeric EsIgLectin displayed stronger antimicrobial activities.

Ig domain–containing proteins such as Dscam promote hemocyte phagocytosis via cell membrane receptors (24), but whether the novel Ig domain in EsIgLectin regulates hemocyte phagocytosis is unknown. rEsIgLectin, rEsIg, and rEsCTLD were injected into the crabs, respectively. Hemolymph was collected at 30 min postinjection, and the hemocytes were isolated for immunohistochemistry assay to detect the binding and cell adhesion property. We observed rEsIgLectin and rEsIg signals at the cell membrane, but not rEsCTLD (Fig. 4A). Moreover, flow cytometry demonstrated that, in dsRNA-mediated EsIgLectin-silenced crabs (Supplemental Fig. 3), the hemocytes had significantly reduced capacity for phagocytosing V. parahaemolyticus (Fig. 4B), suggesting the critical role of EsIgLectin in regulating phagocytosis. We therefore investigated the ability of the Ig domain and C-type lectin–like domain to regulate phagocytosis. For this purpose, we precoated FITC-labeled V. parahaemolyticus with rEsIgLectin, rEsIg, or rEsCTLD and injected them into crab hemolymphs. In vivo phagocytosis assays showed that the rEsIgLectin significantly enhanced V. parahaemolyticus phagocytosis by ∼4-fold, and that rEsIg improved V. parahaemolyticus phagocytosis by >2-fold, but that rEsCTLD had a slight effect on regulating phagocytosis (Fig. 4C). Collectively, the results indicated that bacteria-interacting EsIgLectin may promote hemocyte phagocytosis via interaction of the Ig domain with a yet unidentified receptor (Fig. 4D).

FIGURE 4.

EsIgLectin regulates hemocyte phagocytosis. (A) EsIgLectin affinity with the hemocyte cell membrane. rEsIgLectin, rEsIg, and rEsCTLD were injected into the crabs, respectively. Hemolymph was collected at 30 min postinjection, and the hemocytes were isolated for immunohistochemistry assay to detect the binding and cell adhesion property with an anti-His tag Ab (green). DAPI (blue) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) (red) were used to stain the nuclei and membrane of hemocytes. Three crabs were used for each group. Immunocytochemical images are representative of three independent experiments. Scale bars, 30 μm. (B) Knockdown of EsIgLectin suppressed hemocyte phagocytosis. dsEsIgLectin were abdominally injected into the crab, then heat-inactivated and FITC-labeled V. parahaemolyticus were injected into the hemolymph, and the hemocytes were collected 30 min later for flow cytometric analyses; bacteria alone or dsGFP preinjection was used as the control. A total of 10,000 hemocytes was counted for each sample, which was from three crabs. Data are representative of three independent experiments. (C) Promotion of hemocyte phagocytosis by EsIgLectin. V. parahaemolyticus was heat inactivated and labeled with FITC before coating with rEsIgLectin, rEsIg, and rEsCTLD, respectively. The bacteria were then injected into the hemolymph, and hemocytes were collected 30 min later for flow cytometric analysis; PBS and BSA were used as the controls. A total of 10,000 hemocytes was counted for each sample, which was from three crabs. Data are representative of three independent experiments. (D) Hypothesis of EsIgLectin promotion of hemocyte phagocytosis via interaction of its Ig domain with an unknown receptor. Results shown are the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t test (B and C).

FIGURE 4.

EsIgLectin regulates hemocyte phagocytosis. (A) EsIgLectin affinity with the hemocyte cell membrane. rEsIgLectin, rEsIg, and rEsCTLD were injected into the crabs, respectively. Hemolymph was collected at 30 min postinjection, and the hemocytes were isolated for immunohistochemistry assay to detect the binding and cell adhesion property with an anti-His tag Ab (green). DAPI (blue) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) (red) were used to stain the nuclei and membrane of hemocytes. Three crabs were used for each group. Immunocytochemical images are representative of three independent experiments. Scale bars, 30 μm. (B) Knockdown of EsIgLectin suppressed hemocyte phagocytosis. dsEsIgLectin were abdominally injected into the crab, then heat-inactivated and FITC-labeled V. parahaemolyticus were injected into the hemolymph, and the hemocytes were collected 30 min later for flow cytometric analyses; bacteria alone or dsGFP preinjection was used as the control. A total of 10,000 hemocytes was counted for each sample, which was from three crabs. Data are representative of three independent experiments. (C) Promotion of hemocyte phagocytosis by EsIgLectin. V. parahaemolyticus was heat inactivated and labeled with FITC before coating with rEsIgLectin, rEsIg, and rEsCTLD, respectively. The bacteria were then injected into the hemolymph, and hemocytes were collected 30 min later for flow cytometric analysis; PBS and BSA were used as the controls. A total of 10,000 hemocytes was counted for each sample, which was from three crabs. Data are representative of three independent experiments. (D) Hypothesis of EsIgLectin promotion of hemocyte phagocytosis via interaction of its Ig domain with an unknown receptor. Results shown are the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t test (B and C).

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The intestine is one of the most important immune-related organs in invertebrates (32), and intestine-expressed endogenous EsIgLectin raises the possibility that it may function in regulating microbial homeostasis. Accordingly, we performed 16S rDNA sequencing (the 16S rDNA raw data have been deposited to the NCBI Sequence Read Archive under BioProject accession code PRJNA763732, http://www.ncbi.nlm.nih.gov/bioproject/763732) of intestinal contents from crabs that had been orally injected with BSA, rEsIgLectin, rEsIg, and rEsCTLD. After injecting rEsIgLectin orally according to a previous construct method (Fig. 1J) for 3 d, Western blotting detected high levels of recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD) in the crab intestine (Fig. 5A), indicating the establishment of recombinant protein delivery to the intestine via oral injection. We observed marked differences between BSA-, rEsIgLectin-, rEsIg-, and rEsCTLD-injected crab intestinal contents regarding bacterial diversity based on α-diversity analyses using the Chao1 index (Fig. 5B) and β-diversity analyses using the Bray–Curtis method (Fig. 5C). Moreover, the relative abundance of the top 50 bacteria showed differences between BSA-, rEsIgLectin-, rEsIg-, and rEsCTLD-injected crabs, while the relative abundance of aquatic animal pathogens that included Vibrio and Shewanella were both significantly inhibited in recombinant protein–injected crab intestines, and the abundance of microorganisms that normally live in the intestine and the beneficial intestinal flora (3335), including Bacteroides, Lactococcus, and Prevotella, were increased significantly after recombinant protein injection orally, when compared with BSA (Fig. 5D). These results indicate that EsIgLectin regulates microbiota homeostasis and inhibits the growth of pathogenic bacteria, which is consistent with the antibacterial activity of its C-type lectin–like domain and Ig domains.

FIGURE 5.

