Isolation, Characterization, and biological significance of a novel C-type marine lectin purified from Scylla serrata haemolymph

Isolation, Characterization, and biological significance of a novel C-type marine lectin purified from Scylla serrata haemolymph | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation, Characterization, and biological significance of a novel C-type marine lectin purified from Scylla serrata haemolymph Ritam Guha, Ishwarya Ramachandran, Sivashanmugam Karthikeyan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7249543/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Marine lectins are unique glycoproteins that induce non-specific immunity in fishes and crustaceans. Mud crab Scylla serrata is highly abundant globally and rare, especially in Southeast Asian countries for its economic value. However, the characterisation of the c-type specific lectins is still not explored properly. In this current study, a novel c-type mannose-binding lectin was isolated from the marine crab Scylla serrata haemolymph using affinity chromatography. The purified lectin (Ss-Lec) of a 71kDa showed haemagglutination activity at the lowest concentration, and its homogenous and crystalline nature was evaluated by the HPLC, XRD, FTIR, and MALDI-TOF analysis. Moreover, the Ss-Lec depends on optimum pH, temperature, and calcium chelators. for its improved bioactivity. The purified Ss-Lec showed extensive antimicrobial properties against the important pathogenic aquatic bacteria Streptococcus iniae, Streptococcus agalactiae, Edwardsiella tarda, and, Aeromonas veronii at a concentration of 25-100µg/ml. This highly potential lectin can be further utilized against pathogenic diseases in aquaculture. C-type Lectin Scylla serrata phenoloxidase (PO) activation haemagglutination antibiofilm activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Lectins are typical glycoproteins of non-immune origin that bind to glycoproteins and glycolipids on the cell surface to agglutinate a range of animal cells after recognizing carbohydrate structures (Hirabayashi, 2002). All organisms have lectin-carbohydrate interactions, which are ligand-receptor 2 interactions (Bulgakov et al., 2004). These interactions support a variety of biological functions, including the transport of carbohydrates, glycoproteins, and calcium within cells and tissues (Vasta, 1992), cytolysis and cytotoxicity (Armstrong et al., 1996), and cell adhesion, migration, and apoptosis (Ni Y. & Tizard, 1996). They have been linked to organ morphogenesis, tumor cell metastasis, leukocyte trafficking, immunological response, inflammation, and extracellular matrix recognition. They can also cause cell proliferation, cell arrest, or apoptosis (Sharon & Lis, 2004). Lectins extracted from animal tissues were studied as immunomodulatory, antiviral, anticancer, and apoptotic agents. (Coelho et al., 2017) The immune systems of both vertebrates and invertebrates can use lectins as a nonself-recognition molecule (Renwrantz, 1986; Arason, 1996; Matsushita, 1996; Vasta et al., 1999; Wilson et al., 1999). As the main immunological response to infections, mannose-binding lectin (MBL) has been proposed to be crucial for host defense (Uemura et al., 2002; Ezekowitz, 2003). The finest illustrations of PRP-mediated immunity are in insects and horseshoe crab lectins. Numerous immunological responses, including cytotoxic effects (Ma Y. et al., 1999), phagocytosis enhancement (Kondo et al., 1992), antibacterial activity (Tunkijjanukij & Olafsen, 1998), and nodule formation (Koizumi et al., 1999), are significantly influenced by these lectins. The precise roles of several invertebrate lectins in this nonself-recognition molecule in crustaceans are not well understood, although many of them have been isolated and described (Viswambari et al., 2010). As a result, Marques and Barracco have examined lectin purification, molecular characterization, physiological function, synthesis, and induction upon infection in crustaceans (Marques et al., 2000). The sequence details of several crustacean C-type lectins have been published recently (Wang & Wang, 2013). These reports or studies demonstrate the several immune-related roles that crab lectins play in crustacean immunity, including the capacity to identify nonself-recognition molecules like PRPs. Marine crabs Trichopeltarion nobile (Rama Devi et al., 2012) and Atergatis ocyroe (Elayabharathi et al., 2017) haemolymph contains a calcium-dependent lectin, and marine crabs Portunus pelagicus (Chidhambaradhas et al., 2017; Jayanthi et al., 2017) have shown that lectin possesses antibacterial properties. A lectin was employed as a diagnostic and anticancer drug was obtained from the Korean sea crab Philyra pisum . It detects N-glycolyl neuraminic acid and has antiproliferative properties against cancer cells (Kim et al., 2006). However, the information on the isolation and characterization of mannose-specific C-type lectins from marine crabs is limited. The present study demonstrates the purification and characterization of a novel c-type lectin and its antimicrobial properties from mud crab S. serrata . Materials and Methods S. serrata haemolymph collection In the early morning hours (5.30 am–8.30 am), local fisherman assisted in the collection of 150 g (150 ± 20) mud crabs ( S. serrata ) from the coastal area of Fort Kochi, Ernakulam district, Kerala, India. Sand and planktonic particles were eliminated from the animals by washing them with seawater. Following their cautious transportation to the lab, they were kept in FRP tanks with a flow-through that contained seawater (30 ppt) at 30±2 °C and fed a specially prepared crab diet (Cylla Plus, ICAR-CIBA, India) twice daily. Using a syringe filled with a marine anticoagulant solution (0.45 M NaCl, 0.1 M glucose, 30 mM sodium citrate, 26 mM citric acid, 10 mM EDTA, pH 7.5), haemolymph was quickly extracted from the hemocoel in the arthrodial membrane of the final pair of walking legs (Söderhäll et al., 1983). The haemolymph was centrifuged for 5 minutes at 4 °C at 3300 × g. The cell debris was removed, and the plasma was stored at -80 °C for later use. The experiments were performed following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), application number: 363/GO/Re/S/01/CCSEA/107. The Institutional Animal Ethics Committee of Cochin University of Science and Technology, Kochi, Kerala, India reviewed and approved the protocol. Purification of Ss-Lec With minor adjustments, lectin was purified using the procedures outlined by Rubeena & Preetham (2019). In summary, 10 mg of D+ mannose (Sigma Aldrich) was combined with 7 ml of Sepharose CL-6B beads (Sigma Aldrich) in accordance with manufacturing instructions. The mixture was then maintained at 4ºC and 100 rpm for the entire night in a cooling shaking incubator (Calgon Scientific, India). After a thorough cleaning with sterile water, a 1.5 x 12 cm column (Econo-Pac®, Biorad Laboratory, Canada) was washed six times with TBS/CaCl 2 buffer (10 mM Tris–HCl, 150 mM NaCl, 10 mM CaCl 2 , pH 7.4). The mannose-coupled Sepharose CL-6B beads were then meticulously put inside the column, cleaned three times with TBS/CaCl 2 buffer, and stored at 4ºC for the night. An equivalent amount of TBS/CaCl 2 was then added to the S. serrataa plasma to equilibrate it. Approximately 10 ml of the equilibrated sample was combined with 5 ml of mixing buffer (2.5 M NaCl, 40 mM CaCl2, pH 7.8) containing 20 mM imidazole-HCl and gently swirled for 60 minutes at 24ºC. Gently, the mixes were added to the CL-6B mannose-coupled Sepharose column. The elution buffer (10 mM Tris–HCl, 140 mM NaCl, 3 mM EDTA, pH 8.0) used to elute the purified fractions contained 10ul of 10mM CaCl 2 in each tube. Characterization of purified Ss-Lec SDS-PAGE analysis Eluted fractions were subjected to SDS and native polyacrylamide gel electrophoresis (12%) in the presence and absence of 2-marceptaethanol (Laemmli, 1970). Following electrophoresis, Coomassie Brilliant Blue R-250 (Biorad Laboratories, Canada) was used to stain the gel. By comparing its electrophoretic mobility with that of molecular mass marker proteins (BioRad labs, Canada), the molecular mass of the isolated Ss-Lec was ascertained. The Bradford method (Bradford, 1976) was used to determine the total protein concentration, with bovine serum albumin (BSA) as a reference. High-performance liquid chromatography and Fourier Transform Infrared Spectroscopy (FTIR) analysis HPLC (Agilent Infinity 2000, USA) separations were performed using a reversed-phase C18 column (7.8 mm × 30 cm) at a flow rate of 0.8 ml/min in a previously equilibrated column with TBS-I. For FTIR (Agilent Cary 630, USA) spectrum analysis, 50 µl purified Ss-Lec was placed in a thermostated cell fitted with CaF 2 windows (with 6 µm Teflon spacer for measurements in water). The spectra of biological molecules were recorded at a resolution of 4 cm -1 . X-Ray diffraction (XRD) and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis XRD analysis (Bruker AXS GmbH, D8 QUEST, Germany) was carried out in purified Ss-Lec at 40 kV/20 mA utilising continuous scanning 2θ mode to ascertain the spatial distribution of atomic coordinates and the arrangement of atoms. Scherrer's formula (d = (0.9λ/β cos θ)) is used to calculate the average grain size and shape of the purified Ss-Lec. For MALDI-TOF analysis the Ss-Lec gel bands were carefully trimmed and put into 1.5 ml Eppendorf tubes filled with stain removal solution (100 mM ammonium bicarbonate: 50% acetonitrile (1:1), and vortexed for 45 minutes. Following total stain removal, 100% acetonitrile (ACN) was used to dehydrate the gel fragments at 4ºC. For trypsin digestion and absorption, a trypsin solution was prepared in 40 mM NH 4 HCO 3 , and added to each Eppendorf tube containing gel pieces, and then kept at ice cold. Following absorption, the mixture was incubated for 12–16 hours at 37 °C. 4% formic acid was added to stop the digestion. Gel pieces were washed three times with 4% formic acid (FA) in 50% acetonitrile (ACN) to extract the peptides. The MALDI-TOF/TOF MS (Shimadzu, Biotek Axima Performance, Japan) was used to examine the resulting peptides. Analysis of physiochemical properties The physicochemical characterization of the purified Ss-Lec was evaluated by its haemagglutination property, examining the effects of various physicochemical parameters such as pH, temperature, and cation dependency to determine the optimum activity. These assays were performed following (Vargila et al., 2024) with modified concentrations of the parameters and using human erythrocytes. Functional analysis Haemagglutination (HA) and Haemagglutination inhibition (HAI) assay Haemagglutination assay was performed using 2% human erythrocytes and 25-100 µg/ml purified Ss-Lec. To evaluate the haemagglutination activity equal volume of purified Ss-Lec mixed with 2% RBC and TBS-II (10 mM Tris HCl, 145 mM NaCl, and pH-7.5) in a V-bottom microtiter plate (Greiner, Nunc, Germany). The suspensions of RBCs and lectin were incubated for 45 min at 28º C. These haemagglutinated titres of Ss-Lec were visualized by light microscopy (Nikon microscope Eclipse Ti 100) at the magnification of 40x. Haemagglutination inhibition assay was performed in the presence of carbohydrate competitive inhibitors such as D- glucose, D-galactosamine HCl, D-maltose, N-acetyl D-glucosamine, D-fucose, Lactose, D-Mannose, D-fructose, L-fucose, and sucrose. Serially diluted 400mM carbohydrate solutions were mixed with 100µg/ml purified Ss-Lec and pre-incubated at 28ºC for 45min. Then 2% human erythrocyte suspension was mixed with the Ss-Lec and inhibitor solution and incubated at 28ºC for 2 hours. The button-forming agglutination inhibition points were noted against the serially diluted carbohydrate samples. Yeast Agglutination assay To evaluate the capacity of Ss-Lec to agglutinate yeast cells ( Saccharomyces cerevisiae ), 50μl of purified Ss-Lec at different concentrations (25-100μg/ml) was mixed with an equal volume of TBS I buffer in V-shaped 96-well microtitre plate. Then, an equal volume of yeast suspension (10 6 cells/mL) was added to the wells and incubated for 4 hours at 28ºC. In control, the purified Ss-Lec was replaced by BSA. The yeast agglutination properties of Ss-Lec were visualized by light microscopy (Nikon microscope Eclipse Ti 100) at the magnification of 40x. Encapsulation assay The encapsulation activity of Ss-Lec was performed following previously described method (Rubeena & Preetham, 2019), with minor adjustments. Briefly, equal volume of 2% RBC suspension and CL-6B Sepharose bead suspension, were mixed with Ss-Lec at a concentration of 25, 50, and 100 μg/ml respectively in a V-bottomed microtitre plate and incubated for 45 minutes at 28°C with 15-minute intervals between mixing. TBS I buffer was used in control wells in place of Ss-Lec. On a sterile glass slide, the whole volume of each solution was spread out and left undisturbed for ten minutes. Following that, the slides were examined using a Leica DM IL LED inverted microscope (40X). Phenoloxidase (PO) activity Phenoloxidase (PO) activation activity was assessed according to the previously described method by Sun J et al. (2008) with minor modifications. In brief, purified Ss-Lec (25-100 µg/ml) was preincubated with an