Cholera is one of the most severe diarrheal diseases that affect humans and is responsible for significant morbidity and mortality especially among children in developing countries. It is characterized by numerous, voluminous watery stools, often accompanied by vomiting, and resulting in hypovolemic shock and acidosis. It is caused by certain members of the species Vibrio cholerae which can also cause mild or inapparent infections. Other members of the species may occasionally cause isolated outbreaks of milder Diarrhea whereas others the vast majorities are free-living and not associated with disease. The causative agent of Cholera, Vibrio cholerae, is a Gram-negative highly motile bacterium with a single polar flagellum that inhabits rivers, estuaries or other aquatic environments. Cholera is a water-borne disease and the bacteria are usually transmitted via contaminated food or water. Upon ingestion, the organisms colonize the small intestine where they elaborate the potent CTx (Cholera Toxin) that is directly responsible for the profuse diarrhea characteristic of the disease. The V. cholerae bacteria are shed in large numbers in the typical ‘‘rice water’’ stool into the environment, where they can associate with other members of the ecosystem until they are ingested again, thus completing the life cycle of this organism (Ref.1 & 2).
Vibrio cholerae causes the potentially lethal disease Cholera through the elaboration of the intestinal Secretogen Cholera toxin. However, two other toxins of V. cholerae have been identified. These are ZOT (Zonula Occludens Toxin), which acts by disrupting tight junctions, and ACE (Accessory Cholera Enterotoxin). Cholera toxin contains 5 B (Binding) subunits of 11,500 daltons, an Active (A1) subunit of 23,500 daltons, and a bridging piece (A2) of 5,500 daltons that links A1 to the 5B subunits. When Cholera toxin is released from the bacteria in the infected intestine, it binds to the Intestinal cells known as Enterocytes through the interaction of the pentameric B- subunit of the toxin with the GM1 ganglioside receptor on the intestinal cell, triggering endocytosis of the toxin. V. cholerae NanH (Neuraminidase) facilitates Cholera toxin binding to host intestinal epithelial cells by converting cell surface Polysialogangliosides to GM1 monogangliosides. The ganglioside binds CTx at the PM (Plasma Membrane) and carries the toxin through the TGN (Trans-Golgi Network) to the ER (Endoplasmic Reticulum). Genestein, a Tyrosine kinase inhibitor, inhibits internalization of the Cholera toxin B-subunit. Cholera toxin is first transported to endosomes, and then retrogradely through the Golgi apparatus, where it encounters the KDEL Receptor ((Lys-Asp-Glu-Leu) Endoplasmic reticulum Protein Retention Receptor), which is normally responsible for retrieving lumenal proteins of the ER that have escaped their resident compartment. The A2 chain has a KDEL sequence at its C terminus which is responsible for retrograde transport of the toxin to the ER. Cholera toxin can be taken up by Clathrin-dependent as well as Clathrin- and Caveolae-independent endocytosis in different cell types. In the case of Cholera toxin, after arrival of the intact toxin to the ER, the enzyme PDI (Protein Disulfide Isomerase) ERP70 is involved in the reduction of the disulfide bond of the A-subunit, thus releasing the active A1 subunit, which can then be transported to the cytosol, where it is resistant to Proteasomal degradation, whereas the B subunit remains in the ER. Recent evidence suggests that A1 may be transported through the Sec61 channel. Another protein ERO1L (Endoplasmic Reticulum Oxidoreductin- 1 (S.cerevisiae-like(alpha)) is also believed to play a part in A1 transport. Although this protein-conducting channel has been originally identified in the transport of secretory and other proteins from the cytosol into the ER lumen, there is growing evidence that it may also be used in the reverse direction (Ref.3, 4 & 5).
