In vertebrates, leukocyte-derived chemotaxin-2 (LECT2) is an important immunoregulator with conserved chemotactic and phagocytosis-stimulating activities to leukocytes during bacterial infection. However, whether LECT2 possesses direct antibacterial activity remains unknown. In this article, we show that, unlike tetrapods with a single LECT2 gene, two LECT2 genes exist in teleost fish, named LECT2-a and LECT2-b. Using grass carp as a research model, we found that the expression pattern of grass carp LECT2-a (gcLECT2-a) is more similar to that of LECT2 in tetrapods, while gcLECT2-b has evolved to be highly expressed in mucosal immune organs, including the intestine and skin. Interestingly, we found that gcLECT2-b, with conserved chemotactic and phagocytosis-stimulating activities, can also kill Gram-negative and Gram-positive bacteria directly in a membrane-dependent and a non–membrane-dependent manner, respectively. Moreover, gcLECT2-b could prevent the adherence of bacteria to epithelial cells through agglutination by targeting peptidoglycan and lipoteichoic acid. Further study revealed that gcLECT2-b can protect grass carp from Aeromonas hydrophila infection in vivo, because it significantly reduces intestinal necrosis and tissue bacterial load. More importantly, we found that LECT2 from representative tetrapods, except human, also possesses direct antibacterial activities, indicating that the direct antibacterial property of LECT2 is generally conserved in vertebrates. Taken together, to our knowledge, our study discovered a novel function of LECT2 in the antibacterial immunity of vertebrates, especially teleost fish, greatly enhancing our knowledge of this important molecule.

The intestine of animals harbors trillions of commensal microbes, mostly bacteria (1). In healthy conditions, commensal microbes interact with the intestinal mucosal barrier to maintain immune homeostasis (25). Antimicrobial peptides/proteins are one of the most important components of the intestinal mucosal barrier to defend against pathogens (5, 6). In mammals, numerous intestinal antimicrobial peptides/proteins have been demonstrated to play important roles in maintaining intestinal health (79). However, in teleost fish, studies on intestinal antimicrobial peptides/proteins, especially nonclassical antimicrobial peptides/proteins, are lacking. Thus, the intestine of teleost fish is an ideal organ to identify novel antimicrobial peptides/proteins.

Antibacterial peptides/proteins play a critical role in preventing the infection of microbial pathogens through direct killing (10). Moreover, numerous studies have revealed that, in addition to the direct antimicrobial activities, vertebrate antimicrobial peptides/proteins are pleiotropic molecules with multiple immunoregulatory activities (10, 11). Many antimicrobial peptides (cathelicidins and defensins) and antibacterial proteins (ILs, chemokines, and lectins) can induce the activation of immune cells through direct binding to specific receptors, or through pattern recognition receptors after the formation of complexes with immune ligands such as LPS (6, 1215). Interestingly, some antimicrobial peptides/proteins, such as CXCL20a, thrombin-derived C-terminal peptide, and RNase 3/ECP peptide, can also agglutinate bacteria in a broad spectrum through binding with pathogen-associated molecular patterns (PAMPs), to prevent the invasion of pathogenic bacteria (14, 16, 17). Thus, antimicrobial peptides/proteins can enhance the immunity of the host through a variety of mechanisms.

In 1996, a cytokine, named leukocyte-derived chemotaxin-2 (LECT2), with chemotactic activities of neutrophils was initially purified from PHA-activated human T cell leukemia SKW-3 cells (18). In mammals, LECT2 is mainly produced in the liver and then secreted into the blood; however, in teleost fish, LECT2 shows a wider tissue distribution (1922). Studies have shown that LECT2 plays important roles in controlling inflammation and bacterial infection in both mammals and teleost fish (19, 2325). LECT2 can cause chemotaxis of immune cells from blood to infected tissues and then promote the phagocytic activity of phagocytes to clear the invading pathogens (18, 24, 25). Thus, LECT2 is recognized as a candidate anti-infective and anti-inflammatory drug (25, 26). In addition to immune functions, LECT2 possesses numerous other biological functions, including the regulation of the movement of hematopoietic stem cells (27, 28), the regulation of the growth and differentiation of chondrocytes and osteoblasts, and the repair of tissue damage (26, 2931). However, whether LECT2 possesses direct antibacterial activities remains unknown.

Due to the particularity of the living environment, teleost fish rely heavily on their mucosal immunity to resist pathogens. Using the intestine as the target organ, we isolated and identified multiple potential antibacterial proteins, among which LECT2 caught our attention. We found that, unlike tetrapods with a single LECT2 gene, two LECT2 genes exist in teleost fish, named LECT2-a and LECT2-b. Using grass carp (Ctenopharyngodon idella) as a research model, we further revealed that, unlike LECT2 in mammals, grass carp LECT2-b (gcLECT2-b) has evolved to be highly expressed in mucosal immune organs, which may help to improve mucosal immunity. Surprisingly, to our knowledge, we uncovered that gcLECT2-b, with conserved chemotactic and phagocytosis-stimulating activities, is a novel antibacterial protein with robust direct bactericidal activity. More importantly, we found that the antibacterial activity of LECT2 is conserved in representative vertebrates, except human, indicating that LECT2 is an important bifunctional molecule with conserved immunoregulatory and bactericidal activities in vertebrates, especially teleost fish.

Healthy grass carp (25 ± 5 g) were obtained from a fish farm in Wuhan, China. The experimental protocols were approved by the Animal Ethics Committee of Huazhong Agricultural University (HZAUFI-2021-0011).

The intestinal tissue without muscle layers was homogenized in 5% acetic acid for 10 min, sonicated for 5 min at 40 Hz, shaken overnight at 4°C, and then centrifuged (10,000 × g for 30 min at 4°C) to collect the supernatant (32). Subsequently, gel filtration chromatography with a Sephadex G50-column (2–30 kDa separation range; Huiyan Bio) was used to separate the collected supernatant into 2-mL fractions in an ÄKTA purification system (GE Healthcare), which were then dried with a vacuum freeze drier. Each fraction was dissolved in 100 μl of Tris buffer (20 mM, pH 7.4) and incubated with Escherichia coli (1 × 103 CFUs) at 28°C for 3 h. The treated E. coli was serially diluted and spread onto tryptic soy agar (TSA; Hope Bio) plates to assess the antibacterial activity of each fraction after overnight incubation (33). The fractions with antibacterial activities were subjected to cation exchange chromatography with CM Focurose 6FF matrix (CM matrix, Huiyan Bio) to further separate the potential antibacterial proteins (34). The antibacterial activity of the flow-through and CM matrix-bound fractions was measured as described earlier. The fractions with antibacterial activities were sent to Shanghai Applied Protein Technology Co. Ltd for mass spectrometric identification.

The gcLECT2-a and gcLECT2-b cDNAs were cloned using the primers in Supplemental Table I, sequenced by Tsingke Biotech, and then submitted to the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers MZ160500 and MZ160501, respectively. The phylogenetic tree based on protein sequences was constructed through the minimum-evolution method bootstrapped 1000 times using MEGA software (version 7.0). The multiple sequence alignment was conducted using the ClustalW program (https://www.genome.jp/tools-bin/clustalw) and then visualized by the ESPript program (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The molecular mass, net charge, and hydrophobic content of LECT2 were calculated using DNASTAR software (Madison, WI). The (R+K)% and Pho% (ratio of hydrophobic amino acid residues) of LECT2 were calculated using the online antimicrobial peptide database (http://aps.unmc.edu/AP) (35). The tertiary structure of LECT2 was modeled using online software (http://zhanglab.ccmb.med.umich.edu/I-TASSER/).

To detect the tissue expression patterns of gcLECT2-a and gcLECT2-b, we isolated total RNA from the head-kidney, trunk-kidney, spleen, blood, intestine, skin, gill, liver, muscle, heart, eye, and brain of euthanized grass carp. Meanwhile, two groups of grass carp were injected i.p. with Aeromonas hydrophila (1 × 107 CFUs/ml) or PBS, 100 µl per fish, and total RNA was isolated from the intestine, gill, head-kidney, and liver of fish at 0, 12, 24, 36, and 72 h postinjection. The RNA was reverse transcribed into cDNA using HiScript III Reverse Transcriptase (Vazyme). Quantitative real-time PCR (qPCR) was performed to analyze the expression levels of gcLECT2-a and gcLECT2-b using TSINGKE Master qPCR Mix (Tsingke Biotech) in a CFX Connect Real-Time System (Bio-Rad). The primers used are listed in Supplemental Table I. The expression levels of gcLECT2-a and gcLECT2-b under normal and challenged conditions were analyzed using the 2−ΔCT and 2−ΔΔCT methods, respectively, with the 18S rRNA gene as the internal control (36).