equal volume of laminarin (Sigma-Aldrich, USA) for 5 min at 25 °C. Further, the samples were incubated with 50 µl of 3 µg/ml L-DOPA (L-3,4-dihydroxyphenylalanine, Sigma-Aldrich, USA) for 5 min at 28 °C. The phenoloxidase activity was measured spectrophotometrically at 470 nm at different time points (0 min, 30 min, 60 min, and 90 min) and expressed as units/μg protein. In controls, purified Ss-Lec was replaced by TBS I buffer. Antibacterial analysis of purified Ss- Lec Bacterial strains To evaluate antibacterial effect of purified Ss-Lec, Gram positive Streptococcus agalactiae (OP580171), Streptococcus iniae (NZ_AOCT02000221), and Gram-negative Aeromonas veronii (AY764310), Edwardsiella tarda (MTCC2400) bacterial strains were cultured in tryptic soy broth (TSB) in a shaker for 24 h at 28 °C. Antibacterial activity Purified Ss-Lec's antibacterial activity against both Gram-positive and Gram-negative bacteria was assessed using the agar well diffusion technique. The bacterial broths were centrifuged (6000 x g for 10 min) and resuspended in TBS (50 mM Tris–HCl, 100 mM NaCl, pH 7.5). Further, 1x 10 5 CFU/ml of bacterial cultures were spread on 100 × 20 mm petri plates containing TSB agar, and 10 mm diameter holes were created with per forex. Purified Ss-Lec in varying concentrations (25 µg, 50 µg, and 100 µg) was added to each well, and the plates were then incubated for 24 hours at 28 °C. Biofilm quantification and inhibition assay The formation of biofilm by important aquatic pathogens like Streptococcus agalactiae, Streptococcus iniae, Aeromonas veronii and Edwardsiella tarda against purified Ss-Lec was measured by colorimetric assay (Kumar et al., 2012). In brief, the individual wells of 96 well plates were filled with 150 µL Tryptic soy broth (TSB) followed by inoculation with 10 µL of 1 x 10 9 CFU/ml bacterial culture. Subsequently, 25-100 µg/ml purified Ss-Lec was added to each well and incubated at 28°C for 48 hrs. Non-adherent bacteria were washed using 0.2 ml phosphate buffer saline (PBS, pH 7.2). The adherent bacteria were fixed with sodium acetate (2%) in crystal violet (0.1% w/v). Excessive stains were washed off with distilled water. Then, 0.2ml 95% ethanol was used to dissolve the crystal violet bounded sessile bacteria and the optical density was measured using a microtiter plate reader (Varioskan Lux, Thermofisher Scientific). Purified Ss-Lec's effect on biofilm inhibition was evaluated using previously described method (Bakkiyaraj & Karutha, 2010). Streptococcus agalactiae, Streptococcus iniae, Aeromonas veronii, and Edwardsiella tarda were tested for biofilm inhibition by microscopic visualisation using TSB broth containing the bacterial suspension of 1 x 10 9 CFU/ml. Following the previous biofilm quantification method, various concentrations of purified Ss-Lec (25-100 µg/ml) were added to the wells of a 24-well plate that contained broken glass coverslip pieces. The mixture was then incubated at 28 °C for 48 hours. The biofilm was stained with 0.5 ml of 0.4% crytal violet (w/v) and 0.4% acridine orange (w/v) for 10 minutes at 28ºC. Confocal laser scanning microscopy (Carl Zeiss LSM 710 Germany) examined stained glass pieces at a 20x magnification on slides with the biofilm facing up for the acridine orange stain, and light microscopy (Nikon microscope Eclipse Ti 100) at the magnification of 40x was used to visualize the crystal violet stain. Results Purification of Ss-Lec A C-type lectin from the mud crab S. serrata haemolymph was isolated and purified using affinity chromatography, and 71 kDa molecular mass of the protein was determined using 12% SDS-PAGE (Fig 1) against Coomassie blue staining. The protein concentration was quantified as 104 µg/ml. Characterization of Ss-Lec XRD analysis XRD analysis of the purified Ss-Lec showed one diffraction peak at 31.7°, which shows the purity and crystalline nature of the protein. The single sharp peak depicts the bigger crystalline nature of the pure Ss-Lec. (Fig 2a). HPLC and FTIR analysis Reversed phase HPLC analysis revealed the homogeneity of the purified Ss-Lec as a single peak with a retention time of 5.259 min (Fig 2b and 2c). The FTIR spectrum showed the C=N stretching corresponding to a sharp peak at 1643.08 cm -1 . Followed by 2096.96 cm -1 peak and O-H stretching at 3276.89 cm -1 . MALDI-TOF analysis In MALDI-TOF analysis, the intensity of the peaks corresponding to the 71kDa protein (Fig Sd) was shown. After trypsin digestion, the abundance of the fragments from the parent protein was denoted in peak intensity and their mass-to-charge (m/z) ratio of the molecules. In this study, 12 sharp peaks were observed above 1 x 10 4 intensity, indicating the abundance of the molecules in the Ss-Lec. Physiochemical properties Maximum hemagglutination activity was demonstrated by the Ss-Lec within a limited pH range of 7 to 8 (Fig 3A). The sensitivity of Ss-Lec to temperature showed that its hemagglutination activity decreased at temperatures up to 64 °C, with optimal activity occurring between 30 and 40 °C (Fig 3B). The hemagglutinating activity of Ss-Lec is influenced by divalent cations (Fig 3C). Although increased activity was observed up to 30 mM, hemagglutination activity was reduced at higher concentrations of calcium and magnesium ions. When different amounts of calcium chelators, such as Di and Tetrasodium EDTA, were applied to Ss-Lec, a reduced HA titer was observed up to 1 mM and above 10 mM (Fig 3D). This confirms the lectin's calcium dependence. HA and HAI activity of purified Ss-Lec The haemagglutination (HA) activity of purified Ss-Lec was visualized by light microscope (Fig 4), where a higher quantity of lectin can agglutinate human erythrocytes. In the presence of the carbohydrate inhibitors, the Ss-Lec showed HAI activity where N-acetyl glucosamine, D-mannose, and D- and L-fucose can inhibit the haemagglutination in very minimal concentrations than D-glucose (Table 1). Yeast agglutination capability of Ss-Lec The purified Ss-Lec showed strong agglutination capability towards Saccharomyces cerevisiae (yeast) cells in a concentration dependent pattern where the highest yeast agglutination was observed at 50-100μg/ml concentration (Fig 5) of Ss-Lec. Encapsulation activity The encapsulation assay was used to determine if the pure Ss-Lec could foster cellular encapsulation in vitro. Light microscopy was used to investigate the encapsulation phenomena. The encapsulation activity seen up to a 1-hour incubation period may be similarly stimulated by the Sepharose beads coated with pure Ss-Lec. During the incubation period, the entrance of RBC towards Sepharose beads was seen after 45 minutes, where RBC surrounding the beads was seen (Fig 6). Phenoloxidase (PO) activity The Phenoloxidase activity of the purified ss-Lec was measured in the presence of Laminarin at different time points. A concentration-dependent PO activity was observed. The highest concentration (100μg/ml) of Ss-Lec showed higher PO activity (Fig 7) in all time points from 30 to 90 minutes post-incubation. Similarly, the highest PO activity was also noticed at 90 minutes post-incubation in comparison to other time points. Antibacterial activity of purified Ss-Lec The purified Ss-Lec showed potential antibacterial activity against important gram-positive and gram-negative aquatic pathogens, whereas the zone of bacterial inhibition was measured (Table 2). The higher Ss-Lec at 100μg/ml was able to inhibit a maximum growth of up to 7mm. Antibiofilm properties of Ss-Lec The microtitre plate (MTP) based colorimetric assay was performed to assess the formation of biofilm of Gram-positive bacteria Streptococcus iniae, Streptococcus agalactiae and Gram-negative bacteria Edwardsiella tarda, Aeromonas veronii in the presence and absence of purified Ss-Lec. The biofilm inhibition was increased corresponding to the increased concentration of lectin. At a concentration of 100µg/ml of Ss-Lec, the highest biofilm inhibition of 82% was observed in Edwardsiella tarda following Streptococcus iniae , S. agalactiae and Aeromonas veronii at the rate of 72%, 71%, and 68%, respectively (Fig 8). The anti-biofilm formation was visualized by light and confocal (2D) microscopic methods. Where the different concentrations of purified Ss-Lec can disrupt the growth and formation of biofilms against Streptococcus iniae, Streptococcus agalactiae, Edwardsiella tarda, and Aeromonas veronii compared with controls (Fig 9A & 9B). Discussion Lectins are immunological molecules that are specifically engaged in the removal of external invaders in the crustacean's innate immune system. The majority of lectins found in crustaceans have been identified after the turn of the century, and a multiple number of lectins are extracted and identified from crustacean haemolymph. The mannose-binding lectin, which has a molecular mass of 71 kDa, was isolated and purified from the mud crab S. serrata for the present investigation. The lectin purification from mud crab haemolymph was carried out using the mannose-coupled sepharose CL-6B affinity column. The buffer's calcium ions helped the lectin bind to the mannose ligands in the column, and the EDTA in the elution buffer chelated the calcium ions to release the lectin when it was eluted from the column. To date, several lectins have been isolated from crustaceans with molecular weights ranging from 14 kDa from Tachypleus tridentatus (Tri-spine horseshoe crab) (Inamori et al., 1999), 20 kDa from Scylla serrata (Mud crab) (Jayasankar & Subramoniam, 1999), 55kDa from Scylla serrata (Mercy & Ravindranath,1993) from to a higher range of 452 kDa from Penaeus japonicas (Kuruma shrimp) (Yang et al., 2007) and 220 kDa Litopenaeus schmitti (White shrimp) (Cominetti et al., 2002), the green tiger prawn Penaeus semisulcatus (37 and 118 kDa) (Sivakamavalli & Vaseeharan, 2014), the freshwater crab Paratelphusa jacquemontii (34 kDa) (Denis et al., 2003), the banana prawn Fenneropenaeus merguiensi (32.3 and 30.9 kDa) (Rittidach et al., 2007), and Chinese white shrimp Fenneropenaeus chinensis (168 kDa) (Sun J et al., 2008), blue swimmer crab Portunas pelagicus (155 kDa) (Jayanthi et al., 2017). The purified Ss-Lec was shown to be monovalent, with a decreased affinity for D-glucose, and no activity towards D-galactosamine HCl, Lactose, D-fructose, Sucrose. Whereas, the Ss-Lec had a greater affinity for D-Mannose, D-fucose, L-fucose, N-acetyule D-glucosamine and D-maltose respectively. This aligns with earlier findings by Mitra and Das (2001) and Silva et al. (2012), who discovered that a single lectin protein may show affinity for a variety of sugar compounds. In addition, Jayanthi et al. (2017) previously reported that the crystalline nature and purity were verified by XRD analysis and HPLC analysis, respectively that corelates with our obtained results from the current study. Similarly described by Divya et al. (2018), the functional group analysis and molecular mass confirmation were carried out using FTIR analysis. The ability of lectin to agglutinate against mammalian red blood cells was tested as an agglutinin, and the Ss-Lec successfully agglutinated the RBS at the lowest concentration of 25μg/ml. It is evident from this agglutination response that the purified Ss-Lec can identify foreign invaders via PRPs. Similar agglutination reactions were conducted with P. semisulcatus lectin, which exhibits strong agglutination towards human red blood cells (Sivakamavalli & Vaseeharan, 2014). Additionally, our findings were consistent with previously documented lectins of Litopenaeus setiferus (Alpuche et al., 2005), Macrobrachium rosenbergii (Zenteno et al., 2000), and Sun J. et al. (2008), that the lectin's ability to agglutinate human erythrocytes revealed that it was unable to distinguish between human RBC types. Apart from its function in agglutination, Ss-Lec is also essential for encapsulation and phagocytosis. Encapsulation, only found in invertebrates, and it actively acts to counteract a range of outside dangerous substances. Unlike phagocytosis, which eliminates big foreign, harmful components, encapsulation attempts to surround and completely encase the microorganism intrusions that cause damage within the enclosed cascade. Encapsulation against the Sepharose beads was made easier by Ss-Lec, a characteristic that has already been documented in other invertebrates (Sminia et al., 1974; Sivakamavalli & Vaseeharan, 2014; Jayanthi et al., 2017; Rubeena & Preetham, 2019). Ss-Lec can thus take action by removing and banning alien diseases from prawn aquaculture. Marine white prawn L. vannamei is one of the numerous crustaceans that have been shown to include lectins with a variety of immunological activities and an improved encapsulation process in a number of earlier investigations (Lis et al., 1998; LGuo et al., 2013; Cerenius et al., 2010). Phenoloxidase (PO) is a crucial biological function that is well-known for interacting with marine prawns and other invertebrate immune components. It plays a significant immune-defensive function in several ways, including wound healing and the elimination of microbial invaders, and it activates dormant ProPO by activating the lectin complement pathway (Preetham et al., 2019). As a result of the degranulation of haemoglobin, Sivakamavalli et al. (2014) found that the complex