Comparison of the antibacterial properties of peptides from the hepatopancreas of red king crab and snow crab and development of an approach for red king crab peptide isolation from the hepatopancreas

Preparation of hepatopancreas (HPC) extracts

Hepatopancreas of the red king crab (Paralithodes camtschaticus) and snow crab (Chionoecetes opilio) were collected during scientific voyages in the Barents and Kara Seas on vessels of the Polar Branch of VNIRO (Knipovich PINRO) in 2023–2024. The crabs were caught in the Russian Federation’s territorial waters. This was according to the scientific quota allocated to the institute and the voyage assignments. The hepatopancreas was separated and frozen on the board and delivered to the institute. Afterwards it was kindly provided for our purposes. The laboratory produced samples of crude extracts (acetone powders), which were used for research.

Peptide extraction

Acetonitrile (HPLC grade) and trifluoroacetic acid (extra pure grade) were used for peptide extraction. Low molecular weight protein fractions, containing both proteins and peptides, were extracted from acetone powder derived from the hepatopancreas of both red king crab (Paralithodes camtschaticus) and snow crab (Chionoecetes opilio). The extraction protocols followed established methods: red king crab using method (Molchanov et al., 2023) and snow crab using method (Molchanov et al., 2024). After extraction, all samples were lyophilized and stored frozen at −20 °C until further analysis.

Preparations of the cell wall and cell wall polysaccharide of the gram-positive bacterium M. luteus

The cell wall and polysaccharide of M. luteus were provided by the Experienced Biotechnological Plant (EBP) of IBCh RAS. The samples of cell wall and polysaccharide were obtained as described in Molchanov et al. (2023). The polysaccharide was quantified by anthrone method (by glucose) (Fales, Russell & Fain, 1961). The sample contained polysaccharide at a concentration of 3.3 mg/mL in the following buffer: 20 mM sodium acetate, 0.5 M NaCl, pH 5.5. The buffer reagents were supplied by Sigma-Aldrich (St. Louis, MO, USA).

Polyacrylamide gel zymography with cell wall of M. luteus

Zymography to detect antibacterial activity toward cell wall was performed according to the protocol using cell wall from the gram-positive bacterium M. luteus as substrate (Fukushima & Sekiguchi, 2016). Chemicals used: acrylamide, N,N’-methylenebisacrylamide, ammonium persulfate, glycine, glycerol (Amresco); SDS (Fluka); TEMED, bromophenol blue, Coomassie R-250 (Serva); β-mercaptoethanol, Triton X-100, methylene blue, Tris (Sigma-Aldrich). Hen egg-white lysozyme (HEWL) (Amresco) was used as a positive control for hydrolase activity.

Antibacterial activity was detected after renaturation (3 h, 37 °C) as described in Fukushima & Sekiguchi, (2016). The presence of antibacterial activity was assessed by the absence of background staining. Peptide activity was assessed by zymography at each isolation. For the further experiments, the peptides were eluted from the bands of the zymogram. The spots with the activity in the stained 16% zymogram gel were excised, and peptides extracted as described (Fukutomi et al., 2005). The samples were lyophilized.

Turbidimetric analysis of the activity of peptide samples toward M. luteus cell wall at different pH, ionic strength and in the presence of reducing agent

M. luteus cell wall hydrolysis was assessed via turbidimetry following the protocol in Fukushima & Sekiguchi (2016). The peptide sample (1 o.e./mL) or HEWL (0.02 mg/mL) were added to three mL of cell wall suspension as controls and the samples were incubated at 37 °C. The optical density at 540 nm was measured at certain time intervals during the incubation. Activity at different pHs was tested using 25 mM Tris–HCl (pH 7.2, 9.0) and sodium phosphate buffer (pH 5.5). To prepare the phosphate buffer pH 5.5, one M solutions of Na₂HPO₄ and NaH₂PO₄ (Sigma-Aldrich) were used. To study the effect of ionic conditions, different concentrations of sodium chloride were used: 10, 50 and 150 mM. The effect of the disulfide bond reducing agent on the activity of the peptide was studied in the presence of dithiothreitol (DTT) (Sigma-Aldrich) at concentrations of one mM and 10 mM.

