Cholera toxin (also known ascholeragen and sometimes abbreviated toCTX,Ctx orCT) is anAB5 multimericprotein complex secreted by thebacteriumVibrio cholerae.[1][2] CTX is responsible for the massive, watery diarrhea characteristic ofcholera infection.[3] It is a member of theheat-labile enterotoxin family.
Robert Koch, a German physician and microbiologist, was the first person to postulate the existence of cholera toxin. In 1886, Koch proposed thatVibrio cholerae secreted a substance which caused the symptoms ofcholera.[4] Koch's postulation was proven correct by Indian microbiologistSambhu Nath De, who in 1951 studied and documented the effects of injecting rabbits with heat-killed cholera bacteria.[5] He concluded from this experiment that anendotoxin liberated upon disintegration of the bacteria was the cause of the symptoms of cholera.[5] In 1959, De conducted another experiment, this time using a bacteria-free culture filtrate fromV. cholerae injected into the small intestines of rabbits.[6] The resulting build up of fluid in the intestines conclusively proved the existence of a toxin.[7]
The completetoxin is ahexamer made up of a single copy of the A subunit (part A, enzymatic,P01555), and five copies of the B subunit (part B, receptor binding,P01556), denoted as AB5. Subunit B binds while subunit A activates the G protein which activatesadenylate cyclase. The three-dimensional structure of the toxin was determined usingX-ray crystallography by Zhang et al. in 1995.[8]
The five B subunits—each weighing 11kDa, form a five-membered ring. The A subunit which is 28 kDa, has two important segments. The A1 portion of the chain (CTA1) is a globularenzyme payload thatADP-ribosylatesG proteins, while the A2 chain (CTA2) forms an extendedalpha helix which sits snugly in the central pore of the B subunit ring.[9]
This structure is similar in shape, mechanism, andsequence to theheat-labile enterotoxin secreted by some strains of theEscherichia coli bacterium.
Cholera toxin acts by the following mechanism: First, the B subunit ring of the cholera toxin binds toGM1gangliosides on the surface of target cells. If a cell lacks GM1, the toxin most likely binds to other types ofglycans, such asLewis Y andLewis X, attached to proteins instead of lipids.[10][11][12] Once bound, the entire toxin complex isendocytosed by the cell and the reduction of adisulfide bridge releases the cholera toxin A1 (CTA1) chain. Theendosome is moved to theGolgi apparatus, where the A1 protein is recognized by theendoplasmic reticulum (ER)chaperone,protein disulfide isomerase. The A1 chain is then unfolded and delivered to the membrane, whereEro1 triggers the release of the A1 protein by oxidation of protein disulfide isomerase complex.[13] As the A1 protein moves from the ER into thecytoplasm by theSec61 channel, it refolds and avoids deactivation as a result ofubiquitination.
