Novel Pattern Recognition Receptor Protects Shrimp by Preventing Bacterial Colonization and Promoting Phagocytosis

Xian-Wei Wang, Jie Gao, Yi-Hui Xu, Ji-Dong Xu, Zhen-Xu Fan, Xiao-Fan Zhao and Jin-Xing Wang
J Immunol April 15, 2017, 198 (8) 3045-3057; DOI: https://doi.org/10.4049/jimmunol.1602002

Abstract

The recognition of pathogen-associated molecular patterns is accomplished by the recognition modules of pattern recognition receptors (PRRs). Leucine-rich repeats (LRRs) and C-type lectin-like domain (CTLD) represent the two most universal categories of recognition modules. In the current study, we identified a novel soluble and bacteria-inducible PRR comprising LRRs and a CTLD from the hepatopancreas of kuruma shrimp Marsupenaeus japonicus and named it Leulectin. The module arrangement of Leulectin is unique among all organisms. Both modules, together with the whole molecule, protected shrimp against Vibrio infection. By screening the pathogen-associated molecular patterns that shrimp might encounter, Leulectin was found to sense Vibrio flagellin through the LRRs and to recognize LPS through CTLD. The LRR–flagellin interaction was confirmed by pull-down and far-Western assays and was found to rely on the fourth LRR of Leulectin and the N terminus of flagellin. The recognition of LPS was determined by the long loop region of CTLD in a calcium-independent manner. By sensing the flagellin, LRRs could prevent its attachment to shrimp cells, thereby inhibiting Vibrio colonization. With the ability to recognize LPS, CTLD could agglutinate the bacteria and promote hemocytic phagocytosis. Our study clearly showed the division of labor and the synergy between different recognition modules and provided new insights into the concept of pattern recognition and the function of soluble PRRs in the antibacterial response.

Introduction

Innate pathogen recognition relies primarily on the germline-encoded pattern recognition receptors (PRRs) to recognize the so-called pathogen-associated molecular patterns (PAMPs), such as LPS, lipoteichoic acid, and CpG DNA, which are present on a wide range of pathogens, including bacteria, fungi, virus, and parasites, but are not expressed by host cells (1). Recognition is accomplished through the protein modules of the PRRs. For example, leucine-rich repeat (LRR) domains and carbohydrate-recognition domains (also referred to as C-type lectin-like domain [CTLD]) are most frequently involved in pattern recognition (2).

LRR is a highly conserved sequence found in many proteins of diverse functions. The LRR motif usually contains 20–30 residues that are rich in leucine and will fold into a β strand–α helix structure (3). An LRR domain is built from two or more tandem LRRs to generate a solenoid curve appropriate for protein–protein interaction. In addition to participating in signal transduction, tissue development, cell adhesion, and many other biological processes (4), LRR is the basic recognition module of typical PRRs, such as the vertebrate TLRs, NOD-like receptors, and plant resistance genes (5–7). In jawless vertebrates, the arrangement of LRRs in variable lymphocyte receptors (VLRs) can generate great diversity (8). The PAMPs recognized by these LRR-containing proteins include LPS, dsRNA, zymosan, and bacterial flagellin (9). Although the LRR-containing PRRs are mostly membrane or cytoplasmic proteins, there were some reports that LRR proteins are secreted out of the cells to mediate immune recognition. For example, two LRR proteins, LRIM1 and APL1C, from mosquito Anopheles gambiae circulate in the plasma and form a heterodimeric complex. They protect the host against Plasmodium infection, probably by directing thioester containing protein 1 deposition on parasite surface (10). An LRR protein named Leureptin from Manduca sexta could target bacteria by binding LPS and attaching to the hemocyte membrane upon immune challenge (11). These cases indicate the involvement of soluble LRRs in immune recognition.

CTLD is a double-loop fold and is stabilized by two pairs of disulfide bridges, which are formed by the four most conserved cysteine residues. The inner loop is involved in carbohydrate recognition (12). However, the recognition spectrum of CTLDs is not limited to carbohydrates; it includes proteins, nucleic acids, and even inorganic compounds (13). With the help of CTLDs, many CTLD-containing proteins recognize invading microbes and trigger the corresponding immune responses (14). For example, mammalian mannose-binding lectin is primarily responsible for recognizing the invading microbes in the plasma and can activate the lectin pathway of the complement system, or it can opsonize and clear these particles (15). This protein belongs to the collectin family, a subgroup of CTLD-containing proteins. The CTLD-containing proteins are usually soluble in invertebrates. The genome of mosquito Aedes aegypti encodes >30 galactose-specific binding C-type lectins, and some members of this are critical factors for West Nile virus and dengue virus infection, as well as for gut microbiota homeostasis (16–18). Our previous study also demonstrated that a large portion of the Marsupenaeus japonicus C-type lectin family is soluble in the circulating plasma and plays vital roles in mediating hemocytic phagocytosis, hemolymph microbiota homeostasis, and white spot syndrome virus (WSSV) entry (19–21). These studies indicated that CTLD represents an important class of pattern recognition modules in vertebrates and invertebrates.

Some PRRs, especially soluble ones, contain more than one pattern recognition module of either the same or a different category. For example, many invertebrate C-type lectins harbor two or more CTLDs (22, 23). It is very rare to see the combination of LRRs and CTLD. In the current study, we identified a soluble protein that contains LRRs and a CTLD in the shrimp hepatopancreas and hemolymph and designated it Leulectin. To our knowledge, no such arrangement has been observed before. To study the function of Leulectin, we prepared recombinant proteins containing the two modules and the whole molecule and found that all three recombinant proteins had a protective role against Vibrio infection, suggesting that both the LRRs and CTLD might be involved in pathogen recognition and resistance. The ligands of both modules were determined, and their recognition was characterized in detail. Importantly, by clearly showing the division of labor and synergy between the two modules of Leulectin, this study highlighted the importance of the pattern-recognizing capacity of PRRs and increased our understanding of the significance of tandem expression of pattern recognition modules.

Materials and Methods

Animals and microorganisms

Healthy kuruma shrimp (6–7 g each) were obtained from a farm in Rizhao, Shandong, China and maintained in air-pumped artificial seawater (30 ppt at 25°C) for a week before the experiment. The shrimp were fed with a commercial feed daily.

Vibrio anguillarum (ATCC 43305), V. harveyi (ATCC 33842), V. alginolyticus (ATCC 33840), V. parahaemolyticus (Shandong University Microorganism Culture Collection 210340), Aeromonas hydrophila (isolated from diseased fish), and Escherichia coli DH5α were cultured, collected, and resuspended in sterile PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 [pH 7.4]). The bacterial counts were determined by plating the suspension onto agar plates. WSSV (China isolate) inoculum was plasma from a moribund WSSV-infected kuruma shrimp, diluted by PBS, and stored at −80°C. The virus copy number was determined using real-time PCR, according to a previously described method (24).

