CGL, a Lectin from Crenomytilus grayanus, Exhibits Antibiofilm and Synergistic Antibacterial Activity Against Escherichia coli and Staphylococcus aureus

Abstract

Lectins are carbohydrate-binding proteins that specifically bind to sugar groups associated with other molecules. Several studies have reported that these proteins can also modulate the activity of antibiotics against multidrug-resistant (MDR) strains in addition to interacting with carbohydrates. This study reports that gentamicin exhibits enhanced antibacterial activity against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacterial strains when complexed with Crenomytilus grayanus lectin (CGL). Enzyme-linked lectin, thermofluor, and isothermal titration calorimetry assays revealed that gentamicin interacts with CGL through a domain distinct from the carbohydrate recognition domain. An increase in antibacterial activity was observed when lectin and antibiotic were used together against S. aureus in living systems—specifically, sea urchin (Strongylocentrotus nudus) embryos.

1. Introduction

Due to the rising prevalence of antimicrobial-resistant infections and the slow pace of discovering and developing new antibacterial agents, there has been growing interest in developing alternative antibacterial therapy approaches. One promising alternative is combining antibiotics with lectins. Lectins are a versatile group of proteins that recognize carbohydrates on cell surfaces and play essential roles in biological processes such as cell recognition, adhesion, and immune responses [1]. This combination therapy has several advantages, including a broader spectrum of action, a lower probability of developing resistance, an additive or synergistic effect, reduced side effects, and the ability to overcome drug resistance [2]. Their potential as antibacterial and antibiofilm agents has received increasing attention, especially in light of the urgent need for alternative therapeutic strategies to combat antibiotic-resistant pathogens [3,4].

Despite the extensive research into plant lectins spanning more than a century and a half, recent discoveries have revealed a remarkable diversity of lectins in marine mollusks, each exhibiting unique biological activities. These fascinating proteins participate in complex host-pathogen relationships by acting as key mediators in cellular communication processes. They function as pattern recognition receptors, facilitate cell adhesion, and regulate immune responses within the organism [5].

The intricate molecular mechanisms underlying these interactions hold great promise for advancing medical science. By deciphering how these proteins operate at the molecular level, researchers can pave the way for innovative carbohydrate-based therapies and targeted drug delivery systems capable of combating a wide range of diseases. Among marine mollusks, mussels and oysters have emerged as particularly rich sources of these valuable lectins, making them focal points for ongoing scientific investigation [6].

In our previous study, we identified a novel lectin, CGL, from the sea mussel Crenomytilus grayanus. We established its structure, basic physicochemical properties, and biological activity [7,8]. CGL was found to be a member of the new “mytilectin family” of bivalve mollusks [9]. It is capable of binding galactose (Gal), N-acetylgalactosamine (GalNAc), talose, and larger glycans containing these residues [10]. In addition to possessing antiproliferative [11,12], antibacterial [8], and antifungal [13] properties, the CGL induces macrophages and mononuclear cells in mice to express TNF-α and IL-6, thereby modulating the immune response [14].

Because CGL has previously been reported to exhibit antibacterial and antifungal activities that may be related to killing bacterial pathogens and inhibiting fungal growth in marine mussels, determining its antibiofilm activity is of interest. In addition to these properties, the present study evaluated CGL capacity to modulate antibiotic activity against standard S. aureus (Gram-positive) and E. coli (Gram-negative) reference strains. The study confirmed the effectiveness of CGL when used with the traditional antibiotic gentamicin.

2. Results

2.1. Antibacterial Activity of CGL

The antibacterial activity of CGL was evaluated using the minimum inhibitory concentration (MIC) method. Following the turbidity method yielded similar MIC50 values of 250 µg/mL, indicating its potential as an antibiotic. The MIC50 of gentamicin were significantly lower than those of lectin: 1.05 µg/mL for S. aureus and 2.09 µg/mL for E. coli.

The MIC50 was calculated to determine the combined effect of the CGL and antibiotic on the bacterial cells. The combination of CGL and gentamicin had variable effects depending on the bacteria. A synergistic effect was observed for the CGL-gentamicin combination against S. aureus, while an additive effect was observed against E. coli (Table 1). Combining gentamicin with CGL enhanced the antibiotic activity against S. aureus by decreasing the MIC50 value by a factor of 2.8. These results underscore the context- and bacterium-specific nature of CGL’s antibacterial potential, particularly its promising activity against S. aureus.

Table 1.

Effect of CGL combined with antibiotic on S. aureus and E. coli strains.

