BOL Lectin: A Protein Derived from Cauliflower Exhibits Antibiofilm Activity in In Vitro Assays Against Staphylococcus aureus

Abstract

The BOL lectin, a 34 kDa protein with a hemagglutination titer of 64 hemagglutination units (HU), was extracted from cauliflower (Brassica oleracea spp. botrytis L.), purified by affinity and ion exchange chromatography, and confirmed, in this study, by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The antibiofilm activity of BOL was evaluated at two concentrations (0.1 mg/mL and 1.0 mg/mL) against bacterial strains of importance to human health (Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Streptococcus agalactiae ATCC 12403). In addition to a biofilm formation assay, a pre-formed biofilm assay was conducted, with biofilm structure analyzed by Scanning Electron Microscopy (SEM). The antimicrobial potential of BOL was also investigated using the Minimum Inhibitory Concentration (MIC) assay in 96-well microplates. Among the tested bacterial strains, BOL exhibited activity against S. aureus at 1.0 mg/mL, interfering with both biofilm formation and disrupting pre-formed biofilms, which may be explained by a possible interaction between BOL and the components present in the biofilm matrix. However, no antibiofilm activity was observed against E. coli, B. cereus, or S. agalactiae, possibly due to differences in the composition of their biofilm matrices. Furthermore, BOL showed no detectable bactericidal or bacteriostatic activity in the antimicrobial assays. In conclusion, BOL lectin, at the tested concentrations, does not exhibit direct antimicrobial activity but effectively disrupts the extracellular matrix in S. aureus ATCC 29213.

Keywords: One Health; antimicrobial activity; bacterial resistance; biofilms; biotechnology; plant lectin.

Figure 1

(A) SDS-PAGE (12% acrylamide), stained with Coomassie Blue R-250, showing the fractions collected during the chromatographic purification steps of BOL lectin. MM: molecular weight marker (Thermo Scientific™ PageRuler™ Plus Prestained Protein Ladder); CE: crude extract; PI: fraction obtained from affinity chromatography; PII: fraction obtained from ion exchange chromatography. The purified lectin, indicated by an arrow, is observed as a single band with an apparent molecular weight of 34 kDa. (B) Ribbon diagram of the predicted three-dimensional structure of BOL lectin. The helices are shown in pink, β-sheets in yellow, and loops in gray. The predicted structure reveals a model with 100% of the amino acid residues mapped into two tandemly arrayed MATH domains: domain 1 (residues 14–142) and domain 2 (residues 165–290). (C) Hemagglutination Assay. Hemagglutination activity was evaluated by serial twofold dilutions (from 1:2 to 1:512) of 50 µL of each sample with 25 µL of 2% rabbit erythrocyte suspension in PBS, in U-bottom microtiter plates. After 30 min of incubation at room temperature, agglutination was visually assessed. Panel A1–A10: crude extract; Panel B1–B10: affinity-purified fraction; Panel C1–C10: ion exchange-purified fraction. The endpoint titer was observed in 1:64 in the purified fraction (C), as indicated by the asterisk. The hemagglutination titer is defined as the highest dilution at which visible agglutination still occurs.

Figure 2

Biofilm Inhibition Assay of BOL. NC represents the supplemented TSB medium alone. PC indicates the bacterial strains treated with antimicrobials.

Figure 3

Scanning electron microscopy of Escherichia coli biofilm. The formation of untreated biofilm is observed in the negative control (A), while structural changes are evident in the treatment with Ampicillin (32 µg/mL) (B), BOL (0.1 µg/mL) (C), and BOL (1 µg/mL) (D). Scale bars range from 5 to 10 µm.

Figure 4

Scanning electron microscopy of Staphylococcus aureus biofilm. (A) Negative control, where biofilm formation is observed. (B) Positive control, showing modifications along the structure of the consolidated biofilm caused by Ampicillin (32 µg/mL). (C) Biofilm treated with BOL (0.1 µg/mL), showing no structural changes. (D) Treatment with BOL (1 µg/mL), showing loss of biofilm integrity. Scale bars range from 5 to 10 µm.

Figure 5

Scanning electron microscopy of Streptococcus agalactiae biofilm. (A) Negative control, showing biofilm formation in the absence of treatment. (B) Positive control, with structural modifications in the biofilm caused by Ampicillin (32 µg/mL). (C) Biofilm treated with BOL (0.1 µg/mL), showing no structural changes. (D) Treatment with BOL (1 µg/mL), showing loss of biofilm integrity. Scale bars range from 5 to 10 µm.

Figure 6

Effects of BOL lectin (0.1 mg/mL) on pathogenic bacteria. The positive control indicates only the bacterial inoculum and the culture medium, while the negative well represents the culture medium alone, serving as quality control for the test.

Figure 7

Effects of BOL lectin (1.0 mg/mL) on Escherichia coli and Staphylococcus aureus. The positive control indicates the well with only the bacterial inoculum and culture medium, while the negative well represents the culture medium alone, serving as a quality control for the test.