Immune stimulation and sample collection

Total RNA was extracted using TRIzol Reagent (Invitrogen). Protein samples were collected by homogenizing the tissues in PBS (with 0.5 mM PMSF) and centrifuging at 12,000 × g for 10 min to isolate the supernatant. For immune challenge, the shrimp were infected by i.m. injection at the fourth abdominal segment with the bacterial (1 × 105 CFU per shrimp, 50 μl) or viral (105 copies per shrimp, 50 μl) inocula. PBS (50 μl) was used as the control. Total RNA was collected from hepatopancreas at specific time points postchallenge. At least five shrimp were used for each sample. First-strand cDNA was synthesized with a BioTeke Reverse Transcriptase Kit (Beijing, China), according to the manufacturer’s instructions.

Characterization of the Leulectin sequence

Total RNA from the hepatopancreas of healthy shrimp was sent to the BGI Company (Shenzhen, China) for transcriptome sequencing. A fragment containing a full open-reading frame that encoded a protein with a signal peptide, LRRs, and a CTLD was identified from the sequencing results. The full-length cDNA was then amplified by a pair of gene-specific primers (LeulecGSF/R; Table I) and resequenced to confirm the correct sequence. This gene was designated as Leulectin (leucine-rich repeats containing C-type lectin). The signal peptide was predicted by SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/), and the domain architecture was predicted by SMART (http://smart.embl-heidelberg.de/).

View this table:
Table I. Primers used in this study

Analysis of the expression profiles of Leulectin

The distribution of Leulectin mRNA was determined by RT-PCR, using a pair of primers (LeulecRTF/R; Table I) to generate a 174-bp fragment. A 184-bp fragment of β-actin was amplified with the primers β-actinRTF/R (Table I) as an internal reference. PCR was performed using the following procedure: 94°C for 3 min; multiple cycles of 94°C for 15 s, 54°C for 30 s, and 72°C for 30 s; and 72°C for 5 min. The distribution of Leulectin protein was studied by Western blotting. The protein samples were analyzed by 12.5% SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with 3% nonfat milk in PBS, the membrane was incubated with anti-Leulectin serum (1:300; see source below) and then with HRP-conjugated goat anti-rabbit secondary Ab (1:10,000; Zhongshan, Beijing, China). The target band was visualized by the oxidation of 4-chloro-1-naphthol. The expression of β-actin was detected as the reference.

The temporal expression of Leulectin post–V. anguillarum was determined by real-time RT-PCR using the CFX96 Real-Time System and iQ SYBR Green Supermix (both from Bio-Rad, Hercules, CA). The primers used were the same as for RT-PCR. The reaction conditions were 94°C for 3 min, followed by 40 cycles of 94°C for 10 s and 60°C for 1 min, and then melting from 65 to 95°C. The data were calculated using the 2−ΔΔCt method and normalized to the samples from untreated shrimp. Three independent experiments were performed, and the results represent the mean ± SD.

To determine whether Leulectin was soluble in plasma, the target protein was first enriched to a detectable level using anti-Leulectin Ab. Generally, 100 μg of purified anti-Leulectin Abs (source see below) was coupled with 60 mg of CNBr-activated Sepharose 4B (Amersham Biosciences) with gentle agitation at 25°C for 1 h in coupling buffer (0.1 M NaHCO3, 0.5 M NaCl [pH 8.3]). The resin was washed by coupling buffer and blocked in 0.1 M Tris-HCl (pH 8) at 25°C for 2 h. After four washes alternating between acetic acid buffer (0.1 M NaAC, 0.5 M NaCl [pH 4]) and Tris buffer (0.1 M Tris-HCl, 0.5 M NaCl [pH 8]), the resin was added to shrimp plasma to capture the soluble Leulectin. The mixture was rotated gently at 20°C for 1 h. The resin was washed with Tris buffer five times. Bound proteins were eluted with 0.1 M glycine (pH 2.5) and neutralized by 1 M Tris-HCl (pH 8.5). The eluted samples were detected by Western blotting with anti-Leulectin Abs.

Expression and purification of recombinant proteins

Recombinant LRRs, CTLD, and the whole molecule (LRRs linked to CTLD [LLC]) were expressed after induction with 0.2 mM isopropyl Β-d-thiogalactopyranoside at 28°C for 6 h. The His-tagged proteins were purified using Ni-NTA His Binding resin (Novagen), eluted by 250 mM imidazole, and dialyzed in PBS (with 5% glycerol) to remove the imidazole. The pET32a(+) vector was used to produce a His-Trx (thioredoxin) tag as a control. For the expression of mutated proteins, conventional methods were used to construct the recombinant vector, using the primers listed in Table I. For those proteins that would be applied in vivo, an additional wash with excess 0.1% Triton X-114 at 4°C was performed before the final elution to remove the contamination of endotoxins (25). The endotoxin content of the treated proteins before in vivo application was assessed using a LPS detection kit (GenScript, Nanjing, China) based on the Limulus amebocyte lysate chromogenic assay.

Antiserum preparation and Ab purification

Purified recombinant LLC (rLLC; 2 mg/ml, 1 ml) was mixed with CFA (Sigma) thoroughly and used to immunize a New Zealand white rabbit. The immunization was repeated 25 d later, with adjuvant changed from complete to incomplete. After testing the titer and specificity of the anti-Leulectin Ab by bleeding 7 d later, the rabbit was killed to collect the antiserum.

Purification of the anti-Leulectin Ab was performed using a previously described method, with slight modifications (20). Generally, the antiserum was diluted in 0.15 M NaCl, 20 mM Na2HPO4 (pH 8), filtered through a 0.45-μm filter, and applied to a Protein A resin column (GenScript). The column was washed with the diluting buffer thoroughly and eluted using 0.1 M glycine (pH 2.5). The eluted Abs were neutralized immediately with 1/10 volume of 1 M Tris-HCl (pH 8.5) and then dialyzed in PBS overnight at 4°C. An Ab that did not recognize any shrimp protein was processed similarly as the control IgG.

Survival rate assessments

To determine the possible function of Leulectin in resisting bacterial infection, 2 μg of rLLC was mixed with 5 × 105 CFU of V. anguillarum and injected into shrimp. A total of 30 shrimp was injected, with 36 shrimp injected with the mixture of tag and V. anguillarum as controls. The numbers of surviving shrimp were recorded for 6 d for both groups. The anti-Leulectin Ab was used to neutralize the endogenous protein. Each shrimp was injected with 2 μg of Ab or IgG, and 5 × 105 CFU of V. anguillarum was injected 2 h later. The survival rates were then determined. A rescue experiment was performed by injecting different amounts (1 or 4 μg) of rLLC together with 2 μg of anti-Leulectin Ab, and 5 × 105 CFU of V. anguillarum was injected 2 h later.

Determination of innate immune parameters

The treatment was performed as above with the exception that the bacteria inocula for each shrimp were 105 CFU of V. anguillarum. At 12 h postinfection, shrimp hemolymph was drawn out and mixed with an equal volume of cold anticoagulant (400 mM NaCl, 27 mM trisodium citrate, 100 mM glucose [pH 7.3]). A drop of the mixture was used to determine the total hemocyte count (THC) under the microscope using a hemocytometer. The rest of the mixture was centrifuged at 800 × g for 10 min to separate the plasma and hemocytes. The plasma was centrifuged again at 20,000 × g for 20 min to get the clear supernatant. Plasma phenoloxidase (PO) activity was determined by measuring the conversion of l-3,4-dihydroxyphenylalanine to dopachrome. Plasma (50 μl) was added to 200 μl of the l-3,4-dihydroxyphenylalanine solution (3 mg/ml). The mixture was incubated at 16°C for 15 min, and absorbance at 490 nm was recorded. PO activity was calculated as ΔA490. Plasma lysozyme activity was determined by monitoring the lysis of Micrococcus luteus lyophilized powder (Sigma). The reagent was dissolved in 100 mM PBS (pH 6.4) to a final OD ∼ 0.3 at 570 nm. Fresh plasma (5 μl) was added to 200 μl of the suspension. The mixture was placed at 37°C for 15 min. The absorbance was recorded before (A0) and after (A) the incubation; lysozyme activity was calculated as (A0 − A)/A.

