Cholera toxin (CT) is a protein secreted by the bacterium Vibrio cholerae, known for causing the severe diarrheal disease cholera. Beyond its role in infection, CT is a powerful tool in cell biology research, particularly in cell culture. Its unique properties allow scientists to study fundamental cellular processes, offering insights into how cells communicate and respond to external signals. Researchers leverage this toxin to manipulate specific pathways within cells.
How Cholera Toxin Enters Cells
Cholera toxin is an AB5 multimeric protein, composed of one A subunit and five B subunits. The five B subunits form a ring-like structure that binds to the surface of host cells. These B subunits specifically attach to GM1 ganglioside, a glycolipid found on the outer surface of cell membranes.
After binding to GM1 gangliosides, the entire cholera toxin complex is taken into the cell via endocytosis. The toxin then travels backward through the cell’s internal transport system, moving from the plasma membrane to early endosomes, then to the trans-Golgi network, and finally reaching the endoplasmic reticulum (ER). Once in the ER, the A subunit separates from the B subunits and is transported into the cytosol, where it exerts its effects.
Cholera Toxin’s Cellular Mechanism
Upon reaching the cytosol, the A subunit of cholera toxin, specifically its A1 fragment, acts as an ADP-ribosyltransferase enzyme. This enzyme targets the alpha subunit of the stimulatory G protein (Gsα). The A1 fragment transfers an ADP-ribose group from NAD+ (nicotinamide adenine dinucleotide) to the Gsα protein.
This ADP-ribosylation permanently activates Gsα. Activated Gsα then continuously stimulates adenylyl cyclase, an enzyme in the cell membrane. This sustained activation of adenylyl cyclase leads to a persistent increase in the production of cyclic AMP (cAMP). The elevated cAMP levels then trigger a cascade of downstream cellular responses.
Effects on Cultured Cells
The increase in intracellular cyclic AMP (cAMP) levels induced by cholera toxin alters the behavior of cultured cells. In intestinal epithelial cells, for instance, high cAMP levels activate specific ion channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. This activation leads to an efflux of chloride ions into the intestinal lumen, followed by sodium ions and water to maintain osmotic balance, resulting in the characteristic fluid loss seen in cholera.
Beyond fluid secretion, cholera toxin can influence cell proliferation in various cell types, sometimes stimulating and other times inhibiting cell division, depending on the cell type and toxin concentration. It can also alter gene expression patterns and affect the organization of the cytoskeleton. While cholera toxin causes functional changes, it typically does not kill cultured cells or cause tissue necrosis.
Research Applications of Cholera Toxin
Cholera toxin serves as a valuable reagent in cell biology and neuroscience due to its precise effects on cellular signaling pathways. Its ability to specifically activate the cyclic AMP (cAMP) pathway makes it an effective tool for studying signal transduction mechanisms. Researchers use it to investigate how changes in cAMP levels influence various cellular processes, including cell growth, differentiation, and metabolism.
The B subunit of cholera toxin (CTB) is useful in neuroscience as a neuronal tracer. Because CTB binds with high affinity to GM1 gangliosides on cell membranes, it can be taken up by neurons and transported along their pathways, both backward (retrograde) and forward (anterograde), allowing scientists to map neural connections. Additionally, cholera toxin, or its B subunit, has been explored as a mucosal adjuvant in vaccine development, as it can enhance the immune response to co-administered antigens.