The pET-32a plasmid and BL21(DE3) Rosetta cells were used to produce recombinant LECT2 of representative vertebrates (34). Briefly, the expression of recombinant TrxA-tagged LECT2-b (TrxA-LECT2) was induced with 0.75 mM isopropyl β-D-1-thiogalactopyranoside at 37°C for 6 h, and TrxA-LECT2 was purified by Ni-NTA Resin (GE Healthcare) under native conditions, eluted by 300 mM imidazole, and dialyzed in Tris buffer (20 mM, pH 7.4) to remove the imidazole. Then, the TrxA tag was cleaved by enterokinase (Novoprotein), and LECT2 was purified through cation exchange chromatography using the HiTrap SP HP column (GE Healthcare) in an ÄKTA purification system (GE Healthcare) (34). After that, LECT2 was dialyzed in Tris buffer (20 mM, pH 7.4) and then stored at −80°C until use. The contamination of endotoxin in LECT2 was detected to be negligible (<0.07 endotoxin unit/mg) using an endotoxin detection kit (GenScript).

The CFU assay was used to investigate the antibacterial activity of gcLECT2-b against the following bacteria: E. coli ATCC25922, A. hydrophila ZYAH72, Vibrio mimicus ATCC33847, Pseudomonas fluorescens ATCC17386, Staphylococcus aureus ATCC25923, Streptococcus agalactiae ATCC13813, and Micrococcus luteus ATCC10240 (33). Briefly, the bacteria in log phase (OD600 = 0.5) were harvested and diluted to 5 × 105 CFUs/ml in Tris buffer (20 mM, pH 7.4). Then, 100 μl of bacteria was incubated with 100 μl of gcLECT2-b at the final concentrations of 0, 4, 8, 16, 32, and 64 μM, respectively, at 37°C (except A. hydrophila at 28°C) for 3 h. Following this, 20 μl of treated bacteria was spread onto TSA plates and incubated overnight at 37°C (except A. hydrophila at 28°C) for 12 h, after which the CFU was counted.

The absorbance-based assay was also used to investigate the antibacterial activity of LECT2 in 96-well microtiter plates. Briefly, LECT2 was serially diluted in 100 μl of Tris buffer (20 mM, pH 7.4) containing 20% tryptic soy broth medium to final concentrations ranging from 2 to 32 μM. Then, 100 μl of E. coli (1.5 × 105 CFUs) in 20% tryptic soy broth medium was added to each well and incubated at 37°C (except A. hydrophila at 28°C) for 12 h. Inhibition of bacterial growth was determined at each time point by measuring the absorbance at 600 nm with an Epoch2 microplate spectrophotometer (Bio-Tek). The minimum inhibitory concentration (MIC) was defined as the lowest concentration of gcLECT2-b that inhibited bacterial growth.

The effect of temperature and pH on the antibacterial activity of gcLECT2-b was determined using a previously described method (37). Briefly, gcLECT2-b was incubated at different temperatures (25, 60, 80, and 100°C, respectively) or different pH values (5.5 [10 mM Mes], 7.0 [20 mM Tris], and 9.0 [20 mM Tris], respectively) at room temperature for 1 h. The antibacterial activity was determined by the CFU method as described earlier against the typical strain E. coli ATCC25922.

The cytotoxicities of gcLECT2-b and human antimicrobial peptide LL-37 were measured against C. idella kidney cells by using a CCK8 Kit (Biosharp) according to the manufacturer’s instructions (38). Briefly, cells were seeded onto a 96-well plate at 104 cells/well, and then serially diluted gcLECT2-b and LL-37 were added to each well. As negative and positive controls, PBS and 2% Triton X-100 were added to the control wells, respectively. After 24 h of incubation, the OD490 was measured using a microplate reader (Bio-Tek).

The cytotoxicities of gcLECT2-b and LL-37 were also measured against grass carp erythrocytes as previously described (39). The 8% (v/v) erythrocytes were prepared using our previously described method (33). Subsequently, erythrocytes were incubated with serially diluted gcLECT2-b and LL-37, respectively, for 2 h. As negative and positive controls, PBS and 2% Triton X-100 were added to the control wells, respectively. After being centrifuged at 500 × g for 10 min, the percentage of hemolysis was calculated through measuring the OD405 of the supernatant.

The method described earlier was used to produce gcLECT2-b with hemagglutinin (HA)-tag at the N terminus (HA-gcLECT2-b). Western blot was performed to detect the binding activity of gcLECT2-b to Gram-negative and Gram-positive bacteria as previously described (40). Briefly, after being incubated with 0.1 μM HA-gcLECT2-b, bacteria were washed three times with Tris buffer (20 mM, pH 7.0), and then Western blot analysis was applied using HRP-conjugated mouse anti-HA Ab (ABclonal). Moreover, E. coli and S. aureus were incubated with different concentrations of HA-gcLECT2-b to detect the binding property of gcLECT2-b to bacteria.

The binding activity of gcLECT2-b to PAMPs was examined by ELISA using a previously described method (41, 42). Briefly, ELISA plates were coated with 8 μg LPS (E. coli), peptidoglycan (PGN) (M. luteus), or lipoteichoic acid (LTA) (S. aureus) at 4°C overnight and then blocked with 5% BSA (200 µL/well) at 37°C for 3 h. Serially diluted recombinant HA-gcLECT2-b was added to corresponding wells and incubated at 37°C for 3 h. BSA, instead of HA-gcLECT2-b, was added to control wells. After incubation, plates were washed and incubated with mouse anti-HA Ab (ABclonal) at 37°C for 1 h. After washing, the plates were incubated with HRP-conjugated goat anti-mouse IgG (ABclonal) at 37°C for 45 min. Finally, the plates were washed and incubated with TMB solution (YEASEN) for 5–30 min. The absorbance at 405 nm was read by a plate reader (Bio-Tek).

The localization of gcLECT2-b in bacteria was monitored by structured illumination microscopy (SIM) (43). Briefly, recombinant gcLECT2-b was labeled with FITC using the EZ-Label FITC labeling kit (Pierce, USA) according to the manufacturer's instructions. Then, 107 bacteria were incubated with FITC-labeled gcLECT2-b at a concentration of 0.5 μM at 37°C for a series of time periods. After incubation, bacteria were washed, fixed, stained with DAPI, and then applied onto slides precoated with poly-l-lysine (33). Images were acquired using a SIM (Nikon N-SIM).

A propidium iodide (PI) uptake assay was conducted to assess the bacterial membrane permeability of gcLECT2-b (33). Briefly, bacteria were treated with or without recombinant gcLECT2-b at 37°C for 1 h. Meanwhile, TrxA- and LL37-treated (8 μM) bacteria were included as controls. Then, bacteria were collected and incubated in PBS containing 10 μg/ml PI for 30 min. The influx of PI was measured using the flow cytometer FACSVerse (BD Biosciences).

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed to observe the morphological and microstructural changes of bacteria treated with gcLECT2-b as previously described (44). Briefly, E. coli and S. aureus (5 × 107 CFUs) were treated with recombinant gcLECT2-b (8 μM) in Tris buffer (20 mM, pH 7.0) at 37°C for 1 h. For TEM, treated bacteria were fixed, embedded, sectioned, and then observed with a TEM (Hitachi H-76500). For SEM, treated bacteria were fixed, dehydrated, vacuum dried, sputter coated with gold, and then observed with a field emission SEM (Hitachi S-4800).

Bacterial agglutination activity of gcLECT2-b was detected using a previously described method (45). Briefly, bacteria (1 × 106 CFUs/ml) were incubated in 1 μM recombinant gcLECT2-b at 37°C for 1 h. After staining with DAPI, the agglutination effect was observed under fluorescence microscopy. The minimum agglutination concentration (MAC), which is the lowest concentration that causes bacterial agglutination, of gcLECT2-b was determined with 5-fold serial dilutions of gcLECT2-b. The inhibition effect of different polysaccharides (galactose, mannose, maltose, glucose, lactose, and sucrose) and PAMPs (LPS, PGN, and LTA) on the bacterial agglutination activity of gcLECT2-b was evaluated through competitive combination experiments as previously described (46, 47).

To detect whether gcLECT2-b forms a polymer after binding to bacteria, we performed the protein cross-linking experiment as previously described with minor modifications (7, 48). Briefly, after being incubated with 0.1 μM HA-gcLECT2-b, E. coli was washed three times with PBS. Subsequently, the E. coli was incubated with 2.5 mM BS3 (Thermo Fisher) at room temperature for 1 h. Finally, the mixture was analyzed by Western blot.

