Agglutinating Activity Research Articles

CRISPR/Cas9-mediated generation of auxotrophic Edwardsiella piscicida mutants and immunization in olive flounder (Paralichthys olivaceus)

Edwardsiella piscicida has been a cause of mass mortality in cultured fish. In this study, to produce auxotrophic E. piscicida mutants, a CRISPR/Cas9 system was used instead of the traditional sacB-based allelic exchange method. Under the optimal CRISPR engineering condition, we could efficiently produce either alr or asd gene knockout E. piscicida auxotrophic mutants, and this genome editing process was much simpler and faster than the allelic exchange method. The simultaneous knockout of double auxotrophic genes (alr and asd) and the insertion of a foreign gene expression cassette in E. piscicida chromosome were also successfully performed using the established CRISPR/Cas9 system. Furthermore, to enhance the possibility to get permission as a commercial vaccine, we produced an auxotrophic E. piscicida mutant having only one nucleotide-deleted alr gene (E. piscicida △alr-1). Olive flounder (Paralichthys olivaceus) fingerlings immunized with 1 × 106 and 1 × 105 CFU/fish of E. piscicida △alr-1 showed the superior ability in the induction of serum agglutination activity and in the protection against E. piscicida compared to killed E. piscicida. However, olive flounder immunized with 1 × 107 CFU/fish of E. piscicida △alr-1 showed high mortality far before the challenge, and the isolated E. piscicida from moribund and dead fish had the wild type alr gene, suggesting the reversion of one base-deleted alr gene to original form by a second mutation in olive flounder. Therefore, investigation on the minimum number of edited nucleotide for stable maintenance of E. piscicida mutants should be further conducted.

Exploring New Tetrahydrothienopyridine Derivatives as Platelet Agglutination Inhibitors: Synthesis, Biological Evaluation and In Silico Study

AbstractThe P2Y12 receptor is the major target for antithrombic drugs which plays a key role in platelet activation. New derivatives of 4,5,6,7‐tetrahydrothieno[3,2‐c]pyridine (THP) were designed targeting P2Y12 receptor. An efficient route was developed for synthesis of THP derivatives and subsequently evaluated for their antiplatelet agglutination activity. Amongst the synthesized THP derivatives (4 a–4 g), the compounds 4 a and 4 g displayed significant activity (with 88.25 and 70.17 % inhibition) as compared to other analogs and comparable with that of the reference drugs, aspirin and prasugrel. Data extracted from computational chemistry techniques such as molecular docking, provided the structural rationale for the observed platelet agglutination inhibition by the newly synthesized tetrahydrothienopyridine analogs. We proposed the involvement of residues such as Cys‐194 in the formation of the covalent adduct with the active metabolite of tetrahydrothienopyridine derivatives. This study also put forward the possibility of the existence of an alternate pathway for metabolizing the tetrahydrothienopyridine compounds. The structural data presented in this study is expected to accelerate the research on developing a tetrahydrothienopyridine scaffold as an effective antithrombotic therapeutic modality.

Effect of feeding solid state fermented mahua oil cake (Bassia latifolia) on growth performance, enzymatic and immunological parameters in rohu (Labeo rohita)

Solid-state fermentation of mahua oil cake (Bassia latifolia) was carried out by yeast Saccharomyces cerevisiae and then it was incorporated as feed ingredient for production of fish feed for rohu (Labeo rohita). To evaluate the solid-state fermented mahua oil cake as feed ingredient in rohu, two types of fish feeds were produced i.e. a control feed using raw mahua oil cake and treatment feed using solid-state fermented mahua oil cake. A feeding trial of 60 days’ duration was conducted to know the effect of feeding fish with fermented mahua oil cake during which growth performance, digestibility of nutrients, intestinal enzyme activity and non-specific immune parameter of fish fed on fermented mahua oil cake. Solid-state fermentation of mahua oil cake by yeast improved the quality of mahua oil cake by increase in crude protein (CP), decrease in crude fibre (CF) and anti-nutritional factors like saponin and tannin. Use of fermented mahua oil cake as feed ingredient in rohu resulted in significant improvement (P<0.05) of growth performance and digestibility of nutrients (dry matter, crude protein, crude fibre and ether extract) compared to control fed with raw mahua oil cake. Similarly, significantly higher (P<0.05) intestinal enzyme activity like amylase and protease and non-specific immune parameters like lysozyme, myeloperoxidase, bacterial agglutination, hem-agglutination and respiratory bust activity (P<0.05) were recorded in fish fed on fermented mahua oil cake compared to fish fed on raw mahua oil cake. This showed that, feeding of solid state fermented mahua oil cake increased the growth performance, enzyme activity and immunological parameters in rohu compared to mahua oil cake without solid state fermentation.