Bacterium	Antibiotic		Lectin		ΣFIC 5	Effect 6
	Gentamicin		CGL
	MIC50 1 µg/mL	MIC50 2 µg/mL	MIC50 3 µg/mL	MIC50 4 µg/mL
S. aureus	1.05	0.38	250	31.5	0.48	S
E. coli	2.09	1.55	250	60	0.98	AD

¹ MIC50 value of gentamicin alone; ² MIC value of gentamicin in combination with CGL; ³ MIC value of CGL alone; ⁴ MIC value of CGL in combination with gentamicin; ⁵ ΣFIC = (MIC50 of CGL in combination/MIC50 of CGL alone) + (MIC50 of antibiotic in combination/MIC50 of antibiotic alone). The combinations were classified as synergistic (ΣFIC ≤ 0.5), additive (ΣFIC > 0.5–1.0), indifferent (ΣFIC > 1.0–≤4.0), or antagonistic (ΣFIC > 4.0); ⁶ S, synergistic; AD, additive.

In order to clarify whether CGL would be acting by damaging bacterial structure, a series of experiments were conducted.

The antimicrobial effect of the lectin was further examined using the agar diffusion method. No visible zone of inhibition was observed when 125, 250, 500 or 1000 μg/mL of lectin, or 12.5 or 25 μg/mL of gentamicin, were tested against S. aureus or E. coli. Consequently, the highest concentration of 1000 μg/mL (200 μg per well) was selected for experiments assessing synergy with the antibiotic. The concentration of gentamicin that was found to be appropriate was 50 μg/mL. Visible inhibition zones were observed solely under the combined action of lectin and antibiotic. For S. aureus and E. coli, these zones measured 24.6 mm and 19.4 mm, respectively, compared to 22.4 mm and 17.3 mm for gentamicin alone. It is hypothesized that CGL does not directly kill bacteria; rather, it may inhibit their proliferation by causing agglutination, which in turn blocks nutrient uptake and further cell division (Figure S1).

This assumption is supported by experimental evidence obtained from a study designed to ascertain the leakage of proteins. The amount of protein released from bacterial cells after 24 h of lectin incubation did not increase from the initial (time-zero) level. This finding lends further support to the hypothesis that CGL exerts an inhibitory effect on bacterial growth without causing structural damage to cell membranes. Such damage would undoubtedly be accompanied by protein leakage from bacterial cells.

2.2. Antibiofilm Activity of CGL

We tested the antibiofilm activity of CGL on E. coli and S. aureus bacteria. Biofilm formation was quantified spectrophotometrically using a microtiter plate assay with crystal violet dye. CGL significantly inhibited S. aureus biofilm formation at all tested concentrations. Biofilm biomass decreased across the entire concentration range, reaching 55% at the maximum lectin concentration compared to the control. Lectin had a smaller impact on the biofilm formed by E. coli, reducing its biomass by only 34% (Figure 1A).

Figure 1.

The effect of CGL on (A) the formation of biofilms and (B) the destruction of pre-formed bacterial biofilms. Data are represented as the means ± SD as determined from triplicate experiments. Student’s t-test was used to evaluate the data with the significance level * p ≤ 0.05.

The inhibition of biofilm formation by CGL was observed using fluorescence. In the presence of lectin, a visible decrease in biofilm density was observed for both S. aureus and E. coli. Bacteria within lectin-exposed biofilms stained positively with acridine orange, but negatively with propidium iodide, indicating that lectin does not induce bacterial cell death (Figure S2).

CGL destroyed existing biofilms. At its maximum concentration, the lectin destroyed 58% of S. aureus and 28% of E. coli biofilms (Figure 1B).

2.3. CGL Interaction with Ligands

Enzyme-linked lectin assay (ELLA) was used to obtain quantitative data on the sugar binding and specificity of a CGL. The results showed that PSM, a glycoprotein densely glycosylated with galactose-rich oligosaccharides, was the most potent inhibitor with an MIC of 0.014 mg/mL. The MIC for Gal was 1.4 mg/mL, and Glc (glucose), as non-specific ligand, had no inhibitory effect. Additionally, no significant affinity for antibiotics was observed.

A thermofluor assay was used to screen and characterize the ligand-binding mechanism of CGL since the protein is stabilized against thermal denaturation when interacting with certain ligands. We assessed the interaction of lectin with ligands resulting in a change in melting temperature (ΔTm) in the presence of Gal, Glc, and the aminoglycoside antibiotic gentamicin. As expected, ΔTm in the presence of Gal increased by 10.38 ± 0.9 °C, which could selectively identify galactoside compounds as CGL ligands compared to non-galactoside carbohydrates, such as Glc (0.35 ± 0.14 °C), and gentamicin (−4.43 ± 0.2 °C) (Figure 2).

Figure 2.

Evaluation of CGL-ligands binding using thermofluor assay. The difference ΔTm of CGL in the presence of 5 μL Gal, Glc and gentamicin.