The inhibition effect of gcLECT2-b on the adherence of bacteria to the epithelioma papulosum cyprini (EPC) cells was investigated as previously described (49). Briefly, A. hydrophila was added to EPC cells at a multiplicity of infection of 50. Then, 2-fold serial dilutions of gcLECT2-b were added into each well to the final concentrations of 100, 200, 400, and 800 nM, respectively. After being incubated at 28°C for 1 h, EPC cells were washed, suspended, serially diluted, and plated onto TSA plates to enumerate the total bacteria (Nt) in each sample. To enumerate the number of internalized bacteria (Ni), we treated EPC cells with 200 μg/ml gentamicin for 2 h to kill the bacteria adhered to the cell surface before the washing step. The number of bacteria adhered to the cell surface (Na) was calculated by subtracting the number of internalized bacteria from the number of total bacteria (Na = Nt − Ni).

The chemotactic activity of gcLECT2-b to leukocytes was measured with chemotaxis chambers in 24-well plates (Corning Life Sciences) as previously described (50). Briefly, 600 μl of DMEM containing various concentrations of recombinant gcLECT2-b (0, 10, 100, and 1000 ng/ml) was placed in the bottom of each well. As a positive control, DMEM containing 2% FBS was used. After placing the chemotaxis chambers in each well, 100 μl of peripheral blood leukocytes (PBLs), head-kidney leukocytes (HKLs), and spleen leukocytes (SPLs) isolated from grass carp were added to each chamber, respectively. After being incubated at 28°C for 6 h, the migrated cells in DMEM were analyzed using the flow cytometer FACSVerse with a constant flow time (1 min).

The impact of gcLECT2-b on the phagocytic activity of leukocytes was evaluated as previously described (51, 52). Briefly, grass carp PBLs, HKLs, and SPLs in DMEM were seeded onto 96-well plates (2 × 105 cells/well) and incubated with various concentrations of recombinant gcLECT2-b (0, 10, 100, and 1000 ng/ml) at 28°C for 3 h. The TrxA tag was also included as the negative control. Then, cells were incubated with fluorescent beads (Fluoresbrite Yellow Green Microspheres, 1.0 μm in diameter; Polysciences) at 28°C for 2 h, after which the noningested beads were removed through PBS supplemented with 3% BSA (Thermo Fisher) and 4.5% D-(+)-glucose (Sigma-Aldrich). The phagocytic activity of cells was measured using the flow cytometer FACSVerse.

To test the treatment effect of gcLECT2-b on bacterial infection, we established an enteritis model in grass carp through i.p. injection of A. hydrophila (2 × 106 CFUs/fish) as previously described (33). After 12 h, 100 μl of recombinant gcLECT2-b in PBS (0.25 μg/μl) or PBS alone was i.p. injected into individual fish (24, 49). The survival rate was monitored twice a day for 7 d. At 48 h postinfection, some individuals were anesthetized with 0.02% 3-aminobenzoic acid ethyl ester methanesulfonate (MS-222), and the intestine, spleen, head-kidney, and liver were harvested aseptically. The tissues were weighed and homogenized in PBS (0.1 g/ml) with tissue grinders (Sigma). The tissue homogenate was serially diluted and plated onto Aeromonas spp. selective plates (Rimler-Shotts medium; Hope Bio) to count the number of A. hydrophila. Meanwhile, at 48 h postinfection, the intestinal tissues were fixed in 10% neutral-buffered formalin, sectioned, and stained routinely with H&E to observe the tissue damage.

The p value was analyzed by Student t test using GraphPad Prism 7.0 software (GraphPad). A p value ≤0.05 was considered statistically significant.

The intestinal tissue without muscle layer was homogenized and then centrifuged to collect the supernatant. Subsequently, the supernatant was separated into 88 fractions using gel filtration chromatography (Fig. 1A). Among them, fractions 1–2, 5–6, and 68–70 exhibited high antibacterial activities against E. coli (Fig. 1B). Considering that most antimicrobial peptides/proteins exhibit a cationic nature (44), fractions 1 and 2 were mixed and further separated via cation exchange chromatography. The CM matrix-bound proteins from fractions 1 and 2 were further identified by mass spectrometry because of their higher antibacterial activity. Among the identified proteins, LECT2 caught our attention because it has never been reported as an antimicrobial protein (Fig. 1C).

FIGURE 1.

Isolation and identification of gcLECT2 proteins as potential antibacterial proteins in grass carp intestine. (A) Gel filtration chromatography of intestinal proteins. The proteins were separated into different fractions and collected with tubes (2 ml/fraction). (B) Antibacterial activity of fractions (1–88) collected in (A) against E. coli. The CFU assay was conducted, and the treated bacteria were spread onto TSA plates, incubated, and then counted. The Tris buffer (20 mM, pH 7.4) was used as the negative control (100% growth). The numbers indicate the percentage of bacterial growth relative to the PBS control. (C) Antibacterial activity of the proteins after cation exchange chromatography (left), followed by mass spectrometric identification (right). Fractions 1 and 2 collected in (A) were pooled and further separated via cation-exchange chromatography, and then the antibacterial activity of the flow-through and CM matrix-bound proteins was tested against E. coli using the CFU assay. The CM matrix-bound proteins were identified by mass spectrometry, and the peptide identified from gcLECT2-a and gcLECT2-b is shown in red. (D) Coding sequence alignment of gcLECT2-a and gcLECT2-b. The multiple sequence alignment was conducted using the ClustalW program (https://www.genome.jp/tools-bin/clustalw) and then visualized by the ESPript program (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).

FIGURE 1.

Isolation and identification of gcLECT2 proteins as potential antibacterial proteins in grass carp intestine. (A) Gel filtration chromatography of intestinal proteins. The proteins were separated into different fractions and collected with tubes (2 ml/fraction). (B) Antibacterial activity of fractions (1–88) collected in (A) against E. coli. The CFU assay was conducted, and the treated bacteria were spread onto TSA plates, incubated, and then counted. The Tris buffer (20 mM, pH 7.4) was used as the negative control (100% growth). The numbers indicate the percentage of bacterial growth relative to the PBS control. (C) Antibacterial activity of the proteins after cation exchange chromatography (left), followed by mass spectrometric identification (right). Fractions 1 and 2 collected in (A) were pooled and further separated via cation-exchange chromatography, and then the antibacterial activity of the flow-through and CM matrix-bound proteins was tested against E. coli using the CFU assay. The CM matrix-bound proteins were identified by mass spectrometry, and the peptide identified from gcLECT2-a and gcLECT2-b is shown in red. (D) Coding sequence alignment of gcLECT2-a and gcLECT2-b. The multiple sequence alignment was conducted using the ClustalW program (https://www.genome.jp/tools-bin/clustalw) and then visualized by the ESPript program (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).

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Unlike tetrapods with a single LECT2 gene, two homologous LECT2 genes were identified in grass carp and zebrafish (Danio rerio), named LECT2-a and LECT2-b (Supplemental Fig. 1A). Gene synteny analyses showed that LECT2-a and LECT2-b genes are closely located in the same chromosome in grass carp and zebrafish (Supplemental Fig. 1B). The cDNA sequences of gcLECT2-a and gcLECT2-b both contained a 477-bp open reading frame, which can encode 158 aa. The protein sequences of gcLECT2-a and gcLECT2-b both consisted of a signal peptide (22 aa) and a mature protein (136 aa) (Fig. 1C, 1D). In addition, the gene structures and sequences of gcLECT2-a and gcLECT2-b are highly consistent (Fig. 1C, 1D). Using the specific primers for gcLECT2-a and gcLECT2-b, we further revealed that the expression patterns of gcLECT2-a and gcLECT2-b are significantly different, with gcLECT2-a highly expressed in systemic immune organs, such as that of LECT2 in tetrapods, while gcLECT2-b is highly expressed in mucosal immune organs, including the intestine, skin, and gills (Fig. 2A). After A. hydrophila infection, the expression of gcLECT2-b was significantly increased in mucosal immune organs, including the intestine and gills. On the contrary, the expression of gcLECT2-a was significantly increased in systemic tissues, including the head-kidney and liver (Fig. 2B–E). Therefore, we speculate that gcLECT2-b may play important roles in mucosal immunity.

FIGURE 2.

Gene and protein structures of gcLECT2 and their expression patterns under healthy and infected conditions. (A) Expression patterns of gcLECT2-a and gcLECT2-b in healthy grass carp analyzed by qPCR (n = 12 fish). The expression levels of gcLECT2-a and gcLECT2-b were analyzed using the 2−ΔCT method, with the 18S rRNA gene as the internal control. (BE). Relative expression of gcLECT2-a and gcLECT2-b in intestine (B), gill (C), head-kidney (D), and liver (E) after A. hydrophila infection (n = 5 fish). The fold changes of gcLECT2-a and gcLECT2-b after A. hydrophila infection were calculated by comparing the infected group with the preinfection control using the 2−ΔΔCt method. Data in (B)–(E) are presented as mean ± SE. *p < 0.05, **p < 0.01.