EsIgLectin regulates intestinal microbial homeostasis. (A) Western blotting showing recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD) delivery to the intestine via oral injection in crab. Data are representative of three independent experiments. (B) α-Diversity (Chao1 index) of intestinal contents isolated from BSA (n = 24), rEsIgLectin- (n = 36), rEsIg- (n = 24), and rEsCTLD-injected (n = 30) crabs on day 3, by 16S rDNA sequencing. (C) β-Diversity (Bray–Curtis method) of intestinal contents isolated from BSA- (n = 24), rEsIgLectin- (n = 36), rEsIg- (n = 24), and rEsCTLD-injected (n = 30) crabs on day 3, by 16S rDNA sequencing. (D) Heatmap analysis of the relative abundance of microbiota at the genus level in the intestinal contents samples as in (B) and (C). Data are representative of the average value of samples in different groups.

FIGURE 5.

EsIgLectin regulates intestinal microbial homeostasis. (A) Western blotting showing recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD) delivery to the intestine via oral injection in crab. Data are representative of three independent experiments. (B) α-Diversity (Chao1 index) of intestinal contents isolated from BSA (n = 24), rEsIgLectin- (n = 36), rEsIg- (n = 24), and rEsCTLD-injected (n = 30) crabs on day 3, by 16S rDNA sequencing. (C) β-Diversity (Bray–Curtis method) of intestinal contents isolated from BSA- (n = 24), rEsIgLectin- (n = 36), rEsIg- (n = 24), and rEsCTLD-injected (n = 30) crabs on day 3, by 16S rDNA sequencing. (D) Heatmap analysis of the relative abundance of microbiota at the genus level in the intestinal contents samples as in (B) and (C). Data are representative of the average value of samples in different groups.

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Pathogen infection and deregulation of gut–microbe interactions may lead to intestinal inflammation in both humans and all major model organisms (36). In aquaculture, Gram-negative bacteria usually infect crustaceans such as crabs and shrimp, thereby causing intestinal diseases and leading to weight loss and high mortality. To assess the potential role of EsIgLectin in protecting crab intestines against bacterial infection and inflammation, we established intestinal inflammation and EsIgLectin rescue models. For the intestinal inflammation model, crabs were orally injected with V. parahaemolyticus daily for a total of 12 d (Fig. 6A). For the rescue model, crabs were orally injected with V. parahaemolyticus daily for a total of 12 d, and orally injected with recombinant proteins (i.e., rEsIgLectin, rEsIg, and rEsCTLD) daily from day 6 to 12 (Fig. 6D). The intestinal morphological changes showed that obvious tissue damage was present in the intestine of crabs, which increased during the course of infection. The histological changes, including decrease of epithelial cells density and detachment of epithelial cells from the basement membrane, and a thinner intestinal wall, including basement membrane and visceral muscles, were observed (Fig. 6B). These results were confirmed at higher magnification for histological analysis on day 12 of the intestinal inflammation model (Fig. 6C). After rEsIgLectin, rEsIg, and rEsCTLD had been injected, the morphological changes that included increased epithelial cells density, slight detachment of intestinal epithelial cells from the basement, and a thicker intestinal wall were observed in the rEsIgLectin and rEsIg groups compared with the rEsCTLD groups (Fig. 6E). Considering that the expressions of proinflammatory cytokines in vertebrates and AMPs in Drosophila melanogaster are important factors that trigger intestinal inflammation (37, 38), we detected antimicrobial peptide expression in crab intestines on day 7 of the intestinal inflammation model with or without rEsIgLectin injection on days 6–12 by using the model as shown in (Fig. 6D. The results showed that V. parahaemolyticus significantly induced high AMPs (i.e., ALF1, ALF2, ALF3, Crustin1, Crustin2, DWD, and Lys) expression on day 7 after bacterial infection. In addition, rEsIgLectin injections on day 6 significantly suppressed the expressions of AMPs (Fig. 6G). However, the mechanisms for EsIgLectin to eliminate AMPs expression in the intestinal inflammation still needs further studies.

FIGURE 6.

EsIgLectin protects crab intestine against bacteria-induced inflammation. (A and D) Schematic for establishing the intestinal inflammation (A); recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD) rescue model (D) by oral injection with V. parahaemolyticus. (B) Morphological analysis of crab intestine under the intestinal inflammation model. The intestines were sampled, and images were captured. The histological changes at the same position of the sampled intestines were detected with H&E staining. Obvious tissue damage was present in the intestine of crab at later phase of intestinal inflammation. The histological changes, including decrease of epithelial cells density and detachment of epithelial cells from the basement membrane, and a thinner intestinal wall, including basement membrane and visceral muscles, were observed. Data shown are representative of three independent experiments. Scale bars, 100 μm. (C) Intestinal morphological and histological analysis on day 12 of the intestinal inflammation model. Obvious tissue damage on day 12 was present in the intestine of crab after oral injection with V. parahaemolyticus compared with oral injection with PBS. The histological changes, including decrease of epithelial cells density and detachment of epithelial cells from the basement membrane, and a thinner intestinal wall, including basement membrane and visceral muscles, were observed. Data shown are representative of three independent experiments. Scale bars, 20 μm (left), 100 μm (middle), or 50 μm (right); original magnifications ×200, ×40, and ×100 were used. (E) Intestinal morphological and histological analysis on day 12 of the intestinal inflammation model after oral injection with recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD) on day 6. The morphological changes that included increased epithelial cells density and slight detachment of intestinal epithelial cells from the basement and a thicker intestinal wall were observed in the rEsIgLectin and rEsIg groups compared with rEsCTLD groups. Data shown are representative of three independent experiments. Scale bars, 20 μm (left), 100 μm (middle), or 50 μm (right); original magnifications ×200, ×40, and ×100 were used. (F) qRT-PCR detection of AMPs expression in crab intestines on day 7 of the intestinal inflammation model with or without rEsIgLectin injection. Six crabs were used to prepare each sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student t test (F).

FIGURE 6.

EsIgLectin protects crab intestine against bacteria-induced inflammation. (A and D) Schematic for establishing the intestinal inflammation (A); recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD) rescue model (D) by oral injection with V. parahaemolyticus. (B) Morphological analysis of crab intestine under the intestinal inflammation model. The intestines were sampled, and images were captured. The histological changes at the same position of the sampled intestines were detected with H&E staining. Obvious tissue damage was present in the intestine of crab at later phase of intestinal inflammation. The histological changes, including decrease of epithelial cells density and detachment of epithelial cells from the basement membrane, and a thinner intestinal wall, including basement membrane and visceral muscles, were observed. Data shown are representative of three independent experiments. Scale bars, 100 μm. (C) Intestinal morphological and histological analysis on day 12 of the intestinal inflammation model. Obvious tissue damage on day 12 was present in the intestine of crab after oral injection with V. parahaemolyticus compared with oral injection with PBS. The histological changes, including decrease of epithelial cells density and detachment of epithelial cells from the basement membrane, and a thinner intestinal wall, including basement membrane and visceral muscles, were observed. Data shown are representative of three independent experiments. Scale bars, 20 μm (left), 100 μm (middle), or 50 μm (right); original magnifications ×200, ×40, and ×100 were used. (E) Intestinal morphological and histological analysis on day 12 of the intestinal inflammation model after oral injection with recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD) on day 6. The morphological changes that included increased epithelial cells density and slight detachment of intestinal epithelial cells from the basement and a thicker intestinal wall were observed in the rEsIgLectin and rEsIg groups compared with rEsCTLD groups. Data shown are representative of three independent experiments. Scale bars, 20 μm (left), 100 μm (middle), or 50 μm (right); original magnifications ×200, ×40, and ×100 were used. (F) qRT-PCR detection of AMPs expression in crab intestines on day 7 of the intestinal inflammation model with or without rEsIgLectin injection. Six crabs were used to prepare each sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student t test (F).