formed between PAMPs and lectin might activate the ProPO (inactive form) to PO (active form) (Rubeena and Preetham, 2019). Correlating the previous findings Ss-Lec and laminarin were the triggers for the PO system in this investigation, and PO activity was increased with time. We also documented the antibacterial capabilities of purified Ss-Lec against a broad spectrum of Gram-positive and Gram-negative bacteria in addition to the functional study. Purified Ss-Lec's antibacterial activity was assessed using the agar well diffusion method, demonstrating its comparable bacterial killing capabilities against Gram-negative Edwardsiella tarda and Aeromonas veronii as well as Gram-positive Streptococcus iniae and Streptococcus agalactiae . The chinese prawn F. chinensis's Lectin Fc-hsL shown antibacterial activity against B. subtilis, B. cereus, B. thuringiensis, E. coli , and K. pneumoniae , (Sun YD et al., 2008). In another study (Lai et al., 2013) the recombinant F. chinensis C-Type lectin, or rFcCTL, demonstrated greater antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria. This suggests that lectins are likely involved in the immobilisation of bacteria, binding, and destruction of bacterial cell walls, which results in the inhibition or termination of microorganism growth (Tunkijjanukij & Olafsen, 1998). Purified Ss-Lec was tested for antibiofilm activity after showing promising antibacterial action against bacteria. The unwanted growth of microorganisms on synthetic surfaces submerged in a common matrix is known as biofilm (Yebra et al., 2004). Against the same aquatic pathogens, the efficaciousness of purified Ss-Lec as a degrading peptide in disintegrating biofilm matrix was investigated. According to antibiofilm activity results in current study the increased concentrations of purified Ss-Lec had reduced the production of biofilm. Both Gram-positive and Gram-negative pathogens were shown to be susceptible to the same antibiofilm action of many crustacean proteins, including β-1, 3 glucan binding protein ( P. pelagicus ) (Anjugam et al., 2016), prophenoloxidase ( P. semisulcatus ) (Ishwarya et al., 2016), and crustin ( Episesarma tetragonum ) (Sivakamavalli et al., 2015). Moreover, the purified mannose binding C-type Ss-Lec had a broad range of functional activity which promotes its capability as a potent antimicrobial agent. It is concluded that, under reducing and non-reducing conditions, the affinity chromatography-purified Ss-Lec showed an apparent molecular mass of 71 kDa on SDS-PAGE. The purity, homogeneity, and crystalline nature of Ss-Lec were shown by MAlDI-TOF, HPLC, XRD, and FTIR analysis. Agglutination, phenoloxidase, and encapsulation-enhancing qualities demonstrated Ss-Lec's active immune system participation and immuno-protective function in mud crabs. Therefore, the purified Ss-Lec was projected against a variety of Gram-positive and Gram-negative bacteria to determine its antibiofilm characteristics. Based on all of these results, we concluded that Ss-Lec is found to play a crucial part in the immune system and may be used as a possible antibacterial and therapeutic tool to strengthen crab's defense against pathogens, and it can be used in the aquaculture sector. Declarations Author Contributions Ritam Guha : Conceptualization, Methodology, Data curation, Formal analysis, Writing-original draft, writing review and editing. Ishwarya Ramachandran: Methodology, Data curation, Formal analysis, writing review, and editing. Sivashanmugam Karthikeyan : Data curation, writing review, and editing. Baskaralingam Vaseeharan : Data curation, Writing-review and editing Preetham Elumalai: Conceptualization, writing-review and editing, supervision, project administration, funding acquisition. Competing Interests The authors have no financial interests that are directly or indirectly related to the work submitted for publication. Data availability Data will be available upon request to the corresponding author. Acknowledgments: The authors deeply acknowledge the funding supported by the Department of Science and Technology-Indian National Academy of Engineering (2023/IN-TW/07), India-Taiwan collaboration program, and Centre of Excellence for Aquatic Vaccine Development, Govt. of Kerala at CUSAT. Author RG acknowledges the funding support of the Department of Science and Technology-Innovation in Science Pursuit for Inspired Research Fellowship (IF230205). The author IR deeply acknowledges Indian Council of Medical Research - Department of Health Research, for the encouragement and financial assistance (R.12013/55/2024-HR) towards DHR- Women Scientist Fellowship. References Abdul Salam R, Preetham E (2019) Antimicrobial properties and phenoloxidase activation of the lectin isolated from kadal shrimp (Metapenaeus dobsoni). Fish Shellfish Immunol 90:118–125. https://doi.org/10.1016/j.fsi.2019.04.305 Alpuche J, Pereyra A, Agundis C, Rosas C, Pascual C, Slomianny MC, Zenteno E (2005) Purification and characterization of a lectin from the white shrimp Litopenaeus setiferus (Crustacea decapoda) hemolymph. Biochim Biophys Acta 1724:86–93. https://doi.org/10.1016/j.bbagen.2005.04.014 Arason GJ (1996) Lectins as defence molecules in vertebrates and invertebrates. Fish Shellfish Immunol 6:277–289. https://doi.org/10.1006/fsim.1996.0029 Armstrong PB, Swarnakar S, Srimal S, Misquith S, Hahn EA, Aimes RT, Quigley JP (1996) A cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins. J Biol Chem 271:14717–14721. https://doi.org/10.1074/jbc.271.25.14717 Bakkiyaraj D, Karutha Pandian ST (2010) In vitro and in vivo antibiofilm activity of a coral associated actinomycete against drug resistant Staphylococcus aureus biofilms. Biofouling 26:711–717. https://doi.org/10.1080/08927014.2010.511200 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3 Bulgakov AA, Park KI, Choi KS, Lim HK, Cho M (2004) Purification and characterisation of a lectin isolated from the Manila clam Ruditapes philippinarum in Korea. Fish Shellfish Immunol 16:487–499. https://doi.org/10.1016/j.fsi.2003.08.006 Cerenius L, Jiravanichpaisal P, Liu HP, Soderhall I (2010) Crustacean immunity. In: Söderhäll K (ed) Invertebrate Immunity. Springer, Boston, pp 239–259. https://doi.org/10.1007/978-1-4419-8059-5_13 Chidhambaradhas S, Selvamohan T, Stanislaus PM (2017) Identification and purification of antimicrobial lectins from marine crab Portunus pelagicus (Linneus, 1775). Int J Curr Microbiol Appl Sci 6:2346–2351. https://doi.org/10.20546/ijcmas.2017.606.277 Coelho LCCB, Silva PMDS, Lima VLDM, Pontual EV, Paiva PMG, Napoleão TH, Correia MTDS (2017) Lectins, interconnecting proteins with biotechnological/pharmacological and therapeutic applications. Evid Based Complement Alternat Med 2017:1594074. https://doi.org/10.1155/2017/1594074 Cominetti MR, Marques MRF, Lorenzini DM, Löfgren SE, Daffre S, Barracco MA (2002) Characterization and partial purification of a lectin from the hemolymph of the white shrimp Litopenaeus schmitti. Dev Comp Immunol 26:715–721. https://doi.org/10.1016/s0145-305x(02)00025-3 Da Silva CDC, Coriolano MC, da Silva Lino MA, de Melo CML, de Souza Bezerra R, de Carvalho EVMM, Coelho LCCB (2012) Purification and characterization of a mannose recognition lectin from Oreochromis niloticus (Tilapia Fish): Cytokine production in mice splenocytes. Appl Biochem Biotechnol 166:424–435. https://doi.org/10.1007/s12010-011-9438-1 Denis M, Palatty PM, Bai NR, Suriya SJ (2003) Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii. Eur J Biochem 270:4348–4355. https://doi.org/10.1046/j.1432-1033.2003.03828.x Divya M, Vaseeharan B, Anjugam M, Iswarya A, Karthikeyan S, Velusamy P, Vágvölgyi C (2018) Phenoloxidase activation, antimicrobial, and antibiofilm properties of β-glucan binding protein from Scylla serrata crab hemolymph. Int J Biol Macromol 114:864–873. https://doi.org/10.1016/j.micpath.2020.103992 Elayabharathi JVJM, Mary S, Bai M (2017) Partial purification and characterization of hemolymph lectin of marine crab Atergatis ocyroe by adsorption on formalinized erythrocytes. Int J Biol Res 2:115–119 Ezekowitz RA (2003) Role of the mannose-binding lectin in innate immunity. J Infect Dis 187(Suppl 2):S335–S339. https://doi.org/10.1086/374746 Hirabayashi J (2002) Introduction to lectins. Glycoforum. http://www.glycoforum.gr.jp/science/word/lectin/LEAOO.2002.html Inamori KI, Saito T, Iwaki D, Nagira T, Iwanaga S, Arisaka F, Kawabata SI (1999) A newly identified horseshoe crab lectin with specificity for blood group A antigen recognizes specific O-antigens of bacterial lipopolysaccharides. J Biol Chem 274:3272–3278. https://doi.org/10.1074/jbc.274.6.3272 Ishwarya R, Jayanthi S, Muthulakshmi P, Anjugam M, Jayakumar R, Nazar AK, Vaseeharan B (2016) Immune indices and identical functions of two prophenoloxidases from the haemolymph of green tiger shrimp Penaeus semisulcatus and its antibiofilm activity. Fish Shellfish Immunol 51:220–228. https://doi.org/10.1016/j.fsi.2016.02.011 Jayanthi S, Ishwarya R, Anjugam M, Iswarya A, Karthikeyan S, Vaseeharan B (2017) Purification, characterization and functional analysis of the immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus and their antibiofilm properties. Fish Shellfish Immunol 62:227–237. https://doi.org/10.1016/j.fsi.2017.01.019 Jayasankar V, Subramoniam T (1999) Antibacterial activity of seminal plasma of the mud crab Scylla serrata (Forskal). J Exp Mar Biol Ecol 236:253–259 Kim HH, Jun JI, Kim BS, Cho DH, Min TH, Kim YJ, Ryu CS (2006) U.S. Patent No. 7,015,313. U.S. Patent and Trademark Office, Washington, DC Koizumi N, Imamura M, Kadotani T, Yaoi K, Iwahana H, Sato R (1999) The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydrate-recognition domains. FEBS Lett 443:139–143. https://doi.org/10.1016/S0014-5793(98)01701-3 Kondo M, Matsuyama H, Yano T (1992) The opsonic effect of lectin on phagocytosis by hemocytes of kuruma prawn, Penaeus japonicus. Fish Pathol 27:217–222 Kumar P, Selvi SS, Govindaraju M (2012) In vitro anti-biofilm and anti-bacterial activity of Junceella juncea for its biomedical application. Asian Pac J Trop Biomed 2:930–935. https://doi.org/10.1016/S2221-1691(13)60002-7 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0 Lai X, Kong J, Wang Q, Wang W, Meng X (2013) Identification and molecular characterization of a C-type lectin-like protein from Chinese shrimp (Fenneropenaeus chinensis). Mol Biol Rep 40:2223–2230. https://doi.org/10.1007/s11033-012-2284-6 Guo XN, Jin XK, Li S, Yu AQ, Wu MH, Tan SJ, Wang Q (2013) A novel C-type lectin from Eriocheir sinensis functions as a pattern recognition receptor with antibacterial activity. Fish Shellfish Immunol 35:1554–1565. https://doi.org/10.1016/j.fsi.2013.08.021 Lis H, Sharon N (1998) Lectins: carbohydrate-specific proteins that mediate cellular recognition. Chem Rev 98:637–674. https://doi.org/10.1021/cr940413g Marques MRF, Barracco MA (2000) Lectins, as non-self-recognition factors, in crustaceans. Aquaculture 191:23–44. https://doi.org/10.1016/S0044-8486(00)00417-8 Ma Y, Uemura K, Oka S, Kozutsumi Y, Kawasaki N, Kawasaki T (1999) Antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding protein-dependent cell-mediated cytotoxicity. Proc Natl Acad Sci USA 96:371–375 Anjugam MA, Iswarya AI, Vaseeharan B (2016) Multifunctional role of β-1,3 glucan binding protein purified from the haemocytes of blue swimmer crab Portunus pelagicus and in vitro antibacterial activity of its reaction product. Fish Shellfish Immunol 48:196–205. https://doi.org/10.1016/j.fsi.2015.11.023 Matsushita M (1996) The lectin pathway of the complement system. Microbiol Immunol 40:887–893. https://doi.org/10.1111/j.1348-0421.1996.tb01156.x Mercy PD, Ravindranath MH (1993) Purification and characterization of N-glycolyneuraminic-acid-specific lectin from Scylla serrata. Eur J Biochem 215:697–704. https://doi.org/10.1111/j.1432-1033.1993.tb18081.x Mitra S, Das HR (2001) A novel mannose-binding lectin from plasma of Labeo rohita. Fish Physiol Biochem 25:121–129. https://doi.org/10.1023/A:1020545510295 Ni Y, Tizard I (1996) Lectin-carbohydrate interaction in the immune system. Vet Immunol Immunopathol 55:205–223. https://doi.org/10.1016/S0165-2427(96)05718-2 Preetham E, Rubeena AS, Vaseeharan B, Chaurasia MK, Arockiaraj J, Olsen RE (2019) Anti-biofilm properties and immunological response of an immune molecule lectin isolated from shrimp Metapenaeus monoceros. Fish Shellfish Immunol 94:896–906. https://doi.org/10.1016/j.fsi.2019.09.032 Rama Devi P, Sudhakar GL, Vasudehaven I (2012) Natural agglutinin from crab, Trichopeltarian nobile. Int J Biol Res 5:14–17 Renwrantz L (1986) Lectins in molluscs and arthropods: Their occurrence, origin and roles in immunity. Dev Comp Immunol (Incomplete journal details—please confirm) Rittidach W, Paijit N, Utarabhand P (2007) Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph. Biochim Biophys Acta 1770:106–114. https://doi.org/10.1016/j.bbagen.2006.06.016 Sharon N, Lis H (2004) History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14:53R–62R. https://doi.org/10.1093/glycob/cwh122 Sivakamavalli J, Vaseeharan B (2014) Purification, characterization and functional role of lectin from green tiger shrimp Penaeus semisulcatus. Int J Biol Macromol 67:64–70. https://doi.org/10.1016/j.ijbiomac.2014.03.008 Sivakamavalli J, Nirosha R, Vaseeharan