The thermogram (Figure 3A) and the binding curve (Figure 3B) showed a specific interaction that could be fitted to an independent model. The negative enthalpy change (ΔH = −16.27) implied that the Gal-lectin interaction was exothermic.

Figure 3.

Isothermal titration calorimetry of CGL: (A) thermogram of CGL titrated with Gal; (B) binding isotherm and model fitting for CGL-Gal. Experiments were performed at 25 °C using a Nano Isothermal Titration Calorimeter, and data were fitted to an independent binding model.

The ITC isotherm of gentamicin with CGL showed that the heat released increased with each gentamicin injection. The Kd was determined to be 1.0 × 10⁻³ M, reflecting a lower affinity compared to Gal (Table 2). The thermogram (Figure 4A) and the binding curve (Figure 4B) showed a specific interaction that could be fitted to an independent model.

Table 2.

Thermodynamic binding parameters for CGL with ligands at 25 °C.

Ligand	Kd (M)	n	ΔH (kJ/mol)
Gal	1.796 × 10−4	2.731	−16.27
Gentamicin	1 × 10−3	4.307	100

Figure 4.

Isothermal titration calorimetry of CGL: (A) thermogram of CGL titrated with gentamicin; (B) binding isotherm and model fitting for CGL-gentamicin. Experiments were performed at 25 °C using a Nano Isothermal Titration Calorimeter, and data were fitted to an independent binding model.

Additionally, the positive enthalpy change (ΔH = 100) indicates that the gentamicin-lectin interaction is endothermic, whereas the Gal-lectin interaction is exothermic. The number of binding sites on the lectin (n), or the number of functional valences available to bind with the antibiotic, was approximately 4.0. No detectable changes were observed during the titration of Glc.

2.4. Inhibition of S. aureus Infection by Lectin in Sea Urchin Embryos

We used fertilized eggs from sea urchin Strongylocentrotus nudus to evaluate whether CGL alone or in combination with gentamicin can reduce S. aureus infection in vivo. S. nudus embryos developed normally in the absence of S. aureus. The development of most embryos was delayed by more than 50% or they died after being infected. However, when S. aureus was added with lectin, only 28.6% of the embryos developed abnormally, compared to 94.9% of the control embryos. The same result was obtained when gentamicin was added to embryos infected with bacteria. Embryos treated with both lectin and gentamicin continued to develop normally, with only about 18.6% showing abnormalities. Interestingly, adding either lectin or gentamicin alone to the embryos had no significant effect on their development compared to the control group (Figure 5). E. coli was not used in this experiment, since a synergistic effect was only observed for the CGL-gentamicin combination against S. aureus.

Figure 5.

Inhibitory activity substances on S. aureus infection of S. nudus embryos. S. nudus embryos were infected with S. aureus in the presence or absence of CGL, gentamicin, or a combination of the two. Data are represented as the means ± SD as determined from triplicate experiments. Student’s t-test was used to evaluate the data with the significance level * p ≤ 0.05.

3. Discussion

The binding specificity of lectins is crucial for their biological functions. They participate as mediators in the protein-carbohydrate recognition stage of various biological processes, including cellular communication, host defense, fertilization, cellular development, and parasitic infection [1]. The antibacterial activity of lectins on Gram-positive and Gram-negative bacteria occurs through interaction with components of the bacterial cell wall, such as teichoic and teichuronic acids, peptidoglycans, and lipopolysaccharides [15].

The differential antibiofilm activity of CGL against S. aureus and E. coli may be attributed to structural differences in their cell walls and biofilm matrices. S. aureus possesses a thick peptidoglycan layer with exposed teichoic acids containing Gal or GalNAc, rich in carbohydrate motifs that are likely recognized by CGL [8]. In contrast, E. coli has an outer membrane containing lipopolysaccharides (LPS) with core oligosaccharides that may be less accessible or exhibit lower affinity for CGL. Furthermore, the composition and density of extracellular polymeric substances in biofilms vary between these species, which could influence lectin binding and biofilm disruption efficiency.

The finding that CGL is capable of significantly reducing biofilm biomass and of inhibiting planktonic growth suggests interference with processes closely linked to the cell wall architecture of Gram-positive bacteria, such as early adhesion or extracellular matrix formation. This behavior was particularly evident in S. aureus, where CGL treatment resulted in a significant reduction in biofilm mass. These results lend support to the hypothesis that CGL plays a role in the recognition of conserved bacterial structures involved in pathogenicity and biofilm formation.