FIGURE 2.

Gene and protein structures of gcLECT2 and their expression patterns under healthy and infected conditions. (A) Expression patterns of gcLECT2-a and gcLECT2-b in healthy grass carp analyzed by qPCR (n = 12 fish). The expression levels of gcLECT2-a and gcLECT2-b were analyzed using the 2−ΔCT method, with the 18S rRNA gene as the internal control. (BE). Relative expression of gcLECT2-a and gcLECT2-b in intestine (B), gill (C), head-kidney (D), and liver (E) after A. hydrophila infection (n = 5 fish). The fold changes of gcLECT2-a and gcLECT2-b after A. hydrophila infection were calculated by comparing the infected group with the preinfection control using the 2−ΔΔCt method. Data in (B)–(E) are presented as mean ± SE. *p < 0.05, **p < 0.01.

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The modeled tertiary structure of gcLECT2-b revealed that gcLECT2-b mainly consists of β-sheets and random coils (Fig. 3A). The molecular side of gcLECT2-b mainly composed of β-sheets is intensified by positively charged amino acid residues, while the opposite side is intensified by negatively charged amino acid residues and hydrophobic amino acid residues. These indicated that gcLECT2-b is cationic and amphipathic, which is a typical feature of antimicrobial peptides/proteins (53). To further gain insights into the antibacterial potential of gcLECT2-b, we generated the (R+K)%-Pho% diagram based on the 2339 antimicrobial peptides in the antimicrobial peptide database (Fig. 3B), which depicts the correlation between the hydrophobic content (Pho%) and the percentage of basic amino acids (R+K) of known antimicrobial peptides. When we mapped gcLECT2-b on the (R+K)%-Pho% diagram, we found that gcLECT2-b was close to the (R+K)%-Pho% dots of known antimicrobial peptides, strongly suggesting that gcLECT2-b is a potential antimicrobial protein.

FIGURE 3.

Antibacterial potential and activities of gcLECT2-b. (A) Tertiary structure of gcLECT2-b generated by homology modeling (upper) and electrostatic potential displayed on the molecular surface (lower). The positively charged amino acid residues are shown in blue, the negatively charged amino acid residues are shown in red, and the hydrophobic amino acid residues are shown in white. (B) (R+K)%-Pho% dot plot showing the relationship between gcLECT2-b and known antimicrobial peptides in the antimicrobial peptide database. The 2339 antimicrobial peptides in the antimicrobial peptide database were separated into 10 bins based on the hydrophobic contents (0–10, 11–20, 21–30, 31–40, 41–50, 51–60, 61–70, 71–80, 81–90, and 91–100%), which are represented as 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100, respectively, on the x-axis of the plot. The antimicrobial peptides in the antimicrobial peptide database are shown in black, whereas gcLECT2-b is shown in orange. (C) SDS-PAGE (left) and Western blot (right) analyses of purified gcLECT2-b. Lane M, marker; 1, purified TrxA-gcLECT2-b; 2, enterokinase-digested TrxA-gcLECT2-b; 3, purified gcLECT2-b. The anti-His mAb was used to detect the recombinant proteins. The blot images were split because they were exposed independently. (D) Antibacterial activities of gcLECT2-b against Gram-negative and Gram-positive bacteria. The CFU assay was conducted, and the treated bacteria were spread onto TSA plates, incubated, and then counted. (E) MIC of gcLECT2-b. The absorbance-based assay was conducted in 96-well microtiter plates, and the growth of bacteria was determined by measuring the absorbance at 600 nm. All the experiments were performed in triplicate. The data in (D) are shown as mean ± SD of three independent experiments, and the data in (E) are representative results of three independent experiments.

FIGURE 3.

Antibacterial potential and activities of gcLECT2-b. (A) Tertiary structure of gcLECT2-b generated by homology modeling (upper) and electrostatic potential displayed on the molecular surface (lower). The positively charged amino acid residues are shown in blue, the negatively charged amino acid residues are shown in red, and the hydrophobic amino acid residues are shown in white. (B) (R+K)%-Pho% dot plot showing the relationship between gcLECT2-b and known antimicrobial peptides in the antimicrobial peptide database. The 2339 antimicrobial peptides in the antimicrobial peptide database were separated into 10 bins based on the hydrophobic contents (0–10, 11–20, 21–30, 31–40, 41–50, 51–60, 61–70, 71–80, 81–90, and 91–100%), which are represented as 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100, respectively, on the x-axis of the plot. The antimicrobial peptides in the antimicrobial peptide database are shown in black, whereas gcLECT2-b is shown in orange. (C) SDS-PAGE (left) and Western blot (right) analyses of purified gcLECT2-b. Lane M, marker; 1, purified TrxA-gcLECT2-b; 2, enterokinase-digested TrxA-gcLECT2-b; 3, purified gcLECT2-b. The anti-His mAb was used to detect the recombinant proteins. The blot images were split because they were exposed independently. (D) Antibacterial activities of gcLECT2-b against Gram-negative and Gram-positive bacteria. The CFU assay was conducted, and the treated bacteria were spread onto TSA plates, incubated, and then counted. (E) MIC of gcLECT2-b. The absorbance-based assay was conducted in 96-well microtiter plates, and the growth of bacteria was determined by measuring the absorbance at 600 nm. All the experiments were performed in triplicate. The data in (D) are shown as mean ± SD of three independent experiments, and the data in (E) are representative results of three independent experiments.

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To test the bactericidal activity of gcLECT2-b, we prokaryotically expressed and purified recombinant gcLECT2-b. The purified recombinant gcLECT2-b was examined by SDS-PAGE and Western blot (Fig. 3C). The CFU assay showed that gcLECT2-b possesses dose-dependent broad-spectrum bactericidal activities against Gram-negative (E. coli, A. hydrophila, V. mimicus, and P. fluorescens) and Gram-positive (S. aureus, S. agalactiae, and M. luteus) bacteria (Fig. 3D). The MICs of gcLECT2-b ranged from 2 to 16 μM (Fig. 3E). The biological activities of many proteins are often influenced by temperature and pH (7, 37). However, our study showed that gcLECT2-b has excellent temperature and pH resistance (Supplemental Fig. 2A, 2B). Unlike LL-37 with high cytotoxicity, no significant cytotoxicity of gcLECT2-b to erythrocytes and C. idella kidney cells was observed (Supplemental Fig. 2C, 2D). These findings indicated that gcLECT2-b possesses potent bactericidal activity but low cytotoxicity.

To clarify the bactericidal mechanisms of gcLECT2-b, we investigated the binding ability of recombinant gcLECT2-b (HA-gcLECT2-b) to bacteria and PAMPs. Recombinant gcLECT2-b exhibited binding activity to a variety of bacteria, including four Gram-negative bacteria (V. mimicus, A. hydrophila, P. fluorescens, and E. coli) and three Gram-positive bacteria (S. aureus, M. luteus, and S. agalactiae) (Fig. 4A, 4B). Using E. coli and S. aureus as the representative bacteria, we further found that the binding activity of recombinant gcLECT2-b to bacteria was concentration dependent (Fig. 4C). To explore the molecular basis for the broad-spectrum binding activity of gcLECT2-b to bacteria, we detected the binding activity of gcLECT2-b to various PAMPs, including LPS, PGN, and LTA through ELISA. As shown, HA-gcLECT2-b exhibited concentration-dependent binding activity to a variety of PAMPs, including LPS, PGN, and LTA (Fig. 4D–F).

To further explore the bactericidal mechanisms of gcLECT2-b, we detected the integrity of cell membrane by PI uptake assay. The results revealed that gcLECT2-b can significantly increase the permeability of the cell membrane of E. coli and other Gram-negative bacteria, but not that of S. aureus and other Gram-positive bacteria within 1 h (Fig. 4G–I), suggesting that gcLECT2-b kills Gram-negative and Gram-positive bacteria through different mechanisms. To confirm this conclusion, we observed E. coli and S. aureus under SIM after treatment with FITC-labeled gcLECT2-b from 30 min to 2 h. The results showed that, on FITC-gcLECT2-b treatment, E. coli appeared as hollow rods with a green fluorescence clearly defined bacterial surface within 1 h, suggesting that gcLECT2-b had accumulated on the membrane (Fig. 5A). In addition, the green fluorescence of gcLECT2-b penetrated E. coli cells in a time-dependent manner. On the contrary, on FITC-gcLECT2-b treatment, the green fluorescence was found to penetrate through the membrane of S. aureus and to be located within the cell. After 2 h, the green fluorescence of gcLECT2-b gradually defined the S. aureus surface (Fig. 5B). These results also supported that gcLECT2-b kills Gram-negative and Gram-positive bacteria through different mechanisms. Further, the TEM and SEM observations showed that gcLECT2-b caused severe cell membrane damage and content leakage of E. coli, but not S. aureus. Instead, gcLECT2-b caused severe intracellular damage of S. aureus (Fig. 5C, 5D). These results together strongly demonstrated that gcLECT2-b kills Gram-negative and Gram-positive bacteria in a membrane-dependent and non–membrane-dependent manner, respectively.

