Afil, a Lectin from Aplysina fistularis, Exhibits Antibiofilm and Synergistic Antibacterial Activity Against Resistant Bacteria

Abstract

Lectins from marine sponges have emerged as promising candidates for antimicrobial strategies, particularly against biofilm-forming pathogens. In this study, we report the purification, biochemical characterization, and antibiofilm properties of a new lectin (AfiL) isolated from Aplysina fistularis. AfiL exhibited typical features of sponge lectins, including a β-sheet-rich secondary structure and a predominant oligomeric state in solution. Dynamic light scattering (DLS) analyses confirmed that AfiL predominantly exists as a well-defined oligomer at acidic and neutral pH. Sequence analysis revealed similarity to a putative collectin-like protein from sponge Desydea avara. AfiL selectively agglutinated Staphylococcus aureus strains, correlating with its preferential binding to lipoteichoic acid (LTA). The lectin demonstrated significant antibiofilm activity against S. aureus, S. epidermidis, and Escherichia coli strains, and exhibited synergistic or additive effects when combined with conventional antibiotics against a Methicillin-resistant S. aureus. Isothermal titration calorimetry (ITC) revealed a strong interaction between AfiL and porcine stomach mucin (Kd = 1.71 × 10^-6 M), consistent with multivalent carbohydrate recognition. Overall, our findings highlight the potential of AfiL as a novel antibiofilm agent with species-specific modulatory effects on antibiotic activity and provide new insights into the functional versatility of sponge-derived lectins in microbial control strategies.

Keywords: antibiotic synergy; biofilm inhibition; lectin–carbohydrate interaction; marine sponge lectin; multidrug-resistant bacteria.

Figure 1

SDS–PAGE of purified AfiL. Lane M: molecular weight marker; Lane 1: AfiL under non-reducing conditions; Lane 2: AfiL under reducing conditions (with β-mercaptoethanol).

Figure 2

ITC. Isothermal titration calorimetry (ITC) of AfiL. (A) Thermogram of AfiL titrated with porcine stomach mucin (PSM). (B) Binding isotherm and model fitting for AfiL–PSM interaction; insert: heatmap showing the contributions of enthalpy (ΔH, green), entropy (−TΔS, red), and Gibbs free energy (ΔG, blue) to the binding AfiL–PSM interaction. (C) Thermogram of AfiL titrated with D-lactose. (D) Binding isotherm and model fitting for AfiL–lactose interaction; insert: heatmap representation of integrated heats for AfiL–lactose interaction. Experiments were performed at 25 °C using a MicroCal PEAQ-ITC system, and data were fitted to a one-set-of-sites binding model.

Figure 3

Sequence alignment between AfiL and a putative collectin-like protein from Desydea avara. Amino acid sequence alignment between AfiL (lectin from Aplysina fistularis), partially obtained by MS/MS, and a putative uncharacterized protein encoded in the genome of the marine sponge Desydea avara (GenBank ID: XP_065893106.1). Dark gray shading indicates identical residues; light gray shading indicates similar residues. Amino acids underlined correspond to a predicted fibrillar collagen-like domain.

Figure 4

Effect of AfiL on bacterial cell agglutination. Microscopic visualization of bacterial cell agglutination induced by AfiL. (A) S. aureus in TBS (control); (B) S. aureus with AfiL; (C) S. aureus with AfiL pre-incubated with lactose. Images were acquired using light microscopy at 120× magnification. Agglutination was specifically observed for S. aureus and was inhibited in the presence of 100 mM lactose.

Figure 5

Effect of AfiL on biofilm formation of S. aureus and E. coli strains. Quantification of biofilm biomass (a–e) and viable cell counts (f–j). Bars represent AfiL-treated samples; black bars represent untreated groups (controls). Data are presented as mean ± standard deviation. Statistical significance was calculated in comparison to untreated controls using one-way ANOVA followed by the Bonferroni post hoc test: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).