Bacterial strains and growth conditions

Bacterial strains were sourced from the All-Russian Collection of Microorganisms (VKM) and the American Type Culture Collection (ATCC). Cultivation was carried out in Lysogeny Broth (LB) medium and on LB agar plates. The agar plates contained 1.5% (w/v) agar for the bottom layer and 0.5% (w/v) agar for the top layer. All cultures were incubated at 37 °C.

Lytic activity of peptides

The lytic activity of peptides from red king crab and snow crab was evaluated against the following bacterial strains: Gram-positive bacteria Bacillus tropicus ATCC 4342 and Bacillus subtilis WB800N, as well as the Gram-negative bacterium Escherichia coli MG1655. Peptide solutions (0.1 o.e./ml) were tested on bacterial lawns. For this, 100 µL of the bacterial culture was mixed with four mL of soft agar and poured onto Petri dishes pre-prepared with a solid 1.5% LB agar base. The plates were left at room temperature for 30 min to solidify. Subsequently, 10 µL of each peptide solution was added to the lawn. HEWL solution (three mg/mL) was used as a positive control, and SM+ buffer (50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM MgSO₄, 0.01% gelatin) was used as a negative control. Bacterial cultures were incubated at 37 °C for 18 h in five mL of LB. These cultures were then diluted 1:100 in fresh LB and grown at 37 °C until an optical density (OD₅₉₅) of 0.2 was reached. For the experimental assay, 50 µL of the peptide solution was combined with 50 µL of the bacterial culture suspension. Positive controls consisted of 50 µL of HEWL solution (3 mg/mL) mixed with 50 µL of the bacterial suspension. Negative controls were prepared by mixing 50 µL of the bacterial culture with 50 µL of SM+ buffer. The 96-well plate was incubated at 37 °C in a FilterMax F5 microplate spectrophotometer (Molecular Devices, San Jose, CA, USA), with OD₅₉₅ measurements recorded every 10 min for 9 h. Growth curves were generated using GraphPad Prism version 8.4.343. Error bars on the plots represent the standard deviation, calculated from three independent trials.

Chromatographic purification of red king crab peptide

All procedures were carried out at 4 °C. The following chromatographic media were used in the work: Blue Sepharose 6 Fast Flow (GE HealthCare, Chicago, IL, USA) and MonoQ (GE HealthCare). Both columns were equilibrated with buffer A (10 mM Tris–HCl, pH 7.5). The lyophilized extract of low molecular weight protein fractions from the acetone powder of red king crab HPC was dissolved in 400 µL milliQ water and centrifuged at 12,000 rpm for 5 min. The supernatant was loaded onto the Blue Sepharose 6 Fast Flow column (volume 5 ml). The flow rate was 0.1 ml/min. After complete loading of the sample, the column was washed with buffer A. Elution was performed with buffer A with different NaCl concentrations: 5, 10, 20, 30, 40, 50 mM. After purification on Blue Sepharose 6 Fast Flow, all fractions were lyophilized. The lyophilizates were dissolved in 50 µl of distilled water. Seven µl were taken for analysis of fraction composition by 16% SDS-PAGE (Fales, Russell & Fain , 1961) and seven µL were taken for analysis of the occurrence of antibacterial activity in the fractions by zymography with cell wall (Fukushima & Sekiguchi, 2016). The fraction of unbound proteins was reloaded onto the column Blue Sepharose 6 Fast Flow and eluted with increasing NaCl concentration. The fractions were lyophilized and analyzed as described above. In addition, the fraction eluted in 10 mM NaCl after the first purification and exhibiting activity in the zymogram was loaded onto the MonoQ column. The fractions were eluted with a NaCl gradient and analyzed by 16% SDS-PAGE. The activity of the fractions was evaluated by turbidimetric analysis of the cleavage of the cell wall under the action of the peptide, if it was present in the fractions.

The experiments were independently repeated >3 times (including extraction and analysis). The results of three separate experiments were averaged for the turbidimetric analysis data presented. Error bars represent standard deviation.