B (2015) Purification and characterization of a cysteine-rich 14-kDa antibacterial peptide from the granular hemocytes of mangrove crab Episesarma tetragonum and its antibiofilm activity. Appl Biochem Biotechnol 176:1084–1101. https://doi.org/10.1007/s12010-015-1631-1 Söderhäll K, Smith VJ (1983) Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution. Dev Comp Immunol 7:229–239. https://doi.org/10.1016/0145-305x(83)90004-6 Sun J, Wang L, Wang B, Guo Z, Liu M, Jiang K, Zhang G (2008) Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis. Fish Shellfish Immunol 25:290–297. https://doi.org/10.1016/j.fsi.2008.06.001 Sun YD, Fu LD, Jia YP, Du XJ, Wang Q, Wang YH, Wang JX (2008) A hepatopancreas-specific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity. Mol Immunol 45:348–361. https://doi.org/10.1016/j.molimm.2007.06.355 Sminia T, Borghart-Reinders E, Van AW (1974) Encapsulation of foreign materials experimentally introduced into the freshwater snail Lymnaea stagnalis. Cell Tissue Res 153:307–326. https://doi.org/10.1007/BF00229161 Tunkijjanukij S, Olafsen JA (1998) Sialic acid-binding lectin with antibacterial activity from the horse mussel: further characterization and immunolocalization. Dev Comp Immunol 22:139–150. https://doi.org/10.1016/s0145-305x(98)00017-2 Uemura K, Saka M, Nakagawa T, Kawasaki N, Thiel S, Jensenius JC, Kawasaki T (2002) L-MBP is expressed in epithelial cells of mouse small intestine. J Immunol 169:6945–6950. https://doi.org/10.4049/jimmunol.169.12.6945 Vasta GR (1992) Invertebrate lectins: distribution, synthesis, molecular biology, and function. In: Montreuil J, Vliegenthart JFG, Schachter H (eds) Glycoconjugates: Composition, Structure and Function. Marcel Dekker, New York, 593–634 Vasta GR, Quesenberry M, Ahmed H, O’Leary N (1999) C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway. Dev Comp Immunol 23:401–420. https://doi.org/10.1016/s0145-305x(99)00020-8 Viswambari Devi R, Basilrose MR, Mercy PD (2010) Prospect for lectins in arthropods. Ital J Zool 77:254–260. https://doi.org/10.1080/11250003.2012.671856 Wang XW, Wang JX (2013) Diversity and multiple functions of lectins in shrimp immunity. Dev Comp Immunol 39:27–38. https://doi.org/10.1016/j.dci.2012.04.009 Wilson R, Chen C, Ratcliffe NA (1999) Innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis. J Immunol 162:1590–1596 Yang H, Luo T, Li F, Li S, Xu X (2007) Purification and characterisation of a calcium-independent lectin (PjLec) from the haemolymph of the shrimp Penaeus japonicus. Fish Shellfish Immunol 22:88–97. https://doi.org/10.1016/j.fsi.2006.03.015 Yebra DM, Kiil S, Dam-Johansen K (2004) Antifouling technology—past, present and future steps towards efficient and environmentally friendly antifouling coatings. Prog Org Coat 50:75–104. https://doi.org/10.1016/j.porgcoat.2003.06.001 Zenteno R, Vazquez L, Sierra C, Pereyra A, Slomianny MC, Bouquelet S, Zenteno E (2000) Chemical characterization of the lectin from the freshwater prawn Macrobrachium rosenbergii (De Man) by MALDI-TOF. Comp Biochem Physiol B 127:243–250. https://doi.org/10.1016/s0305-0491(00)00260-1 Vargila F, Mettilda Bai SM, Josephine Mary JV, Citarasu T (2024) Isolation, characterization and antimicrobial properties of hepatopancreas lectin of the freshwater crab Oziotelphusa naga. Protein Expr Purif 222:106536. https://doi.org/10.1016/j.pep.2024.106536 Tables Table 1: The minimal inhibitory concentration corresponds to the lowest carbohydrate concentration able to neutralize the hemagglutinating activity of Ss-Lec. The values are expressed in millimolars, and the highest carbohydrate concentration used was 400 mM. N-acetyl glucosamine, D-mannose, and D- and L- fucose could inhibit haemagglutination at a very low concentration. Sugar Maximum Concentration of sugar (mM) Minimum inhibitory Concentration of sugar (mM) D- glucose 400 12.5 D-galactosamine HCl 400 - D-maltose 400 0.781 N-acetyl D-glucosamine 400 0.390 D-fucose 400 0.390 Lactose 400 - D-Mannose 400 0.195 D-fructose 400 - L-fucose 400 0.390 Sucrose 400 - Table 2: Antibacterial activity of purified Ss-Lec. The zone of inhibition values are expressed as ‘ - =No zone of inhibition; + = ≤ 5mm ; ++ = ≤ 7 mm; +++ = ≥ 7mm’. Pathogen TBS buffer Ss-Lec 25µg/ml Ss-Lec 50µg/ml Ss-Lec 100µg/ml Streptococcus agalactiae - - + ++ Streptococcus iniae - - + ++ Aeromonas veronii - - + ++ Edwardsiella tarda - - ++ +++ Additional Declarations No competing interests reported. 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13:51:47","extension":"xml","order_by":67,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166649,"visible":true,"origin":"","legend":"","description":"","filename":"3caea69095554b6a902b5710800f926e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/63ebf85f2782d803395b1e26.xml"},{"id":94865360,"identity":"f69ec615-9008-4598-be48-e97e89eed90b","added_by":"auto","created_at":"2025-10-31 13:51:47","extension":"html","order_by":68,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":183134,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/dfb2116f3ac66cb7222849dc.html"},{"id":94865274,"identity":"ac5d541b-6993-41a4-9dcc-46da28bfaf2a","added_by":"auto","created_at":"2025-10-31 13:51:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":590019,"visible":true,"origin":"","legend":"\u003cp\u003ePolyacrylamide gel electrophoresis (12%) in the presence of sodium dodecyl sulphate (SDS) of Ss-Lec purified by Mannose-Sepharose CL 6B affinity chromatography. Lane A contains reducing pattern and Lane B contains non-reducing pattern of purified Ss-Lec (71kDa) from \u003cem\u003eScylla serrata\u003c/em\u003ehaemolymph. The protein bands were stained with Coomassie brilliant blue – R 250.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/19ae773e07b3f42d58a6f542.png"},{"id":94865276,"identity":"5639dac4-5fbd-4501-a61b-a2f1907dee44","added_by":"auto","created_at":"2025-10-31 13:51:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":651225,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of purified Ss-Lec. (a) XRD analysis of purified \u003cem\u003eSs\u003c/em\u003e-Lec showed a diffraction peak at 31.70˚ which indicates the crystalline nature of the protein, (b) HPLC analysis of purified \u003cem\u003eSs\u003c/em\u003e-Lec was analyzed using a reversed-phase C\u003csub\u003e18\u003c/sub\u003e column and the isolated protein emerged as a single peak with a retention time of 5.2 min, (c) FTIR analysis of purified \u003cem\u003eSs\u003c/em\u003e-Lec for functional group identification. and (d) MALDI-TOF analysis of Purified Ss-Lec\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/d06fca48ffeb8c5aadeb9bc7.png"},{"id":94986537,"identity":"00cd482e-ecff-444c-8d4f-ebcc9055f80d","added_by":"auto","created_at":"2025-11-03 07:00:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":216591,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pH (A), Temperature (B), Divalent cations (C), Calcium chelators (D) on the hemagglutinating activity of the purified Ss-Lec to determine the physiochemical properties.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/0f725511e53c64218ba0d7cd.png"},{"id":94985607,"identity":"22d128b6-ff57-417e-9161-6f759b6dd383","added_by":"auto","created_at":"2025-11-03 06:58:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":519213,"visible":true,"origin":"","legend":"\u003cp\u003eHaemagglutination activity of purified Ss-Lec against 2% human RBCs. The purified Ss-Lec showed the strongest agglutination at 100μg/ml concentration.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/0c11d354a6dedbcfc1d06367.png"},{"id":94865290,"identity":"a9fd3016-99d1-48e2-9d6a-e8a61111904a","added_by":"auto","created_at":"2025-10-31 13:51:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":534033,"visible":true,"origin":"","legend":"\u003cp\u003eAgglutination activity of purified Ss-Lec against \u003cem\u003eSaccharomyces cerevisiae (\u003c/em\u003eyeast) cells. The purified Ss-Lec showed the strongest agglutination at 100μg/ml concentration.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/8715513873ead22ee8b063a4.png"},{"id":94865281,"identity":"d8d54a03-a97a-4565-a763-17d1c295c354","added_by":"auto","created_at":"2025-10-31 13:51:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":856309,"visible":true,"origin":"","legend":"\u003cp\u003eEncapsulation activity of purified Ss-Lec in Sepharose CL-6B beads. The purified Ss-Lec showed the strongest encapsulation activity at 100μg/ml concentration.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/97bb9cf720ce71c057c4d141.png"},{"id":94865295,"identity":"0012e101-541d-46a7-b8d1-0156b0b440b5","added_by":"auto","created_at":"2025-10-31 13:51:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":144733,"visible":true,"origin":"","legend":"\u003cp\u003ePhenoloxidase (PO) activity of the purified ss-Lec. The purified Ss-Lec showed the strongest PO activity at 100μg/ml concentration in time dependent manner.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/ceac30dba1b70f8f25ff02ab.png"},{"id":94986169,"identity":"b989bc90-790f-4f88-97a4-c740e96b5c71","added_by":"auto","created_at":"2025-11-03 07:00:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":156524,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro quantification of biofilm inhibition by purified \u003cem\u003eSs\u003c/em\u003e-Lec against \u003cem\u003eEdwardsiella tarda, Aeromonas veronii, Streptococcus iniae \u003c/em\u003eand\u003cem\u003e Streptococcus agalactiae.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/c7fea8ce8bf6a3d356b8e3fd.png"},{"id":94865289,"identity":"ca90047b-caeb-4b78-bb6d-d8774cbf4e96","added_by":"auto","created_at":"2025-10-31 13:51:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3446657,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003eAntibiofilm activity of purified \u003cem\u003eSs\u003c/em\u003e-Lec in 2.5D confocal view of the biofilm inhibition against Gram-positive bacteria \u003cem\u003eStreptococcus iniae, Streptococcus agalactiae \u003c/em\u003eand\u003cem\u003e \u003c/em\u003eGram-negative\u003cem\u003e \u003c/em\u003ebacteria\u003cem\u003e Edwardsiella tarda, Aeromonas veronii.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB:\u003c/strong\u003eAntibiofilm activity of purified \u003cem\u003eSs\u003c/em\u003e-Lec in 2D light microscopic view against Gram-positive bacteria \u003cem\u003eStreptococcus iniae, Streptococcus agalactiae \u003c/em\u003eand\u003cem\u003e \u003c/em\u003eGram-negative\u003cem\u003e \u003c/em\u003ebacteria\u003cem\u003e Edwardsiella tarda, Aeromonas veronii.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/559e302c4fb3881c60c8ca47.png"},{"id":94990462,"identity":"62f9ab04-80ab-49cd-8db5-b77b4ea9af71","added_by":"auto","created_at":"2025-11-03 07:17:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7851874,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7249543/v1/134778e8-3963-4367-90c3-5c8c85481739.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation, Characterization, and biological significance of a novel C-type marine lectin purified from Scylla serrata haemolymph","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLectins are typical glycoproteins of non-immune origin that bind to glycoproteins and glycolipids on the cell surface to agglutinate a range of animal cells after recognizing carbohydrate structures (Hirabayashi, 2002). All organisms have lectin-carbohydrate interactions, which are ligand-receptor 2 interactions (Bulgakov et al., 2004). These interactions support a variety of biological functions, including the transport of carbohydrates, glycoproteins, and calcium within cells and tissues (Vasta, 1992), cytolysis and cytotoxicity (Armstrong et al., 1996), and cell adhesion, migration, and apoptosis (Ni Y. \u0026amp; Tizard, 1996). They have been linked to organ morphogenesis, tumor cell metastasis, leukocyte trafficking, immunological response, inflammation, and extracellular matrix recognition. They can also cause cell proliferation, cell arrest, or apoptosis (Sharon \u0026amp; Lis, 2004). Lectins extracted from animal tissues were studied as immunomodulatory, antiviral, anticancer, and apoptotic agents. (Coelho et al., 2017) The immune systems of both vertebrates and invertebrates can use lectins as a nonself-recognition molecule (Renwrantz, 1986; Arason, 1996; Matsushita, 1996; Vasta et al., 1999; Wilson et al., 1999). As the main immunological response to infections, mannose-binding lectin (MBL) has been proposed to be crucial for host defense (Uemura et al., 2002; Ezekowitz, 2003). The finest illustrations of PRP-mediated immunity are in insects and horseshoe crab lectins. Numerous immunological responses, including cytotoxic effects (Ma Y. et al., 1999), phagocytosis enhancement (Kondo et al., 1992), antibacterial activity (Tunkijjanukij \u0026amp; Olafsen, 1998), and nodule formation (Koizumi et al., 1999), are significantly influenced by these lectins. The precise roles of several invertebrate lectins in this nonself-recognition molecule in crustaceans are not well understood, although many of them have been isolated and described (Viswambari et al., 2010). As a result, Marques and Barracco have examined lectin purification, molecular characterization, physiological function, synthesis, and induction upon infection in crustaceans (Marques