Only a few marine invertebrate lectins have been reported to have similar biofilm-inhibiting properties. These include sponge-derived lectins such as AfiL, ALL, AFL, AcrL, and HOL-18 [3,16,17,18]; OXYL, which is derived from a feather star [19]; and MytiLec-1, which is the only lectin derived from a bivalve [9]. MytiLec-1 inhibited the formation of biofilms by Pseudomonas aeruginosa bacteria at a concentration of 200 μg/mL. Compared to the control, there was a 15% reduction in biofilm production. The antibiofilm activity of other members of the Mytilectin family (MTL and MCL) has not yet been studied.

Based on the biofilm inhibition and destruction activity of CGL, we hypothesized that the lectin has a synergistic effect with antibiotics against S. aureus and E. coli. The observed synergy between CGL and the conventional antibiotic gentamicin further underscores CGL potential biomedical applications. CGL enhanced gentamicin efficacy against E. coli and demonstrated a synergistic effect against S. aureus (Table 1). These results support the potential of marine-derived lectins as selective antibiotic adjuvants. These proteins may enhance the efficacy of conventional antibiotics in a strain-specific manner, potentially reducing required dosages and delaying the onset of resistance in targeted clinical settings.

Most studies investigating the synergistic interaction between lectins and antibiotics have focused on plant lectins (Table 3).

Table 3.

Comparative overview of key lectins with synergistic antibacterial activity.

Source	Lectin	Bacterium	Antibiotic	MIC50 Reduction (Fold) 1	ΣFIC 2	Effect 3	References
Sea mussel Crenomytilus grayanus	CGL	S. aureus	Gentamicin	2.8	0.48	S	Current research
		E. coli	Gentamicin	1.34	0.98	AD
Inflorescence bracts of Alpinia purpurata (Viell.)	ApuL	S. aureus	Oxacillin	1	1	AD	[20]
		Pseudomonas aeruginosa	Ceftazidime	1	1	AD
Plant Myracrodruon urundeuva	MuBL	S. aureus	Cefoxitin	2	0.7	AD	[21]
			Cefotaxime	10	0.3	S
	MuHL	S. aureus	Cefoxitin	10	0.2	S
			Cefotaxime	10	0.1	S
	MuLL	S. aureus	Cefoxitin	5	0.2	S
			Cefotaxime	2	0.5	S
Calliandra surinamensis leaves	CasuL	S. aureus	Tetracycline	4	0.250	S	[22]
			Ampicillin	0.5	2	I
Canavalia ensiformis seeds	ConA	S. aureus	Gentamicin	5	NC	-	[23]
		E coli	Gentamicin	1.6	NC	-
Vatairea macrocarpa seeds	VML	S. aureus	Gentamicin	1.3	NC	-
			Norfloxacin	5	NC	-
			Penicillin	1.2	NC	-	[24]
		E. coli	Gentamicin	1	NC	-
			Norfloxacin	0.36	NC	-
			Penicillin	1	NC	-

¹ Reduction in MIC value of a lectin in combination with an antibiotic; ² ΣFIC = (MIC50 of lectin in combination/MIC50 of lectin alone) + (MIC50 of antibiotic in combination/MIC50 of antibiotic alone). The combinations were classified as synergistic (ΣFIC ≤ 0.5), additive (ΣFIC > 0.5–1.0), indifferent (ΣFIC > 1.0–≤4.0), or antagonistic (ΣFIC > 4.0); NC, not calculated; ³ S, synergistic; AD, additive; I, indifferent.

Several studies have examined lectins from marine sponges [3,16,25] and the hemolymph of the horseshoe crab, Tachypleus tridentatus [26]. The lectins demonstrated significant antibiofilm activity and exhibited synergistic or additive effects when combined with conventional antibiotics.

The mechanism by which lectins synergize with antibiotics is not fully understood. One theory suggests that antibiotics bind to lectins through their CRD. Santos et al. [24] demonstrated that combining VML with the antibacterial drugs gentamicin, norfloxacin, and penicillin significantly increased antibiotic activity against S. aureus. Gentamicin inhibition of hemagglutinating activity revealed its interaction with the CRD of VML (MIC = 50 mM). However, norfloxacin, penicillin, and mannose (the negative control) could not inhibit hemagglutinating activity and thus could not interact with the VML CRD. Aminoglycoside antibiotics (AGAs), such as gentamicin, are carbohydrate mimetics with a general structural motif consisting of an inositol derivative linked to at least one amino sugar [27]. Given the sugar-based structure of AGAs, it was expected that these molecules could compete with carbohydrates for CRD binding, as was observed with gentamicin binding to VML. Lectins may interact with antibiotics within the internal cavity of dimeric or tetrameric structures without affecting the CRD. Interactions at the hydrophobic sites of lectins may explain the increased activity of antibiotics that do not exhibit hemagglutinating activity and do not bind to CRD. These interactions at hydrophobic sites may possibly explain the increased antibiotic activity of norfloxacin and penicillin in the presence of VML [24]. Gentamicin was found to not inhibit the binding activity of CGL, unlike Gal, a specific lectin monosaccharide. It is likely that CGL does not interact with gentamicin through the CRD. It is known that Mytilectins have a strong tendency to oligomerize [10]. Therefore, the formation of such hydrophobic sites in the oligomeric form of CGL is quite possible. The ability of lectins to interact with bacterial cell wall polysaccharides or LPS likely facilitates the transport of antibiotics across bacterial membranes. Alternatively, after binding to a CRD on the lectin, the antibiotic passes through the biofilm layer due to the lectin targeted action and attacks the bacterial cell wall directly. Thus, a significant increase in antibiotic activity is achieved [23,24]. However, elucidating the 3D-structure of these lectins in complex with gentamicin is necessary for a better understanding of the molecular basis of these interactions.

