Mechanism of action of Cholera toxin and Pertussis toxin (Action on G protein alpha subunit)
- Bacteria that are pathogenic to humans can release poisons that interrupt specific G protein linked receptor function, leading to illnesses such as pertussis, botulism, and cholera.
Cholera toxin (CT): Produced by Gram negative water-borne bacterium Vibrio cholerae.
Structure of Cholera toxin:
- Multimeric enterotoxin that consists of a single A subunit surrounded by five B subunits.
- Cholera toxin (also known as choleragen) is an 87kD protein composed of two functional units:
1) Enzymatic A subunit (240 residues)
- It has 2 important segments (CTA1 and CTA2).
- A1 domain containing the enzymatic active site, and an A2 domain that has an a-helical tail that interact with B subunit.
- The CTA1 fragment catalyses ADP-ribosylation of the Gs alpha subunit (Gαs) proteins using NAD.
2) Intestinal receptor binding B subunit (103 residues each)
- B subunit contains five identical peptides that assemble into a pentameric ring surrounding a central pore.
Mechanism of action:
Cholera Toxin Stimulates Adenylate Cyclase by Permanently Activating Gsα
- When cholera toxin is released from the bacteria in the infected intestine, it binds to the small intestinal cells.
- Following binding to the membrane of enteric cells, the toxin is endocytosed and travels to the endoplasmic reticulum.
- It is then cleaved at a single site by a bacterial protease to yield two fragments, A1 (the N-terminal ~195 residues) and A2 (the C-terminal ~45 residues), that remain joined by a disulfide bond.
- Enzymatically active A subunit (CTA1) is translocated to the cytosol.
- In the cytoplasm, A1 catalyzes the irreversible transfer of the ADP–ribose unit from NAD to a specific Arg side chain of Gs (ADP-ribosylation of the Gsα protein).
- The ADP-ribosylation causes the Gαs subunit to lose its catalytic activity of inactivating GTP by hydrolyzing it to GDP + Pi
- ADP- ribosylated Gsα GTP can activate AC but is incapable of hydrolyzing its bound GTP. As a consequence, the AC remains “locked” in its active state.
- As a result there occurs ~100-fold rise in intracellular cAMP concentration.
- The high cAMP levels causes the phosphorylation and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein causing a dramatic efflux of ions (sodium and potassium) and water from infected enterocytes into the intestinal lumen leading to the severe diarrhea.
Figure 2: ADP – ribosylation at an arginine residue of the stimulatory Gsα subunit of heterotrimeric G protein
ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes.
Anti – diarrhoea treatment:
- Stimulation of enkephalins (regulate intestinal secretion by acting directly on enterocytes).
- Enkephalins bind to the opioid receptors on enterocytes (which act through G proteins) to inhibit the stimulation of cAMP synthesis induced by cholera toxin, thereby directly controlling ion transport.
Pertussis toxin (PT)
- Exotoxin produced by the bacterium Bordetella pertussis ( bacterium that commonly infects the respiratory tract) which causes whooping cough.
Structure: AB5 type exotoxin (formed of 2 subunits). The “A” subunit possesses enzyme activity.
Mechanism of action:
- PT is released from bacteria in an inactive form.
- When PT binds to a cell membrane receptor, it is taken up in an endosome.
- Then it is transported to trans-Golgi network and endoplasmic reticulum. During this transport, the A subunit becomes activated.
- Pertussis toxin catalyzes ADP-ribosylation at a cysteine residue of the inhibitory Giα, making it incapable of exchanging GDP for GTP. The inhibitory pathway is blocked.
- This prevents the G proteins from interacting with G protein-coupled receptors on the cell membrane.
- The Gi subunits remain locked in their GDP-bound state (inactive state).
- As consequence they are unable to inhibit adenylate cyclase activity, leading to increased cellular concentrations of cAMP.
- Increase in cAMP in epithelial cells of the airways promotes loss of fluids and electrolytes and mucus secretion.