FIGURE 4.

Binding activity of gcLECT2-b to bacteria and PAMPs, as well as bacterial membrane permeability of gcLECT2-b. (A) SDS-PAGE and Western blot analyses of purified HA-gcLECT2-b. (B) Purified HA-gcLECT2-b (0.1 μM) was incubated with Gram-negative (E. coli, A. hydrophila, V. mimicus, and P. fluorescens) and Gram-positive (S. aureus, S. agalactiae, and M. luteus) bacteria at 37°C for 1 h. Western blot was then conducted to identify the HA-gcLECT2-b bound to bacteria. TrxA purified with the same method was used as a control. (C) Concentration-dependent binding activity of HA-gcLECT2-b to E. coli and S. aureus determined by Western blot. Purified TrxA was used as a control. (DF) Concentration-dependent binding activity of HA-gcLECT2-b to LPS (D), PGN (E), and LTA (F) determined by ELISA. (G and H) Membrane permeability of Gram-negative (G) and Gram-positive (H) bacteria treated with gcLECT2-b. The bacteria were incubated with gcLECT2-b at 37°C for 1 h, and then the influx of PI was detected by flow cytometry. (I) The data in (G) and (H) were analyzed statistically. All experiments were performed in triplicate. The data in (B) and (C) are representative results of three independent experiments, and the data in (D)–(F) are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.

FIGURE 4.

Binding activity of gcLECT2-b to bacteria and PAMPs, as well as bacterial membrane permeability of gcLECT2-b. (A) SDS-PAGE and Western blot analyses of purified HA-gcLECT2-b. (B) Purified HA-gcLECT2-b (0.1 μM) was incubated with Gram-negative (E. coli, A. hydrophila, V. mimicus, and P. fluorescens) and Gram-positive (S. aureus, S. agalactiae, and M. luteus) bacteria at 37°C for 1 h. Western blot was then conducted to identify the HA-gcLECT2-b bound to bacteria. TrxA purified with the same method was used as a control. (C) Concentration-dependent binding activity of HA-gcLECT2-b to E. coli and S. aureus determined by Western blot. Purified TrxA was used as a control. (DF) Concentration-dependent binding activity of HA-gcLECT2-b to LPS (D), PGN (E), and LTA (F) determined by ELISA. (G and H) Membrane permeability of Gram-negative (G) and Gram-positive (H) bacteria treated with gcLECT2-b. The bacteria were incubated with gcLECT2-b at 37°C for 1 h, and then the influx of PI was detected by flow cytometry. (I) The data in (G) and (H) were analyzed statistically. All experiments were performed in triplicate. The data in (B) and (C) are representative results of three independent experiments, and the data in (D)–(F) are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.

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FIGURE 5.

Observation of bacteria treated with gcLECT2-b. (A and B) Localization of FITC-gcLECT2-b in E. coli (A) and S. aureus (B). The bacteria were treated with FITC-labeled gcLECT2-b for different times and then observed with SIM. (C and D) TEM and SEM imaging of E. coli (C) and S. aureus (D) treated with gcLECT2-b. The bacteria were treated with gcLECT2-b for 1 h and then fixed and processed for ultrastructural observation. The white arrows indicate the leakage of intracellular contents. The images shown are representative results of three independent experiments.

FIGURE 5.

Observation of bacteria treated with gcLECT2-b. (A and B) Localization of FITC-gcLECT2-b in E. coli (A) and S. aureus (B). The bacteria were treated with FITC-labeled gcLECT2-b for different times and then observed with SIM. (C and D) TEM and SEM imaging of E. coli (C) and S. aureus (D) treated with gcLECT2-b. The bacteria were treated with gcLECT2-b for 1 h and then fixed and processed for ultrastructural observation. The white arrows indicate the leakage of intracellular contents. The images shown are representative results of three independent experiments.

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In addition to direct antimicrobial activity, some antimicrobial proteins protect the host through agglutination of bacteria at low concentrations (54). As shown in (Fig. 6A–C, we found that gcLECT2-b possesses broad-spectrum agglutination activity to Gram-negative and Gram-positive bacteria in a dose-dependent manner. The MACs of gcLECT2-b ranged from 0.04 to 1 μM (Fig. 6D). Previous studies have shown that lectins agglutinate bacteria through the recognition of surface polysaccharides (47, 55, 56). Unlike the mechanism of lectins, we found that PGN and LTA, but not galactose, mannose, maltose, glucose, lactose, sucrose, or LPS, could competitively inhibit the bacterial agglutination activity of gcLECT2-b (Fig. 6E). Interestingly, we found that partial gcLECT2-b formed a dimer when interacting with bacteria (Fig. 6F). Considering that the agglutination of bacteria can inhibit the adhesion of pathogenic bacteria to cells, we also examined the inhibitory effect of gcLECT2-b on bacterial adhesion. We found that gcLECT2-b could significantly inhibit the adhesion of A. hydrophila to EPC cells at low concentrations (100–800 nM) that do not inhibit bacterial growth but can agglutinate bacteria (Fig. 6G, 6H). These results indicated that gcLECT2-b can prevent the adhesion of A. hydrophila to EPC cells through agglutination at low concentrations.

LECT2 is a well-known cytokine, and its immunoregulatory activities to leukocytes were studied extensively (18, 24, 25, 57). Using the Transwell system, we evaluated the chemotactic activity of gcLECTC2-b to leukocytes isolated from grass carp blood, spleen, and head-kidney. Like LECT2-a in teleost fish (25, 57) and LECT2 in tetrapods (18, 24), gcLECT2-b also possesses immunoregulatory activity (Fig. 7). gcLECT2-b could chemoattract leukocytes, including lymphoid and myeloid cells, from various tissues at a wide range of concentrations (10–1000 ng/ml) (Fig. 7A–C). Moreover, gcLECT2-b could significantly increase the uptake of fluorescent beads by lymphoid and myeloid cells from various tissues (Fig. 7D–I), with the optimum concentration of ∼400 nM (Supplemental Fig. 3A, 3B). These results demonstrated that, in addition to direct bactericidal activity, gcLECT2-b also possesses immunoregulatory activity.

FIGURE 6.

gcLECT2-b possesses bacterial agglutination activity and inhibits bacterial adhesion to EPC cells. (A and B) gcLECT2-b possesses broad-spectrum agglutination activity to Gram-negative (A) and Gram-positive (B) bacteria. The Tris buffer (20 mM, pH 7.4) was set as the negative control. Each image is a representative result of three independent experiments. (C) Data in (A) and (B) were calculated using ImageJ software and analyzed statistically. (D) Minimal agglutinating concentrations of gcLECT2-b to various bacteria. The MAC of gcLECT2-b was determined with 5-fold serial dilutions of gcLECT2-b. (E) Inhibitory effect of different polysaccharides and PAMPs on bacterial agglutination activity of gcLECT2-b. Different polysaccharides (5 mg/ml) and PAMPs (5 mg/ml) were added to inhibit the bacterial agglutination activity of gcLECT2-b (0.1 μM) through competitive combination experiments. The Tris buffer (20 mM, pH 7.4) and gcLECT2-b (0.1 μM) were set as the negative and positive controls, respectively. (F) Part of gcLECT2-b dimerized after binding to E. coli. HA-gcLECT2-b was incubated with E. coli; then the mixture was cross-linked with BS3 and finally analyzed by SDS-PAGE and Western blot. (G) Inhibitory effects of gcLECT2-b on growth of A. hydrophila at different concentrations. The absorbance-based assay was conducted in 96-well microtiter plates, and the growth of bacteria was determined by measuring the absorbance at 600 nm. (H) gcLECT2-b can significantly inhibit the adhesion of A. hydrophila to EPC cells at subinhibitory concentrations. A. hydrophila was added to EPC cells, followed by gcLECT2-b. After being incubated at 28°C for 1 h, EPC cells were washed, suspended, serially diluted, and plated onto TSA plates to enumerate the bacteria. All the experiments were performed in triplicate. The data in (D)–(G) are representative results of three independent experiments, and the data in (C) and (H) are presented as mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001.