Preparation of the ligands for screening

The carbohydrate ligands LPS, peptidoglycan, lipoteichoic acid, mannan, and dextran were purchased from Sigma (Table II) and dissolved in distilled water to a final concentration of 40 μg/ml.

The protein ligands used were expressed under isopropyl β-d-thiogalactopyranoside induction, purified by affinity chromatography using GST resin (GenScript), dialyzed in PBS, and adjusted to a final concentration of 50 μg/ml. The primers used for the recombinant expression are listed in Table II.

Bacterial genomic DNA was obtained using a MagExtractor-Genome-NPK1000 Kit (Toyobo); the virulent plasmids were extracted using a SanPrep Colum Plasmid Mini-Prep Kit (Sangon, Shanghai, China). The concentration of the DNA was adjusted to 5 μg/ml.

ELISA

The 96-well plates were coated with the ligands. For carbohydrate ligands, 50 μl of the solution was used to coat the 96-well plates, and the plates were air-dried at 37°C; for the protein ligands, the plates were coated with 100 μl of the solution overnight at 4°C. The coating by DNA was performed according to a previous method, with some modification: the plates were precoated by polylysine in PBS (50 μg/ml) for 2 h at 28°C, washed, and then coated by 50 μl of the DNA solution overnight (26, 27).

All wells were then blocked by 5% BSA in PBS at 37°C for 2 h. After washing three times with PBS with 0.05% Tween 20 (PBST), recombinant LRRs (rLRRs), recombinant CTLD (rCTLD), rLLC, or the tag was added in a volume of 100 μl and a concentration of 20 pM. The plates were maintained at 28°C for 3 h. After washing four times with PBST, 100 μl of the anti-His tag Ab dilution (1:1000 in PBS) was added to each well and incubated at 37°C for 1 h. Then plates were washed with PBST four times. The alkaline phosphatase–conjugated horse anti-mouse secondary Ab (1:10,000 in PBS; Zhongshan) was added in a volume of 100 μl. Four additional washings with PBST were performed. Finally, 50 μl of the p-nitro-phenyl phosphate (1 mg/ml in 10 mM diethanolamine with 0.5 mM MgCl2) was added to each well. The incubation was maintained for 30 min at 25°C, and absorbance was measured using a BioTek plate reader at 405 nm. The data are presented as the mean ± SD derived from at least three independent tests. The difference was determined by one-way ANOVA, followed by the Tukey multiple-comparison test. A significant difference was accepted at p < 0.05.

The dose-dependent recognition of LPS by rCTLD was studied by ELISA in a similar manner. For experiments requiring CaCl2 or EDTA, the two reagents were added to the incubations of proteins and ligands to a final concentration of 5 mM.

Pull-down assay

The pull-down assay was performed to check the interaction between V. anguillarum flagellin A (FlaA) and Leulectin. Generally, the purified recombinant FlaA (rFlaA; 20 μg) was incubated with rLRRs, rCTLD, or rLLC (20 μg) in a total volume of 1 ml PBS with gentle rotation at 4°C for 3 h. GST resin (20 μl) was added to the mixture and incubated for another 1 h at 4°C. The resin was washed five times with PBS by centrifugation at 3000 × g for 2 min each. Glutathione solution (10 mM, 20 μl) was added to elute the proteins. The samples were analyzed by SDS-PAGE and stained by Coomassie brilliant blue R250. The GST tag and His-Trx tag were used as controls. The interactions between other recombinant proteins were performed similarly.

For the pull-down of native Leulectin, rFlaA (4 μg) was incubated with 20 ml of the plasma from the bacterial-challenged shrimp overnight. The rFlaA–Leulectin complex was captured using 20 μl of GST resin, washed, eluted using 50 μl of glutathione solution, and analyzed by Western blotting.

Far-Western assay

The far-Western assay was performed according to a previous method, with some modifications (28). Briefly, 2 μg of rFlaA was separated by 12.5% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was washed with denaturation buffer (6 M guanidine-HCl in basic buffer [20 mM HEPES (pH 7.5), 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.1% Triton X-100, 5% glycerol]) at 4°C for 10 min with gentle agitation. Half of the volume of the denaturation buffer was replaced with the basic buffer and the wash was continued for another 10 min. Serial dilution and washing were performed five more times until the concentration of guanidine-HCl reached 90 mM. Then basic buffer was used to wash the membrane for 10 min. The blocking buffer (3% nonfat milk in basic buffer) was added to block the membrane for 2 h. Then rLRRs, rCTLD, rLLC (1 μg), or the shrimp plasma containing the native Leulectin (5 ml) was added to the interaction buffer (1% milk in basic buffer). After rotation at 4°C overnight, PBST was used to wash off the noninteracting proteins, and the bound proteins were analyzed by Western blotting, as described above, and detected by anti-Leulectin Abs.

Flagellin-attachment assay

rFlaA (5 μg), together with the tested proteins (5 μg), was injected into shrimp. An hour later, the hepatopancreas was fixed by Davidson’s AFA fixative (30% ethanol, 22% formalin, and 11.5% acetic acid) for 48 h and subjected to paraffin sectioning. Meanwhile, hemocytes were collected, spread onto glass slides, and fixed with 4% paraformaldehyde. Slides were blocked using 3% BSA and incubated with 1:500 diluted anti-GST serum overnight at 4°C. The slides were washed with PBS and incubated with 1:1000 diluted Alexa Fluor 488 (Molecular Probes) at 37°C for 1 h. The nuclei were stained with DAPI. The slides were observed under a fluorescence microscope (Olympus BX51).

Bacterial-colonization assay

Three bacterial strains that had been confirmed as resistant to several antibiotics were used to check whether Leulectin could influence the bacterial colonization in the shrimp hepatopancreas. About 2 × 105 CFU V. harveyi (ATCC 33842) or V. alginolyticus (ATCC 33840) or 2 × 106 CFU V. tasmaniensis (separated from shrimp intestines) was mixed with 4 μg of rLRRs or the mutated protein rLRRΔ4 and injected into shrimp. The hepatopancreases were homogenized in PBS with 0.45 M NaCl (mPBS) 24 h later. The homogenate was plated onto Luria-Bertani agar plates (3% NaCl, containing specific antibiotics) to determine the colony number. V. harveyi and V. alginolyticus colonies were also randomly picked out for PCR identification using specific primers for FlaA (Table I).