Preliminary report on the hemagglutinating activity of the Scorpaena plumieri fish venom

The scorpionfish Scorpaena plumieri is one of the most venomous fish species in the Brazilian coast. Amongst many biological activities, the S. plumieri fish venom (SpV) promotes hemagglutination. Although this activity appears to be associated to the presence of C-type lectins in the venom, it has not yet been chemically or functionally characterized. In the present work we sought to advance the characterization of the hemagglutinating activity associated to this venom. By fractionating SpV through saline precipitation followed by size exclusion chromatography we obtained two purified fractions - HF1 and HF3 - with Ca2+-dependent agglutinating activity against rabbit erythrocytes, which remained stable upon storage at 4 and -80oC. HF1 and HF3 were bacteriostatic against Gram-positive bacteria (Staphylococcus aureus), displaying minimum inhibitory concentration (MIC) of 50 and 200 μg/mL, respectively. In addition, a resazurin-based viability assay revealed that both fractions, at doses up to 370 μg/mL, were cytotoxic against tumor and non-tumor cell lines. Finally, a tendency towards edema formation could be detected when the fractions - particularly HF1 - were injected into mice footpads. We believe our data contribute to a better understanding of the biological properties of the so often neglected fish venoms.

Effects of vitamin C on haemolymph activity and haemocyte structure of Cherax quadricarinatus after white spot syndrome virus infection

In this study, we used enzymatic chemistry, histology and molecular biology to systematically study the effects of vitamin C (VC) in feed on haemocyte number and haemolymph activity in Cherax quadricarinatus. We also compared the effects of VC on the number of haemocytes, the haemocyte microstructure and the ultrastructure of C. quadricarinatus haemocytes after White spot syndrome virus (WSSV) infection. When feed was supplemented with 800 mg/kg VC (C5 group), the number of haemocytes increased by 42.26% compared with the control group (C1 group); and the haemolymph of the C5 group had the highest agglutination activity and anti-bacterial rate, which was significantly higher than that of the C1 group (p < 0.05). WSSV infection significantly reduced the total number of haemocytes, and the number of haemocytes was significantly higher in the C1 group than in the than C5 (p < 0.05) for up to 96 h. At 96 h, transmission electron microscopy revealed crushed perinuclear material, cell lysis, and damaged cell membranes in the C1 group, and large apparently damaged particles in the C5 group, with clear cell swelling and small particles, although the cell structure was intact. These results showed that VC enhanced the immune function of crayfish to a certain extent, maintained the integrity of the haemocyte structure and conferred an immunoprotective effect on C. quadricarinatus against WSSV infection.

The Protective Effects of Cath-MH With Anti-Propionibacterium Acnes and Anti-Inflammation Functions on Acne Vulgaris.