CTA1 is then free to bind with a human partner protein calledARF6 (ADP-ribosylation factor 6); binding to Arf6 drives a change in the shape of CTA1 which exposes its active site and enables its catalytic activity.[14] The CTA1 fragment catalysesADP-ribosylation of theGs alpha subunit (Gαs) proteins usingNAD. The ADP-ribosylation causes the Gαs subunit to lose its catalytic activity of GTP hydrolysis into GDP + Pi, thus maintaining Gαs in its activated state. Increased Gαs activation leads to increasedadenylate cyclase activity, which increases the intracellular concentration of3',5'-cyclic AMP (cAMP) to more than 100-fold over normal and over-activatescytosolicPKA. These active PKA then phosphorylate thecystic fibrosis transmembrane conductance regulator (CFTR)chloride channel proteins, which leads toATP-mediatedefflux ofchlorideions and leads to secretion ofH2O,Na+,K+, andHCO3− into theintestinal lumen. In addition, the entry of Na+ and consequently the entry of water intoenterocytes are diminished. The combined effects result in rapid fluid loss from the intestine, up to 2 liters per hour, leading to severedehydration and other factors associated with cholera, including a rice-waterstool.[15]
Thepertussis toxin (also an AB5 protein) produced byBordetella pertussis acts in a similar manner with the exception that it ADP-ribosylates theGαi subunit, rendering it unable to inhibit cAMP production.[16]
The gene encoding the cholera toxin was introduced intoV. cholerae byhorizontal gene transfer.Virulent strains ofV. cholerae (the O1 and O139serogroups) hold genes from a virus known as theCTXφ bacteriophage.[17] The integrated CTXφ genome contains many of the genes of RS1, a filamentous "satellite" phage, including elements for replication (RstA), integration (RstB), preventing repression of CTXφ replication (RstC) and regulation of gene expression (RstR), as well as genes coding for proteins needed for phage packaging and secretion (Psh, Cep, OrfU, Ace and Zot), which are very similar to the genes ofFf filamentous coliphages.[17] These genes (and others) enable the replication and later secretion of the CTXφ bacteriophage, facilitating the horizontal gene transfer of CTXφ to other susceptible cells without requiring excision of the prophage from the original host bacterium, transmitting the gene encoding CTX together with the remainder of the phage genome.[17]
Because the B subunit appears to be relatively non-toxic, researchers have found a number of applications for it in cell and molecular biology. It is routinely used as aneuronal tracer.[18]
Treatment of cultured rodent neural stem cells with cholera toxin induces changes in the localization of the transcription factorHes3 and increases their numbers.[19]
GM1 gangliosides are found inlipid rafts on the cell surface. B subunit complexes labelled with fluorescent tags or subsequently targeted with antibodies can be used to identify rafts.
There are currently two vaccines for cholera: Dukoral and Shanchol. Both vaccines use whole killedV. cholerae cells however, Dukoral also contains recombinant cholera toxin β (rCTB). Some studies suggest that the inclusion of rCTB may improve vaccine efficacy in young children (2-10) and increase the duration of protection. This is countered by the costs of protecting and storing rCTB against degradation.[20]
Another application of the CTB subunit may be as a vaccineadjuvant to other vaccines. It has been shown that coupling CTB and antigens improves the response of the vaccine. Currently the adjuvant potential of CTB has been shown in large animal models so further research is needed. This may allow for CTB to be used as an adjuvant for vaccinating against many kinds of diseases. This may include bacterial and viral infections, allergy and diabetes. Of note, as CTB has shown to inducemucosalhumoral immune responses, vaccines against mucosal viruses such as HIV are a potential target.[20]
Since cholera toxin has been shown to preferentially bind to GM1 gangliosides, this characteristic can be utilized for membrane studies. Lipid rafts are difficult to study as they vary in size and lifetime, as well being part of an extremely dynamic component of cells. Using cholera toxin β as a marker, we can get a better understanding of the properties and functions of lipid rafts.[21]
Endocytosis is broadly divided intoclathrin-dependent and clathrin-independent process, and the cholera toxin utilizes both pathways. Cholera toxin has been shown to enter cells viaendocytosis in multiple pathways. These pathways includecaveolae, clathrin-coated pits,clathrin-independent carriers (CLICs), andGPI-Enriched Endocytic Compartments (GEECs) pathway,ARF6-mediated endocytosis andFast Endophilin-Mediated Endocytosis (FEME). How cholera toxin triggers these endocytosis pathways is not fully understood, but the fact that cholera toxin triggers these pathways shows the use of the toxin as an important marker to investigate these mechanisms.[21]
One of the most important aspects of cholera toxin is the retrograde traffic mechanism that transports the toxin from the cell membrane back to the trans-Golgi network and the endoplasmic reticulum. Since both cholera toxin and GM1 species can be tagged with a fluorescent tags, the mechanism of retrograde traffic can be monitored. This opens up the potential to monitor the mechanism in real time. This may open up new discoveries on how intracellular transport works and how protein and lipid sorting work in theendocytotic pathway.[21]