Bacterial-agglutination assay

Overnight-cultured V. anguillarum was labeled with 2 mg/ml of FITC for 2 h, washed, and resuspended in TBS to a final OD600 of 0.5. Equal volumes (30 μl) of bacterial suspension and recombinant protein solutions in TBS (final concentrations from 1.875 to 240 μg/ml) were mixed and maintained at 28°C for 1 h, and agglutination was observed under a fluorescence microscope. For the LPS-inhibitory test, 20 μg of LPS was added to the incubation.

Bacterial-binding (inhibitory) assay

To test whether rCTLD or the mutated proteins could bind to V. anguillarum, the proteins (4 μg) were incubated with 108 CFU overnight-cultured bacteria in 1 ml of PBS at 25°C for 30 min with gentle agitation. The bacteria were washed with 1 ml of PBST three times, resuspended in 50 μl of PBS, and subjected to Western blotting using anti-His Ab (1: 2000; Zhongshan). For the LPS-inhibitory test, 20 μg of LPS was incubated with rCTLD and mutated rCTLD at 25°C for 1 h with rotation. Thereafter, binding assay was performed as described above.

Phagocytosis assay

Overnight-cultured V. anguillarum was heat killed and labeled with FITC. The bacterial suspension (1 × 108 microbes per milliliter, 1 ml) in mPBS was mixed with 100 μg of recombinant proteins to ensure full coating. The bacteria were pelleted and washed three times with mPBS by centrifugation. The suspension was adjusted to 5 × 109 microbes per milliliter.

For the in vivo analysis, the bacterial suspension (20 μl) was injected into shrimp. The hemocytes were collected 40 min later and resuspended in 100 μl of mPBS. Flow cytometry was performed to detect the hemocytic phagocytosis using CellQuest software (Becton Dickinson). Ten thousand cells were counted for each sample, which was from 10 shrimp. For the in vitro analysis, 2 × 105 hyaline cells and semigranular cells were mixed with 5 × 106 coated labeled microbes in a total volume of 50 μl of mPBS. The mixture was incubated for 1 h in dark at 25°C, with the exception of the blank group, which was maintained on ice. Thereafter, the hemocytes were obtained by centrifugation at 500 × g for 8 min, washed, and resuspended in 50 μl of mPBS. Phagocytosis was detected by flow cytometry. Two thousand cells were counted for each sample. For the in vivo and in vitro analyses, extracellular fluorescence was quenched by adding 1 μl of 0.5% trypan blue to the cell suspension. The experiments were repeated three times.

Separation of shrimp hemocytes

The separation of shrimp hemocytes was performed according to a previously reported protocol, with slight modifications (29). Generally, the hemolymph was collected and mixed with cold anticoagulant at a ratio of 2:1. The mixture (5 ml) was immediately loaded onto 18 ml of continuous gradient of 65% Percoll (GE Pharmacia) in 0.45 M NaCl, which was pregenerated by centrifugation at 50,000 × g for 1 h. Different classes of hemocytes were obtained by centrifugation at 2000 × g for 20 min. The band containing hyaline cells and semigranular cells was collected carefully with a Pasteur pipette, washed and resuspended in mPBS, and immediately subjected to phagocytosis analysis.

Results

Leulectin is a novel PRR with unique domain architecture

The full-length cDNA of Leulectin was 1310 bp, with an open-reading frame of 1185 bp, encoding a polypeptide of 394 residues (GenBank accession number AFJ59945, https://www.ncbi.nlm.nih.gov/protein/387165442/). The Leulectin protein contained a signal peptide, six LRRs, and a CTLD (Fig. 1A, 1B). The LRRs of Leulectin showed some similarity to an LRR-containing G protein–coupled receptor 4 from Menidia estor (ACS45391, 43% identity) and VLR from Petromyzon marinus (AAT70334, 42% identity). Notably, the latter protein is the AgR in jawless vertebrates, with LRR as the basic recognition module (8). A phylogenetic analysis of the LRRs of Leulectin and some representative LRR-containing proteins that were proved to participate in the immune response was performed, and the result again suggested the relatively close relationship between Leulectin and VLRs (Fig. 1C). The homology with VLR suggested that Leulectin LRRs might be involved in immune recognition. Leulectin contained a typical CTLD, with the additional two cysteine residues conserved in crustacean CTLDs. No glycosylation sites were predicted in CTLD. The mature protein had a predicted molecular mass of 41.5 kDa and a predicted isoelectric point of 4.32.

FIGURE 1.
FIGURE 1.

Sequence information for Leulectin. (A) The cDNA sequence of Leulectin. The sequence was subjected to online SMART analysis. The amino acids are numbered. The signal peptide is in italics, the LRRs module is boxed and the CTLD module is underlined. (B) Schematic illustration of Leulectin’s structure. The signal peptide, LRRs, CTLD, and LLR are illustrated. Numbers in the LRRs indicate the single repeat. The three conserved disulfide bridges of CTLD are shown. The sites for mutation of the CTLD are also labeled under the domain. (C) Phylogenetic analysis of the LRRs of Leulectin and some representative LRR-containing proteins. The neighbor-joining phylogenetic tree was built by MEGA 6, with bootstrap of 1000.

Leulectin responds to Vibrio infection and is soluble in plasma

Leulectin mRNA was primarily expressed in the hepatopancreas, with much lower levels in hemocytes, gills, stomach, and intestines. In addition, the protein was only detected in hepatopancreas (Fig. 2A). To check whether Leulectin was involved in the immune response, its expression was studied after challenge with diverse pathogens. The results showed that Leulectin could respond to stimulation by bacteria, especially pathogenic bacteria (Vibrio), but not to the serious shrimp virus WSSV. V. anguillarum induced Leulectin expression most effectively (Fig. 2B). The temporal expression of Leulectin after V. anguillarum challenge was then studied. As shown in Fig. 2C, the expression of Leulectin showed an early decrease and then an obvious increase (∼5-fold compared with control) from 6 to 24 h postchallenge. The expression of Leulectin then recovered until 48 h postinfection. Its expression level in circulating plasma also was evaluated by immunoprecipitation enrichment. The results showed that Leulectin was a soluble protein, and V. anguillarum invasion could influence its synthesis and secretion into plasma, with a visibly obvious induction at 24 h postinfection (Fig. 2D). This suggested that Leulectin might function not only in the hepatopancreas, but also in the whole body by spreading through the hemolymph.

FIGURE 2.
FIGURE 2.

Leulectin was induced by Vibrio infection. (A) Tissue distribution of Leulectin. RNA and protein samples were extracted from healthy shrimp. Expression was studied by RT-PCR and Western blotting with β-actin as the internal reference. Each sample was from at least three animals, and the data are representative of two independent repeats. (B) Pathogen specificity of Leulectin. Shrimp were infected by different bacterial strains (105 CFU) or WSSV (105 copies). RNAs in the hepatopancreas were extracted 12 h later. RT-PCR was performed. Data are representative of two independent repeats, with at least five shrimp for each sample. (C) Expression profiles of Leulectin mRNA after V. anguillarum infection in the hepatopancreas. RNA was extracted at each time point. Quantitative RT-PCR was conducted to check the expression of Leulectin in each sample, with β-actin as the reference. The expression level was normalized to that in untreated shrimp. Results are shown as the mean ± SD. Three independent repeats were performed with at least five shrimp for each sample. (D) Expression profiles of Leulectin in plasma. The Leulectin Abs were coupled with CNBr-activated Sepharose 4B beads to immunoprecipitate soluble Leulectin in plasma. The immunoprecipitates were analyzed by Western blotting with anti-Leulectin Abs. The data are representative of two independent experiments. **p < 0.01, *p < 0.05, Student t test.