FIGURE 6.

gcLECT2-b possesses bacterial agglutination activity and inhibits bacterial adhesion to EPC cells. (A and B) gcLECT2-b possesses broad-spectrum agglutination activity to Gram-negative (A) and Gram-positive (B) bacteria. The Tris buffer (20 mM, pH 7.4) was set as the negative control. Each image is a representative result of three independent experiments. (C) Data in (A) and (B) were calculated using ImageJ software and analyzed statistically. (D) Minimal agglutinating concentrations of gcLECT2-b to various bacteria. The MAC of gcLECT2-b was determined with 5-fold serial dilutions of gcLECT2-b. (E) Inhibitory effect of different polysaccharides and PAMPs on bacterial agglutination activity of gcLECT2-b. Different polysaccharides (5 mg/ml) and PAMPs (5 mg/ml) were added to inhibit the bacterial agglutination activity of gcLECT2-b (0.1 μM) through competitive combination experiments. The Tris buffer (20 mM, pH 7.4) and gcLECT2-b (0.1 μM) were set as the negative and positive controls, respectively. (F) Part of gcLECT2-b dimerized after binding to E. coli. HA-gcLECT2-b was incubated with E. coli; then the mixture was cross-linked with BS3 and finally analyzed by SDS-PAGE and Western blot. (G) Inhibitory effects of gcLECT2-b on growth of A. hydrophila at different concentrations. The absorbance-based assay was conducted in 96-well microtiter plates, and the growth of bacteria was determined by measuring the absorbance at 600 nm. (H) gcLECT2-b can significantly inhibit the adhesion of A. hydrophila to EPC cells at subinhibitory concentrations. A. hydrophila was added to EPC cells, followed by gcLECT2-b. After being incubated at 28°C for 1 h, EPC cells were washed, suspended, serially diluted, and plated onto TSA plates to enumerate the bacteria. All the experiments were performed in triplicate. The data in (D)–(G) are representative results of three independent experiments, and the data in (C) and (H) are presented as mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001.

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FIGURE 7.

Chemotactic and phagocytosis-stimulating activities of gcLECT2-b to grass carp leukocytes. (AC) Chemotactic activity of gcLECT2-b to leukocytes. Different concentrations of gcLECT2-b (0, 10, 100, and 1000 ng/ml) were placed in the bottom of each well, and then 100 μl of PBLs, HKLs, and SPLs were added into each chamber, respectively. After being incubated at 28°C for 6 h, the migrated cells in DMEM were analyzed by flow cytometry with a constant flow time (1 min). As a positive control, 2% FBS was used. (DF) Phagocytosis-stimulating activity of gcLECT2-b to leukocytes. PBLs, HKLs, and SPLs were stimulated with gcLECT2-b (400 nM) for 3 h and then incubated with FITC-labeled beads for 2 h. Thereafter, the phagocytic activity of leukocytes was measured by flow cytometry. (GI) The data in (D)–(F) were analyzed statistically. All the experiments were performed in triplicate, and the data in (A)–(C) and (G)–(I) are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.

FIGURE 7.

Chemotactic and phagocytosis-stimulating activities of gcLECT2-b to grass carp leukocytes. (AC) Chemotactic activity of gcLECT2-b to leukocytes. Different concentrations of gcLECT2-b (0, 10, 100, and 1000 ng/ml) were placed in the bottom of each well, and then 100 μl of PBLs, HKLs, and SPLs were added into each chamber, respectively. After being incubated at 28°C for 6 h, the migrated cells in DMEM were analyzed by flow cytometry with a constant flow time (1 min). As a positive control, 2% FBS was used. (DF) Phagocytosis-stimulating activity of gcLECT2-b to leukocytes. PBLs, HKLs, and SPLs were stimulated with gcLECT2-b (400 nM) for 3 h and then incubated with FITC-labeled beads for 2 h. Thereafter, the phagocytic activity of leukocytes was measured by flow cytometry. (GI) The data in (D)–(F) were analyzed statistically. All the experiments were performed in triplicate, and the data in (A)–(C) and (G)–(I) are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.

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After we found that gcLECT2-b possesses antibacterial, chemotactic, and phagocytosis-stimulating activities in vitro, we wondered whether gcLECT2-b has a protective effect in vivo. Therefore, we established an enteritis model in grass carp through i.p. injection of A. hydrophila. Postinfection, grass carp were treated with gcLECT2-b at 12 h. The results showed that the survival rate of grass carp treated with gcLECT2-b (73%) was much higher than those treated with PBS (0%) (Fig. 8A). To further examine the effects of gcLECT2-b on intestinal damage and tissue bacterial clearance during A. hydrophila infection, we observed the pathological changes of intestine and counted the bacterial loads in organs after A. hydrophila infection. The results showed that gcLECT2-b significantly reduced the bacterial load in intestine, spleen, head-kidney, and liver (Fig. 8B). Moreover, H&E staining showed that gcLECT2-b significantly attenuated the damage of A. hydrophila to the intestine of grass carp at 2 d postinfection (Fig. 8C). These results indicated that gcLECT2-b plays important protective roles in vivo.

FIGURE 8.

Protective effect of gcLECT2-b in grass carp. (A) The therapeutic effect of gcLECT2-b in grass carp after A. hydrophila infection. Diagram showing procedure of therapeutic experiment (left). Grass carp were injected i.p. with a lethal dose of A. hydrophila (2 × 106 CFUs/fish), followed by a single dose of gcLECT2-b (1 μg/g body weight) or an equal volume of PBS at 12 h postinfection (n = 30 fish). (B) gcLECT2-b reduced the bacterial loads in grass carp tissues after A. hydrophila infection. At 48 h postinfection, the grass carp tissues were homogenized, serially diluted, and plated onto plates to count the number of A. hydrophila (n = 5 fish). (C) gcLECT2-b reduced the intestinal damage in grass carp after A. hydrophila infection. At 2 d postinfection, the intestinal tissues were fixed, sectioned, and stained with H&E to observe the tissue damage. **p < 0.01.

FIGURE 8.

Protective effect of gcLECT2-b in grass carp. (A) The therapeutic effect of gcLECT2-b in grass carp after A. hydrophila infection. Diagram showing procedure of therapeutic experiment (left). Grass carp were injected i.p. with a lethal dose of A. hydrophila (2 × 106 CFUs/fish), followed by a single dose of gcLECT2-b (1 μg/g body weight) or an equal volume of PBS at 12 h postinfection (n = 30 fish). (B) gcLECT2-b reduced the bacterial loads in grass carp tissues after A. hydrophila infection. At 48 h postinfection, the grass carp tissues were homogenized, serially diluted, and plated onto plates to count the number of A. hydrophila (n = 5 fish). (C) gcLECT2-b reduced the intestinal damage in grass carp after A. hydrophila infection. At 2 d postinfection, the intestinal tissues were fixed, sectioned, and stained with H&E to observe the tissue damage. **p < 0.01.

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Because gcLECT2-b has direct antibacterial activity, we considered whether gcLECT2-a and its homologs also have direct antibacterial activity in other vertebrates. To explore the evolution of the bactericidal activity of LECT2 in vertebrates, we analyzed the biochemical properties of representative LECT2s were analyzed across species. The results showed that vertebrate LECT2 proteins are alkaline proteins with small molecular mass (<15.3 kDa) and strong positive charges (calculated charge ≥ +8.0 [pH 7]) (Table I). After we found that grass carp, a lower vertebrate, possesses bactericidal LECT2-b, we wondered whether the bactericidal activity of LECT2 emerged as a unique event in teleost fish or exists widely in vertebrates. Thus, we chose grass carp, American alligator (Alligator mississippiensis), ruddy duck (Oxyura jamaicensis), long-finned pilot whale (Globicephala melas), and human as the representative species of fish, reptiles, birds, aquatic mammals, and terrestrial mammals, respectively, to compare the antibacterial activities of LECT2 protein. To gain insights into the similarity of these representative LECT2 proteins, we conducted the multiple sequence alignment analysis. As shown in (Fig. 9A, the LECT2 proteins are highly conserved (identity > 52%), especially the cysteine residues critical to form the intramolecular disulfide bonds. However, the distribution of the charged and hydrophobic amino acid residues is not conserved. As shown in (Fig. 9B–F, one molecular side of LECT2 proteins from grass carp (LECT2-a), American alligator, ruddy duck, and long-finned pilot whale, but not human, is intensified by positively charged amino acid residues, while the opposite side is intensified by negatively charged amino acid residues and hydrophobic amino acid residues. These results revealed that LECT2 proteins from grass carp, American alligator, ruddy duck, and long-finned pilot whale, but not human, formed cationic amphiphilic structures. These are common characteristics of antimicrobial peptides/proteins that target negatively charged bacterial membranes (53).

