The latent ADP-ribosyltransferase activity of cholera toxin (CT) that is activated after proteolytic nicking and reduction is associated with the CT A1 subunit (CTA1) polypeptide. This activity is stimulated in vitro by interaction with eukaryotic proteins termed ADP-ribosylation factors (ARFs). We analyzed this interaction in a modified bacterial two-hybrid system in which the T18 and T25 fragments of the catalytic domain of Bordetella pertussis adenylate cyclase were fused to CTA1 and human ARF6 polypeptides, respectively. Direct interaction between the CTA1 and ARF6 domains in these hybrid proteins reconstituted the adenylate cyclase activity and permitted cAMP-dependent signal transduction in an Escherichia coli reporter system. We constructed improved vectors and reporter strains for this system, and we isolated variants of CTA1 that showed greatly decreased ability to interact with ARF6. Amino acid substitutions in these CTA1 variants were widely separated in the primary sequence but were contiguous in the three-dimensional structure of CT. These residues, which begin to define the ARF interaction motif of CTA1, are partially buried in the crystal structure of CT holotoxin, suggesting that a change in the conformation of CTA1 enables it to bind to ARF. Variant CTA polypeptides containing these substitutions assembled into holotoxin as well as wild-type CTA, but the variant holotoxins showed greatly reduced enterotoxicity. These findings suggest functional interaction between CTA1 and ARF is required for maximal toxicity of CT in vivo.
C holera, caused by the Gram-negative intestinal pathogenVibrio cholerae, remains a significant cause of morbidity and mortality in the developing world. Cholera toxin (CT), the primary virulence factor produced by the bacterium, is responsible for the massive dehydrating diarrhea characteristic of the disease (1). CT is secreted by the bacterium into the external milieu as an oligomeric complex of a single enzymatic A subunit (CTA) noncovalently coupled to a pentamer (CTB) of identical B subunits, and it has latent ADP-ribosyltransferase activity. The A subunit has a single intramolecular disulfide bond between Cys-187 and Cys-199, forming a protease-sensitive loop (2). Nicking within this loop by bacterial or host proteases generates the CTA1 and CTA2 polypeptides. CTA1 contains the enzyme active site, and CTA2 tethers the A1 polypeptide to CTB. Both nicking and reduction of the disulfide bond are required for full enzymatic activity (3). CTB binds to its receptor, ganglioside GM 1 , on the surface of enterocytes. After internalization of CT and reduction of CTA, CTA1 gains access to the cell cytosol and catalyzes the NAD-dependent ADP ribosylation of the ␣ subunit of the regulatory heterotrimeric G protein, Gs, inhibiting its GTPase activity and locking it in an active form. The consequence is constitutive stimulation of adenylate cyclase activity and increased intracellular cAMP levels (4), leading to fluid and electrolyte secretion by the affected enterocytes and the mas...