To confirm the absence of interaction between the CRD of the lectin and the antibiotics observed in the inhibition assay, the interaction between gentamicin and CGL was analyzed using a thermofluor assay. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon. Upon ligand binding, the protein complex denaturates at a higher temperature and the difference in the Tm value in the presence and absence of the compound reflects ligand binding. Thus, the thermal shift assay can serve as a tool to seek for stabilizing reagents, and to identify natural ligands that provide insight into the biological function of the protein. As expected, the increase in ΔTm in the presence of Gal was able to selectively identify galactoside compounds as CGL ligands compared to non-galactoside carbohydrates such as Glc and gentamicin. These data correlate well with those obtained using other biophysical methods. Serendipitously, BJcuL, a snake venom Gal-binding lectin isolated from Bothrops jararacussu, was identified as a ligand of gentamicin. BJcuL increased protein stability in the thermofluor assay despite having no inhibitory effect in the hemagglutination method [28].

ITC was used to better understand the nature of the lectin-antibiotic interaction. ITC measures the heat absorbed or released during biomolecular interactions. A series of titrations is performed, during which a binding ligand (titrant) is added in aliquots to a sample cell containing its binding partner, all at a constant temperature. The heat released or absorbed is measured against a reference cell containing only the medium. This allows one to derive the complete thermodynamic parameters of the binding event, including enthalpy (ΔH), entropy (ΔS), the binding constant, and stoichiometry (n) [29]. ITC data revealed that CGL has a higher affinity for Gal than for gentamicin. Additionally, positive enthalpy changes implied that the gentamicin-CGL interaction was endothermic, whereas the Gal-CGL interaction was exothermic. The number of binding sites on the lectin, or the number of functional valences available to bind with the antibiotic, was greater than the number of CRDs per CGL subunit. Therefore, it can be assumed that gentamicin interacts with amino acid residues of the protein that do not belong to the CRD, which confirms the results of previous methods.

The synergistic effect of lectin and gentamicin on bacterial cells, which have an opposite effect on CGL stability (Gal stabilizes and gentamicin destabilizes), can be explained by independent, yet complementary, pathways of bacterial cell damage. Binding to the natural ligand, galactose, stabilizes CGL in its functional conformation. This increases the lifetime of the protein active form and its binding efficiency to glycoconjugates on bacterial cell surfaces. Gentamicin indirectly facilitates CGL action by reducing the production of extracellular polymers and the metabolism and virulence of bacteria. Gentamicin acts directly on the bacterial cell, impairing protein synthesis, causing ribosomal dysfunction, and inducing a stress state. Although gentamicin destabilizes the globular structure of CGL (leftward shift in Tm and endothermicity in ITC), the functionally crucial CRD may still be capable of binding Gal and matrix glycans, particularly at moderate concentrations and temperatures.

In recent years, the sea urchin has become an important model organism for studying many diseases, including pathogen infections. Sea urchin embryogenesis is an excellent model for studying the effects of natural products or engineered drugs, temperature, and other factors on cell differentiation in vivo because it can be observed over a short period of time [30,31]. For instance, the embryotoxic activity of plant-derived lectins on the embryos of the sea urchin Lytechinus variegatus was examined. IC₅₀ doses for toxicity on larval development ranged from 0.6 to 96.3 μg/mL [32]. In the present study, at a subinhibitory concentration of 250 μg/mL, CGL demonstrated no effect on S. nudus embryos. However, the combined action of the lectin and antibiotic appeared to offer protection to the embryos against bacterial infection.

Thus, this study provides the experimental in vivo demonstration of synergy between a lectin and an antibiotic. In vivo studies focusing specifically on lectins remain limited, with more research being conducted on antimicrobial peptides or plant phenolics [33]. Further research is needed to substantiate conclusions and formulate evidence-based recommendations for using natural compounds in aquaculture to reduce antibiotic dosages.