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Finding that full-length EsIgLectin and the Ig domain bound to the hemocyte membrane and promoted phagocytosis prompted us to screen for the EsIgLectin receptor. After constructing rEsIg (Fig. 1B), we used a T7 phage display library expressing crab genes that had been previously used (29) to screen potential Ig-binding proteins. We identified some possible binding proteins (Fig. 7A), with polymeric Ig receptor (pIgR) being of particular interest because of its roles as a cell membrane receptor and in active phagocytosis. The Ig domains are the signatures of pIgR in both vertebrates and invertebrates; phylogenetic tree construction that contained the EsIgLectin Ig domain, the pIgR Ig domains, and other proteins with Ig domains showed that the EsIgLectin Ig domain was on the same branch as invertebrate pIgR and had a closer genetic relationship with the pIgR Ig1 domain (Fig. 7B).

FIGURE 7.

EsIgLectin interacts with EspIgR via the Ig domain. (A) The rEsIg protein–based T7 phage library screening suggesting the potential interaction between EsIgLectin and EspIgR. (B) Phylogenetic analysis of the Ig domains from EsIgLectin and pIgR across vertebrate and invertebrate species. (C) Recombinant expression and purification of Ig1, Ig2, Ig3, and SC from EspIgR. Constructs pGEX4T-1-EspIgR-SC, -Ig1, -Ig2, -Ig3, or empty pGEX4T-1 were transformed into E. coli, and the expression of proteins was induced by IPTG and analyzed by SDS-PAGE. Lane M, protein markers; lane 1, proteins from E. coli with pGEX4T-1-EspIgR-SC, -Ig1, -Ig2, -Ig3, or empty pGEX4T-1 without IPTG induction; lane 2, proteins from E. coli with pGEX4T-1-EspIgR-SC, -Ig1, -Ig2, -Ig3, or empty pGEX4T-1 with 0.5 mM IPTG induction for 6 h at 37°C; lane 3, the supernatant of the E. coli with treatment by ultrasound; lane 4, the precipitation of the E. coli with treatment by ultrasound; lane 5, purified recombinant protein pGEX4T-1-EspIgR-SC, -Ig1, -Ig2, -Ig3, or empty pGEX4T-1. (D) GST pull-down assay analysis of the interaction of EspIgR-SC, -Ig1, -Ig2, and -Ig3 with full-length EsIgLectin. (E) GST pull-down assay analysis of the interaction of EspIgR-SC, -Ig1, -Ig2, and -Ig3 with the EsIgLectin Ig domain. (F) GST pull-down assay analysis of the interaction of EspIgR-SC, -Ig1, -Ig2, and -Ig3 with the EsIgLectin C-type lectin–like domain. Data are representative of three independent experiments (D–F).

FIGURE 7.

EsIgLectin interacts with EspIgR via the Ig domain. (A) The rEsIg protein–based T7 phage library screening suggesting the potential interaction between EsIgLectin and EspIgR. (B) Phylogenetic analysis of the Ig domains from EsIgLectin and pIgR across vertebrate and invertebrate species. (C) Recombinant expression and purification of Ig1, Ig2, Ig3, and SC from EspIgR. Constructs pGEX4T-1-EspIgR-SC, -Ig1, -Ig2, -Ig3, or empty pGEX4T-1 were transformed into E. coli, and the expression of proteins was induced by IPTG and analyzed by SDS-PAGE. Lane M, protein markers; lane 1, proteins from E. coli with pGEX4T-1-EspIgR-SC, -Ig1, -Ig2, -Ig3, or empty pGEX4T-1 without IPTG induction; lane 2, proteins from E. coli with pGEX4T-1-EspIgR-SC, -Ig1, -Ig2, -Ig3, or empty pGEX4T-1 with 0.5 mM IPTG induction for 6 h at 37°C; lane 3, the supernatant of the E. coli with treatment by ultrasound; lane 4, the precipitation of the E. coli with treatment by ultrasound; lane 5, purified recombinant protein pGEX4T-1-EspIgR-SC, -Ig1, -Ig2, -Ig3, or empty pGEX4T-1. (D) GST pull-down assay analysis of the interaction of EspIgR-SC, -Ig1, -Ig2, and -Ig3 with full-length EsIgLectin. (E) GST pull-down assay analysis of the interaction of EspIgR-SC, -Ig1, -Ig2, and -Ig3 with the EsIgLectin Ig domain. (F) GST pull-down assay analysis of the interaction of EspIgR-SC, -Ig1, -Ig2, and -Ig3 with the EsIgLectin C-type lectin–like domain. Data are representative of three independent experiments (D–F).

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To test the interaction between EsIgLectin and EspIgR, we constructed GST-tagged recombinant proteins that included Ig1, Ig2, and Ig3, and SC that included Ig1–3 from EspIgR for pull-down assays (Fig. 7C). The results showed that only EspIgR-SC or EspIgR-Ig1 interacted with full-length EsIgLectin (Fig. 7D), the EsIgLectin Ig domain interacted with EspIgR-SC or EspIgR-Ig1 (Fig. 7E), and the EsIgLectin C-type lectin–like domain did not interact with any pIgR-related recombinant protein (Fig. 7F), indicating that the EsIgLectin Ig domain interacted with the EspIgR Ig1 domain.

To test the role of the EsIgLectin–EspIgR axis on hemocyte phagocytosis, we analyzed the expression pattern of EspIgR in hemocytes, which showed significantly induced EspIgR expression at 6 h after abdominal injection of V. parahaemolyticus (Fig. 8A). We therefore determined whether EspIgR affected EsIgLectin regulation of hemocyte phagocytosis. FITC-labeled V. parahaemolyticus was precoated with rEsIgLectin, rEsIg, or rEsCTLD, and then abdominally injected into crab with GFP RNAi or EspIgR RNAi (Fig. 8B) for in vivo phagocytosis assays, which showed that rEsIgLectin- or rEsIg-regulated phagocytosis was significantly suppressed in EspIgR-silenced hemocytes, but that EspIgR had only a slight effect on rEsCTLD-regulated phagocytosis (Fig. 8C). The reason why rEsCTLD-regulated phagocytosis was also suppressed in EspIgR-silenced hemocytes might have been because of the phagocytic role of EspIgR. Taken together, these results showed that EsIgLectin promoted phagocytosis in hemocytes using EspIgR as the receptor.

FIGURE 8.