et al., 2000). The sequence details of several crustacean C-type lectins have been published recently (Wang \u0026amp; Wang, 2013). These reports or studies demonstrate the several immune-related roles that crab lectins play in crustacean immunity, including the capacity to identify nonself-recognition molecules like PRPs. Marine crabs \u003cem\u003eTrichopeltarion nobile\u003c/em\u003e (Rama Devi et al., 2012) and \u003cem\u003eAtergatis ocyroe\u003c/em\u003e (Elayabharathi et al., 2017) haemolymph contains a calcium-dependent lectin, and marine crabs \u003cem\u003ePortunus pelagicus\u003c/em\u003e (Chidhambaradhas et al., 2017; Jayanthi et al., 2017) have shown that lectin possesses antibacterial properties. A lectin was employed as a diagnostic and anticancer drug was obtained from the Korean sea crab \u003cem\u003ePhilyra pisum\u003c/em\u003e. It detects N-glycolyl neuraminic acid and has antiproliferative properties against cancer cells (Kim et al., 2006). However, the information on the isolation and characterization of mannose-specific C-type lectins from marine crabs is limited. The present study demonstrates the purification and characterization of a novel c-type lectin and its antimicrobial properties from mud crab \u003cem\u003eS. serrata\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. serrata\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;haemolymph collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the early morning hours (5.30 am\u0026ndash;8.30 am), local fisherman assisted in the collection of 150 g (150 \u0026plusmn; 20) mud crabs (\u003cem\u003eS. serrata\u003c/em\u003e) from the coastal area of Fort Kochi, Ernakulam district, Kerala, India. Sand and planktonic particles were eliminated from the animals by washing them with seawater. Following their cautious transportation to the lab, they were kept in FRP tanks with a flow-through that contained seawater (30 ppt) at 30\u0026plusmn;2 \u0026deg;C and fed a specially prepared crab diet (Cylla Plus, ICAR-CIBA, India) twice daily. Using a syringe filled with a marine anticoagulant solution (0.45 M NaCl, 0.1 M glucose, 30 mM sodium citrate, 26 mM citric acid, 10 mM EDTA, pH 7.5), haemolymph was quickly extracted from the hemocoel in the arthrodial membrane of the final pair of walking legs (S\u0026ouml;derh\u0026auml;ll et al., 1983). The haemolymph was centrifuged for 5 minutes at 4 \u0026deg;C at 3300 \u0026times; g. The cell debris was removed, and the plasma was stored at -80 \u0026deg;C for later use.\u003c/p\u003e\n\u003cp\u003eThe experiments were performed following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), application number: 363/GO/Re/S/01/CCSEA/107. The Institutional Animal Ethics Committee of Cochin University of Science and Technology, Kochi, Kerala, India reviewed and approved the protocol. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurification of Ss-Lec\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith minor adjustments, lectin was purified using the procedures outlined by Rubeena \u0026amp; Preetham (2019). In summary, 10 mg of D+ mannose (Sigma Aldrich) was combined with 7 ml of Sepharose CL-6B beads (Sigma Aldrich) in accordance with manufacturing instructions. The mixture was then maintained at 4\u0026ordm;C and 100 rpm for the entire night in a cooling shaking incubator (Calgon Scientific, India). After a thorough cleaning with sterile water, a 1.5 x 12 cm column (Econo-Pac\u0026reg;, Biorad Laboratory, Canada) was washed six times with TBS/CaCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ebuffer (10 mM Tris\u0026ndash;HCl, 150 mM NaCl, 10 mM CaCl\u003csub\u003e2\u003c/sub\u003e, pH 7.4). The mannose-coupled Sepharose CL-6B beads were then meticulously put inside the column, cleaned three times with TBS/CaCl\u003csub\u003e2\u003c/sub\u003e buffer, and stored at 4\u0026ordm;C for the night. An equivalent amount of TBS/CaCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas then added to the \u003cem\u003eS. serrataa\u003c/em\u003e plasma to equilibrate it. Approximately 10 ml of the equilibrated sample was combined with 5 ml of mixing buffer (2.5 M NaCl, 40 mM CaCl2, pH 7.8) containing 20 mM imidazole-HCl and gently swirled for 60 minutes at 24\u0026ordm;C. Gently, the mixes were added to the CL-6B mannose-coupled Sepharose column. The elution buffer (10 mM Tris\u0026ndash;HCl, 140 mM NaCl, 3 mM EDTA, pH 8.0) used to elute the purified fractions contained 10ul of 10mM CaCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ein each tube.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of purified Ss-Lec\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS-PAGE analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEluted fractions were subjected to SDS and native polyacrylamide gel electrophoresis (12%) in the presence and absence of 2-marceptaethanol (Laemmli, 1970). Following electrophoresis, Coomassie Brilliant Blue R-250 (Biorad Laboratories, Canada) was used to stain the gel. By comparing its electrophoretic mobility with that of molecular mass marker proteins (BioRad labs, Canada), the molecular mass of the isolated Ss-Lec was ascertained. The Bradford method (Bradford, 1976) was used to determine the total protein concentration, with bovine serum albumin (BSA) as a reference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh-performance liquid chromatography and Fourier Transform Infrared Spectroscopy (FTIR) analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHPLC (Agilent Infinity 2000, USA) separations were performed using a reversed-phase C18 column (7.8 mm \u0026times; 30 cm) at a flow rate of 0.8 ml/min in a previously equilibrated column with TBS-I. For FTIR (Agilent Cary 630, USA) spectrum analysis, 50 \u0026micro;l purified Ss-Lec was placed in a thermostated cell fitted with CaF\u003csub\u003e2\u003c/sub\u003e windows (with 6 \u0026micro;m Teflon spacer for measurements in water). The spectra of biological molecules were recorded at a resolution of 4 cm\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX-Ray diffraction (XRD) and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXRD analysis (Bruker AXS GmbH, D8 QUEST, Germany) was carried out in purified Ss-Lec at 40 kV/20 mA utilising continuous scanning 2\u0026theta; mode to ascertain the spatial distribution of atomic coordinates and the arrangement of atoms. Scherrer\u0026apos;s formula (d = (0.9\u0026lambda;/\u0026beta; cos \u0026theta;)) is used to calculate the average grain size and shape of the purified Ss-Lec.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor MALDI-TOF analysis the Ss-Lec gel bands were carefully trimmed and put into 1.5 ml Eppendorf tubes filled with stain removal solution (100 mM ammonium bicarbonate: 50% acetonitrile (1:1), and vortexed for 45 minutes. Following total stain removal, 100% acetonitrile (ACN) was used to dehydrate the gel fragments at 4\u0026ordm;C. For trypsin digestion and absorption, a trypsin solution was prepared in 40 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e, and added to each Eppendorf tube containing gel pieces, and then kept at ice cold. Following absorption, the mixture was incubated for 12\u0026ndash;16 hours at 37 \u0026deg;C. 4% formic acid was added to stop the digestion. Gel pieces were washed three times with 4% formic acid (FA) in 50% acetonitrile (ACN) to extract the peptides. The MALDI-TOF/TOF MS (Shimadzu, Biotek Axima Performance, Japan) was used to examine the resulting peptides.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of physiochemical properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physicochemical characterization of the purified Ss-Lec was evaluated by its haemagglutination property, examining the effects of various physicochemical parameters such as pH, temperature, and cation dependency to determine the optimum activity. These assays were performed following (Vargila et al., 2024) with modified concentrations of the parameters and using human erythrocytes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHaemagglutination (HA) and Haemagglutination inhibition (HAI) assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaemagglutination assay was performed using 2% human erythrocytes and 25-100 \u0026micro;g/ml purified Ss-Lec. To evaluate the haemagglutination activity equal volume of purified Ss-Lec mixed with 2% RBC and TBS-II (10 mM Tris HCl, 145 mM NaCl, and pH-7.5) in a V-bottom microtiter plate (Greiner, Nunc, Germany). The suspensions of RBCs and lectin were incubated for 45 min at 28\u0026ordm; C. These haemagglutinated titres of Ss-Lec were visualized by light microscopy (Nikon microscope Eclipse Ti 100) at the magnification of 40x.\u003c/p\u003e\n\u003cp\u003eHaemagglutination inhibition assay was performed in the presence of carbohydrate competitive inhibitors such as D- glucose, D-galactosamine HCl, D-maltose, N-acetyl D-glucosamine, D-fucose, Lactose, D-Mannose, D-fructose, L-fucose, and sucrose. \u0026nbsp;Serially diluted 400mM carbohydrate solutions were mixed with 100\u0026micro;g/ml purified Ss-Lec and pre-incubated at 28\u0026ordm;C for 45min. Then 2% human erythrocyte suspension was mixed with the Ss-Lec and inhibitor solution and incubated at 28\u0026ordm;C for 2 hours. The button-forming agglutination inhibition points were noted against the serially diluted carbohydrate samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast Agglutination assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the capacity of Ss-Lec to agglutinate yeast cells (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e), 50\u0026mu;l of purified Ss-Lec at different concentrations (25-100\u0026mu;g/ml) was mixed with an equal volume of TBS I buffer in V-shaped 96-well microtitre plate. Then, an equal volume of yeast suspension (10\u003csup\u003e6\u003c/sup\u003e cells/mL) was added to the wells and incubated for 4 hours at 28\u0026ordm;C. In control, the purified Ss-Lec was replaced by BSA. The yeast agglutination properties of Ss-Lec were visualized by light microscopy (Nikon microscope Eclipse Ti 100) at the magnification of 40x.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEncapsulation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe encapsulation activity of Ss-Lec was performed following previously described method (Rubeena \u0026amp; Preetham, 2019), with minor adjustments. Briefly, equal volume of 2% RBC suspension and CL-6B Sepharose bead suspension, were mixed with Ss-Lec at a concentration of 25, 50, and 100 \u0026mu;g/ml respectively in a V-bottomed microtitre plate and incubated for 45 minutes at 28\u0026deg;C with 15-minute intervals between mixing. TBS I buffer was used in control wells in place of Ss-Lec. On a sterile glass slide, the whole volume of each solution was spread out and left undisturbed for ten minutes. Following that, the slides were examined using a Leica DM IL LED inverted microscope (40X).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenoloxidase (PO) activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhenoloxidase (PO) activation activity was assessed according to the previously described method by Sun J et al. (2008) with minor modifications. In brief, purified Ss-Lec (25-100 \u0026micro;g/ml) was preincubated with an equal volume of laminarin (Sigma-Aldrich, USA) for 5 min at 25 \u0026deg;C. Further, the samples were incubated with 50 \u0026micro;l of 3 \u0026micro;g/ml L-DOPA (L-3,4-dihydroxyphenylalanine, Sigma-Aldrich, USA) for 5 min at 28 \u0026deg;C. The phenoloxidase activity was measured spectrophotometrically at 470 nm at different time points (0 min, 30 min, 60 min, and 90 min) and expressed as units/\u0026mu;g protein. In controls, purified Ss-Lec was replaced by TBS I buffer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial analysis of purified Ss- Lec\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial strains \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To evaluate antibacterial effect of purified Ss-Lec, Gram positive \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e (OP580171), \u003cem\u003eStreptococcus iniae\u003c/em\u003e (NZ_AOCT02000221), and Gram-negative \u003cem\u003eAeromonas veronii\u003c/em\u003e (AY764310), \u003cem\u003eEdwardsiella tarda\u003c/em\u003e (MTCC2400) bacterial strains were cultured in tryptic soy broth (TSB) in a shaker for 24 h at 28 \u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePurified Ss-Lec\u0026apos;s antibacterial activity against both Gram-positive and Gram-negative bacteria was assessed using the agar well diffusion technique. The bacterial broths were centrifuged (6000 x g for 10 min) and resuspended in TBS (50 mM Tris\u0026ndash;HCl, 100 mM NaCl, pH 7.5). Further, 1x 10\u003csup\u003e5\u003c/sup\u003e CFU/ml of bacterial cultures were spread on 100 \u0026times; 20 mm petri plates containing TSB agar, and 10 mm diameter holes were created with per forex. Purified Ss-Lec in varying concentrations (25 \u0026micro;g, 50 \u0026micro;g, and 100 \u0026micro;g) was added to each well, and the plates were then incubated for 24 hours at 28 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm quantification and inhibition assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe formation of biofilm by important aquatic pathogens like \u003cem\u003eStreptococcus agalactiae, Streptococcus iniae, Aeromonas veronii\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Edwardsiella tarda\u003c/em\u003e against purified Ss-Lec was measured by colorimetric assay (Kumar et al., 2012). In brief, the individual wells of 96 well plates were filled with 150 \u0026micro;L Tryptic soy broth (TSB) followed by inoculation with 10 \u0026micro;L of 1 x 10\u003csup\u003e9\u003c/sup\u003e CFU/ml bacterial culture. Subsequently, 25-100 \u0026micro;g/ml purified Ss-Lec was added to each well and incubated at 28\u0026deg;C for 48 hrs. Non-adherent bacteria were washed using 0.2 ml phosphate buffer saline (PBS, pH 7.2). The adherent bacteria were fixed with sodium acetate (2%) in crystal violet (0.1% w/v). Excessive stains were washed off with distilled water. Then, 0.2ml 95% ethanol was used to dissolve the crystal violet bounded sessile bacteria and the optical density was measured using a microtiter plate reader (Varioskan Lux, Thermofisher Scientific).