Acne vulgaris is a common adolescent skin condition which is mainly caused by Propionibacterium acnes overcolonization and subsequent inflammation. Our previous studies have demonstrated that Cath-MH, an antimicrobial peptide from the skin of the frog Microhyla heymonsivogt, possesses potential antimicrobial, LPS-binding, and anti-septicemic properties. However, its protective effects and potential mechanisms against acne vulgaris are still unclear. In the present study, its anti-P. acnes effects were measured by two-fold broth dilution method, agglutination assay, scanning electron microscopy and confocal laser scanning microscopy experiments. Its treatment potential for acne vulgaris was further evaluated in mice ear inoculated by P. acnes. In addition, the binding ability between Cath-MH and LTA was measured by the Circular Dichroism and antibacterial assay. Moreover, the anti-inflammatory efficiency of Cath-MH was evaluated in LTA- and LPS-induced RAW 264.7 macrophage cells. Cath-MH was found to kill P. acnes with a MIC value of about 1.56 μM by membrane disruption mechanism. It also exhibited agglutination activity against P. acnes. Cath-MH was able to bind LTA as well as LPS, inhibit LTA/LPS-stimulated TLR2/4 expression, and subsequently decreased the inflammatory response in RAW 264.7 cells. As expected, Cath-MH alleviated the formation of edema and the infiltration of inflammatory cells in acne mouse model with concurrent suppression of P. acnes growth and inflammatory cytokines expression in vivo. The potent P. acnes inhibition activity combined with powerful anti-inflammatory effect of Cath-MH indicates its potential as a novel therapeutic option for acne vulgaris.

Probiotic potential of Weissella paramesenteroides MYPS5.1 isolated from customary dairy products and its therapeutic application.

The online version contains supplementary material available at 10.1007/s13205-021-03074-2.

Expression and functional characterization of peptidoglycan recognition protein-S6 involved in antibacterial responses in the razor clam Sinonovacula constricta

It has been recognized that peptidoglycan recognition proteins (PGRPs), structurally conserved molecules, play crucial roles in the innate immunity of invertebrate. However, few studies have been taken to explore their potential functions. In this study, a novel PGRP from the razor clam Sinonovacula constrict designated as ScPGRP-S6 was identified and characterized. The open reading frame (ORF) of ScPGRP-S6 was 666 bp in length, encoding a protein of 221 amino acid with a signal peptide (1–30) and a typical PGRP domain (39–187). The sequence alignment combined with phylogenetic analysis collectively confirmed that ScPGRP-S6 was a novel member belonging to PGRP-S family. The mRNA transcript of ScPGRP-S6 in the hepatopancreases was significantly up-regulated after peptidoglycan (PGN) stimulation, while it was moderately up-regulated after lipopolysaccharide (LPS) stimulation. The result of immunofluorescence detection demonstrated that the positive signal enhanced obviously after Vibrio parahaemolyticus challenge. Notably, the recombinant protein of ScPGRP-S6 (designed as rScPGRP-S6) exhibited high agglutination activity towards V. parahaemolyticus but weak to Staphylococcus aureus. Furthermore, rScPGRP-S6 showed strong amidase and antibacterial activity in the presence of Zn2+. Collectively, our results manifested that ScPGRP-S6 could act as a scavenger in the innate immune response of S. constricta.

Isolation and Characterization of Human Monoclonal Antibodies to Pneumococcal Capsular Polysaccharide 3.

ABSTRACTThe current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human ST3 pneumococcal capsular polysaccharide (PPS3) antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven PPS3-specific human monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination in vitro, and four mediated both activities. Two humAbs, namely, C10 and C27, that used the same variable heavy (VH) and light (VL) chain domains (VH3-9*01/VL2-14*03) both altered ST3 gene expression in vitro; however, C10 had fewer VL somatic mutations, higher PPS3 affinity, and promoted in vitro ST3 opsonophagocytic and agglutinating activity, whereas C27 did not. In C57BL/6 mice, both humAbs reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. After performing VL swaps, C10VH/C27VL exhibited reduced ST3 binding and agglutination, but C27VH/C10VL binding was unchanged. However, both humAbs lost the ability to reduce colonization in vivo when their light chains were replaced. Our findings associate the ability of PPS3-specific humAbs to reduce colonization with ST3 agglutination and opsonophagocytic activity, and reveal an unexpected role for the VL in their functional activity in vitro and in vivo. These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3.IMPORTANCE Despite the global success of vaccination with pneumococcal conjugate vaccines, serotype 3 (ST3) pneumococcus remains a leading cause of morbidity and mortality. In comparison to other vaccine-included serotypes, the ST3 pneumococcal capsular polysaccharide (PPS3) induces a weaker opsonophagocytic response, which is considered a correlate of vaccine efficacy. Previous studies of mouse PPS3 monoclonal antibodies identified ST3 agglutination as a correlate of reduced ST3 nasopharyngeal colonization in mice; however, neither the agglutinating ability of human vaccine-elicited PPS3 antibodies nor their ability to prevent experimental murine nasopharyngeal colonization has been studied. We generated and analyzed the functional and in vivo efficacy of human vaccine-elicited PPS3 monoclonal antibodies and found that ST3 agglutination associated with antibody affinity, protection in vivo, and limited somatic mutations in the light chain variable region. These findings provide new insights that may inform the development of antibody-based therapies and next-generation vaccines for ST3.