EsIgLectin regulates hemocyte phagocytosis via EspIgR. (A) Expression patterns of EspIgR in the hemocytes of crabs abdominally injected with bacteria; PBS and untreated crab hemocytes were used as the controls. Six crabs were used to prepare each sample, and the data are representative of three independent experiments. (B) RNAi of the expression of EspIgR in crabs; dsGFP are used as the controls. Three crabs were used to prepare each sample, and the data are representative of three independent experiments. (C) Promotion of hemocyte phagocytosis by the EsIgLectin–EspIgR axis. V. parahaemolyticus were heat inactivated and FITC labeled before coating with rEsIgLectin, rEsIg, and rEsCTLD. The bacteria were then injected into crab hemolymph pretreated with dspIgR or dsGFP, and the hemocytes were collected 30 min later for flow cytometric analysis; PBS and BSA were used as the controls. A total of 10,000 hemocytes from three crabs was counted for each sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001 using Student t test (A–C).

FIGURE 8.

EsIgLectin regulates hemocyte phagocytosis via EspIgR. (A) Expression patterns of EspIgR in the hemocytes of crabs abdominally injected with bacteria; PBS and untreated crab hemocytes were used as the controls. Six crabs were used to prepare each sample, and the data are representative of three independent experiments. (B) RNAi of the expression of EspIgR in crabs; dsGFP are used as the controls. Three crabs were used to prepare each sample, and the data are representative of three independent experiments. (C) Promotion of hemocyte phagocytosis by the EsIgLectin–EspIgR axis. V. parahaemolyticus were heat inactivated and FITC labeled before coating with rEsIgLectin, rEsIg, and rEsCTLD. The bacteria were then injected into crab hemolymph pretreated with dspIgR or dsGFP, and the hemocytes were collected 30 min later for flow cytometric analysis; PBS and BSA were used as the controls. A total of 10,000 hemocytes from three crabs was counted for each sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001 using Student t test (A–C).

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Intestinal infection after oral intake of bacteria and hemolymph infection after limb injury appear to be two independent events, but the common open circulatory system in crustaceans and insects means that the two infection pathways in nature may have some correlation. Combined with the findings that the interaction between Drosophila hemocytes and intestinal stem cells promoted infection resistance (39), and the observation that mammalian pIgR transports IgA and IgM to the intestine (40), we speculated that EsIgLectin may establish the immune axis between the intestine and hemocytes through EspIgR (Fig. 9A). To test this hypothesis, we first determined the mRNA expression levels of EspIgR in the intestine and EsIgLectin in hemocytes after orally injected V. parahaemolyticus–induced intestinal inflammation. The results showed that the expression of intestinal-derived EspIgR was quickly induced at the early phase (6 h after oral injection) of intestinal inflammation (Fig. 9B), whereas the expression of hemocyte-derived EsIgLectin was also significantly induced at the later phase (3 d after oral injection) of intestinal inflammation (Fig. 9C), which may have been caused by damaged intestinal walls, leading to the release of orally injected bacteria into the hemolymph (Fig. 9D), as was detected by the bacterial FITC signal, thereby activating EsIgLectin expression in hemocytes. Together, these results suggested that hemocyte-produced EsIgLectin may execute transcytosis into the intestine during intestinal inflammation.

FIGURE 9.

Hemocyte-derived EsIgLectin transcytosis into the intestine at the later phase of intestinal inflammation. (A) Hypothesis of intestine and hemocytes interactions through the EsIgLectin–EspIgR axis in the situation of intestinal inflammation. At the early phase, hemocytes maintained the low expression of EsIgLectin and could not enter the intestine because of the barrier of a healthy intestinal wall. At the later phase, bacteria may have been released from the intestine into hemolymph through a damaged intestinal wall, thus stimulating high expression of EsIgLectin in hemocytes. Hemocyte-derived EsIgLectin may have entered the intestine through the damaged intestinal wall, and thus import into the apical membrane via EspIgR may have occurred. (B and C) The expression patterns of EspIgR in the intestine (B) and EsIgLectin in hemocytes (C) at the different phases of intestinal inflammation. Crabs received oral injections of V. parahaemolyticus or PBS; the control was untreated crab. Six crabs were used to prepare each sample, and the data are representative of three independent experiments. (D) Detection of the orally injected FITC-labeled V. parahaemolyticus signal in the hemolymph at the early and later phase of intestinal inflammation. (E) The rEsIgLectin was abdominally injected into crabs under oral bacterial injection-induced intestinal inflammation at days 3 and 7, respectively, and the protein level of rEsIgLectin in the intestine was then detected by Western blotting. (F) Protein levels of abdominally injected rEsIgLectin in the intestine at the early and later phases of intestinal inflammation. After 12 h of rEsIgLectin injection, the protein levels of rEsIgLectin in the intestine were detected by Western blotting with anti-His tag Ab. Three crabs were pooled together as one sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t test (B and C).

FIGURE 9.

Hemocyte-derived EsIgLectin transcytosis into the intestine at the later phase of intestinal inflammation. (A) Hypothesis of intestine and hemocytes interactions through the EsIgLectin–EspIgR axis in the situation of intestinal inflammation. At the early phase, hemocytes maintained the low expression of EsIgLectin and could not enter the intestine because of the barrier of a healthy intestinal wall. At the later phase, bacteria may have been released from the intestine into hemolymph through a damaged intestinal wall, thus stimulating high expression of EsIgLectin in hemocytes. Hemocyte-derived EsIgLectin may have entered the intestine through the damaged intestinal wall, and thus import into the apical membrane via EspIgR may have occurred. (B and C) The expression patterns of EspIgR in the intestine (B) and EsIgLectin in hemocytes (C) at the different phases of intestinal inflammation. Crabs received oral injections of V. parahaemolyticus or PBS; the control was untreated crab. Six crabs were used to prepare each sample, and the data are representative of three independent experiments. (D) Detection of the orally injected FITC-labeled V. parahaemolyticus signal in the hemolymph at the early and later phase of intestinal inflammation. (E) The rEsIgLectin was abdominally injected into crabs under oral bacterial injection-induced intestinal inflammation at days 3 and 7, respectively, and the protein level of rEsIgLectin in the intestine was then detected by Western blotting. (F) Protein levels of abdominally injected rEsIgLectin in the intestine at the early and later phases of intestinal inflammation. After 12 h of rEsIgLectin injection, the protein levels of rEsIgLectin in the intestine were detected by Western blotting with anti-His tag Ab. Three crabs were pooled together as one sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t test (B and C).

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To determine whether hemocyte-derived EsIgLectin executes transcytosis to the intestine, we abdominally injected rEsIgLectin into the hemolymph of crabs during the early or later phase of intestinal inflammation (Fig. 9E). Western blotting showed that rEsIgLectin was involved in transcytosis into the intestine at the later phase, rather than the early phase (Fig. 9F). Taken together, these results showed that hemocyte-derived EsIgLectin entered the damaged intestinal wall, and thus may execute transcytosis into the intestinal mucosa through EspIgR, thereby establishing the axis between the intestine and hemocytes during the later phase of intestinal inflammation.