4. Materials and Methods

4.1. Materials

CGL was isolated as described previously [6]. Gram-positive Staphylococcus aureus KMM 434 and Gram-negative Escherichia coli KMM 8450 bacteria were obtained from the Collection of Marine Microorganisms (KMM) of G.B. Elyakov Pacific Institute of Bioorganic Chemistry (Vladivostok, Russia). Tryptic soy agar (TSA) was from HIMEDIA (Mumbai, India); tryptic soy broth (TSB) was from Oxoid (Basingstoke, UK); crystal violet was purchased from Servicebio (Wuhan, China); formaldehyde solution was purchased from Thermo Fisher Scientific (Waltham, MA, USA); gentamicin, mucin from porcine stomach, type III (PSM), bovine serum albumin (BSA), 3,3′,5,5′-tetramethylbenzidine (TMB) were from Sigma-Aldrich (St Louis, MO, USA); SYPRO orange was from Lumiprobe Corporation (Westminster, MD, USA); acridine orange, D-galactose and D-glucose were from Sigma-Aldrich (St Louis, MO, USA).

4.2. Preparation and Standardization of the Inoculum

Bacterial strains were initially cultured in TSA at 37 °C for 24 h. Isolated colonies were then transferred TSB and incubated for an additional 24 h under the same conditions, with stirring at 120 RPM. After cultivation, the bacteria were placed in test tubes with 5 mL of sterile PBS.

4.3. Minimum Inhibitory Concentration (MIC) Test

MIC was determined by assays of microdilution in 96-well microplates. CGL and gentamicin solutions in sterile PBS were serially diluted at 50 µL per well. The bacterial culture density was adjusted turbidimetrically to 2 × 10⁶ CFU/mL in sterile PBS at 600 nm (OD600), after which 10% TSA was added. Subsequently, 50 µL of bacterial inoculum were added to each well of the plate, and the cells were incubated for 24 h. The MIC50 was defined as the lowest CGL concentration capable of inducing a reduction of more than 50% in OD600 compared to the 100% growth control. The measurement was conducted using a microplate spectrophotometer (Synergy H1, BioTek, Winooski, VT, USA) at 0 and 24 h. Additionally, the MIC50 value for gentamicin was determined.

4.4. Synergy Assay

To evaluate the effect of CGL in combination with antibiotic, we followed [34]. 100 μL of inoculum containing 10% TSA at a final concentration of 1 × 10⁶ CFU/mL and 50 μL of CGL with a subinhibitory concentration were added to the wells of the plate to which 50 μL of the antibiotic had been previously added by microdilution. The plates were then incubated at 37 °C for 24 h and the results were read at 600 nm. Controls were prepared with only 100 μL of inoculum with 10% TSA at a concentration of 1 × 10⁶ CFU/mL + 100 μL sterile PBS.

The evaluation of the interaction between the different treatments was performed by determining the sum of fractional inhibitory concentration index (ΣFIC), as follows: ΣFIC = (MIC50 of CGL in combination/MIC50 of CGL alone) + (MIC50 of antibiotic in combination/MIC50 of antibiotic alone). The combinations were classified as synergistic (ΣFIC ≤ 0.5), additive (ΣFIC > 0.5–1.0), indifferent (ΣFIC > 1.0–≤4.0), or antagonistic (ΣFIC > 4.0).

4.5. Antibiofilm Activity

A bacterial suspension with a final concentration of 1 × 10⁸ CFU/mL (50 μL) was mixed with an equal volume of CGL in 96-well microtiter plates at various concentrations (0–500 μg/mL). The plates were then incubated for 24 h at 37 °C to allow the formation of biofilms. After the incubation period, the wells were washed with 200 μL of PBS to remove any free-floating bacteria. The biofilms formed in the wells were stained with 0.1% crystal violet for 15 min and then washed with PBS to remove the excess dye. Then, the samples were treated with 7% acetic acid to release the dissolved dye. The absorbance values of each well were measured by an automated microtiter plate reader at 570 nm. The inhibition of biofilm formation resulting from lectin treatment was calculated relative to the control samples as follows: % biofilm formation = (OD570 experiment/OD570 control) × 100%.

To determine the ability of lectin to destroy the structure of an already formed biofilm, 100 μL of bacterial suspension at a final concentration of 1.0 × 10⁸ CFU/mL per well and 100 μL of sterile PBS were added to 96-well plates to form a biofilm. The plate was then incubated at 37 °C for 24 h under static conditions. After incubation, the biofilms in the wells were washed three times with 200 μL of sterile PBS to remove any remaining planktonic cells. Then, 200 μL of lectin solutions at different final concentrations (0–500 μg/mL), were added to the plate. After incubating at 37 °C for 2 h, the wells were washed three times with 200 μL sterile PBS. Staining and measurement were carried out as described above.