Leulectin protects shrimp from Vibrio infection

The domain architecture and expression profiles suggested a possible role for Leulectin during bacterial infection; therefore, recombinant proteins, including the full-length Leulectin (LLC) and the two modules (LRRs and CTLD), were expressed, purified, and applied in vivo to investigate their functions in bacterial resistance (Fig. 3A). The endotoxin level of these proteins was <3 EU/mg, and this was lower than the standard for some pharmaproteins administered in humans (25). Thus, the potential interference from endotoxin contamination to protein was excluded. As shown in Fig. 3B, rLLC could rescue the shrimp from death caused by V. anguillarum infection. The protective effect was obvious from the very beginning. The survival rate was ∼80% in the rLLC group in the first 2 d, whereas it was only ∼50 and 30% in the control groups. Although much less death occurred at the late stage, the survival rate for the rLLC group was always significantly higher than for the control group because of the protective effect of rLLC at the early stage of infection. In addition, both modules showed protective roles in the resistance against V. harveyi infection but with weaker abilities than the full protein (Fig. 3C).

FIGURE 3.
FIGURE 3.

Protecting host from Vibrio infection by Leulectin. (A) Recombinant expression and purification of Leulectin and its two modules. The proteins were expressed from the pET32a (+) vector in Rosetta (DE3) cells and purified by affinity chromatography. (B) Protective role of rLLC against V. anguillarum. Shrimp were injected with a mixture of rLLC (2 μg) and V. anguillarum (5 × 105 CFU). The survival rate was recorded for 6 d. (C) Protective role of rLLC, rLRRs, and rCTLD against V. harveyi. Shrimp were injected with the mixture of each protein (2 μg) and V. harveyi (5 × 105 CFU), and the survival rate was recorded. (D) Purification of Leulectin Abs. The antiserum was generated by immunizing a rabbit with rLLC. Abs were purified from the antiserum using Protein A resin. (E) Suppression of the host resistance against V. anguillarum upon neutralization of endogenous Leulectin using Abs. Leulectin Abs (2 μg) were injected into shrimp 2 h before the bacterial infection (5 × 105 CFU). The survival rate was recorded. (F) Rescue of the neutralization by the recombinant protein. Different amounts of rLLC (1 or 4 μg) were injected together with Leulectin Ab. The infection was performed as above. (G) Influence on innate immune responses by Leulectin. Three parameters (THC, plasma PO activity, and plasma lysozyme activity) were determined following the different treatments. For each group, six shrimp were used. The results are expressed as the mean ± SEM (for THC) or mean ± SD (for PO activity and lysozyme activity) derived from three independent repeats. (H) Morphological analysis showing the protective role of rLLC. Treated shrimp hepatopancreases were sectioned and stained with H&E for histological examination. B, B cells; DHT, damage to the hepatopancreatic tube; HE, hemocyte encapsulation; S, sloughing of hepatopancreatic cells. Data are representative of two independent repeats. Scale bars, 50 μm. For the survival rates, results were analyzed by the log-rank (Mantel–Cox) test using GraphPad Prism. *0.01 < p < 0.05, **0.001 < p < 0.01, Student t test.

The expression of Leulectin could not be silenced by dsRNA application (data not shown); therefore, anti-Leulectin Ab was purified and injected into shrimp hemolymph to neutralize the native protein (Fig. 3D). The results showed that the neutralization of Leulectin significantly suppressed the host resistance against V. anguillarum (Fig. 3E). The survival rate was lower upon bacteria injection after Leulectin neutralization. To confirm the result, rLLC was used to rescue the effect of neutralization. The addition of rLLC to Leulectin-neutralized shrimp increased the survival rate in a dose-dependent manner (Fig. 3F).

In addition to the survival rate, other immune parameters, including THC, plasma PO activity, and plasma lysozyme activity, were noted. The addition of rLLC alleviated the decrease in THC and strengthened the induction of PO activity and lysozyme activity caused by bacterial infection. Conversely, Leulectin neutralization aggravated the decrease in THC and weakened PO lysozyme induction (Fig. 3G). These data further suggested a protective role for Leulectin.

Histological analysis also was performed to confirm the protective role of Leulectin against V. anguillarum. As shown in Fig. 3H, bacterial infection led to hemocyte encapsulation (infiltration) (H&E) at 6 h postinfection, followed by sloughing of the hepatopancreatic cells and damage to the hepatopancreatic tube at 48 h postinfection. These effects are the typical histopathology of a bacterially infected shrimp hepatopancreas. However, the shrimp treated with V. anguillarum plus rLLC exhibited healthy tissue morphology after treatment. Taken together, these results showed that Leulectin protected shrimp from Vibrio infection.

Leulectin recognizes Vibrio flagellin through its LRRs

Leulectin contains two typical pattern recognition modules; therefore, we screened for the potential PAMPs to which they might bind to reveal the origin of Leulectin’s function. The microorganism carbohydrates (LPS, peptidoglycan, lipoteichoic acid, dextran, and mannan), bacterial critical virulent proteins (flagellin, pilin, porin, and outer membrane proteins), WSSV major envelope proteins (VP28, VP26, and VP24), and bacterial genomic DNA and virulence plasmids were used to represent the usual PAMPs that shrimp might encounter and to determine the binding spectrum of Leulectin. Using an ELISA screen, rLRRs and rLLC were found to bind FlaA, which is a key virulence factor of V. anguillarum. In addition, rCTLD was found to bind LPS (Tables I, II). We concluded that the FlaA- and LPS-binding activity of the whole molecule was due to the LRR and CTLD modules, respectively.

View this table:
Table II. ELISA screening results

Pull-down assays were performed to confirm the interaction between Leulectin and FlaA. As shown in Fig. 4A, rFlaA could pull down rLRRs and rLLC but not rCTLD or the control His-Trx tag. In addition, rFlaA could pull down native Leulectin from shrimp plasma (Fig. 4B). A Far-Western assay was also performed to further confirm the Leulectin–FlaA interaction; the results indicated that rFlaA could bind rLRRs, rLLC, and native Leulectin but not rCTLD (Fig. 4C). These data demonstrated that Leulectin could recognize V. anguillarum FlaA through its LRR module.

FIGURE 4.
FIGURE 4.