To verify the role of EsIgLectin–EspIgR interactions in innate immunity, we injected abdominally dsEsIgLectin (Fig. 10A) or dsEspIgR (Fig. 10B) into crab hemolymphs to knock down expressions of EsIgLectin and EspIgR, respectively, including, but not limited to, the intestine and hemocytes. Oral bacterial infection was performed in the intestine, and the survival assay showed that intestinal EsIgLectin- or EspIgR-silenced crabs had significantly suppressed survival rates after V. parahaemolyticus infection, whereas orally injected rEsIgLectin, rEsIg, and rEsCTLD significantly increased survival (Fig. 10C). Abdominal bacterial infection was performed in hemolymph, and the survival assay showed that hemocyte EsIgLectin- or EspIgR-silenced crabs had significantly suppressed survival after abdominal infection of V. parahaemolyticus and abdominally injected rEsIgLectin and rEsIg, and rEsCTLD significantly increased the survival, while rEsIgLectin significantly increased the survival in an EspIgR-dependent manner (Fig. 10D). These results suggested that the EsIgLectin–EspIgR axis played critical roles in the antibacterial immune responses (Fig. 11).

FIGURE 10.

The EsIgLectin–EspIgR axis protects crabs against oral and abdominal bacterial infection. (A) RNAi of EsIgLectin expression in crabs. Three crabs were used to prepare each sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. **p < 0.01, ***p < 0.001, analyzed by Student t test. (B) RNAi of EspIgR expression in crabs. Three crabs were used to prepare each sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. **p < 0.01, ***p < 0.001, analyzed by Student t test. (C) EsIgLectin- and EspIgR-protected crabs from intestinal infection. Crabs were abdominally injected with dsEsIgLectin or dsEspIgR, or orally injected with recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD), or with dsGFP or BSA as the controls, and their 6-d survival post–V. parahaemolyticus infection was recorded. Each group consisted of 20 crabs. (D) Recombinant protein regulated crab susceptibility to V. parahaemolyticus abdominal infection via EspIgR. Crabs were abdominally injected with dsEsIgLectin and dsEspIgR, with or without recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD); dsGFP or BSA served as the controls; and their 6-d survival post–V. parahaemolyticus infection was recorded. Each group consisted of 20 crabs. For the survival rates, results were analyzed using the log rank (Mantel–Cox) test in the GraphPad Prism software. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t test.

FIGURE 10.

The EsIgLectin–EspIgR axis protects crabs against oral and abdominal bacterial infection. (A) RNAi of EsIgLectin expression in crabs. Three crabs were used to prepare each sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. **p < 0.01, ***p < 0.001, analyzed by Student t test. (B) RNAi of EspIgR expression in crabs. Three crabs were used to prepare each sample, and the data are representative of three independent experiments. Results shown are the mean ± SD. **p < 0.01, ***p < 0.001, analyzed by Student t test. (C) EsIgLectin- and EspIgR-protected crabs from intestinal infection. Crabs were abdominally injected with dsEsIgLectin or dsEspIgR, or orally injected with recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD), or with dsGFP or BSA as the controls, and their 6-d survival post–V. parahaemolyticus infection was recorded. Each group consisted of 20 crabs. (D) Recombinant protein regulated crab susceptibility to V. parahaemolyticus abdominal infection via EspIgR. Crabs were abdominally injected with dsEsIgLectin and dsEspIgR, with or without recombinant protein (i.e., rEsIgLectin, rEsIg, rEsCTLD); dsGFP or BSA served as the controls; and their 6-d survival post–V. parahaemolyticus infection was recorded. Each group consisted of 20 crabs. For the survival rates, results were analyzed using the log rank (Mantel–Cox) test in the GraphPad Prism software. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t test.

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FIGURE 11.

Schematic of EsIgLectin–EspIgR axis–regulated antibacterial immunity in hemocytes and the intestine. (A) Abdominal and oral bacterial injection, imitating injury-caused hemolymph infection and ingestion-caused intestinal infection in nature, respectively. (B) Hemolymph bacterial infection induced high expression of EsIgLectin and EspIgR in hemocytes. Secreted EsIgLectin regulated antimicrobial activities in hemolymph and promoted bacterial phagocytosis via EspIgR in hemocytes. (C) Acute oral bacterial infection induced high expression of EsIgLectin in intestine. EsIgLectin regulated microbiota homeostasis and protected the intestine from inflammation through intestinal epithelial cell–expressed EsIgLectin. (D) Intestinal inflammation recruiting both intestinal-derived and hemocyte-derived EsIgLectin to protect intestines from infection. Hemocyte-derived EsIgLectin could enter the intestine through inflammation-damaged and thinner intestinal walls, and thus transcytosis may occur through intestinal epithelial cells into the apical membrane via EspIgR. The EsIgLectin–EspIgR axis therefore protected the crabs from bacterial infection.

FIGURE 11.

Schematic of EsIgLectin–EspIgR axis–regulated antibacterial immunity in hemocytes and the intestine. (A) Abdominal and oral bacterial injection, imitating injury-caused hemolymph infection and ingestion-caused intestinal infection in nature, respectively. (B) Hemolymph bacterial infection induced high expression of EsIgLectin and EspIgR in hemocytes. Secreted EsIgLectin regulated antimicrobial activities in hemolymph and promoted bacterial phagocytosis via EspIgR in hemocytes. (C) Acute oral bacterial infection induced high expression of EsIgLectin in intestine. EsIgLectin regulated microbiota homeostasis and protected the intestine from inflammation through intestinal epithelial cell–expressed EsIgLectin. (D) Intestinal inflammation recruiting both intestinal-derived and hemocyte-derived EsIgLectin to protect intestines from infection. Hemocyte-derived EsIgLectin could enter the intestine through inflammation-damaged and thinner intestinal walls, and thus transcytosis may occur through intestinal epithelial cells into the apical membrane via EspIgR. The EsIgLectin–EspIgR axis therefore protected the crabs from bacterial infection.

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The decapod crustacean circulatory system is traditionally classified as an open system, which permits hemolymphs to fully communicate with other organs, such as the intestine, to circulate and transport hormones, hemocytes, and proteins to and from cells in the body to provide nourishment and help fight diseases and maintain homeostasis (41). In this study, we showed the immune functions of EsIgLectin in crab on injury or oral bacterial infection, involving strong antibacterial activity, which regulated phagocytosis in hemocytes, as well as microbiota homeostasis regulation and inflammation control in the intestine. Furthermore, we found EsIgLectin-regulated hemocyte phagocytosis through its receptor EspIgR, and hemocyte-derived EsIgLectin could execute transcytosis to the intestine at the later phase of intestinal inflammation. We therefore provide evidence for the axis of hemocytes and the intestine in systemic antibacterial responses.

As an important member of non-TLR pathogen recognition receptors, C-type lectins play crucial roles in innate immunity, such as pathogen recognition and clearance of invading microorganisms, by recognizing and noncovalently binding to specific sugar moieties and agglutinating pathogens by binding to cell surface glycoproteins and glycoconjugates (9). In this study, we showed that the EsIgLectin C-type lectin–like domain was required for bacterial binding, bacterial agglutination, bacterial clearance, and bacterial growth inhibition in a QPD motif–dependent manner, which is similar to the findings for insects (42) and shrimp (43). In mammals, Ig protein binds to complementary molecules of pathogens and links them together to cause agglutination (44); however, few studies have reported the function of Ig in invertebrates. In this study, we found that the EsIgLectin Ig domain bound with many different bacterial strains, resulting in antibacterial activity, which largely enhanced the antibacterial function of EsIgLectin by combining use of the abilities of the C-type lectin–like domain. Vertebrate IgM could form polymeric structure through the well-defined disulfide bonds involving cysteine residues at positions 337, 414, and 575 of the A chains (45). Similar with that, EsIgLectin formed polymer structures that included monomers, dimers, trimers, and even tetramers via the cysteine residues in its Ig domain. Therefore, EsIgLectin shared conservation with vertebrate Abs at the level of polymeric protein structures that were constructed by cysteine residues.