Purification and characterization of a highly thermostable GlcNAc-binding lectin from Collaea speciosa seeds

Lectins from plants of the Diocleinae subtribe often exhibit specificity towards mannose/glucose and derived sugars, with some plants also displaying a second lectin specific to lactose/GalNAc. Here, we present a novel lectin from Collaea speciosa, named CsL, that displays specificity for GlcNAc/glucose. The lectin was extracted from Collaea speciosa seeds and purified by a single chromatographic step on a Sephadex G-50 matrix. In solution, the lectin appears as a dimeric protein composed of 25 kDa monomers. The protein is stable at pH 7–8 and dependent on divalent cations. CsL maintained its agglutination activity after heating to 90 °C for 1 h. Glycan array studies revealed that CsL binds to N-glycans with terminal GlcNAc residues, chitobiose and chitotriose moieties. The partial amino acid sequence of the lectin is similar to that of some lactose-specific lectins from the same subtribe. In contrast to other ConA-like lectins, CsL is not toxic to Artemia. Because of its remarkably different properties and specificity, this lectin could be the first member of a new group inside the Diocleinae lectins.

F-type lectin from serum of the Antarctic teleost fish Trematomus bernacchii (Boulenger, 1902): Purification, structural characterization, and bacterial agglutinating activity.

The increasing availability of sequenced genomes has enabled a deeper understanding of the complexity of fish lectin repertoires involved in early development and immune recognition. The teleost fucose-type lectin (FTL) family includes proteins that preferentially bind fucose and display tandemly arrayed carbohydrate-recognition domains (CRDs) or are found in mosaic combinations with other domains. They function as opsonins, promoting phagocytosis and the clearance of microbial pathogens. The Antarctic fish Trematomus bernacchii is a Perciforme living at extremely low temperatures (-1.68°C) which is considered a model for studying adaptability to the variability of environmental waters. Here, we isolated a Ca++-independent fucose-binding protein from the serum of T. bernacchii by affinity chromatography with apparent molecular weights of 32 and 30kDa under reducing and non-reducing conditions, respectively. We have characterized its carbohydrate binding properties, thermal stability and potential ability to recognize bacterial pathogens. In western blot analysis, the protein showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, its molecular and structural aspects, showing that it contains two CRD-FTLs confirmed that T. bernacchii FTL (TbFTL) is a bona fide member of the FTL family, with binding activity at low temperatures and the ability to agglutinate bacteria, thereby suggesting it participates in host-pathogen interactions in low temperature environments.

An l-rhamnose-binding lectin from Nile tilapia (Oreochromis niloticus) possesses agglutination activity and regulates inflammation, phagocytosis and respiratory burst of monocytes/macrophages

Rhamnose-binding lectins (RBLs), a Ca2+-independent lectin family, are widely present in vertebrates and invertebrates, which involve in the innate immune response. However, the functional characterization and related regulation mechanisms of RBLs remain unclear in teleost fish. In this study, an l-rhamnose-binding lectin-like (OnRBL-L) was identified and functionally characterized from Nile tilapia (Oreochromis niloticus). The open reading frame of OnRBL-L is 678 bp encoding 225 aa. The sequence of OnRBL-L has relatively conservative characteristic peptide motifs, including YGR, DPC, and KYL-motif. Expression analysis showed that OnRBL-L was abundantly distributed in intestine tissue, and widely existed in all detected tissues. Meanwhile, the expression of OnRBL-L increased significantly in vivo (liver, spleen, head kidney, intestine, gills and peripheral blood) and in vitro (monocytes/macrophages) following challenges with two important tilapia pathogenic bacteria Streptococcus agalactiae and Aeromonas hydrophila. In addition, the recombinant OnRBL-L was found to bind and agglutinate S. agalactiae and A. hydrophila. Furthermore, OnRBL-L could participate in non-specific cellular immune defense, including reducing the expression of pro-inflammatory factors (IL-6、IL-8 and TNF-α), and enhancement of the phagocytosis and respiratory burst of MO/MФ. Overall, our results provide new insights into the understanding of RBL as an important pattern recognition molecule and regulator in non-specific cell immunity in an early vertebrate.