4.6. Fluorescence Microscopy

Biofilm formation was performed as described above. The formed biofilms were washed and a dye solution in PBS was added at working concentrations of 5 μg/mL for acridine orange and 3 μg/mL for propidium iodide. The cells were incubated with the dye in darkness for 15 min. Microscopic examination using a fluorescence microscope (MIB-2-FL, LOMO, St. Petersburg, Russia) was performed as soon as possible after incubation.

4.7. Evaluation of Protein Leakage

The leakage of proteins from microbial cells was evaluated according to [20]. The CGL concentration was adjusted to the MIC50 value for the respective bacteria in a total volume of 200 µL, corresponding to 100 µL of medium containing 0.9% saline and the lectin, and 100 µL of inoculum (10⁶ CFU/mL). For negative controls, the lectin was replaced with 0.9% saline. The assays were maintained in a shaking incubator at 37 °C for 0 and 24 h. After this period, the samples were centrifuged at 300× g for 10 min at 25 °C, and the protein concentration of the resulting supernatant was evaluated using the Bradford method [35]. The amount of leaked protein was calculated by subtracting the protein content at time zero from the content at the end of the assay. The assays were performed in triplicate.

4.8. Zone of Inhibition (ZI) Assay

The antibacterial activity was verified using a ZI assay with S. aureus and E. coli bacterial strains. Once the agar had solidified in the plate, 8 mm diameter wells were made in the solidified agar and 100 μL of culture (0.5 OD at 600 nm) was spread on the plate. The wells were then filled with either 200 μL of CGL at concentrations of 125, 250, 500 or 1000 μg/mL, or 200 μL of gentamicin at concentrations of 12.5, 25 or 50 μg/mL as a positive control. All experiments were performed in triplicate. The plates were then incubated for 24 h at 28 °C. The ZI around the wells that were free of visible bacterial colonies was then measured.

A synergy assay was performed to evaluate the effect of CGL in combination with the antibiotic. Nutrient agar plates with wells were prepared as described above. The wells were then filled with 200 μL of CGL (at a concentration of 1000 μg/mL), 200 μL of gentamicin (at a concentration of 50 μg/mL) as a positive control, or 200 μL of CGL (at a final concentration of 1000 μg/mL) with gentamicin (at a final concentration of 50 μg/mL). A negative control (200 μL of 0.9% saline) was also included on the same plate. All experiments were performed in triplicate. The plates were then incubated for 24 h at 28 °C. The ZI around the wells that were free of visible bacterial colonies was measured.

4.9. Enzyme-Linked Lectin Assay (ELLA)

ELLA was performed to analyze the inhibition of CGL binding to PSM, a glycoprotein to which the lectin exhibits the greatest activity, and various ligands. The wells of a microtiter plate (Nunc, Roskilde, Denmark) were coated with 100 μL of PSM (5 μg/mL) in PBS and incubated overnight at 4 °C. The plates were emptied and rinsed five times with 0.05% Tween-20 in PBS. Then, the wells were blocked with 250 μL of 1% BSA in PBS for 2 h at room temperature. After washing, 50 μL of twofold serial dilutions of ligands at a concentration of 0.5 mg/mL of PSM, D-galactose (positive control), D-glucose (negative control), and gentamicin were added to the wells. Then, 50 μL of CGL conjugated with horseradish peroxidase (CGL-HRP), diluted 1:500, was added. The microtiter plate incubated for 2 h at room temperature with stirring. After washing, the bound CGL-HRP was detected using TMB as the substrate. The reaction was stopped by adding 50 μL of 5% sulfuric acid. The end product was measured at 450 nm. The CGL-HRP conjugate was obtained as followed [36].

4.10. Thermofluor Assay

The Thermofluor assay, also referred to as differential scanning fluorimetry (DSF), monitors the effects of ligands on temperature-dependent protein unfolding by measuring the melting temperature (Tm) of the protein. CGL thermostability was evaluated in the presence of different sugars (galactose and glucose) and the aminoglycoside antibiotic gentamicin. 2 μL CGL (117.6 mM), 5 μL glycosidic ligands (125 mM), 5 μL gentamicin (0.096 mM) and 2 μL 5x SYPRO orange, 2.5 μL PBS pH 8.0 and 13 μL ultrapure water were placed in the wells of a 96-well qPCR plate, which was then sealed. SYPRO Orange fluorescence was measured as a function of a temperature gradient from 25 to 96 °C using a qPCR DTlite (DNA-Technology, Moscow, Russia). Three measurements were carried out at the end of a 1 min equilibration time for each 1 °C temperature increment. Tm is defined as the inflection point at which 50% of the protein is unfolded.