FIGURE 9.

The protein sequence and structures of LECT2 proteins in representative vertebrates. (A) Protein sequence alignment of LECT2 proteins from representative vertebrates. The multiple sequence alignment was conducted using the ClustalW program (https://www.genome.jp/tools-bin/clustalw) and then visualized by the ESPript program (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The GenBank accession numbers of LECT2 proteins used are as follows: Ctenopharyngodon idella LECT2-a, MZ160500; Globicephala melas LECT2, XM_030869919.1; Homo sapiens LECT2, NM_002302.3; Alligator mississippiensis LECT2, XM_006276412.2; Oxyura jamaicensis LECT2, XP_035194646.1. (BF) Tertiary structure of representative LECT2 generated by homology modeling (upper) and electrostatic potential displayed on molecular surface (lower). The grass carp LECT2-a was used for this comparison. The positively charged amino acid residues are shown in blue, the negatively charged amino acid residues are shown in red, and the hydrophobic amino acid residues are shown in white.

FIGURE 9.

The protein sequence and structures of LECT2 proteins in representative vertebrates. (A) Protein sequence alignment of LECT2 proteins from representative vertebrates. The multiple sequence alignment was conducted using the ClustalW program (https://www.genome.jp/tools-bin/clustalw) and then visualized by the ESPript program (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The GenBank accession numbers of LECT2 proteins used are as follows: Ctenopharyngodon idella LECT2-a, MZ160500; Globicephala melas LECT2, XM_030869919.1; Homo sapiens LECT2, NM_002302.3; Alligator mississippiensis LECT2, XM_006276412.2; Oxyura jamaicensis LECT2, XP_035194646.1. (BF) Tertiary structure of representative LECT2 generated by homology modeling (upper) and electrostatic potential displayed on molecular surface (lower). The grass carp LECT2-a was used for this comparison. The positively charged amino acid residues are shown in blue, the negatively charged amino acid residues are shown in red, and the hydrophobic amino acid residues are shown in white.

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Table I.

Biochemical properties of LECT2 proteins from representative vertebrates

SpeciesLength (aa)Molecular Mass (kDa)PICharge
Homo sapiens 133 14.572 9.31 9.39 
Mus musculus 133 14.623 9.13 8.39 
Macaca nemestrina 133 14.598 9.39 9.39 
Oryctolagus cuniculus 133 14.355 8.39 8.39 
Globicephala melas 133 14.668 9.42 10.55 
Tursiops truncatus 133 14.796 9.31 9.56 
Phoca vitulina 133 14.629 9.29 9.39 
Oxyura jamaicensis 133 14.279 9.44 10.55 
Calypte anna 133 14.491 9.36 9.39 
Strigops habroptila 133 14.464 9.33 9.38 
Alligator mississippiensis 133 14.632 9.38 9.72 
Ctenopharyngodon idella (LECT2-a) 136 14.895 9.43 11.21 
Ctenopharyngodon idella (LECT2-b) 136 15.283 9.86 14.38 
SpeciesLength (aa)Molecular Mass (kDa)PICharge
Homo sapiens 133 14.572 9.31 9.39 
Mus musculus 133 14.623 9.13 8.39 
Macaca nemestrina 133 14.598 9.39 9.39 
Oryctolagus cuniculus 133 14.355 8.39 8.39 
Globicephala melas 133 14.668 9.42 10.55 
Tursiops truncatus 133 14.796 9.31 9.56 
Phoca vitulina 133 14.629 9.29 9.39 
Oxyura jamaicensis 133 14.279 9.44 10.55 
Calypte anna 133 14.491 9.36 9.39 
Strigops habroptila 133 14.464 9.33 9.38 
Alligator mississippiensis 133 14.632 9.38 9.72 
Ctenopharyngodon idella (LECT2-a) 136 14.895 9.43 11.21 
Ctenopharyngodon idella (LECT2-b) 136 15.283 9.86 14.38 

Further experiments confirmed that, like gcLECT2-b, gcLECT2-a and LECT2 proteins of American alligator, ruddy duck, and long-finned pilot whale, but not human, also possess bactericidal activity against E. coli (Fig. 10A–F). Although the LECT2 from long-finned pilot whale possesses antibacterial activity, the effect is weak. Considering that some antibacterial proteins function in acidic conditions (7), we further examined the antibacterial activity of human LECT2 in different pH conditions, and the results confirmed that human LECT2 has no antibacterial activity (Fig. 10G). As expected, like gcLECT2-b, gcLECT2-a and LECT2 proteins of American alligator, ruddy duck, and long-finned pilot whale, but not human, could dramatically increase the cell membrane permeability of E. coli (Fig. 10H). Moreover, TEM observation further showed that LECT2 proteins of grass carp, American alligator, and ruddy duck could dramatically damage E. coli and cause the leakage of cell contents (Fig. 10J). However, like human LECT2, long-finned pilot whale LECT2 also could not damage bacteria because of the weak antibacterial activity. These results indicated that the bactericidal activity of LECT2 is generally conserved in vertebrates, especially in lower vertebrates (Fig. 11).

FIGURE 10.

Antibacterial activity of LECT2 proteins from representative vertebrates. (AE) Antibacterial activity of grass carp LECT2-a (A), American alligator LECT2 (B), ruddy duck LECT2 (C), long-finned pilot whale LECT2 (D), and human LECT2 (E) against E. coli. The absorbance-based assay was conducted in 96-well microtiter plates, and the growth of bacteria was determined by measuring the absorbance at 600 nm. (F) Bactericidal activity of LECT2 proteins from grass carp (LECT2-a), American alligator, ruddy duck, long-finned pilot whale, and human against E. coli. The CFU assay was conducted, and the treated bacteria were spread onto TSA plates, incubated, and then counted. (G) Antibacterial activity of human LECT2 under different pH conditions. The CFU assay was conducted, and the treated bacteria were spread onto TSA plates, incubated, and then counted. (H) Membrane permeability of E. coli after being treated with LECT2. The bacteria were incubated with LECT2 at 37°C for 1 h, and then the influx of PI was detected by flow cytometry. The representative results of three independent experiments were shown. (I) Statistical analysis of the percentage of PI-positive E. coli tested in (H). (J) TEM imaging of E. coli treated with LECT2 proteins from grass carp (LECT2-a), American alligator, ruddy duck, long-finned pilot whale, and human. The bacteria were treated with LECT2 for 1 h and then fixed and processed for TEM observation. All the experiments were performed in triplicate. The data in (A)–(E) and (J) are representative results of three independent experiments, and the data in (F) and (I) are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01. NS, not significant.

FIGURE 10.

Antibacterial activity of LECT2 proteins from representative vertebrates. (AE) Antibacterial activity of grass carp LECT2-a (A), American alligator LECT2 (B), ruddy duck LECT2 (C), long-finned pilot whale LECT2 (D), and human LECT2 (E) against E. coli. The absorbance-based assay was conducted in 96-well microtiter plates, and the growth of bacteria was determined by measuring the absorbance at 600 nm. (F) Bactericidal activity of LECT2 proteins from grass carp (LECT2-a), American alligator, ruddy duck, long-finned pilot whale, and human against E. coli. The CFU assay was conducted, and the treated bacteria were spread onto TSA plates, incubated, and then counted. (G) Antibacterial activity of human LECT2 under different pH conditions. The CFU assay was conducted, and the treated bacteria were spread onto TSA plates, incubated, and then counted. (H) Membrane permeability of E. coli after being treated with LECT2. The bacteria were incubated with LECT2 at 37°C for 1 h, and then the influx of PI was detected by flow cytometry. The representative results of three independent experiments were shown. (I) Statistical analysis of the percentage of PI-positive E. coli tested in (H). (J) TEM imaging of E. coli treated with LECT2 proteins from grass carp (LECT2-a), American alligator, ruddy duck, long-finned pilot whale, and human. The bacteria were treated with LECT2 for 1 h and then fixed and processed for TEM observation. All the experiments were performed in triplicate. The data in (A)–(E) and (J) are representative results of three independent experiments, and the data in (F) and (I) are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01. NS, not significant.

Close modal
FIGURE 11.

Schematic diagram showing the functions of LECT2 in vertebrates. LECT2 exhibits conserved immunoregulatory activities in vertebrates, including chemotactic and phagocytosis-stimulating activities to leukocytes. Notably, two LECT2 proteins exist in teleost fish, with one (LECT2-b) specialized in mucosal immune organs. Interestingly, LECT2-b of grass carp and LECT2 of representative tetrapods, except human, exhibit direct antibacterial activities, including bactericidal and agglutination activities. These results together indicate that LECT2 can protect the host from bacterial infection through multiple functions.