Amphioxus ribosomal proteins RPS15, RPS18, RPS19 and RPS30-precursor act as immune effectors via killing or agglutinating bacteria

Previous studies show that some ribosomal proteins perform immune effector functions via killing bacteria directly. However, it remains largely unknown about other effector functions of ribosomal proteins during a bacterial infection. In this study, we expressed and purified four ribosomal proteins of the amphioxus Branchiostoma japonicum, termed rBjRPS15, rBjRPS18, rBjRPS19 and rBjRPS30-precursor (rBjRPS30P). They all exhibited bactericidal activity against Gram-positive Staphylococcus aureus, and with the exception of rBjRPS19 and rBjRPS30P, were capable of killing Gram-negative Escherichia coli. Importantly, rBjRPS15, rBjRPS19 and rBjRPS30P were able to agglutinate S. aureus in the presence of Mg2+, but none of them could agglutinate E. coli even in the presence of Mg2+ or Ca2+. Moreover, the S. aureus agglutination was achieved by the binding of these three proteins to the peptidoglycan component of the bacterial cell wall. This is the first report showing that some ribosomal proteins possess bacterial agglutinating activity, and these data provide a new angle to the roles of ribosomal proteins in immune defense.

A multi‐epitope chimeric protein elicited a strong antibody response and partial protection against Edwardsiella ictaluri in Nile tilapia

Edwardsiella ictaluri infects several fish species and protection of the all the susceptible fish hosts from the pathogen using a monovalent vaccine is impossible because the species is composed of host-based genotypes that are genetic, serological and antigenic heterogenous. Here, immunoinformatic approach was employed to design a cross-immunogenic chimeric EiCh protein containing multi-epitopes. The chimeric EiCh protein is composed of 11 B-cell epitopes and 7 major histocompatibility complex class II epitopes identified from E.ictaluri immunogenic proteins previously reported. The 49.32kDa recombinant EiCh protein was expressed in vitro in Escherichia coli BL-21 (DE3) after which inclusion bodies were successfully solubilized and refolded. Ab initio protein modelling revealed secondary and tertiary structures. Secondary structure was confirmed by circular dichroism spectroscopy. Antigenicity of the chimeric EiCh protein was exhibited by strong reactivity with serum from striped catfish and Nile tilapia experimentally infected with E.ictaluri. Furthermore, immunogenicity of the chimeric EiCh protein was investigated in vivo in Nile tilapia juveniles and it was found that the protein could strongly induce production of specific antibodies conferring agglutination activity and partially protected Nile tilapia juveniles with a relative survival percentage (RPS) of 42%. This study explored immunoinformatics as reverse vaccinology approach in vaccine design for aquaculture to manage E.ictaluri infections.

Haem agglutination Activity and Biofilm Formation from Clinical Isolates of Enterococcus and Their Characterization in a Tertiary Care Centre in Eastern India

Haem agglutination Activity and Biofilm Formation from Clinical Isolates of Enterococcus and Their Characterization in a Tertiary Care Centre in Eastern India