Comparison of the activity of peptides from the red king crab and the snow crab at different pH values

We have previously investigated the activity of the extract of low molecular weight protein fractions from the HPC of the red king crab at different pH values (Molchanov et al., 2023). In the present paper we compared the activity of peptide preparations isolated from the gel bands with activity of red king crab and snow crabs at different pH. HEWL was used as a control for cell wall degradation. The general scheme for isolating the peptides used in the studies is shown in Fig. 1. The obtained results are presented on Fig. 2. The analysis of kinetics of cell wall degradation reveals that both peptides exhibit maximum activity at pH 7.2, which is close to HEWL. Incubation for one hour at pH 9.0 showed that red king crab peptide is less active than snow crab peptide and HEWL. After 2 h of incubation, the activity of all three samples became nearly the same. Both peptides have the lowest activity at pH 5.5.

Investigation of the influence of ionic strength and disulphide reducing agents on the activities of peptides from red king crab and snow crab

AMPs are known to be very sensitive to salt content (Yu et al., 2011; Bals et al., 1998). The effect of different concentrations of NaCl on the activity of the peptides was analyzed. The obtained data are shown in Fig. 3. It was found that NaCl at any concentration reduced the activity of the red king crab peptide, whereas the activity of the snow crab peptide even slightly increased in the presence of salt, as with HEWL.

It is known that many AMPs have cysteine-rich regions (Moe et al., 2018; Sperstad et al., 2009) and are therefore very sensitive to the action of a reducing agent. The effect of a reducing agent of disulfide bonds, dithiothreitol, on activity was studied. The activity was assessed by measuring the change in optical density of the cell wall suspension at 540 nm during incubation with the peptide at 37 °C at pH 7.2 at DTT concentrations of 1 and 10 mM. It was shown (Fig. 4) that the presence of DTT strongly suppressed the activity of the red king crab peptide, whereas it had almost no effect on the snow crab peptide, as with HEWL, and even slightly increased the activity of the snow crab peptide during the first hours of incubation.

Lytic activity of peptides

The lytic activity of peptides from red king crab and snow crab was assessed on bacterial lawns, where both peptides demonstrated pronounced lytic activity against Gram-positive Bacillus species (Figs. 5A–5C). Notable zones of lysis appeared on the lawns of Bacillus tropicus ATCC 4342 and Bacillus subtilis WB800N at the areas where the peptides were applied (Figs. 5B and 5C). These findings confirm the peptides strong bactericidal effects against these Gram-positive bacteria. In comparison, HEWL exhibited activity exclusively against Bacillus subtilis WB800N (Fig. 5C), highlighting the strain-specific susceptibility of Bacillus subtilis to HEWL. No lytic activity from either peptides or HEWL was observed on the lawn of the Gram-negative strain Escherichia coli MG1655 (Fig. 5A).

Further investigation revealed that the peptides from red king crab and snow crab demonstrate potent lytic effects against the Gram-positive Bacillus tropicus ATCC 4342 and Bacillus subtilis WB800N strains (Figs. 5E and 5F), resulting in complete inhibition of bacterial growth in liquid medium. These results underscore the strong antimicrobial potential of these peptides against certain Gram-positive bacteria, consistent with the findings in the agar-based lawn assay. However, as observed in the previous experiment with agar lawns, the peptides showed no lytic activity against the Gram-negative Escherichia coli MG1655 (Fig. 5D). Interestingly, rather than inhibiting growth, the peptides seemed to promote the growth of E. coli in the liquid medium. This phenomenon could be explained by the bacteria utilizing the peptides as a source of nutrients or energy, thereby enhancing their metabolic activity.

In contrast to the peptides, HEWL, which is often used as a reference antimicrobial agent for its ability to degrade peptidoglycan in bacterial cell walls, exhibited only a modest inhibitory effect on the growth of Escherichia coli MG1655, as compared to the positive control (Fig. 5D). The minimal impact of HEWL on E. coli may be due to the species’ intrinsic resistance mechanisms or the inability of HEWL to effectively penetrate the outer membrane of Gram-negative bacteria. Regarding Bacillus tropicus ATCC 4342, HEWL only temporarily inhibited bacterial growth, suggesting the emergence of resistant mutants during the culture process (Fig. 5E). This transient effect may indicate a partial resistance of the strain to HEWL, which could be due to natural variation in the strain or specific adaptive mechanisms developed under the experimental conditions. Interestingly, for Bacillus subtilis WB800N, HEWL exhibited a lytic effect comparable to that of the tested peptides, making it clear that this strain is highly susceptible to both HEWL and the peptides (Fig. 5F). The results from both the agar-based and liquid culture experiments highlight the significant antimicrobial potential of the red king crab and snow crab peptides against specific Gram-positive bacteria, while also providing insight into the resistance mechanisms and susceptibility profiles of different bacterial strains.