\u003c/p\u003e\n\u003cp\u003ePurified Ss-Lec\u0026apos;s effect on biofilm inhibition was evaluated using previously described method (Bakkiyaraj \u0026amp; Karutha, 2010). \u003cem\u003eStreptococcus agalactiae, Streptococcus iniae, Aeromonas veronii, and Edwardsiella tarda\u003c/em\u003e were tested for biofilm inhibition by microscopic visualisation using TSB broth containing the bacterial suspension of 1 x 10\u003csup\u003e9\u003c/sup\u003e CFU/ml. Following the previous biofilm quantification method, various concentrations of purified Ss-Lec (25-100 \u0026micro;g/ml) were added to the wells of a 24-well plate that contained broken glass coverslip pieces. The mixture was then incubated at 28 \u0026deg;C for 48 hours. The biofilm was stained with 0.5 ml of 0.4% crytal violet (w/v) and 0.4% acridine orange (w/v) for 10 minutes at 28\u0026ordm;C. Confocal laser scanning microscopy (Carl Zeiss LSM 710 Germany) examined stained glass pieces at a 20x magnification on slides with the biofilm facing up for the acridine orange stain, and light microscopy (Nikon microscope Eclipse Ti 100) at the magnification of 40x was used to visualize the crystal violet stain.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePurification of Ss-Lec\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA C-type lectin from the mud crab \u003cem\u003eS. serrata\u003c/em\u003e haemolymph was isolated and purified using affinity chromatography, and 71 kDa molecular mass of the protein was determined using 12% SDS-PAGE (Fig 1) against Coomassie blue staining. The protein concentration was quantified as 104 \u0026micro;g/ml.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of Ss-Lec\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXRD analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXRD analysis of the purified Ss-Lec showed one diffraction peak at 31.7\u0026deg;, which shows the purity and crystalline nature of the protein. The single sharp peak depicts the bigger crystalline nature of the pure Ss-Lec. (Fig 2a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC and FTIR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReversed phase HPLC analysis revealed the homogeneity of the purified Ss-Lec as a single peak with a retention time of 5.259 min (Fig 2b and 2c). The FTIR spectrum showed the C=N stretching corresponding to a sharp peak at 1643.08 cm\u003csup\u003e-1\u003c/sup\u003e. Followed by 2096.96 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003epeak and O-H stretching at 3276.89 cm\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMALDI-TOF analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn MALDI-TOF analysis, the intensity of the peaks corresponding to the 71kDa protein (Fig Sd) was shown. After trypsin digestion, the abundance of the fragments from the parent protein was denoted in peak intensity and their mass-to-charge (m/z) ratio of the molecules. In this study, 12 sharp peaks were observed above 1 x 10\u003csup\u003e4\u003c/sup\u003e intensity, indicating the abundance of the molecules in the Ss-Lec.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiochemical properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaximum hemagglutination activity was demonstrated by the Ss-Lec within a limited pH range of 7 to 8 (Fig 3A). The sensitivity of Ss-Lec to temperature showed that its hemagglutination activity decreased at temperatures up to 64 \u0026deg;C, with optimal activity occurring between 30 and 40 \u0026deg;C (Fig 3B). The hemagglutinating activity of Ss-Lec is influenced by divalent cations (Fig 3C). Although increased activity was observed up to 30 mM, hemagglutination activity was reduced at higher concentrations of calcium and magnesium ions. When different amounts of calcium chelators, such as Di and Tetrasodium EDTA, were applied to Ss-Lec, a reduced HA titer was observed up to 1 mM and above 10 mM (Fig 3D). This confirms the lectin\u0026apos;s calcium dependence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHA and HAI activity of purified Ss-Lec\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe haemagglutination (HA) activity of purified Ss-Lec was visualized by light microscope (Fig 4), where a higher quantity of lectin can agglutinate human erythrocytes. In the presence of the carbohydrate inhibitors, the Ss-Lec showed HAI activity where N-acetyl glucosamine, D-mannose, and D- and L-fucose can inhibit the haemagglutination in very minimal concentrations than D-glucose (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast agglutination capability of Ss-Lec\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe purified Ss-Lec showed strong agglutination capability towards \u003cem\u003eSaccharomyces cerevisiae\u0026nbsp;\u003c/em\u003e(yeast)\u003cem\u003e\u0026nbsp;\u003c/em\u003ecells in a concentration dependent pattern where the highest yeast agglutination was observed at 50-100\u0026mu;g/ml concentration (Fig 5) of Ss-Lec.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEncapsulation activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe encapsulation assay was used to determine if the pure Ss-Lec could foster cellular encapsulation in vitro. Light microscopy was used to investigate the encapsulation phenomena. The encapsulation activity seen up to a 1-hour incubation period may be similarly stimulated by the Sepharose beads coated with pure Ss-Lec. During the incubation period, the entrance of RBC towards Sepharose beads was seen after 45 minutes, where RBC surrounding the beads was seen (Fig 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenoloxidase (PO) activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Phenoloxidase activity of the purified ss-Lec was measured in the presence of Laminarin at different time points. A concentration-dependent PO activity was observed. The highest concentration (100\u0026mu;g/ml) of Ss-Lec showed higher PO activity (Fig 7) in all time points from 30 to 90 minutes post-incubation. Similarly, the highest PO activity was also noticed at 90 minutes post-incubation in comparison to other time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial activity of purified Ss-Lec\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe purified Ss-Lec showed potential antibacterial activity against important gram-positive and gram-negative aquatic pathogens, whereas the zone of bacterial inhibition was measured (Table 2). The higher Ss-Lec at 100\u0026mu;g/ml was able to inhibit a maximum growth of up to 7mm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibiofilm properties of Ss-Lec\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microtitre plate (MTP) based colorimetric assay was performed to assess the formation of biofilm of Gram-positive bacteria \u003cem\u003eStreptococcus iniae, Streptococcus agalactiae\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003eGram-negative\u003cem\u003e\u0026nbsp;\u003c/em\u003ebacteria\u003cem\u003e\u0026nbsp;Edwardsiella tarda, Aeromonas veronii\u0026nbsp;\u003c/em\u003ein the presence and absence of purified Ss-Lec. The biofilm inhibition was increased corresponding to the increased concentration of lectin. At a concentration of 100\u0026micro;g/ml of Ss-Lec, the highest biofilm inhibition of 82% was observed in \u003cem\u003eEdwardsiella tarda\u003c/em\u003e following \u003cem\u003eStreptococcus iniae\u003c/em\u003e, \u003cem\u003eS. agalactiae\u003c/em\u003e and \u003cem\u003eAeromonas veronii\u003c/em\u003e at the rate of 72%, 71%, and 68%, respectively (Fig 8).\u003c/p\u003e\n\u003cp\u003eThe anti-biofilm formation was visualized by light and confocal (2D) microscopic methods. Where the different concentrations of purified Ss-Lec can disrupt the growth and formation of biofilms against \u003cem\u003eStreptococcus iniae, Streptococcus agalactiae, Edwardsiella tarda,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAeromonas veronii\u0026nbsp;\u003c/em\u003ecompared with controls (Fig 9A \u0026amp; 9B).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLectins are immunological molecules that are specifically engaged in the removal of external invaders in the crustacean\u0026apos;s innate immune system. The majority of lectins found in crustaceans have been identified after the turn of the century, and a multiple number of lectins are extracted and identified from crustacean haemolymph. The mannose-binding lectin, which has a molecular mass of 71 kDa, was isolated and purified from the mud crab \u003cem\u003eS. serrata\u003c/em\u003e for the present investigation. The lectin purification from mud crab haemolymph was carried out using the mannose-coupled sepharose CL-6B affinity column. The buffer\u0026apos;s calcium ions helped the lectin bind to the mannose ligands in the column, and the EDTA in the elution buffer chelated the calcium ions to release the lectin when it was eluted from the column. To date, several lectins have been isolated from crustaceans with molecular weights ranging from 14 kDa from \u003cem\u003eTachypleus tridentatus\u003c/em\u003e (Tri-spine horseshoe crab) (Inamori et al., 1999), 20 kDa from \u003cem\u003eScylla serrata\u003c/em\u003e (Mud crab) (Jayasankar \u0026amp; Subramoniam, 1999), 55kDa from \u003cem\u003eScylla serrata\u0026nbsp;\u003c/em\u003e(Mercy \u0026amp; Ravindranath,1993)\u003cem\u003e\u0026nbsp;\u003c/em\u003efrom \u0026nbsp;to a higher range of 452 kDa from \u003cem\u003ePenaeus japonicas\u003c/em\u003e (Kuruma shrimp) (Yang et al., 2007) and 220 kDa \u003cem\u003eLitopenaeus schmitti\u003c/em\u003e (White shrimp) (Cominetti et al., 2002), the green tiger prawn \u003cem\u003ePenaeus semisulcatus\u003c/em\u003e (37 and 118 kDa) (Sivakamavalli \u0026amp; Vaseeharan, 2014), the freshwater crab \u003cem\u003eParatelphusa jacquemontii\u003c/em\u003e (34 kDa) (Denis et al., 2003), the banana prawn \u003cem\u003eFenneropenaeus merguiensi\u003c/em\u003e (32.3 and 30.9 kDa) (Rittidach et al., 2007), and Chinese white shrimp \u003cem\u003eFenneropenaeus chinensis\u003c/em\u003e (168 kDa) (Sun J et al., 2008), blue swimmer crab \u003cem\u003ePortunas pelagicus\u003c/em\u003e (155 kDa) (Jayanthi et al., 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe purified Ss-Lec was shown to be monovalent, with a decreased affinity for D-glucose, and no activity towards D-galactosamine HCl, Lactose, D-fructose, Sucrose. Whereas, the Ss-Lec had a greater affinity for D-Mannose, D-fucose, L-fucose, N-acetyule D-glucosamine and D-maltose respectively. This aligns with earlier findings by Mitra and Das (2001) and Silva et al. (2012), who discovered that a single lectin protein may show affinity for a variety of sugar compounds. \u0026nbsp;In addition, Jayanthi et al. (2017) previously reported that the crystalline nature and purity were verified by XRD analysis and HPLC analysis, respectively that corelates with our obtained results from the current study. Similarly described by Divya et al. (2018), the functional group analysis and molecular mass confirmation were carried out using FTIR analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe ability of lectin to agglutinate against mammalian red blood cells was tested as an agglutinin, and the Ss-Lec successfully agglutinated the RBS at the lowest concentration of 25\u0026mu;g/ml. It is evident from this agglutination response that the purified Ss-Lec can identify foreign invaders via PRPs. Similar agglutination reactions were conducted with \u003cem\u003eP. semisulcatus\u003c/em\u003e lectin, which exhibits strong agglutination towards human red blood cells (Sivakamavalli \u0026amp; Vaseeharan, 2014). Additionally, our findings were consistent with previously documented lectins of \u003cem\u003eLitopenaeus setiferus\u003c/em\u003e (Alpuche et al., 2005),\u0026nbsp;\u003cem\u003eMacrobrachium rosenbergii\u003c/em\u003e (Zenteno et al., 2000), and Sun J. et al. (2008), that the lectin\u0026apos;s ability to agglutinate human erythrocytes revealed that it was unable to distinguish between human RBC types. Apart from its function in agglutination, Ss-Lec is also essential for encapsulation and phagocytosis. Encapsulation,\u003cbr\u003e\u0026nbsp;only found in invertebrates, and it actively acts to counteract a range of\u0026nbsp;\u003cbr\u003e\u0026nbsp;outside dangerous substances. Unlike phagocytosis, which