The production of hyperimmune pseudotuberculosis sera

Background . Pseudotuberculosis remains a serious public health problem, which dictates the need to develop simple and rapid diagnostic methods for this disease. The effectiveness of the latter largely depends on the activity and specificity of the diagnostic sera. Currently, in our country, a diagnostic serum for Yersinia pseudotuberculosis O-monovalent (serotype I, III) is produced with a regulated area of application: for an approximate agglutination reaction on glass (Pasteur NIIEM, St. Petersburg). Preparation of pseudotuberculosis sera for a wider scope of their use, in particular, as a source of specific antibodies in the design of diagnostic immunobiological preparations and test systems for pseudotuberculosis, is relevant and in demand in healthcare practice. Aims . To obtain hyperimmune pseudotuberculosis sera, promising for use in the practice of laboratory studies for pseudotuberculosis. Materials and methods. Chinchilla rabbits aged 3–6 months, weighing 2.5–3.0 kg served as animal producers of pseudotuberculosis sera. As immunogens, we used a corpuscular antigen (CAg) - a suspension of cells of the epidemically significant Yersinia pseudotuberculosis 3704 O: 1b strain isolated by an outbreak of pseudotuberculosis in the town of Zima, Irkutsk region, inactivated by boiling, and a preparation obtained from the outer membranes, containing the main surface immunogen of the strain. For the adsorption of experimental sera, in order to minimize the nonspecific response, we used heterologous microorganisms Escherichiacoli, Salmonella typhimurium, Shigella flexneri and Y. enterocolitica O:3, which have a similarity of surface antigenic structures with the causative agent of pseudotuberculosis. The presence of specific antibodies in experimental pseudotuberculosis sera was determined in a volumetric agglutination reaction. Results . Optimal schemes of rabbit immunization were selected, which made it possible to obtain hyperimmune sera against Y. pseudotuberculosis 3704 O:1b antigens with an agglutination activity of 1:3200-1:6400. In low dilutions of experimental sera (1:100–1:400), obtained against corpuscular antigen and outer membrane antigens (Escherichia coli, Salmonella typhimurium, Shigella flexneri , Y. enterocolitica О:3, S . enteritidis Gartnery) was observed in the agglutination reaction (AR). After the adsorption of experimental pseudotuberculosis sera by cells of heterologous strains, there was no cross-reaction with the indicated microorganisms in the agglutination reaction. Conclusions . The obtained hyperimmune adsorbed sera against the boiling-inactivated Y. pseudotuberculosi s 3704 O: 1b corpuscular antigen and outer membrane antigens can be used as a source of specific antibodies in the design of diagnostic immunobiological preparations for the detection of pseudotuberculosis microbe, as well as in the monitoring of the epidemic situation.

Two Amphioxus ApeC-Containing Proteins Bind to Microbes and Inhibit the TRAF6 Pathway.

The apextrin C-terminal (ApeC) domain is a class of newly discovered protein domains with an origin dating back to prokaryotes. ApeC-containing proteins (ACPs) have been found in various marine and aquatic invertebrates, but their functions and the underlying mechanisms are largely unknown. Early studies suggested that amphioxus ACP1 and ACP2 bind to bacterial cell walls and have a role in immunity. Here we identified another two amphioxus ACPs (ACP3 and ACP5), which belong to the same phylogenetic clade with ACP1/2, but show distinct expression patterns and sequence divergence (40-50% sequence identities). Both ACP3 and ACP5 were mainly expressed in the intestine and hepatic cecum, and could be up-regulated after bacterial challenge. Both prokaryotic-expressed recombinant ACP3 and ACP5 could bind with several species of bacteria and yeasts, showing agglutinating activity but no microbicidal activity. ELISA assays suggested that their ApeC domains could interact with peptidoglycan (PGN), but not with lipoteichoic acid (LTA), lipopolysaccharides (LPS) and zymosan A. Furthermore, they can only bind to Lys-type PGN from Staphylococcus aureus, but not to DAP-type PGN from Bacillus subtilis and not to moieties of PGN such as MDPs, NAMs and NAGs. This recognition spectrum is different from that of ACP1/2. We also found that when expressed in mammalian cells, ACP3 could interact with TRAF6 via a conserved non-ApeC region, which inhibited the ubiquitination of TRAF6 and hence suppressed downstream NF-κB activation. This work helped define a novel subfamily of ACPs, which have conserved structures, and have related yet diversified molecular functions. Its members have dual roles, with ApeC as a lectin and a conserved unknown region as a signal transduction regulator. These findings expand our understanding of the ACP functions and may guide future research on the role of ACPs in different animal clades.