Phagocytosis is a multistep and receptor-mediated process initiated directly or indirectly by particle recognition, and it can be separated into steps that include particle attachment to the cell surface and particle ingestion by cells (46). Fc receptors, specifically the Fcγ receptor subtypes that recognize IgG, have been well studied for their role in phagocytosis (47). However, other Igs, including IgM and IgA, also opsonize pathogens and play a role in phagocytosis (48, 49). In this study, we found that the EsIgLectin Ig domain, rather than C-type lectin–like domain, had the ability to bind to the surface of hemocytes, thereby contributing to the cell binding activity of EsIgLectin. Flow cytometry showed that the full-length EsIgLectin and its Ig domain regulated the rate of hemocyte bacterial phagocytosis positively, while the C-type lectin–like domain had no role in this process, highlighting the important function of the Ig domain in phagocytosis but also raising the question of identity of its phagocytic receptor. A protein interaction assay showed that the EsIgLectin Ig domain bound with the Ig1 of EspIgR, and that EspIgR-silenced hemocytes did not show a significant phagocytosis during rEsIgLectin and rEsIg stimulations. Despite the finding that the EsIgLectin–EspIgR axis regulated hemocyte phagocytosis, the detailed signaling mechanisms activated on protein interaction on the cell surface require further investigation.

Several soluble C-type lectins were identified as critical factors that regulated the intestinal microbiota through C-type lectin–like domain–mediated bacterial recognition (22, 23). In contrast with previous studies, this study presented evidence that a novel Ig domain in C-type lectin also regulated intestinal microbiota homeostasis and protected the host from V. parahaemolyticus–induced inflammation. The intestinal epithelium is the major barrier of defense against pathogenic microorganisms. It manages the beneficial interaction between the host and commensal bacteria through highly coordinated and regulated stress and immune responses (50). Dysfunction in this signaling may result in intestinal dysbiosis and chronic inflammation, whereby these pathologies can in turn negatively influence intestinal homeostasis to cause diseases (51). Further studies are needed to determine whether the other Ig domain–containing proteins in invertebrates have similar functions and how the Ig domain regulates intestinal microbiota homeostasis and AMPs expression during inflammation. Notably, our results showed independence and interactions between the intestine and hemocytes during different phases of intestinal inflammation. During the early phase, intestinal cells expressing EsIgLectin may directly regulate microbiota homeostasis and inflammation. At the later phase, intestinal walls are damaged and become thinner, leading to the release of intestinal bacteria into the hemolymph (Fig. 10D), to stimulate high expression of EsIgLectin in the hemocytes and subsequent secretion, thus entering the damaged intestinal wall. In mammals, Ab delivery to the mucosal surface requires transport across epithelial layers, which is dependent on pIgR recognizing polymerized IgA and IgM, transporting the Abs across the epithelial cells, and then secreting and releasing them into the luminal space (40). From the perspective of the pIgR transport mode for Abs, the core structure of pIgR recognition is the Ig functional domain, which means the transcytosis function of pIgR on the Ig functional domain may be conserved in vertebrates and invertebrates. Considering the open circulatory system of arthropods (41), several Ig domain–containing proteins produced by hemocytes may be accessible through pIgR to the intestinal lumen at specific time points, to form an interactive immune response network of hemocytes, hemolymph, and intestines. Based on that, we speculate that hemocyte-derived EsIgLectin may execute transcytosis into the intestinal mucosa through EspIgR, which may remedy the deficiency of intestinal endogenous EsIgLectin abundance, to protect crabs from intestinal inflammation.

In conclusion, both abdominal and oral bacterial injection induced high EsIgLectin expressions in hemocytes and the intestine, respectively (Fig. 11A). Soluble EsIgLectin bound to different bacterial strains, promoted bacteria agglutination, inhibited bacterial growth, and regulated bacterial clearance efficiently in both C-type lectin–like domain– and Ig domain–dependent manners (Fig. 11B). Moreover, EsIgLectin bound to EspIgR through the interaction of its Ig domain with the EspIgR Ig1 domain (Fig. 11B). In hemocytes, EsIgLectin regulated bacterial phagocytosis via its cell membrane receptor EspIgR (Fig. 11B). In the intestine, EsIgLectin maintained microbiota homeostasis and protected the crab from bacteria-induced inflammation via intestinal endogenous EsIgLectin (Fig. 11C) and possibly EspIgR-mediated transcytosis of hemocyte-derived EsIgLectin into the intestinal mucosa (Fig. 11D).

We thank the Experimental Platform for Molecular Zoology (East China Normal University, Shanghai, China) for providing the instruments essential for conducting this study.

This work was supported by the Shanghai Rising-Star Program (Grant 20QA1403000 to W.L.) and the National Natural Science Foundation of China (Grant 31972820 to W.L.; Grant 31970490 to Q.W.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

W.L., K. Zhou, and Q.W. designed research; K. Zhou, Y.Q., and Y.S. performed research; K. Zhao and X.N. performed bioinformatics analyses; W.P. and Y.W. contributed new reagent/analytic tools; W.L. and K. Zhou analyzed data; W.L. wrote the paper; and W.L. supervised the study.

The raw data presented in this article have been submitted to the National Center for Biotechnology Information Sequence Read Archive (http://www.ncbi.nlm.nih.gov/bioproject/763732) under accession number PRJNA763732.

The online version of this article contains supplemental material.

Abbreviations used in this article:

CBB

Coomassie brilliant blue

dsEsIgLectin

dsRNA against EsIgLectin

dsEspIgR

dsRNA against EspIgR

EsIgLectin

Eriocheir sinensis (H. Milne-Edwards) (Decapoda: Grapsidae) Ig domain–containing C-type lectin