Once inside the cytoplasm, CTxA1 interacts with a protein known as Adenosine Diphosphate
ARF6 ((ADP)-Ribosylation Factor-6), which enhances the activity of the toxin. CTxA1 makes two large conformational changes when interacting with ARF6-GTP, both in loop regions. The CTxA1 activation loop changes from a structured loop to an amphipathic (polar) helix to make direct contacts to ARF6-GTP. Further, the open active-site loop conformation creates a knob on the surface of CTxA1 near a motif (the "ADP-ribosylating turn-turn" motif) known in functionally related toxins to be involved in binding to the human protein targeted by the toxin. Active A1 catalyzes the transfer of the ADP-ribosyl moiety of NAD to a component of the AC (Adenylate Cyclase) system. The process is complex. AC
is activated normally by a regulatory protein (GN-AlphaS) and GTP; however activation is normally brief because another regulatory protein (GN-AlphaI
), hydrolyzes GTP. The A1 fragment catalyzes the attachment of ADPR (ADP-Ribose) to the regulatory protein forming GN-AlphaS-ADPR from which GTP cannot be hydrolyzed. This renders AC
constitutively active, thereby increasing the intracellular level of cAMP (Cyclic Adenosine 3,5-monophosphate). The high cAMP levels activate the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) through the activation of AKs (Adenylate Kinases), stimulating mucosal cells to pump large amounts of Cl- (Chloride Ions) into the intestinal contents. H2O (Water), Na+ (Sodium Ions), K+ (Potassium ions), HCO3- (Bicarbonate Ions) and other electrolytes follow due to the osmotic and electrical gradients caused by the loss of Cl-. The lost H2O and electrolytes in mucosal cells are replaced from the blood. Thus, the toxin-damaged cells become pumps for water and electrolytes causing the diarrhea, loss of electrolytes, and dehydration that are characteristic of Cholera. Vibrio cholerae ACE stimulates Ca2+-dependent Cl- /HCO3- secretion in intestinal cells (Ref.2, 6 & 7).
Vibrio cholerae produces a variety of extracellular products including ZOT. The ZOT gene, along with other genes encoding virulence factors such as CTxA, CTxB, and ACE, is part of the chromosomally integrated genome of a filamentous phage designated CTX-Fi. The high concurrence among V. cholerae strains of the ZOT gene and the CTX genes also suggests a possible synergistic role of ZOT in the causation of acute dehydrating diarrhea typical of Cholera. Beside its role in phage morphogenesis, ZOT also increases the permeability of the small intestine by affecting the structure of the Intercellular tight junctions. ZOT also possesses a cell specificity related to the toxin interaction with a specific receptor whose surface expression differs on various cells. ZOT induces modifications of cytoskeletal organization that lead to the opening of tight junctions secondary to the transmembrane PLC (Phospholipase-C) and subsequent PKC-Alpha (Protein Kinase C-Alpha)-dependent polymerization of Actin
filaments strategically localized to regulate the paracellular pathway. The effect of ZOT on tight junctions might lead to intestinal secretion after the permeation of the intercellular space. This modulation is reversible, time- and dose-dependent, and confined to the small intestine because ZOT does not affect colon permeability. Furthermore, the number of ZOT receptors seems to decrease along the intestinal villous axis (Ref.4, 8 & 9).
In addition to Cholera toxin, V.cholerae also secrete certain exotoxins including HlyA (Haemolysin-A) and RTX toxin. HlyA possess membrane-targeting cytolytic activity. HlyA causes lysis after cell swelling (by colloid osmosis) due to an elevation of cation permeability. On the other hand, the secreted RTX toxin could enter the intestinal cells gaining direct access to the cytoplasmic G-Actin. The RTX toxin could thus be the enzyme that then covalently cross-links G-Actin, disrupting the equilibrium between F-Actin
and G-Actin, leading to the depolymerization of stress fibers. Alternatively, RTX could activate an endogenous cross-linking protein, which then carries out the cross-linking reaction. Finally, RTX may function either from within or from outside the cell to activate signal pathways to activate endogenous proteins for both depolymerization and cross-linking. Infection with V. cholerae results in a spectrum of responses ranging from life-threatening secretory diarrhea to mild or unapparent infections of no manifestation except a serologic response. Treatment of Cholera involves the rapid intravenous replacement of the lost fluid and ions. Following this replacement, administration of isotonic maintenance solution should continue until the Diarrhea ceases. Most antibiotics and chemotherapeutic agents have no value in cholera therapy, although a few (e.g. Tetracyclines) may shorten the duration of diarrhea and reduce fluid loss (Ref.1, 10 & 11).