Recognition of flagellins by Leulectin LRR modules. (A) Determination of the rLLC/rLRR–FlaA interaction by a pull-down assay. The purified proteins (20 μg each) were incubated in 1 ml of PBS with rotation at 4°C for 3 h. GST resin was then added to pull down the complex containing rFlaA. The His-Trx tag and GST tag were used as controls. Data are representative of two independent repeats. (B) Determination of the interaction between rFlaA and native Leulectin. rFlaA (4 μg) was incubated with 20 ml of shrimp plasma overnight. GST resin was used to pull down the protein complex, which was analyzed by Western blotting and detected using anti-Leulectin Abs. (C) Confirmation of the Leulectin–rFlaA interaction by far-Western assay. rFlaA (2 μg each lane) was run on an SDS-PAGE gel and transferred onto a nitrocellulose membrane. The proteins were renatured on the membrane by adding a series of concentrations of guanidine-HCl. rLLC/rLRRs/rCTLD (1 μg) or shrimp plasma (5 ml) was added to recognize rFlaA. Attached proteins were detected by anti-Leulectin Abs. (D and E) Determination of the interaction between rLRRs and other flagellins by a pull-down assay. The pull-down assay was performed as above. (F) Illustration of the mutated rLRRs. (G) Determination of the key region influencing the rLLR–rFlaA interaction in the LRR modules using a pull-down assay. (H) Illustration of truncated rFlaA. (I) Determination of the key region of FlaA involved in rLRR–rFlaA interaction by a pull-down assay. rFlaAN shared a similar m.w. to rLRR; therefore, Western blotting was performed to detect the rFlaAN-pulling sample using anti-Leulectin Abs.

In addition to FlaA, the V. anguillarum genome encodes four polar flagellins. To check whether Leulectin could also recognize them, we recombinantly expressed flagellin B, flagellin C, flagellin D, and flagellin E. The pull-down test showed that all of these proteins could interact with rLRRs (Fig. 4D). In addition, flagellin is a characteristic and critical protein of many shrimp bacterial pathogens. To determine whether the Leulectin–flagellin interaction was a common mechanism, FlaAs from V. alginolyticus and A. hydrophila, both of which are serious pathogens of shrimp, were used to perform the pull-down experiment. As shown in Fig. 4E, both FlaAs could interact with rLRRs. All of the above data suggested that Leulectin could bind flagellins through its LRRs.

To uncover the biochemical basis of the Leulectin–FlaA interaction, mutated proteins of LRRs and truncated proteins of FlaA were expressed to reveal the critical regions for the interaction (Fig. 4F, 4H). As shown in Fig. 4G, when the fourth LRR was deleted from the LRR module, the mutated protein could not be pulled down by rFlaA, suggesting that the fourth LRR greatly influenced the recognition of FlaA. In addition, only the truncated FlaA proteins that harbored the Flagellin_N domain could pull down rLRRs (Fig. 4I), indicating that this domain might be the target site of Leulectin.

Leulectin binds LPS through CTLD

To characterize the rCTLD–LPS interaction, the dose-dependent recognition was studied using wild-type rCTLD. As shown in Fig. 5A, rCTLD bound LPS in a dose-dependent manner. The binding was saturable, with the maximum at 20 μg/ml of rCTLD. The binding curve fitted a one-site binding model with R2 > +0.997. The maximum binding (Bmax) and dissociation constant (Kd) were 1.006 and 1.284 μg/ml, respectively. To check whether calcium was necessary for the binding, external calcium or EDTA was added to the incubation to increase or eliminate the ion effect. The result showed that, although the CTLD–LPS interaction was calcium independent, calcium could enhance the binding capacity by reducing the protein amount to reach saturation (Fig. 5B).

FIGURE 5.
FIGURE 5.

Recognition of LPS by the Leulectin CTLD module. (A–F) Recognition of LPS by wild-type and mutated rCTLD. LPS (2 μg per well) was coated in the 96-well plates and air-dried at 37°C. After the wells were blocked with 5% BSA, serial concentrations of proteins were added to the well to bind LPS for 3 h at 28°C. The bound proteins were detected using conventional ELISA with an anti-His tag Ab. For the tests needing CaCl2 or EDTA, the reagents were added to the binding incubation to a final concentration of 5 mM. The results are expressed as the mean ± SD derived from three independent repeats. (G) SDS-PAGE result of the proteins used in the LPS-recognition test.

A previous study suggested some critical regions and sites for the ligand binding of a CTLD module; however, firm evidence was lacking. In this study, rCTLD–LPS interaction was used as a model to illustrate the significance of these sites. Fig. 5C shows that the second loop of the CTLD fold, which is also referred to as the long loop region (LLR) and is flexible for ligand binding (30), determined the LPS-binding ability of rCTLD. Deletion of LLR fully abolished LPS binding. Fig. 5D suggested that the two pairs of disulfide bridges (C284–C391, C367–C383) were critical for the function of the CTLD fold. Mutation of the cysteines for the two bridges significantly reduced the binding ability of the whole domain. However, an additional disulfide bridge (C255–C266), which is common in the long form of CTLD, did not seem to be relevant for ligand binding.

There is a conserved WIGL-like motif in the β2 strand of many CTLDs whose function has remained unknown until now (13). In this study, deletion of this motif reduced the LPS-binding ability of rCTLD (Fig. 5E); however, the molecular mechanism still needs to be determined. As shown in Fig. 5F, the three residues that were predicted to contribute to carbohydrate binding (E358 and L360 in the EPN-like motif and E365) were all important for the interaction with LPS. Mutation of these residues suppressed the binding ability significantly. In addition, mutation of a site that is involved in calcium binding (DD379–380) had a slight influence on LPS binding, consistent with the result of the calcium-elimination experiment shown in Fig. 5B. The recombinant proteins used in this section are presented in Fig. 5G.

LRRs prevent flagellin attachment and bacterial colonization through sensing flagellin

Flagellin is the principle component of flagella, which are vital for the initial attachment of bacteria to host cells; therefore, we investigated whether the recognition of flagellin by Leulectin would influence the attachment of flagellin to shrimp cells and the colonization of Vibrio in vivo. A preliminary study showed that recombinant flagellin could bind to the hemocyte surface when applied in vivo (Fig. 6A). When the flagellin was injected together with rLLR or rLLC, no positive signal was observed. These results suggested that the interaction of FlaA with rLLRs or rLLC could block the attachment of flagellin to hemocytes. In addition, the proteins that could not recognize flagellin, including recombinant LRRΔ4, rCTLD, and recombinant LLCΔ4, did not block the attachment of flagellin to the hemocytes (Fig. 6B). Further study showed that the prevention of attachment was not limited to hemocytes but extended throughout the hepatopancreas. The presence of the two flagellin-recognizing proteins, rLLR or rLLC, eliminated the attachment of flagellin to hepatopancreas cells (Fig. 6B). These data suggested a possible mechanism for how Leulectin protected shrimp from bacterial infection.

FIGURE 6.
FIGURE 6.

Prevention of FlaA-mediated attachment and bacterial colonization by Leulectin LRR modules. (A) FlaA attachment to hemocytes and hepatopancreatic cells. rFlaA (5 μg) was injected into shrimp, and the hemocytes were collected and fixed. One hour later, the hepatopancreas was fixed and subjected to paraffin sectioning. The slides were analyzed by immunohistochemistry and detected using an anti-GST Ab. Data are representative of two independent repeats. Original magnification ×400 for hemocytes and ×200 for hepatopancreas. (B) Prevention of FlaA attachment by Leulectin LRR module. rFlaA was injected into shrimp together with the tested proteins (5 μg). Hemocytes and hepatopancreases were detected as above. Results are representative of two independent tests. (C) Prevention of FlaA-mediated bacterial colonization by rLRRs. Wild-type or mutated rLRRs (4 μg) were injected into shrimp together with several strains of bacteria (V. harveyi, 2 × 105 CFU; V. alginolyticus, 2 × 105 CFU; V. tasmaniensis, 2 × 106 CFU). The hepatopancreases were homogenized, and the products were plated onto agar plates to determine the bacterial numbers. ***p < 0.001.