4.11. Isothermal Titration Calorimetry (ITC)

ITC experiments were performed using a Nano Isothermal Titration Calorimeter (TA Instruments-Waters LLC, New Castle, DE, USA). CGL and the ligands were solubilized in PBS at the final concentrations of 100 µM CGL, 16 mM Gal, 16 mM Glc, and 50 mM gentamicin.

Each ligand solution was titrated into the CGL solution in 1.96 μL aliquots using an automated microsyringe (TA Instruments-Waters LLC, USA), with 200 s intervals between injections. A total of 25 injections were made into 190 μL of CGL solution. During the entire experiment, the sample cell was stirred at 300 rpm and maintained at 25 °C. Control experiments were performed by titrating ligand into PBS alone under identical conditions.

Thermodynamic parameters, including dissociation constant (Kd), binding stoichiometry (n), enthalpy change (ΔH) were calculated using the NanoAnalyze Data Analysis software v3.11.0 (TA Instruments-Waters LLC), applying the “Independent” binding model with fitted offset correction, as recommended by the manufacturer.

4.12. Sea Urchin Capture and Egg Fertilization

The sea urchins (Strongylocentrotus nudus) were collected at the Marine Experimental Station of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry in Troitsy Bay (Posyet Bay, Sea of Japan).

The sea urchins were handled in reservoirs maintained with seawater until arrival at the laboratory. The water was kept clear and oxygenated with a filter at 22 °C and salinity adjusted to 39–45 ppm. For fertilization, gametes were collected after stimulating the males and females by administering 3 mL of 0.5 M KCl into their coelomic cavities. The eggs were collected in filtered seawater, passed through an 80-μm mesh filter to remove debris, and washed three times by decantation. The eggs were suspended in 50 mL of filtered seawater, and the number of eggs was counted using a Biolam light microscope (LOMO, Russia). The eggs were then adjusted to a concentration of 2000 eggs/mL.

Sperm were collected similarly but recovered with a Pasteur pipette and maintained at a low temperature (4 °C) until use. Fertilization was performed by mixing 0.05 mL of concentrated sperm with 2.45 mL of seawater and adding it to the egg cell suspension with light agitation for 5 min. All experiments were conducted at 18 °C under a 12-h light/dark photoperiod. Samples were checked for fertilization by microscopy as described above.

4.13. Embryotoxic Evaluation

The effect of the lectin on sea urchin embryogenesis was assessed prior to the formation of 16 blastomeres in the control group. The assay was carried out in a 24-well plate. First, 500 µL of a solution containing a mixture of a 2 × 10⁶ CFU/mL inoculum and lectin (or gentamicin, or a combination of lectin and gentamicin) at subinhibitory concentrations in filtered seawater was added to the wells of the plate. Immediately post-fertilization, 500 µL of the eggs were added to the plate. Controls were performed using only eggs and filtered seawater. The effective infection dose per embryo was 10³ CFU. The time interval between infection and fixation was 3 h. The assays were stopped by the addition of 10% formaldehyde, after which the number of normal embryos was counted in comparison to the controls. A total of 50 to 100 embryos were selected from each well three times, and the number of normal and abnormal embryos was counted. Each experiment was carried out in triplicate. Embryos were classified as normal or abnormal based on morphological criteria such as deformation, developmental delay, and cessation of blastula division compared to the control group.

4.14. Statistical Analysis

Experimental results are presented as the mean ± SD. Differences between the means were evaluated by two-tailed Student’s t-test, with p < 0.05 considered to be statistically significant.

5. Conclusions

These findings establish CGL as a promising candidate for developing alternative antibiofilm strategies, especially for preventing biofilm formation in clinically relevant bacteria. The results also reinforce the biotechnological relevance of bivalve-derived lectins as selective modulators of antibiotic activity that enhance efficacy in a species-specific manner. A better understanding of the mechanisms underlying lectin-mediated processes could lead to novel therapeutic strategies and biotechnological innovations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27041961/s1.

Author Contributions

Conceptualization, I.V.C. and T.O.M.; investigation, I.V.C., T.O.M., O.I.N. and L.S.; methodology, I.V.C., T.O.M. and O.I.N.; writing—original draft, I.V.C., T.O.M. and O.V.C.; visualization, T.O.M.; writing—review and editing, K.-F.H., X.J., Y.Z. and O.V.C.; project administration, O.V.C. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available on request.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was supported by the Russian Science Foundation (grant no. 25-24-20131) and by the Ministry of Vocational Education and Employment of Primorsky Krai (agreement no. 30-2025-005020).