eliminates big foreign, harmful components, encapsulation attempts to surround and completely encase the microorganism\u0026nbsp;\u003cbr\u003eintrusions that cause damage within the enclosed cascade. Encapsulation against the Sepharose beads was made easier by Ss-Lec, a characteristic that has already been documented in other invertebrates (Sminia et al., 1974; Sivakamavalli \u0026amp; Vaseeharan, 2014; Jayanthi et al., 2017; Rubeena \u0026amp; Preetham, 2019). Ss-Lec can thus take action by removing and banning alien diseases from prawn aquaculture. Marine white prawn \u003cem\u003eL. vannamei\u003c/em\u003e is one of the numerous crustaceans that have been shown to include lectins with a variety of immunological activities and an improved encapsulation process in a number of earlier investigations (Lis et al., 1998; LGuo et al., 2013; Cerenius et al., 2010). Phenoloxidase (PO) is a crucial biological function that is well-known for interacting with marine prawns and other invertebrate immune components. It plays a significant immune-defensive function in several ways, including wound healing and the elimination of microbial invaders, and it activates dormant ProPO by activating the lectin complement pathway (Preetham et al., 2019). As a result of the degranulation of haemoglobin, Sivakamavalli et al. (2014) found that the complex formed between PAMPs and lectin might activate the ProPO (inactive form) to PO (active form) (Rubeena and Preetham, 2019). Correlating the previous findings Ss-Lec and laminarin were the triggers for the PO system in this investigation, and PO activity was increased with time. We also documented the antibacterial capabilities of purified Ss-Lec against a broad spectrum of Gram-positive and Gram-negative bacteria in addition to the functional study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePurified Ss-Lec\u0026apos;s antibacterial activity was assessed using the agar well diffusion method, demonstrating its comparable bacterial killing capabilities against Gram-negative \u003cem\u003eEdwardsiella tarda and Aeromonas veronii\u0026nbsp;\u003c/em\u003eas well as Gram-positive \u003cem\u003eStreptococcus iniae and Streptococcus agalactiae\u003c/em\u003e. The chinese prawn \u003cem\u003eF. chinensis\u0026apos;s\u003c/em\u003e Lectin Fc-hsL shown antibacterial activity against \u003cem\u003eB. subtilis, B. cereus, B. thuringiensis, E. coli\u003c/em\u003e, and \u003cem\u003eK. pneumoniae\u003c/em\u003e, (Sun YD et al., 2008). In another study (Lai et al., 2013) the recombinant \u003cem\u003eF. chinensis\u0026nbsp;\u003c/em\u003eC-Type lectin, or rFcCTL, demonstrated greater antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria. This suggests that lectins are likely involved in the immobilisation of bacteria, binding, and destruction of bacterial cell walls, which results in the inhibition or termination of microorganism growth (Tunkijjanukij \u0026amp; Olafsen, 1998). Purified Ss-Lec was tested for antibiofilm activity after showing promising antibacterial action against bacteria. The unwanted growth of microorganisms on synthetic surfaces submerged in a common matrix is known as biofilm (Yebra et al., 2004). Against the same aquatic pathogens, the efficaciousness of purified Ss-Lec as a degrading peptide in disintegrating biofilm matrix was investigated. According to antibiofilm activity results in current study the increased concentrations of purified Ss-Lec had reduced the production of biofilm. Both Gram-positive and Gram-negative pathogens were shown to be susceptible to the same antibiofilm action of many crustacean proteins, including \u0026beta;-1, 3 glucan binding protein (\u003cem\u003eP. pelagicus\u003c/em\u003e) (Anjugam et al., 2016), prophenoloxidase (\u003cem\u003eP. semisulcatus\u003c/em\u003e) (Ishwarya et al., 2016), and crustin (\u003cem\u003eEpisesarma tetragonum\u003c/em\u003e) (Sivakamavalli et al., 2015). Moreover, the purified mannose binding C-type Ss-Lec had a broad range of functional activity which promotes its capability as a potent antimicrobial agent.\u003c/p\u003e\n\u003cp\u003eIt is concluded that, under reducing and non-reducing conditions, the affinity chromatography-purified Ss-Lec showed an apparent molecular mass of 71 kDa on SDS-PAGE. The purity, homogeneity, and crystalline nature of Ss-Lec were shown by MAlDI-TOF, HPLC, XRD, and FTIR analysis. Agglutination, phenoloxidase, and encapsulation-enhancing qualities demonstrated Ss-Lec\u0026apos;s active immune system participation and immuno-protective function in mud crabs. Therefore, the purified Ss-Lec was projected against a variety of Gram-positive and Gram-negative bacteria to determine its antibiofilm characteristics. Based on all of these results, we concluded that Ss-Lec is found to play a crucial part in the immune system and may be used as a possible antibacterial and therapeutic tool to strengthen crab\u0026apos;s defense against pathogens, and it can be used in the aquaculture sector.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRitam Guha\u003c/strong\u003e: Conceptualization, Methodology, Data curation, Formal analysis, Writing-original draft, writing review and editing. \u003cstrong\u003eIshwarya Ramachandran:\u0026nbsp;\u003c/strong\u003eMethodology, Data curation, Formal analysis, writing review, and editing. \u003cstrong\u003eSivashanmugam Karthikeyan\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Data curation, writing review, and editing. \u0026nbsp;\u003cstrong\u003eBaskaralingam Vaseeharan\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eData curation, Writing-review and editing\u003cstrong\u003e\u0026nbsp;Preetham Elumalai:\u003c/strong\u003e Conceptualization, writing-review and editing, supervision, project administration, funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no financial interests that are directly or indirectly related to the work submitted for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors deeply acknowledge the funding supported by the Department of Science and Technology-Indian National Academy of Engineering (2023/IN-TW/07), India-Taiwan collaboration program, and Centre of Excellence for Aquatic Vaccine Development, Govt. of Kerala at CUSAT. Author RG acknowledges the funding support of the Department of Science and Technology-Innovation in Science Pursuit for Inspired Research Fellowship (IF230205). The author IR deeply acknowledges Indian Council of Medical Research - Department of Health Research, for the encouragement and financial assistance (R.12013/55/2024-HR) towards DHR- Women Scientist Fellowship.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdul Salam R, Preetham E (2019) Antimicrobial properties and phenoloxidase activation of the lectin isolated from kadal shrimp (Metapenaeus dobsoni). Fish Shellfish Immunol 90:118\u0026ndash;125. https://doi.org/10.1016/j.fsi.2019.04.305\u003c/li\u003e\n\u003cli\u003eAlpuche J, Pereyra A, Agundis C, Rosas C, Pascual C, Slomianny MC, Zenteno E (2005) Purification and characterization of a lectin from the white shrimp Litopenaeus setiferus (Crustacea decapoda) hemolymph. Biochim Biophys Acta 1724:86\u0026ndash;93. https://doi.org/10.1016/j.bbagen.2005.04.014\u003c/li\u003e\n\u003cli\u003eArason GJ (1996) Lectins as defence molecules in vertebrates and invertebrates. Fish Shellfish Immunol 6:277\u0026ndash;289. https://doi.org/10.1006/fsim.1996.0029\u003c/li\u003e\n\u003cli\u003eArmstrong PB, Swarnakar S, Srimal S, Misquith S, Hahn EA, Aimes RT, Quigley JP (1996) A cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins. J Biol Chem 271:14717\u0026ndash;14721. https://doi.org/10.1074/jbc.271.25.14717\u003c/li\u003e\n\u003cli\u003eBakkiyaraj D, Karutha Pandian ST (2010) In vitro and in vivo antibiofilm activity of a coral associated actinomycete against drug resistant Staphylococcus aureus biofilms. Biofouling 26:711\u0026ndash;717. https://doi.org/10.1080/08927014.2010.511200\u003c/li\u003e\n\u003cli\u003eBradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248\u0026ndash;254. https://doi.org/10.1016/0003-2697(76)90527-3\u003c/li\u003e\n\u003cli\u003eBulgakov AA, Park KI, Choi KS, Lim HK, Cho M (2004) Purification and characterisation of a lectin isolated from the Manila clam Ruditapes philippinarum in Korea. Fish Shellfish Immunol 16:487\u0026ndash;499. https://doi.org/10.1016/j.fsi.2003.08.006\u003c/li\u003e\n\u003cli\u003eCerenius L, Jiravanichpaisal P, Liu HP, Soderhall I (2010) Crustacean immunity. In: S\u0026ouml;derh\u0026auml;ll K (ed) Invertebrate Immunity. Springer, Boston, pp 239\u0026ndash;259. https://doi.org/10.1007/978-1-4419-8059-5_13\u003c/li\u003e\n\u003cli\u003eChidhambaradhas S, Selvamohan T, Stanislaus PM (2017) Identification and purification of antimicrobial lectins from marine crab Portunus pelagicus (Linneus, 1775). Int J Curr Microbiol Appl Sci 6:2346\u0026ndash;2351. https://doi.org/10.20546/ijcmas.2017.606.277\u003c/li\u003e\n\u003cli\u003eCoelho LCCB, Silva PMDS, Lima VLDM, Pontual EV, Paiva PMG, Napole\u0026atilde;o TH, Correia MTDS (2017) Lectins, interconnecting proteins with biotechnological/pharmacological and therapeutic applications. Evid Based Complement Alternat Med 2017:1594074. https://doi.org/10.1155/2017/1594074\u003c/li\u003e\n\u003cli\u003eCominetti MR, Marques MRF, Lorenzini DM, L\u0026ouml;fgren SE, Daffre S, Barracco MA (2002) Characterization and partial purification of a lectin from the hemolymph of the white shrimp Litopenaeus schmitti. Dev Comp Immunol 26:715\u0026ndash;721. https://doi.org/10.1016/s0145-305x(02)00025-3\u003c/li\u003e\n\u003cli\u003eDa Silva CDC, Coriolano MC, da Silva Lino MA, de Melo CML, de Souza Bezerra R, de Carvalho EVMM, Coelho LCCB (2012) Purification and characterization of a mannose recognition lectin from Oreochromis niloticus (Tilapia Fish): Cytokine production in mice splenocytes. Appl Biochem Biotechnol 166:424\u0026ndash;435. https://doi.org/10.1007/s12010-011-9438-1\u003c/li\u003e\n\u003cli\u003eDenis M, Palatty PM, Bai NR, Suriya SJ (2003) Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii. Eur J Biochem 270:4348\u0026ndash;4355. https://doi.org/10.1046/j.1432-1033.2003.03828.x\u003c/li\u003e\n\u003cli\u003eDivya M, Vaseeharan B, Anjugam M, Iswarya A, Karthikeyan S, Velusamy P, V\u0026aacute;gv\u0026ouml;lgyi C (2018) Phenoloxidase activation, antimicrobial, and antibiofilm properties of \u0026beta;-glucan binding protein from Scylla serrata crab hemolymph. Int J Biol Macromol 114:864\u0026ndash;873. https://doi.org/10.1016/j.micpath.2020.103992\u003c/li\u003e\n\u003cli\u003eElayabharathi JVJM, Mary S, Bai M (2017) Partial purification and characterization of hemolymph lectin of marine crab Atergatis ocyroe by adsorption on formalinized erythrocytes. Int J Biol Res 2:115\u0026ndash;119\u003c/li\u003e\n\u003cli\u003eEzekowitz RA (2003) Role of the mannose-binding lectin in innate immunity. J Infect Dis 187(Suppl 2):S335\u0026ndash;S339. https://doi.org/10.1086/374746\u003c/li\u003e\n\u003cli\u003eHirabayashi J (2002) Introduction to lectins. Glycoforum. http://www.glycoforum.gr.jp/science/word/lectin/LEAOO.2002.html\u003c/li\u003e\n\u003cli\u003eInamori KI, Saito T, Iwaki D, Nagira T, Iwanaga S, Arisaka F, Kawabata SI (1999) A newly identified horseshoe crab lectin with specificity for blood group A antigen recognizes specific O-antigens of bacterial lipopolysaccharides. J Biol Chem 274:3272\u0026ndash;3278. https://doi.org/10.1074/jbc.274.6.3272\u003c/li\u003e\n\u003cli\u003eIshwarya R, Jayanthi S, Muthulakshmi P, Anjugam M, Jayakumar R, Nazar AK, Vaseeharan B (2016) Immune indices and identical functions of two prophenoloxidases from the haemolymph of green tiger shrimp Penaeus semisulcatus and its antibiofilm activity. Fish Shellfish Immunol 51:220\u0026ndash;228. https://doi.org/10.1016/j.fsi.2016.02.011\u003c/li\u003e\n\u003cli\u003eJayanthi S, Ishwarya R, Anjugam M, Iswarya A, Karthikeyan S, Vaseeharan B (2017) Purification, characterization and functional analysis of the immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus and their antibiofilm properties. Fish Shellfish Immunol 62:227\u0026ndash;237. https://doi.org/10.1016/j.fsi.2017.01.019\u003c/li\u003e\n\u003cli\u003eJayasankar V, Subramoniam T (1999) Antibacterial activity of seminal plasma of the mud crab Scylla serrata (Forskal). J Exp Mar Biol Ecol 236:253\u0026ndash;259\u003c/li\u003e\n\u003cli\u003eKim HH, Jun JI, Kim BS, Cho DH, Min TH, Kim YJ, Ryu CS (2006) U.S. Patent No. 7,015,313. U.S. Patent and Trademark Office, Washington, DC\u003c/li\u003e\n\u003cli\u003eKoizumi N, Imamura M, Kadotani T, Yaoi K, Iwahana H, Sato R (1999) The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydrate-recognition domains. FEBS Lett 443:139\u0026ndash;143. https://doi.org/10.1016/S0014-5793(98)01701-3\u003c/li\u003e\n\u003cli\u003eKondo M, Matsuyama H, Yano T (1992) The opsonic effect of lectin on phagocytosis by hemocytes of kuruma prawn, Penaeus japonicus. Fish Pathol 27:217\u0026ndash;222\u003c/li\u003e\n\u003cli\u003eKumar P, Selvi SS, Govindaraju M (2012) In vitro anti-biofilm and anti-bacterial activity of Junceella juncea for its biomedical application. Asian Pac J Trop Biomed 2:930\u0026ndash;935. https://doi.org/10.1016/S2221-1691(13)60002-7\u003c/li\u003e\n\u003cli\u003eLaemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680\u0026ndash;685. https://doi.org/10.1038/227680a0\u003c/li\u003e\n\u003cli\u003eLai