To test whether the ability to recognize and prevent flagellin attachment was indeed related to the protective role of Leulectin, the bacteria colonization level in the hepatopancreas was studied. As shown in Fig. 6C, when V. anguillarum was injected together with rLLRs, the number of colonized bacteria in the hepatopancreas was significantly lower than in controls. In addition, the mutated protein, rLRRΔ4, which had lost the ability to recognize flagellin, did not possess such a colonization-inhibiting effect. Because the number of colonized externally injected bacteria was determined primarily based on the colony morphology, V. harveyi and V. alginolyticus colonies were picked randomly for identification using specific primers for FlaA, and the result was consistent with that in Fig. 6C (data not shown). All data in this section indicated that Leulectin prevented the initial adherence and subsequent colonization of bacteria in the host through recognizing bacterial flagellin via the LRR module.

CTLD agglutinates bacteria and promotes phagocytosis by recognizing LPS

To find out how rCTLD exerts its protective role, the ability of this protein to agglutinate the bacteria was determined, because agglutination is an original function of a lectin and would prevent the spreading of bacteria in the hemolymph. FITC-labeled V. anguillarum was incubated with the tested proteins to see the possible consequence. As shown in Fig. 7A, rCTLD caused obvious agglutination of V. anguillarum. In addition, preincubation with LPS eliminated the effect of rCTLD. Furthermore, the mutated protein rCTLDΔLLR, which could not bind LPS, did not possess the ability to agglutinate bacteria. These results indicated that the LPS-binding activity conferred agglutinating ability to rCTLD. It was notable that rLLRs also caused slight bacterial agglutination, possibly because of their flagellin-recognizing activity. However, the agglutination caused by rLRRs might also originate from the protein aggregates, because slight aggregation was detected in the rLRR solution (Supplemental Fig. 1). The two separate domains conferred strong agglutination ability on the full length protein rLLC. The minimal agglutinating concentration of rCTLD to kinds of bacteria was tested, and the concentration for Gram-negative bacteria was much lower than for the Gram-positive bacteria, further suggesting the importance of LPS binding ability for agglutination induction (Fig. 7B).

FIGURE 7.
FIGURE 7.

Promotion of bacterial agglutination and hemocytic phagocytosis by Leulectin CTLD module. (A) Bacterial agglutination caused by recombinant Leulectin proteins. V. anguillarum was labeled with 1 mg/ml of FITC for 1 h. The bacteria were incubated with the tested proteins (final concentration with 15 μg/ml) at 28°C for 1 h. The incubation was observed under a fluorescence microscopy. For a test with LPS, LPS (20 μg) was added to the incubation. The data shown are representative of two independent repeats. (B) Minimal agglutinating concentration of rCTLD for different kinds of bacteria. The bacteria were incubated with rCTLD (1.875–240 μg/ml) to determine the minimal concentration that caused bacterial agglutination. The experiments were repeated independently twice. (C) LPS-mediated bacteria binding by rCTLD. Wild-type or mutated rCTLD (4 μg) was incubated with 108 CFU V. anguillarum at 25°C for 1 h. After washing, the bacterial pellet was subjected to Western blotting and detected with an anti-His Ab. For the test with LPS (20 μg), it was added to the protein solution before adding the bacteria. (D) Opsonin effect of rCTLD in vivo. V. anguillarum was heat-killed, labeled with FITC, and fully coated by wild-type or mutated rCTLD. Then the bacteria (1 × 108 CFU) were injected into shrimp. Hemocytes were collected 40 min later and subjected to flow cytometry analysis. Ten thousand hemocytes were counted for each sample. The graphs are representative of three independent assays, and the phagocytosis ratios were calculated from those three tests. (E) Separation of shrimp hemocytes. Hemolymph was loaded onto a pregenerated continuous gradient of 65% Percoll and centrifuged at 2000 × g for 20 min. The bands containing hyaline cells and semigranular cells were collected. (F) Opsonin effect of rCTLD in vitro. The bacteria (5 × 106 CFU) were incubated with 2 × 105 hyaline cells and semigranular cells at 25°C for 1 h. The mixture was centrifuged at 500 × g for 8 min to obtain the hemocytes. After washing, the hemocytes were analyzed by flow cytometry. Two thousand hemocytes were counted for each sample. The phagocytosis ratios were calculated from three independent experiments, and the graphs are representative of the three repeats.

Previous studies showed that some C-type lectins could function as opsonins. To test whether opsonization was a way for rCTLD to participate in the immune response against Vibrio, the protein-bacteria interaction was studied. The results showed that rCTLD could bind to the surface of V. anguillarum, and the binding could be inhibited by adding LPS. In addition, the mutated protein rCTLDΔLLR, which could not recognize LPS, lost the bacteria-binding ability (Fig. 7C). After confirming the labeling ability of rCTLD, flow cytometry was performed to study whether rCTLD could promote hemocytic phagocytosis. As shown in Fig. 7D, rCTLD coating led to a higher percentage of hemocyte phagocytosis (∼16%) compared with the controls (∼9%) in vivo. Because a previous study claimed that crustacean hyaline cells and semigranular cells are the main participators in phagocytosis, we isolated these two classes through a classical Percoll-separation procedure (Fig. 7E). The in vitro test using these cells showed that rCTLD coating could also promote phagocytosis. The percentage of phagocytosis was ∼59% in the rCTLD group, which was higher than in the control groups in which the expression tag (∼27%) or mutated rCTLD (∼29%) was used (Fig. 7F). The above data suggested that the protective role of the CTLD module in the anti-Vibrio response operated via agglutinating and opsonizing invading bacteria.

PAMP-recognition ability determines the protective roles of Leulectin

The above results showed that the two pattern recognition modules of Leulectin exerted synergic protective roles in the antibacterial response, which generally relies on the ability to recognize PAMPs. To confirm this, the roles of mutated proteins in resisting bacterial infection were investigated. As shown in Fig. 8A, mutated rLRRs, which could not recognize flagellin, lost their protective role. When V. anguillarum was injected together with rLRRs, the survival rate was >60 and 50% after the first and fifth day postinjection, respectively. However, the survival rate was only ∼30 and 20% for the mutated rLRR group (rLRRsΔ4). Similar results were observed for mutated rCTLD in Fig. 8B. When the LPS-recognition motif of CTLD was deleted, the protective role of the mutated protein (rCTLDΔLLR) was always 30% lower than that of the wild-type protein (rCTLD). For full-length rLLC, deletion of either recognition motif (rLLCΔ4 or rLLCΔLLR) led to partial loss of the protective role. Furthermore, the protective role was severely compromised when both recognition motifs were deleted (rLLCΔ4ΔLLR) (Fig. 8C). These data further confirmed that the immune-recognition ability originated from the two modules, which determined the protective activity of Leulectin during bacterial infection.

FIGURE 8.
FIGURE 8.

Pattern-recognition ability-mediated protective role of Leulectin. LRR (A), CTLD (B), LLC (C) and the mutated proteins were tested. Shrimp were injected with a mixture of the tested proteins (2 μg) and V. anguillarum (5 × 105 CFU). The survival rates were recorded.