EspIgR

Eriocheir sinensis polymeric Ig receptor

FISH

fluorescence in situ hybridization

Ig1

first Ig domain

IPTG

isopropyl-β-d-thiogalactopyranoside

LB

Luria–Bertani

NR

nonreducing

pIgR

polymeric Ig receptor

qRT-PCR

quantitative RT-PCR

R

reducing

rEsIgΔCys42, 102

EsIgLectin position 42 and 102 cysteine residue–deleted Ig domain recombinant protein

rEsIgLectinΔCys42, 102

full-length EsIgLectin mutated protein

RNAi

RNA interference

1.
Zelensky
A. N.
,
J. E.
Gready
.
2005
.
The C-type lectin-like domain superfamily.
FEBS J.
272
:
6179
6217
.
2.
Brown
G. D.
,
J. A.
Willment
,
L.
Whitehead
.
2018
.
C-type lectins in immunity and homeostasis.
Nat. Rev. Immunol.
18
:
374
389
.
3.
Gerardo
N. M.
,
B.
Altincicek
,
C.
Anselme
,
H.
Atamian
,
S. M.
Barribeau
,
M.
de Vos
,
E. J.
Duncan
,
J. D.
Evans
,
T.
Gabaldón
,
M.
Ghanim
, et al
2010
.
Immunity and other defenses in pea aphids, Acyrthosiphon pisum.
Genome Biol.
11
:
R21
.
4.
Rao
X. J.
,
X.
Cao
,
Y.
He
,
Y.
Hu
,
X.
Zhang
,
Y. R.
Chen
,
G.
Blissard
,
M. R.
Kanost
,
X. Q.
Yu
,
H.
Jiang
.
2015
.
Structural features, evolutionary relationships, and transcriptional regulation of C-type lectin-domain proteins in Manduca sexta.
Insect Biochem. Mol. Biol.
62
:
75
85
.
5.
Rao
X. J.
,
T.
Shahzad
,
S.
Liu
,
P.
Wu
,
Y. T.
He
,
W. J.
Sun
,
X. Y.
Fan
,
Y. F.
Yang
,
Q.
Shi
,
X. Q.
Yu
.
2015
.
Identification of C-type lectin-domain proteins (CTLDPs) in silkworm Bombyx mori.
Dev. Comp. Immunol.
53
:
328
338
.
6.
Waterhouse
R. M.
,
E. V.
Kriventseva
,
S.
Meister
,
Z.
Xi
,
K. S.
Alvarez
,
L. C.
Bartholomay
,
C.
Barillas-Mury
,
G.
Bian
,
S.
Blandin
,
B. M.
Christensen
, et al
2007
.
Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes.
Science
316
:
1738
1743
.
7.
Wang
X. W.
,
J. X.
Wang
.
2013
.
Diversity and multiple functions of lectins in shrimp immunity.
Dev. Comp. Immunol.
39
:
27
38
.
8.
Zhang
X. W.
,
Y.
Wang
,
X. W.
Wang
,
L.
Wang
,
Y.
Mu
,
J. X.
Wang
.
2016
.
A C-type lectin with an immunoglobulin-like domain promotes phagocytosis of hemocytes in crayfish Procambarus clarkii.
Sci. Rep.
6
:
29924
.
9.
Xia
X.
,
M.
You
,
X. J.
Rao
,
X. Q.
Yu
.
2018
.
Insect C-type lectins in innate immunity.
Dev. Comp. Immunol.
83
:
70
79
.
10.
Williams
A. F.
,
A. N.
Barclay
.
1988
.
The immunoglobulin superfamily—domains for cell surface recognition.
Annu. Rev. Immunol.
6
:
381
405
.
11.
Watson
F. L.
,
R.
Püttmann-Holgado
,
F.
Thomas
,
D. L.
Lamar
,
M.
Hughes
,
M.
Kondo
,
V. I.
Rebel
,
D.
Schmucker
.
2005
.
Extensive diversity of Ig-superfamily proteins in the immune system of insects.
Science
309
:
1874
1878
.
12.
Kang
J. S.
,
T.
Oohashi
,
Y.
Kawakami
,
Y.
Bekku
,
J. C.
Izpisúa Belmonte
,
Y.
Ninomiya
.
2004
.
Characterization of dermacan, a novel zebrafish lectican gene, expressed in dermal bones.
Mech. Dev.
121
:
301
312
.
13.
Yamaguchi
Y.
2000
.
Lecticans: organizers of the brain extracellular matrix.
Cell. Mol. Life Sci.
57
:
276
289
.
14.
Pancer
Z.
,
B.
Diehl-Seifert
,
B.
Rinkevich
,
W. E.
Müller
.
1997
.
A novel tunicate (Botryllus schlosseri) putative C-type lectin features an immunoglobulin domain.
DNA Cell Biol.
16
:
801
806
.
15.
Guillou
F.
,
G.
Mitta
,
C.
Dissous
,
R.
Pierce
,
C.
Coustau
.
2004
.
Use of individual polymorphism to validate potential functional markers: case of a candidate lectin (BgSel) differentially expressed in susceptible and resistant strains of Biomphalaria glabrata.
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
138
:
175
181
.
16.
Pan
G.
,
J.
Bao
,
Z.
Ma
,
Y.
Song
,
B.
Han
,
M.
Ran
,
C.
Li
,
Z.
Zhou
.
2018
.
Invertebrate host responses to microsporidia infections.
Dev. Comp. Immunol.
83
:
104
113
.
17.
Little
T. J.
,
D.
Hultmark
,
A. F.
Read
.
2005
.
Invertebrate immunity and the limits of mechanistic immunology.
Nat. Immunol.
6
:
651
654
.
18.
Zhu
Y.
,
X.
Yu
,
G.
Cheng
.
2020
.
Insect C-type lectins in microbial infections.
Adv. Exp. Med. Biol.
1204
:
129
140
.
19.
Luo
T.
,
H.
Yang
,
F.
Li
,
X.
Zhang
,
X.
Xu
.
2006
.
Purification, characterization and cDNA cloning of a novel lipopolysaccharide-binding lectin from the shrimp Penaeus monodon.
Dev. Comp. Immunol.
30
:
607
617
.
20.
Wei
X.
,
L.
Wang
,
W.
Sun
,
M.
Zhang
,
H.
Ma
,
Y.
Zhang
,
X.
Zhang
,
S.
Li
.
2018
.
C-type lectin B (SpCTL-B) regulates the expression of antimicrobial peptides and promotes phagocytosis in mud crab Scylla paramamosain.
Dev. Comp. Immunol.
84
:
213
229
.
21.
Vaishnava
S.
,
M.
Yamamoto
,
K. M.
Severson
,
K. A.
Ruhn
,
X.
Yu
,
O.
Koren
,
R.
Ley
,
E. K.
Wakeland
,
L. V.
Hooper
.
2011
.
The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine.
Science
334
:
255
258
.
22.
Pang
X.
,
X.
Xiao
,
Y.
Liu
,
R.
Zhang
,
J.
Liu
,
Q.
Liu
,
P.
Wang
,
G.
Cheng
.
2016
.
Mosquito C-type lectins maintain gut microbiome homeostasis.
Nat. Microbiol.
1
:
16023
.
23.
Zhang
Y. X.
,
M. L.
Zhang
,
X. W.
Wang
.
2021
.
C-type lectin maintains the homeostasis of intestinal microbiota and mediates biofilm formation by intestinal bacteria in shrimp.
J. Immunol.
206
:
1140
1150
.
24.
Li
X. J.
,
L.
Yang
,
D.
Li
,
Y. T.
Zhu
,
Q.
Wang
,
W. W.
Li
.
2018
.
