Contribution of individual disulfide bonds to biological action of Escherichia coli heat-stable enterotoxin B

Abstract

Heat-stable enterotoxins (STs) of Escherichia coli are peptides which alter normal gut physiology by stimulating the loss of water and electrolytes. The action of heat-stable toxin B (STb) is associated with an increase in levels of lumenal 5-hydroxytryptamine and prostaglandin E 2 , known mediators of intestinal secretion. In addition, the toxin is responsible for elevation of cytosolic calcium ion levels in cultured cells. STb is a 48-amino-acid basic peptide containing four cysteine residues and two disulfide bonds. Previous work indicates that disulfide bonds are required for intestinal secretory activity, and yet the relative contribution of the two bonds to toxin stability and action is presently unclear. Site-directed mutagenesis was used to alter the cysteine residues of STb to assess the role of the individual disulfide bonds in toxin activity. Our results indicate that loss of a single disulfide bond was sufficient to abolish the intestinal secretory and G protein-coupled calcium ion influx activities associated with STb toxicity. Loss of toxin action was not a function of increased sensitivity of STb mutants to proteolysis, since mutant toxins displayed proteolytic decay rates equivalent to that of wild-type STb. Circular dichroism spectroscopy of mutant STb toxins indicated that single-disulfide-bond elimination did not apparently affect the toxin secondary structure of one mutant, STbC33S,C71S. In contrast, the ␣-helical content of the other disulfide bond mutant, STbC44S,C59G, was significantly altered, as was that of reduced and alkylated authentic STb. Since both Cys-Cys mutant STbs were completely nontoxic, the absence of biological activity cannot be explained by dramatic secondary structural changes alone; keys to the conformational requirements for STb toxicity undoubtedly reside in the three-dimensional structure of this peptide.

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells

“…The amphipathic N-terminal (residues 10 to 22) and hydrophobic C-terminal (residues 38 to 44) helices are connected by a 16-amino-acid flexible loop which contains a cluster of hydrophobic residues (46). The two disulfide bonds are critical for the biological activity of STb, as are the polar residues (Arg29 and Asp30) of the flexible loop (2,6,7,46). In vitro experiments indicate that following the initial binding of STb to cells through an uncharacterized process, STb activates a pertussis toxin-sensitive G protein (G i3 ) in several different cell lines of intestinal and nonintestinal origin, resulting in calcium ion entry through an apparent ligand-gated calcium ion channel (5,20,21).…”

Effect ofEscherichia coliSTb toxin on NIH-3T3 cells

“…The STb tridimensional structure shows two antiparallel α helices (Cys10‐Lys22, amphipathic, and Gly38‐Ala44, hydrophobic) connected by a glycine‐rich loop (Sukumar et al , 1995). Two disulfide bridges (Cys10‐Cys48 and Cys21‐Cys36) stabilize the structure (Sukumar et al , 1995) and both bridges are necessary for enterotoxicity (Arriaga et al , 1995; Okamoto et al , 1995). Moreover, in vitro STb oligomerization, as hexamers and heptamers, seems to be important for toxicity expression (Labrie et al , 2001b).…”

Cell type-dependent internalization of theEscherichia coliSTb enterotoxin

“…The two helices are linked by two disulfide bonds and separated by a glycine‐rich loop (Sukumar et al , 1995). This specific structure is required for enterotoxicity (Arriaga et al , 1995; Okamoto et al , 1995).…”