Discussion

By sensing pathogens before they enter host cells, soluble PRRs play important roles in immune defense. In the current study, we identified Leulectin as a soluble PRR in shrimp plasma that contains both of the typical pattern recognition modules. To our knowledge, such a unique arrangement has not been observed in other organisms. The coexistence of two recognition modules of different categories in a single PRR probably expands the target range and enhances the function of the protein. Moreover, the appearance of Leulectin in healthy shrimp plasma and the induction of its titer by bacterial challenge further suggested its role in the anti-Vibrio response. Both modules were found to have protective roles solely against Vibrio; therefore, the potential PAMPs that these two modules might recognize were screened to determine the origin of their functions. We found that LRRs and CTLD mainly recognize flagellin and LPS, respectively. Flagellin and LPS are the key PAMPs of bacterial pathogens, and the recognition targets of Leulectin correlated well with the results that its expression was induced significantly by bacterial, but not viral, pathogens. In addition, the mutated modules that lost their recognition abilities also lost their protective role, highlighting again that the recognition capacity of a PRR is the determinant of its role in host defense.

The flagellum is vital for the motility of flagellated bacteria. It makes the bacteria spread easily, causing extensive dissemination. In addition, bacteria use their flagella to attach to the host cells to initiate invasion (31, 32). Flagellin, the primary component of a flagellum, is one of the most abundant proteins and is also necessary for infection by flagellated bacteria (33). As a serious pathogen against shrimp aquaculture, V. anguillarum encodes five flagellin genes (34, 35). A previous study showed that FlaA was the most important one: it mostly determined the bacterial motility and is essential for the bacteria to break through the host integument (35). In addition, flagellin D and flagellin E are involved in virulence (34). Furthermore, bacterial flagellins were proved to be the principle immunogenic determinants and cause many kinds of immune responses (36). For these reasons, sensing flagellin could be a way for shrimp to discriminate the bacterial pathogens. Flagellin was identified as the ligand of several mammalian PRRs, such as TLR5 and NLRC4/Ipaf/CLAN/CARD12 (37, 38). In the current study, using ELISA screening and pull-down and far-Western assays, we proved that Leulectin could recognize flagellin. To the best of our knowledge, this is the first flagellin-recognizing molecule found in invertebrates. Although Leulectin is not a true membrane receptor, like the mammalian TLR5, the sensing capacity might confer a vital role to Leulectin by disrupting flagellin and flagellum-mediated pathogenesis. In addition, the flagellins recognized by Leulectin included several types (FlaA and flagellin B–E) from different species (V. anguillarum, V. alginolyticus, and A. hydrophila), indicating that Leulectin might participate in resistance to a wide range of bacterial pathogens through sensing flagellins.

In addition to preventing flagellin-mediated bacteria adherence and colonization through the recognition capacity of the LRR module, Leulectin labeled the bacteria via the CTLD module, which led to bacterial agglutination and enhanced hemocytic phagocytosis. Many studies suggested a close relationship between agglutination and protection (39, 40). For example, secretory IgA could protect intestinal epithelial cells from Shigella flexneri through its agglutinating ability (39). In addition, the human secreted IgG Ab could agglutinate Streptococcus pneumoniae, and the agglutination protected the mouse upper respiratory tract from bacterial colonization by blocking bacterial access to the epithelial surface (40). In this study, the CTLD module of Leulectin also caused agglutination of V. anguillarum, suggesting a possible role in the prevention of bacterial colonization. In this sense, the CTLD module functioned similarly to the LRR module. However, the consequence of agglutination is not limited to effects on colonization. The agglutination of bacteria into large clusters could also inhibit bacterial transmission along with the hemolymph circulation and allow for effective mechanical elimination by the host. This also suggested a contribution of the CTLD module to the protective role of Leulectin through its agglutinating ability. Phagocytosis is the principle way to clear invading exogenous bacteria (41). Previous studies showed that hyaline cells are the main type of shrimp hemocytes responsible for phagocytosis. In addition, granular cells are involved in phagocytosis (42). Phagocytosis is usually trigged by the recognition of the targets. The recognition is either direct via cell membrane receptors or indirect and mediated via opsonization factors (opsonins) (43–46). The mammalian Ab and MBL are classical opsonins, and some C-type lectins from invertebrates were also identified as opsonins by binding and presenting the bacterial pathogens to phagocytes (15, 20, 47, 48). In the current study, we found that coating by the CTLD module could promote hemocytes to phagocytose V. anguillarum, in vivo and in vitro, indicating the involvement of Leulectin in phagocytosis through the opsonic ability of the CTLD module.

It is not rare for a soluble PRR to harbor more than one pattern recognition module of either the same or a different category. The tandem expression of these modules effectively expands the recognition spectrum and enhances the immune function of the whole molecule. Such synergistic effects were studied for some PRRs, especially the C-type lectins with dual CTLDs. For example, Bombyx mori expresses a multibinding protein (BmMBP) that contains two CTLDs, both of which contribute to the wide binding spectra of sugars and microorganisms. The multirecognition characteristic of BmMBP is important to inhibit pathogens during the early stage of the infection (49). Compared with these reports, Leulectin identified in this study is unique in its architecture, with a tandem arrangement of LRRs and CTLD, two typical pattern-recognition modules. To our knowledge, such an arrangement has not been reported in other organisms. More particularly, the two domains showed considerable coordination during the anti-Vibrio response by preventing colonization and favoring phagocytosis, respectively. This distinct division of labor toward the same goal represents a paradigm for the synergy between different pattern recognition modules and provided new insights into understanding the concept of pattern recognition.

In recent years, C-type lectin-like receptors (CTLRs) were proved to be one of the most important groups of PRRs in shrimp (50). Different from the reported CTLRs that recognize pathogens solely by CTLD, Leulectin represents a new member of the shrimp CTLR family and expands the target spectrum of this type of PRRs. Previous studies suggested that the functional diversity of shrimp CTLRs relies mainly on the sequence/structure diversity of CTLD. Our findings in Leulectin highlighted the contribution of modules other than CTLD to the functional diversity of CTLRs.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by the National Natural Science Foundation of China (Grants 31302217, 31622058, and 31630084), the Young Scholars Program of Shandong University (Grant 2015WLJH26), and by Fundamental Research Funds of Shandong University (Grant 2015TB018) (to X.-W.W. and J.-X.W.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CTLD
    C-type lectin-like domain
    CTLR
    C-type lectin-like receptor
    FlaA
    flagellin A
    LLC
    LRR linked to CTLD
    LLR
    long loop region
    LRR
    leucine-rich repeat
    mPBS
    PBS with 0.45 M NaCl
    PAMP
    pathogen-associated molecular pattern
    PBST
    PBS with 0.05% Tween 20
    PO
    phenoloxidase
    PRR
    pattern recognition receptor
    rCTLD
    recombinant CTLD
    rFLaA
    recombinant FlaA
    rLLC
    recombinant LLC
    rLLR
    recombinant LLR
    THC
    total hemocyte count
    VLR
    variable lymphocyte receptor
    WSSV
    white spot syndrome virus.

  • Received November 28, 2016.
  • Accepted February 6, 2017.

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