Mechanism of action of cholera toxin: Studies on the lag period

Abstract

The lag period for activation of adenylate cyclase by choleragen was shorter in mouse neuroblastoma N18 cells than in rat glial C6 cells. N18 cells have 500-fold more toxin receptors than C6 cells. Treatment of C6 cells with ganglioside GM1 increased the number of toxin receptors and decreased the lag phase. Choleragen concentration also effected the lag phase, which increased as the toxin concentration and the amount of toxin bound decreased. The concentration, however, required for half-maximal activation of adenylate cyclase depended on the exposure time; at 1.5, 24, and 48 hr, the values were 200, 1.1, and 0.35 PM, respectively. Under the latter conditions, each cell was exposed to 84 molecules to toxin. The length of the lag period was temperature-dependent. When exposed to choleragen at 37, 24, and 20 degrees C, C6 cells began to accumulate cyclic AMP after 50, 90, and 180 min, respectively. In GM1-treated cells, the corresponding times were 35, 60, and 120 min. Cells treated with toxin at 15 degrees C for up to 22 hr did not accumulate cAMP, whereas above this temperature they did. Antiserum to choleragen, when added prior to choleragen, completely blocked the activation of adenylate cyclase. When added after the toxin, the antitoxin lost its inhibitory capability in a time and temperature-dependent manner. Cells, however, could be preincubated with toxin at 15 degrees C, and the antitoxin was completely effective when added before the cells were warmed up. Finally, cells exposed to choleragen for less than 10 min at 37 degrees C accumulated cyclic AMP when shifted to 15 degrees C. Under optimum conditions at 37 degrees C, the minimum lag period for adenylate cyclase activation in these cells was 10 min. These findings suggest that the lag period for choleragen action represents a temperature-dependent transmembrane event, during which the toxin (or its active component) gains access to adenylate cyclase.

Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.

“…In cells treated with CT, cAMP concentrations increased only after a delay of 30 min and did not reach a plateau until 90min. The initial lag is a consistent feature of the response of intact cells to CT (22). In contrast, the cells exposed to PA and EF showed a rapid increase in cAMP concentrations, with no indication of a lag.…”

“…3 Two other bacterial toxins that act by increasing cellular cAMP concentrations have been well characterized. CT and Escherichia coli heat-labile enterotoxin bind to ganglioside GM1 cell surface receptors and enter the cytoplasm by a mechanism that seems to involve membrane penetration (20)(21)(22). Proteolytic activation and disulfide bond reduction of CT free the Mr 21,000 Al peptide, which is an ADP-ribosyl transferase.…”

“…Ribosylation ofthe nucleotide regulatory subunit ofadenylate cyclase converts it to a permanently active form. At least one of these steps occurs at a slow rate because a lag is invariably seen between CT addition and the initial rise in cAMP content (22).…”

Application of Polarization Modulation Infrared Reflection Absorption Spectrosocpy Under Electrochemical Control for Structural Studies of Biomimetic Assemblies

“…The five subunits of cholera toxin bind specifically to GM 1 gangliosides on the cell surface [158]. Upon binding, the toxin translocate across the membrane and initiates infection via enzymatic reactions [159]. In order to prevent the infection, eukaryote have developed a large variety of transmembrane proteins such as siglecs which, reversibly and weakly bind to gangliosides on the cell surface [157,160,161].…”

Inhibition by Apple Polyphenols of ADP‐Ribosyltransferase Activity of Cholera Toxin and Toxin‐Induced Fluid Accumulation in Mice

“…mediates the binding of the toxin to a specific receptor, G M1 monosialoganglioside, on the surface of target eukaryotic cells (3). The bound toxin is then endocytosed in endosomal vesicles and transported intracellularly through the Golgi apparatus to the endoplasmic reticulum, where the A subunit is translocated out of the vesicle into cytosol (11).…”