FIGURE 11.

Schematic diagram showing the functions of LECT2 in vertebrates. LECT2 exhibits conserved immunoregulatory activities in vertebrates, including chemotactic and phagocytosis-stimulating activities to leukocytes. Notably, two LECT2 proteins exist in teleost fish, with one (LECT2-b) specialized in mucosal immune organs. Interestingly, LECT2-b of grass carp and LECT2 of representative tetrapods, except human, exhibit direct antibacterial activities, including bactericidal and agglutination activities. These results together indicate that LECT2 can protect the host from bacterial infection through multiple functions.

Close modal

In vertebrates, LECT2 is an important immunoregulator with conserved chemotactic and phagocytosis-stimulating activities. In this study, we show that, unlike tetrapods with a single LECT2 gene, two LECT2 genes exist in teleost fish, including grass carp and zebrafish. Gene synteny analyses revealed that the two LECT2 genes in teleost fish should arise by single gene duplication. In mammals, LECT2 is mainly produced in the liver and then secreted into the blood; however, in grass carp, we found that gcLECT2-a is highly expressed in systemic immune organs, such as the kidney and liver, while gcLECT2-b is highly expressed in mucosal immune organs, such as the intestine and skin (19). Thus, although evolved from the common ancestor, the expression patterns and functions of gcLECT2-a and gcLECT2-b might evolve divergently.

The origin and specialization of gcLECT2-b in mucosal immunity gave rise to our research interest. The structural and biochemical analyses revealed that gcLECT2-b possesses a cationic amphiphilic feature like most of the cationic antimicrobial peptides/proteins (53). Functional analyses further confirmed that gcLECT2-b is a novel, broad-spectrum, and potent antibacterial protein. Due to their special evolutionary status and living environment, teleost fish rely heavily on antibacterial molecules in mucosal immune organs to maintain their health (58). Thus, in concert with antimicrobial peptides/proteins, gcLECT2-b may play important roles in preventing bacterial infections through mucosal tissues.

Natural antimicrobial peptides/proteins, such as gcCXCL20a (14), thrombin-derived C-terminal peptide (59), and IL-26 (13), are commonly characterized by positive charges and amphiphilicity. The positive charges promote the electrostatic attraction of antimicrobial peptides/proteins to various negatively charged membrane components of bacteria (60, 61). Like these antimicrobial peptides/proteins, gcLECT2-b can bind to various bacterial cell wall components, including LPS, LTA, and PGN, suggesting that gcLECT2-b recognizes bacteria by targeting PAMPs. Interestingly, gcLECT2-b kills Gram-negative bacteria through disrupting the cell membrane, like most of the classical antimicrobial peptides/proteins (13, 62), which was supported by the membrane permeability assay, SIM, TEM, as well as SEM observations. However, like some antimicrobial peptides/proteins, gcLECT2-b kills Gram-positive bacteria in a non–membrane-dependent manner, suggesting that gcLECT2-b may interfere with bacterial metabolism by binding to intracellular targets (63, 64). The specific mechanism used by gcLECT2-b to kill Gram-positive bacteria remains to be elucidated.

Previous studies have proved that the bacterial agglutination activities of antimicrobial peptides/proteins can prevent bacterial infections and facilitate the clearance of pathogenic bacteria by host immune cells (54, 6571). Our results showed that gcLECT2-b possesses broad-spectrum agglutination activity to bacteria at nanomolar concentrations. Competitive combination experiments further revealed that gcLECT2-b agglutinates various bacteria through specific binding to PGN and/or LTA, rather than nonspecific recognition of polysaccharides on the cell surface. The mechanism is different from that of lectins, which agglutinate bacteria by binding to polysaccharides on the bacterial cell surface (47, 55, 56). This may be because gcLECT2-b does not contain a carbohydrate recognition domain, which is used by lectins to interact with glycans (47, 55, 56). Studies have shown that antimicrobial peptides/proteins can exhibit considerable bacterial agglutination activity by cross-linking the surface components of bacteria and forming crystal lattices, such as crustin, thanatin, chemokines, and thrombin-derived peptides (14, 17, 71, 72). To clarify the mechanism of the bacterial agglutination activity of gcLECT2-b, we conducted the protein cross-linking experiment. The result showed that part of gcLECT2-b dimerized after binding to bacteria. This should be the main mechanism by which gcLECT2-b agglutinates bacteria. More importantly, our study showed that gcLECT2-b can inhibit the adhesion of A. hydrophila to EPC cells in vitro at subinhibitory concentrations. Consistent with this, gcLECT2-b can protect grass carp from A. hydrophila infection in vivo, indicating that LECT2 is a candidate anti-infective and anti-inflammatory drug in teleost fish.

Like LECT2 of tetrapods, LECT2-a of teleost fish possesses conserved immunoregulatory activities, including chemotactic and phagocytosis-stimulating activities to leukocytes (24, 25). Interestingly, we found that LECT2-b, specifically evolved in teleost fish, also possesses immunoregulatory activities in grass carp, like that of LECT2-a. Thus, we speculate that in healthy conditions or the early stage of infections, low concentrations of gcLECT2-b may prevent the invasion of pathogenic bacteria through agglutination and immunoregulation, while in the late stage of infections, high concentrations of gcLECT2-b can defend pathogenic bacteria through direct killing.

Because we found that gcLECT2-b has direct antibacterial activity, we considered whether LECT2 proteins in other vertebrates also have direct antibacterial activity. Therefore, in addition to gcLECT2-a, LECT2 proteins from representative tetrapods were selected to explore the evolution of the antibacterial activity of LECT2 in vertebrates. Interestingly, like gcLECT2-b, gcLECT2-a and LECT2 proteins from American alligator and ruddy duck also formed a typical cationic amphipathic feature, which was responsible for the antibacterial activity. However, the cationic amphipathic feature of the LECT2 from the long-finned pilot whale was not obvious, because of the ambiguity of the hydrophobic surface. Unexpectedly, the LECT2 from human was not found to possess the cationic amphipathic feature. Consistent with the cationic amphipathic feature, gcLECT2-a and LECT2 proteins from American alligator, ruddy duck, and long-finned pilot whale, but not human, could kill bacteria directly, with the activity of gcLECT2-b being the highest. These results suggest that the bactericidal activity of LECT2 is more conserved in lower vertebrates.

Taken together, two LECT2 proteins were found in teleost fish, with LECT2-a being more similar to the LECT2 in tetrapods and LECT2-b having evolved to be specialized in the mucosal immune organs of fish (Fig. 11). The newly discovered bactericidal and agglutination activities of LECT2-b make it a fascinating molecule that deserves further study. More interestingly, we found that the bactericidal activity of LECT2 is generally conserved in vertebrates, which greatly enhanced our knowledge of this important molecule.

We thank Zhe Hu (Huazhong Agricultural University) for assistance in SIM and Limin He (Huazhong Agricultural University) for assistance in TEM.

This work was supported by the National Key Research and Development Program of China (2018YFD0900505), the National Natural Science Foundation of China (31602184), the Laboratory of Lingnan Modern Agriculture Project (NT2021008), the China Agriculture Research System (CARS-46), and Fundamental Research Funds for the Central Universities (2662018QD053).

The online version of this article contains supplemental material.

The gcLECT2-a and gcLECT2-b cDNAs were submitted to the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers MZ160500 and MZ160501.

Abbreviations used in this article:

     
  • EPC

    epithelioma papulosum cyprini

  •  
  • gcLECT2-a

    grass carp LECT2-a

  •  
  • HA

    hemagglutinin

  •  
  • HKL

    head-kidney leukocyte

  •  
  • LECT2

    leukocyte-derived chemotaxin-2

  •  
  • LTA

    lipoteichoic acid

  •  
  • MAC

    minimum agglutination concentration

  •  
  • MIC

    minimum inhibitory concentration

  •  
  • PAMP

    pathogen-associated molecular pattern

  •  
  • PBL

    peripheral blood leukocyte

  •  
  • PGN

    peptidoglycan

  •  
  • PI

    propidium iodide

  •  
  • qPCR

    quantitative real-time PCR

  •  
  • SEM

    scanning electron microscopy

  •  
  • SIM

    structured illumination microscopy

  •  
  • SPL

    spleen leukocyte

  •  
  • TEM

    transmission electron microscopy

  •  
  • TSA

    tryptic soy agar

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The authors have no financial conflicts of interest.

Supplementary data