ANALYSIS OF SHIGA TOXIN SUBUNIT ASSOCIATION BY USING HYBRID-A POLYPEPTIDES AND SITE-SPECIFIC MUTAGENESIS

Shiga toxin (STX), a bacterial toxin produced by Shigella dysenteriae type 1, is a hexamer composed of five receptor-binding B subunits whic h encircle an alpha-helix at the carboxyl terminus of the enzymatic A polypeptide. Hybrid toxins constructed by fusing the A polypeptide seq uences of STX and Shiga-like toxin type II were used to confirm that t he carboxyl terminus of the A subunits governs association with the B pentamers, The alpha-helix of the 293-amino-acid STX A subunit contain s nine residues (serine 279 to methionine 287) which penetrate the non polar pore of the B-subunit pentamer. Site directed mutagenesis was us ed to establish the involvement of two residues bordering this alpha-h elix aspartic acid 278 and arginine 288, in coupling the C terminus of StxA to the B pentamer, Amino acid substitutions at StxB residues arg inine 33 and tryptophan 34, which are on the membrane-contacting surfa ce of the pentamer, reduced cytotoxicity without affecting holotoxin f ormation. Although these B-subunit mutations did not involve receptor- binding residues, they may have induced an electrostatic repulsion bet ween the holotoxin and the mammalian cell membrane or disrupted cytopl asmic translocation.