Vicia ervilia lectin (VEA) has an antibiofilm effect on both Gram-positive and Gram-negative pathogenic bacteria

Abstract

Bacterial growing resistance to antibiotics poses a critical threat to global health. This study investigates, for the first time, the antibiofilm properties of Vicia ervilia agglutinin (VEA) from six different V. ervilia accessions against pathogenic bacteria, and the yeast Candida albicans. In the absence of antimicrobial properties, purified VEA significantly inhibited biofilm formation, both in Gram-positive and Gram-negative bacteria, but not in C. albicans. With an inhibitory concentration ranging from 100 to 500 µg/ml, the VEA antibiofilm activity was more relevant against the Gram-positive bacteria Streptococcus aureus and Staphylococcus epidermidis, whose biofilm was reduced up to 50% by VEA purified from accessions #5 and #36. VEA antibiofilm variability between accessions was observed, likely due to co-purified small molecules rather than differences in VEA protein sequences. In conclusion, VEA seed extracts from the accessions with the highest antibiofilm activity could represent a valid approach for the development of an effective antibiofilm agent.

Keywords: Vicia ervilia; Antibiotic resistance; Antimicrobial; Biofilm; Lectin; Lectin gene.

Fig. 1

Chromatogram of VEA purification trough affinity chromatography. a Example of affinity chromatography elution profile at 280 nm for one of the six V. ervilia accessions utilized in this work. The first peak (fraction 3–10) corresponds to unbound proteins eluted from the column with PBS buffer. The second peak (fractions 27–31) results from protein elution after the addiction of PBS buffer with 0.1 M glucose to the column. b-c SDS-PAGE followed by Coomassie staining of the first (b) and the second (c) affinity chromatography peak. Numbers of the affinity chromatography fractions analyzed by SDS-PAGE are reported below gels. Twenty µl of each elution fraction were loaded on a 15% polyacrylamide denaturing gel. The black arrow marks the 21-kDa beta-chain subunit of the purified VEA; the arrowhead indicates a protein of around 30 kDa that likely represents uncleaved lectin precursor. Numbers at left indicate molecular mass markers (Mk) expressed in kDa

Fig. 2

SDS-PAGE analysis to confirm VEA purification for all the six V. ervilia accessions (#5, #12, #21, #23, #36, #46). Twenty µg of proteins from crude extract (CE) (in figure reported only for the #5 accession, as an example), or from 30–70% ammonium sulphate precipitation samples, or two µg of proteins purified by affinity chromatography (P), were loaded on a polyacrylamide gel and elettrophoretically separated before Coomassie staining. Black arrow: 21-kDa subunit of the purified VEA. The arrowhead indicates a protein of around 30 kDa that likely represents uncleaved lectin precursor. Numbers at left indicate molecular mass markers (Mk) expressed in kDa

Fig. 3

The effect of VEA lectins on biofilm formation. P. aeruginosa (a), S. aureus (b), S. epidermidis (c), C. albicans (d) were inoculated into a 96-well plate containing crude extract (CE) and purified VEA lectin (P) at 100 or 500 µg/ml and incubated for 24 h. Biofilm biomass was quantified by crystal violet assay (absorbance 570 nm). As positive control, gentamicin, and fluconazole for the yeast, were used. Antibiofilm lectin activity was even determined with PHA at the concentration of 50 and 100 µg/ml. Data represent the mean ± SD of three independent experiments performed in quadrupled. *P < 0.05, **P < 0.01 (treated versus untreated microorganisms)

Fig. 4

Protein alignment of lectin amino acid sequences. Lectin proteins from two species of the Fabeae tribe, VVA from Vicia villosa and PSA from Pisum sativum, are shown for comparison. The arrow indicates the internal cleavage site for removal of the signal peptide. In the rectangle, the A (Ala) and Q (Glu) substitutions in accession #36 are reported with respect to the other V. ervilia accessions (#5–12-21–23-46) that have an identical amino acid sequence. Harrowheads indicate conserved amino acid residues for carbohydrate binding. The SL/VEEN stretch, marked by two arrows, represents the six amino acid peptide between the mature protein alfa and beta chains. “*”: amino acidic residues are identical in all sequences in the alignment; “:” conserved substitutions, i.e. the amino acid is replaced by one having similar characteristics;”.” semi-conserved substitutions, i.e., amino acids having similar shape