A galactose-binding lectin isolated from Aplysia kurodai (sea hare) eggs inhibits streptolysin-induced hemolysis

Abstract

A specific galactose-binding lectin was shown to inhibit the hemolytic effect of streptolysin O (SLO), an exotoxin produced by Streptococcus pyogenes. Commercially available lectins that recognize N-acetyllactosamine (ECA), T-antigen (PNA), and Tn-antigen (ABA) agglutinated rabbit erythrocytes, but had no effect on SLO-induced hemolysis. In contrast, SLO-induced hemolysis was inhibited by AKL, a lectin purified from sea hare (Aplysia kurodai) eggs that recognizes α-galactoside oligosaccharides. This inhibitory effect was blocked by the co-presence of d-galactose, which binds to AKL. A possible explanation for these findings is that cholesterol-enriched microdomains containing glycosphingolipids in the erythrocyte membrane become occupied by tightly stacked lectin molecules, blocking the interaction between cholesterol and SLO that would otherwise result in penetration of the membrane. Growth of S. pyogenes was inhibited by lectins from a marine invertebrate (AKL) and a mushroom (ABA), but was promoted by a plant lectin (ECA). Both these inhibitory and promoting effects were blocked by co-presence of galactose in the culture medium. Our findings demonstrate the importance of glycans and lectins in regulating mechanisms of toxicity, creation of pores in the target cell membrane, and bacterial growth.

Figure 1

Purified AKL (left) and SLO (right) were subjected to SDS-PAGE under reducing condition. The standard marker proteins (M) used were phosphorylase b (96 kDa), bovine serum albumin (66 kDa), ovalbumin (42 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (21 kDa), and lysozyme (14 kDa).

Figure 2

Stable absorbance of hemin chloride and optical detection of SLO-induced hemolysis. (A) 1 mL of RBCs ruptured by distilled water (dotted line), or RBCs hemolyzed by SLO (solid line), was mixed with 1 mL HCl (2%) in acetone to convert heme to hemin chloride. Arrow: optimal absorbance (wavelength 510 nm); (B) Serially 2-fold diluted SLO was mixed with ruptured RBCs and HCl (2%) in acetone, and absorbance at 510 nm was measured.

Figure 3

Effects of lectins on SLO-induced hemolysis. RBCs were pre-mixed with serially diluted lectins (AKL: solid line; ECA: dotted line; ABA: small dashed line; PNA: large dashed line). SLO was added to each sample, mixed with HCL/acetone, and inhibition of hemolysis was assessed by measurement of absorbance at 510 nm as in Figure 2. Error bars: SE from three independent experiments. * p < 0.01 in comparison with negative control.

Figure 4

SEM imaging of RBCs agglutinated by AKL in the presence of SLO. (A) AKL-induced hemagglutination; (B) negative control (RBCs without AKL or SLO). Scale bars: 20 µm.

Figure 5

Pretreatment with MβCD suppresses the inhibitory effect of AKL on SLO-induced hemolysis. White columns: hemagglutination activity of AKL in RBCs pretreated with (+) or without (−) 12.5 mM of MβCD. Shaded columns: inhibitory effect of pretreatment with 12.5 mM of MβCD on hemolytic activity of 2 mg/mL AKL in RBCs in the presence of SLO. Error bars: SE from three independent cell preparations assayed individually. * p < 0.01 in comparison with negative control.

Figure 6

Effects of added lectins on growth of S. pyogenes in culture. S. pyogenes was grown in liquid culture with each of the indicated lectins (200 μg) for 18 h. The turbidity of the culture medium was assessed by measurement of absorbance at 650 nm. TBS: negative control, AKL: Aplysia kurodai galactose-binding lectin, AKL + Gal: AKL with 100 mM galactose, ECA: Erythrina cristagalli agglutinin, ECA + Lac: ECA with 100 mM lactose (Galâ1-4Glc), ABA: Agaricus bisporus agglutinin, PNA: peanut (Arachis hypogaea) agglutinin.

Figure 7

Stimulatory and inhibitory effects of ECA and AKL on growth of S. pyogenes. Culture media containing ECA, AKL, or TBS (as in Figure 6) were diluted 10-fold with saline. Each of the three media (100 μL) was spread on agar plates containing 5% rabbit RBCs and incubated for 18 h. Arrows indicate colonies.

Figure 8

Delay of bacterial growth by addition of AKL. S. pyogenes cells (10³/mL) were incubated with (solid line) or without (dotted line) 2 mg/mL AKL. Growth was assessed based on turbidity (absorbance at 650 nm).