Pathogen-specific binding soluble down syndrome cell adhesion molecule (Dscam) regulates phagocytosis via membrane-bound Dscam in Crab.
Front. Immunol.
9
:
801
.
25.
Yang
L.
,
X.
Li
,
X.
Qin
,
Q.
Wang
,
K.
Zhou
,
H.
Li
,
X.
Zhang
,
Q.
Wang
,
W.
Li
.
2019
.
Deleted in azoospermia-associated protein 2 regulates innate immunity by stimulating Hippo signaling in crab.
J. Biol. Chem.
294
:
14704
14716
.
26.
Reichelt
P.
,
C.
Schwarz
,
M.
Donzeau
.
2006
.
Single step protocol to purify recombinant proteins with low endotoxin contents.
Protein Expr. Purif.
46
:
483
488
.
27.
Sun
W.
,
H.
Li
,
Y.
Zhao
,
L.
Bai
,
Y.
Qin
,
Q.
Wang
,
W.
Li
.
2021
.
Distinct vitellogenin domains differentially regulate immunological outcomes in invertebrates.
J. Biol. Chem.
296
:
100060
.
28.
Wang
X. W.
,
J.
Gao
,
Y. H.
Xu
,
J. D.
Xu
,
Z. X.
Fan
,
X. F.
Zhao
,
J. X.
Wang
.
2017
.
Novel pattern recognition receptor protects shrimp by preventing bacterial colonization and promoting phagocytosis.
J. Immunol.
198
:
3045
3057
.
29.
Zhao
Y. H.
,
H.
Li
,
H.
Zhao
,
W. K.
Sun
,
Q.
Wang
,
W. W.
Li
.
2021
.
An ancient interleukin-16-like molecule regulates hemocyte proliferation via integrin β1 in invertebrates.
J. Biol. Chem.
297
:
100943
.
30.
Chapuis
R. M.
,
M. E.
Koshland
.
1974
.
Mechanism of IgM polymerization.
Proc. Natl. Acad. Sci. USA
71
:
657
661
.
31.
Halpern
M. S.
,
M. E.
Koshland
.
1973
.
The stoichiometry of J chain in human secretory IgA.
J. Immunol.
111
:
1653
1660
.
32.
Guo
Z.
,
E.
Lucchetta
,
N.
Rafel
,
B.
Ohlstein
.
2016
.
Maintenance of the adult Drosophila intestine: all roads lead to homeostasis.
Curr. Opin. Genet. Dev.
40
:
81
86
.
33.
Li
K.
,
W.
Guan
,
G.
Wei
,
B.
Liu
,
J.
Xu
,
L.
Zhao
,
Y.
Zhang
.
2007
.
Phylogenetic analysis of intestinal bacteria in the Chinese mitten crab (Eriocheir sinensis).
J. Appl. Microbiol.
103
:
675
682
.
34.
Zhang
M.
,
Y.
Sun
,
L.
Chen
,
C.
Cai
,
F.
Qiao
,
Z.
Du
,
E.
Li
.
2016
.
Symbiotic bacteria in gills and guts of Chinese mitten crab (Eriocheir sinensis) differ from the free-living bacteria in water.
PLoS One
11
:
e0148135
.
35.
Wong
A. C.
,
J. M.
Chaston
,
A. E.
Douglas
.
2013
.
The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis.
ISME J.
7
:
1922
1932
.
36.
Kim
S. H.
,
W. J.
Lee
.
2014
.
Role of DUOX in gut inflammation: lessons from Drosophila model of gut-microbiota interactions.
Front. Cell. Infect. Microbiol.
3
:
116
.
37.
Lhocine
N.
,
P. S.
Ribeiro
,
N.
Buchon
,
A.
Wepf
,
R.
Wilson
,
T.
Tenev
,
B.
Lemaitre
,
M.
Gstaiger
,
P.
Meier
,
F.
Leulier
.
2008
.
PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling.
Cell Host Microbe
4
:
147
158
.
38.
Paredes
J. C.
,
D. P.
Welchman
,
M.
Poidevin
,
B.
Lemaitre
.
2011
.
Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection.
Immunity
35
:
770
779
.
39.
Ayyaz
A.
,
H.
Li
,
H.
Jasper
.
2015
.
Haemocytes control stem cell activity in the Drosophila intestine.
Nat. Cell Biol.
17
:
736
748
.
40.
Wei
H.
,
J. Y.
Wang
.
2021
.
Role of polymeric immunoglobulin receptor in IgA and IgM transcytosis.
Int. J. Mol. Sci.
22
:
2284
.
41.
McGaw
I. J.
2005
.
The decapod crustacean circulatory system: a case that is neither open nor closed.
Microsc. Microanal.
11
:
18
36
.
42.
Li
J.
,
J.
Bi
,
P.
Zhang
,
Z.
Wang
,
Y.
Zhong
,
S.
Xu
,
L.
Wang
,
B.
Li
.
2021
.
Functions of a C-type lectin with a single carbohydrate-recognition domain in the innate immunity and movement of the red flour beetle, Tribolium castaneum.
Insect Mol. Biol.
30
:
90
101
.
43.
Senghoi
W.
,
P.
Runsaeng
,
P.
Utarabhand
.
2017
.
FmLC5, a putative galactose-binding C-type lectin with two QPD motifs from the hemocytes of Fenneropenaeus merguiensis participates in shrimp immune defense.
J. Invertebr. Pathol.
150
:
136
144
.
44.
Hammes
H. P.
,
V.
Kiefel
,
H.
Laube
,
K.
Federlin
.
1990
.
Impaired agglutination of IgM resulting from non-enzymatic glycation in diabetes mellitus.
Diabetes Res. Clin. Pract.
9
:
37
42
.
45.
Davis
A. C.
,
K. H.
Roux
,
J.
Pursey
,
M. J.
Shulman
.
1989
.
Intermolecular disulfide bonding in IgM: effects of replacing cysteine residues in the mu heavy chain.
EMBO J.
8
:
2519
2526
.
46.
Stuart
L. M.
,
R. A.
Ezekowitz
.
2008
.
Phagocytosis and comparative innate immunity: learning on the fly.
Nat. Rev. Immunol.
8
:
131
141
.
47.
Raghavan
M.
,
P. J.
Bjorkman
.
1996
.
Fc receptors and their interactions with immunoglobulins.
Annu. Rev. Cell Dev. Biol.
12
:
181
220
.
48.
Ogden
C. A.
,
R.
Kowalewski
,
Y.
Peng
,
V.
Montenegro
,
K. B.
Elkon
.
2005
.
IGM is required for efficient complement mediated phagocytosis of apoptotic cells in vivo.
Autoimmunity
38
:
259
264
.
49.
Shen
L.
1992
.
Receptors for IgA on phagocytic cells.
Immunol. Res.
11
:
273
282
.
50.
Fanning
S.
,
L. J.
Hall
,
D.
van Sinderen
.
2012
.
Bifidobacterium breve UCC2003 surface exopolysaccharide production is a beneficial trait mediating commensal-host interaction through immune modulation and pathogen protection.
Gut Microbes
3
:
420
425
.
51.
Yoo
J. Y.
,
M.
Groer
,
S. V. O.
Dutra
,
A.
Sarkar
,
D. I.
McSkimming
.
2020
.
Gut microbiota and immune system interactions.
Microorganisms
8
:
1587
.

The authors have no financial conflicts of interest.

Supplementary data