The B subunit of pirABvp toxin secreted from vibrio parahaemolyticus causing AHPND is an amino sugar specific lectin

Abstract

Vibrio parahaemolyticus (Vp) is the etiological agent of the acute hepatopancreatic necrosis disease (AHPND) in Penaeus vannamei shrimp. Vp possesses a 63–70 kb conjugative plasmid that encodes the binary toxin PirAvp/PirBvp. The 250 kDa PirABvp complex was purified by affinity chromatography with galactose-sepharose 4B and on a stroma from glutaraldehyde-fixed rat erythrocytes column, as a heterotetramer of PirAvp and PirBvp subunits. In addition, recombinant pirB (rPirBvp) and pirA (rPirAvp) were obtained. The homogeneity of the purified protein was determined by SDS-PAGE analysis, and the yield of protein was 488 ng/100 μg of total protein of extracellular products. The PirABvp complex and the rPirBvp showed hemagglutinating activity toward rat erythrocytes. The rPirAvp showed no hemagglutinating capacity toward the animal red cells tested. Among different mono and disaccharides tested, only GalNH2 and GlcNH2 were able to inhibit hemagglutination of the PirABvp complex and the rPirBvp. Glycoproteins showed inhibitory specificity, and fetuin was the glycoprotein that showed the highest inhibition. Other glycoproteins, such as mucin, and glycosaminoglycans, such as heparin, also inhibited the activity. Desialylation of erythrocytes enhanced the hemagglutinating activity. This confirms that Gal or Gal (β1,4) GlcNAc are the main ligands for PirABvp. The agglutinating activity of the PirABvp complex and the rPirBvp is not dependent on cations, because addition of Mg2+ or Ca2+ showed no effect on the protein capacity. Our results strongly suggest that the PirBvp subunit is a lectin, which is part of the PirA/PirBvp complex, and it seems to participate in bacterial pathogenicity.