X, Kong J, Wang Q, Wang W, Meng X (2013) Identification and molecular characterization of a C-type lectin-like protein from Chinese shrimp (Fenneropenaeus chinensis). Mol Biol Rep 40:2223\u0026ndash;2230. https://doi.org/10.1007/s11033-012-2284-6\u003c/li\u003e\n\u003cli\u003eGuo XN, Jin XK, Li S, Yu AQ, Wu MH, Tan SJ, Wang Q (2013) A novel C-type lectin from Eriocheir sinensis functions as a pattern recognition receptor with antibacterial activity. Fish Shellfish Immunol 35:1554\u0026ndash;1565. https://doi.org/10.1016/j.fsi.2013.08.021\u003c/li\u003e\n\u003cli\u003eLis H, Sharon N (1998) Lectins: carbohydrate-specific proteins that mediate cellular recognition. Chem Rev 98:637\u0026ndash;674. https://doi.org/10.1021/cr940413g\u003c/li\u003e\n\u003cli\u003eMarques MRF, Barracco MA (2000) Lectins, as non-self-recognition factors, in crustaceans. Aquaculture 191:23\u0026ndash;44. https://doi.org/10.1016/S0044-8486(00)00417-8\u003c/li\u003e\n\u003cli\u003eMa Y, Uemura K, Oka S, Kozutsumi Y, Kawasaki N, Kawasaki T (1999) Antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding protein-dependent cell-mediated cytotoxicity. Proc Natl Acad Sci USA 96:371\u0026ndash;375\u003c/li\u003e\n\u003cli\u003eAnjugam MA, Iswarya AI, Vaseeharan B (2016) Multifunctional role of \u0026beta;-1,3 glucan binding protein purified from the haemocytes of blue swimmer crab Portunus pelagicus and in vitro antibacterial activity of its reaction product. Fish Shellfish Immunol 48:196\u0026ndash;205. https://doi.org/10.1016/j.fsi.2015.11.023\u003c/li\u003e\n\u003cli\u003eMatsushita M (1996) The lectin pathway of the complement system. Microbiol Immunol 40:887\u0026ndash;893. https://doi.org/10.1111/j.1348-0421.1996.tb01156.x\u003c/li\u003e\n\u003cli\u003eMercy PD, Ravindranath MH (1993) Purification and characterization of N-glycolyneuraminic-acid-specific lectin from Scylla serrata. Eur J Biochem 215:697\u0026ndash;704. https://doi.org/10.1111/j.1432-1033.1993.tb18081.x\u003c/li\u003e\n\u003cli\u003eMitra S, Das HR (2001) A novel mannose-binding lectin from plasma of Labeo rohita. Fish Physiol Biochem 25:121\u0026ndash;129. https://doi.org/10.1023/A:1020545510295\u003c/li\u003e\n\u003cli\u003eNi Y, Tizard I (1996) Lectin-carbohydrate interaction in the immune system. Vet Immunol Immunopathol 55:205\u0026ndash;223. https://doi.org/10.1016/S0165-2427(96)05718-2\u003c/li\u003e\n\u003cli\u003ePreetham E, Rubeena AS, Vaseeharan B, Chaurasia MK, Arockiaraj J, Olsen RE (2019) Anti-biofilm properties and immunological response of an immune molecule lectin isolated from shrimp Metapenaeus monoceros. Fish Shellfish Immunol 94:896\u0026ndash;906. https://doi.org/10.1016/j.fsi.2019.09.032\u003c/li\u003e\n\u003cli\u003eRama Devi P, Sudhakar GL, Vasudehaven I (2012) Natural agglutinin from crab, Trichopeltarian nobile. Int J Biol Res 5:14\u0026ndash;17\u003c/li\u003e\n\u003cli\u003eRenwrantz L (1986) Lectins in molluscs and arthropods: Their occurrence, origin and roles in immunity. Dev Comp Immunol (Incomplete journal details\u0026mdash;please confirm)\u003c/li\u003e\n\u003cli\u003eRittidach W, Paijit N, Utarabhand P (2007) Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph. Biochim Biophys Acta 1770:106\u0026ndash;114. https://doi.org/10.1016/j.bbagen.2006.06.016\u003c/li\u003e\n\u003cli\u003eSharon N, Lis H (2004) History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14:53R\u0026ndash;62R. https://doi.org/10.1093/glycob/cwh122\u003c/li\u003e\n\u003cli\u003eSivakamavalli J, Vaseeharan B (2014) Purification, characterization and functional role of lectin from green tiger shrimp Penaeus semisulcatus. Int J Biol Macromol 67:64\u0026ndash;70. https://doi.org/10.1016/j.ijbiomac.2014.03.008\u003c/li\u003e\n\u003cli\u003eSivakamavalli J, Nirosha R, Vaseeharan B (2015) Purification and characterization of a cysteine-rich 14-kDa antibacterial peptide from the granular hemocytes of mangrove crab Episesarma tetragonum and its antibiofilm activity. Appl Biochem Biotechnol 176:1084\u0026ndash;1101. https://doi.org/10.1007/s12010-015-1631-1\u003c/li\u003e\n\u003cli\u003eS\u0026ouml;derh\u0026auml;ll K, Smith VJ (1983) Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution. Dev Comp Immunol 7:229\u0026ndash;239. https://doi.org/10.1016/0145-305x(83)90004-6\u003c/li\u003e\n\u003cli\u003eSun J, Wang L, Wang B, Guo Z, Liu M, Jiang K, Zhang G (2008) Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis. Fish Shellfish Immunol 25:290\u0026ndash;297. https://doi.org/10.1016/j.fsi.2008.06.001\u003c/li\u003e\n\u003cli\u003eSun YD, Fu LD, Jia YP, Du XJ, Wang Q, Wang YH, Wang JX (2008) A hepatopancreas-specific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity. Mol Immunol 45:348\u0026ndash;361. https://doi.org/10.1016/j.molimm.2007.06.355\u003c/li\u003e\n\u003cli\u003eSminia T, Borghart-Reinders E, Van AW (1974) Encapsulation of foreign materials experimentally introduced into the freshwater snail Lymnaea stagnalis. Cell Tissue Res 153:307\u0026ndash;326. https://doi.org/10.1007/BF00229161\u003c/li\u003e\n\u003cli\u003eTunkijjanukij S, Olafsen JA (1998) Sialic acid-binding lectin with antibacterial activity from the horse mussel: further characterization and immunolocalization. Dev Comp Immunol 22:139\u0026ndash;150. https://doi.org/10.1016/s0145-305x(98)00017-2\u003c/li\u003e\n\u003cli\u003eUemura K, Saka M, Nakagawa T, Kawasaki N, Thiel S, Jensenius JC, Kawasaki T (2002) L-MBP is expressed in epithelial cells of mouse small intestine. J Immunol 169:6945\u0026ndash;6950. https://doi.org/10.4049/jimmunol.169.12.6945\u003c/li\u003e\n\u003cli\u003eVasta GR (1992) Invertebrate lectins: distribution, synthesis, molecular biology, and function. In: Montreuil J, Vliegenthart JFG, Schachter H (eds) Glycoconjugates: Composition, Structure and Function. Marcel Dekker, New York, 593\u0026ndash;634\u003c/li\u003e\n\u003cli\u003eVasta GR, Quesenberry M, Ahmed H, O\u0026rsquo;Leary N (1999) C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway. Dev Comp Immunol 23:401\u0026ndash;420. https://doi.org/10.1016/s0145-305x(99)00020-8\u003c/li\u003e\n\u003cli\u003eViswambari Devi R, Basilrose MR, Mercy PD (2010) Prospect for lectins in arthropods. Ital J Zool 77:254\u0026ndash;260. https://doi.org/10.1080/11250003.2012.671856\u003c/li\u003e\n\u003cli\u003eWang XW, Wang JX (2013) Diversity and multiple functions of lectins in shrimp immunity. Dev Comp Immunol 39:27\u0026ndash;38. https://doi.org/10.1016/j.dci.2012.04.009\u003c/li\u003e\n\u003cli\u003eWilson R, Chen C, Ratcliffe NA (1999) Innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis. J Immunol 162:1590\u0026ndash;1596\u003c/li\u003e\n\u003cli\u003eYang H, Luo T, Li F, Li S, Xu X (2007) Purification and characterisation of a calcium-independent lectin (PjLec) from the haemolymph of the shrimp Penaeus japonicus. Fish Shellfish Immunol 22:88\u0026ndash;97. https://doi.org/10.1016/j.fsi.2006.03.015\u003c/li\u003e\n\u003cli\u003eYebra DM, Kiil S, Dam-Johansen K (2004) Antifouling technology\u0026mdash;past, present and future steps towards efficient and environmentally friendly antifouling coatings. Prog Org Coat 50:75\u0026ndash;104. https://doi.org/10.1016/j.porgcoat.2003.06.001\u003c/li\u003e\n\u003cli\u003eZenteno R, Vazquez L, Sierra C, Pereyra A, Slomianny MC, Bouquelet S, Zenteno E (2000) Chemical characterization of the lectin from the freshwater prawn Macrobrachium rosenbergii (De Man) by MALDI-TOF. Comp Biochem Physiol B 127:243\u0026ndash;250. https://doi.org/10.1016/s0305-0491(00)00260-1\u003c/li\u003e\n\u003cli\u003eVargila F, Mettilda Bai SM, Josephine Mary JV, Citarasu T (2024) Isolation, characterization and antimicrobial properties of hepatopancreas lectin of the freshwater crab Oziotelphusa naga. Protein Expr Purif 222:106536. https://doi.org/10.1016/j.pep.2024.106536\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e The minimal inhibitory concentration corresponds to the lowest carbohydrate concentration able to neutralize the hemagglutinating activity of Ss-Lec. The values are expressed in millimolars, and the highest carbohydrate concentration used was 400 mM. N-acetyl glucosamine, D-mannose, and D- and L- fucose could inhibit haemagglutination at a very low concentration.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eSugar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eMaximum Concentration of sugar (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003eMinimum inhibitory Concentration of sugar (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eD- glucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eD-galactosamine HCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eD-maltose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e0.781\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eN-acetyl D-glucosamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e0.390\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eD-fucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e0.390\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eLactose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eD-Mannose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e0.195\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eD-fructose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eL-fucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e0.390\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003eSucrose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Antibacterial activity of purified Ss-Lec. The zone of inhibition values are expressed as \u0026lsquo; - =No zone of inhibition; + = \u0026le; 5mm ; ++ = \u0026le; 7 mm; +++ = \u0026ge; 7mm\u0026rsquo;.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"593\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePathogen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTBS buffer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSs-Lec\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e25\u0026micro;g/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSs-Lec\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e50\u0026micro;g/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSs-Lec\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e100\u0026micro;g/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus agalactiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus iniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eAeromonas veronii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eEdwardsiella tarda\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"C-type Lectin, Scylla serrata, phenoloxidase (PO) activation, haemagglutination, antibiofilm activity","lastPublishedDoi":"10.21203/rs.3.rs-7249543/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7249543/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMarine lectins are unique glycoproteins that induce non-specific immunity in fishes and crustaceans. Mud crab \u003cem\u003eScylla serrata\u003c/em\u003e is highly abundant globally and rare, especially in Southeast Asian countries for its economic value. However, the characterisation of the c-type specific lectins is still not explored properly. In this current study, a novel c-type mannose-binding lectin was isolated from the marine crab \u003cem\u003eScylla serrata\u003c/em\u003e haemolymph using affinity chromatography. The purified lectin (Ss-Lec) of a 71kDa showed haemagglutination activity at the lowest concentration, and its homogenous and crystalline nature was evaluated by the HPLC, XRD, FTIR, and MALDI-TOF analysis. Moreover, the Ss-Lec depends on optimum pH, temperature, and calcium chelators. for its improved bioactivity. The purified Ss-Lec showed extensive antimicrobial properties against the important pathogenic aquatic bacteria \u003cem\u003eStreptococcus iniae, Streptococcus agalactiae, Edwardsiella tarda, \u003c/em\u003eand,\u003cem\u003e Aeromonas veronii \u003c/em\u003eat a concentration of 25-100µg/ml. This highly potential lectin can be further utilized against pathogenic diseases in aquaculture.\u003c/p\u003e","manuscriptTitle":"Isolation, Characterization, and biological significance of a novel C-type marine lectin purified from Scylla serrata haemolymph","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 13:51:39","doi":"10.21203/rs.3.rs-7249543/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-28T23:16:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-27T06:28:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182311194983788887606407991093516608006","date":"2025-09-05T08:07:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-30T22:10:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-11T05:52:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-11T05:51:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biotechnology","date":"2025-07-30T07:01:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d43115b2-11d9-4d2e-97da-834f061744a8","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-31T13:51:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 13:51:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7249543","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7249543","identity":"rs-7249543","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}