Characterization and functional study of a chimera galectin from yellow drum Nibea albiflora

Galectins are protein that participates in a variety of immune responses in the process of pathogenic infections. In the present study, a chimera galectin gene was screened from the transcriptome database of Nibea albiflora, which was named as YdGal-3. The results of qRT-PCR showed that the mRNA transcripts of YdGal-3 were ubiquitously distributed in all the detected tissues. After infection with Vibrio harveyi, the expression of YdGal-3 in liver, spleen, and head kidney increased significantly. Immunohistochemistry showed that YdGal-3 protein was widely expressed in the head kidney. The purified YdGal-3 protein by prokaryotic expression agglutinated red blood cells. Sugar inhibition assay showed that the agglutinating activity of YdGal-3 protein was inhibited by different sugars including lactose, D-galactose, and lipopolysaccharide. In addition, we mutated YdGal-3 His 294 into proline (P), alanine (A), glycine (G), and aspartic acid (D), it was further proved that the residue plays a key role in agglutination. YdGal-3 agglutinated some gram-negative bacteria including Pseudomonas plecoglossicida, Vibrio parahemolyticus, V. harveyi, and Aeromonas hydrophila, and exhibited antibacterial activity. These results suggested that YdGal-3 protein played an important role in the innate immunity of N. albiflora.

The N‐ and C‐terminal carbohydrate recognition domains of galectin‐9 from Carassius auratus contribute differently to its immunity functions to Aeromonas hydrophila and Staphylococcus aureus

Galectin-9, an important pathogen recognition receptor (PRR), could recognize and bind pathogen-associated molecular patterns (PAMPs) on the surface of invading microorganisms, initiating the innate immune responses. A galectin-9 was identified from Qihe crucian carp Carassius auratus and designated as CaGal-9. The predicted CaGal-9 protein contained two non-identical carbohydrate recognition domains (CRDs), namely, N-CRD and C-CRD. The recombinant proteins (rCaGal-9, rN-CRD and rC-CRD) were purified from Escherichia coli BL21 (DE3) and exhibited strong agglutinating activity with erythrocytes of rabbit. The haemagglutination was inhibited by D-galactose, α-lactose and N-acetyl-D-galactose. Results of microbial agglutination assay showed that three recombinant proteins agglutinated Gram-negative bacterium Aeromonas hydrophila and Gram-positive bacterium Staphylococcus aureus. With regard to the binding activity, each recombinant protein could bind to LPS, PGN and the examined microorganisms (A. hydrophila and S. aureus) with different binding affinities. The integrated analyses suggested that CaGal-9 with two CRD domains could play an important role in immune defence against pathogenic microorganisms for C. auratus.

Expression and functional characterization of an L-rhamnose-binding lectin from Nile tilapia (Oreochromis niloticus) in host defense against bacterial infection

Rhamnose-binding lectins (RBLs), a Ca2+-independent lectin family, are able to recognize varieties of pathogens and involve in the innate immune response. In this study, an L-rhamnose-binding lectin-like (OnRBL-1) was identified and functionally characterized from Nile tilapia (Oreochromis niloticus). The open reading frame of OnRBL-1 is 702 bp encoding 233 amino acids. The sequence of OnRBL-1 has relatively conservative characteristic peptide motifs, including YGR, DPC, and KYL-motif. Expression analysis showed that OnRBL-1 was abundantly distributed in liver tissue, and widely existed in all detected tissues. Meanwhile, the expressions of OnRBL-1 increased significantly in vivo (liver, spleen, head kidney, intestine, gills and peripheral blood) and in vitro (hepatocytes) following challenges with two important tilapia pathogenic bacteria Streptococcus agalactiae and Aeromonas hydrophila. Moreover, the recombinant OnRBL-1 was expressed and purified, which was found to bind and agglutinate S. agalactiae and A. hydrophila. Overall, This study indicated that OnRBL-1 may function as an important pattern recognition molecule, possessing recognition and agglutination activity to bacterial pathogens, thus involving in the innate immune defense of O. niloticus.