Development of an approach for the chromatographic purification of an antibacterial peptide from an extract of low molecular weight protein fractions from the HPC of the red king crab

It is important to obtain a pure preparation of the antibacterial peptide in order to analyze its cytotoxic properties and to identify the amino acid sequence of the peptide. Our previous attempts to purify the peptide from the extract of low molecular weight protein fractions of the red king crab hepatopancreas using gel filtration on Sephadex G-75 and Sephacryl S-200 HR did not result in any significant purification of the peptide from other proteins, although a peptide band in the five kDa region appeared on SDS-PAGE. The assumption of affinity binding to heparin-sepharose due to affinity for the polysaccharide was also not confirmed. Zymographic analysis revealed good binding of the peptide to the heterocyclic dye methylene blue, which may be due to hydrophobic interactions. It has three basic rings: two aromatic carbon rings and one containing nitrogen and sulphur.

It was suggested that the peptide could be retained on the Cibacron Blue 3G-A dye, which contains an anthraquinone residue that also contains three rings, two of which are aromatic. This dye is covalently linked to agarose in the chromatographic resin Blue Sepharose 6 Fast Flow. Blue Sepharose 6 Fast Flow is known to bind many proteins such as albumin, interferon, lipoproteins, coagulation factors and some enzymes including kinases, dehydrogenases and enzymes requiring adenyl-containing cofactors. We attempted to purify the peptide from the extract of low-molecular-weight protein fractions of the red king crab HPC on Blue Sepharose 6 Fast Flow. A lyophilized sample of the extract of low-molecular-weight protein fractions from the hepatopancreas was dissolved in 400 µL of loading buffer and loaded onto a column with Blue Sepharose 6 Fast Flow. Elution was performed with a NaCl gradient. The fractions were analyzed in 16% SDS-PAGE (Laemmli, 1970).

It was found that most of the proteins were in the fraction of unbound proteins and the column capacity was not sufficient. However, the peptide band was detected in the fractions eluted with 10–50 mM NaCl, most of which was in the 50 mM NaCl fraction (Fig. 6). Thus, the peptide was weakly retained on the Blue Sepharose 6 Fast Flow column. The weak interaction is probably due to the presence of a large amount of salt in the extract itself before loading, which was visible on the conductometric curve. Analysis of activity by zymography using a cell wall showed that the fractions with the activity were the fraction of unbound proteins and the fraction in 10 mM NaCl (Fig. 7). Since the column capacity was not sufficient in the first purification, a second purification was performed on Sepharose 6 Fast Flow for the fraction of unbound proteins (fraction 4, see Fig. 6). The fractions were analyzed in 16% SDS-PAGE. It was found that the 10 mM NaCl fraction contained almost pure peptide (Fig. 8).

In parallel, to remove higher molecular weight protein contaminants from the sample of 10 mM NaCl fraction obtained during the first purification on Blue Sepharose 6 Fast Flow (fraction 4, see Fig. 6), an attempt was made to purify it on the MonoQ anion chromatography column with NaCl gradient. The fractions were analyzed in 16% SDS-PAGE (Fig. 9). It was found that this step did not lead to a significant purification from impurities, but the analysis of peptide activity towards the cell wall, carried out by the turbidimetric method (Fukushima & Sekiguchi, 2016), revealed that the activity of the peptide in the fraction with 10 mM NaCl increased by one and a half times compared to the fraction of unbound proteins (Fig. 10). Thus, the scheme for obtaining a pure peptide preparation from red king crab HPC can be presented as follows (Fig. 11). It should be noted that for the snow crab peptide, this scheme did not enable its complete purification. This proves that despite the common property—the cleavage of the cell wall polysaccharide (Molchanov et al., 2023; Molchanov et al., 2024)—they have different nature, which is also confirmed by the results on the effect of ionic strength and a disulfide bond reducing agent on their activity.