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Tag words: Vibrio cholerae, V cholerae, V cholerae O139, cholera, diarrhea.

Vibrio cholerae

Kingdom: Bacteria
Phylum: Proteobacteria
Class: Gamma Proteobacteria
Order: Vibrionales
Family: Vibrionaceae
Genus: Vibrio
Species: V. cholerae

Kenneth Todar currently teaches Microbiology 100 at the University of Wisconsin-Madison.  His main teaching interests include general microbiology, bacterial diversity, microbial ecology and pathogenic bacteriology.

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Vibrio cholerae and Asiatic Cholera (page 3)

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© Kenneth Todar, PhD

Cholera Toxin

Cholera toxin activates the adenylate cyclase enzyme in cells of the intestinal mucosa leading to increased levels of intracellular cAMP, and the secretion of H20, Na+, K+, Cl-, and HCO3- into the lumen of the small intestine. The effect is dependent on a specific receptor, monosialosyl ganglioside (GM1 ganglioside) present on the surface of intestinal mucosal cells. The bacterium produces an invasin, neuraminidase, during the colonization stage which has the interesting property of degrading gangliosides to the monosialosyl form, which is the specific receptor for the toxin.

The toxin has been characterized and contains 5 binding (B) 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. Once it has entered the cell, the A1 subunit enzymatically transfers ADP ribose from NAD to a protein (called Gs or Ns), that regulates the adenylate cyclase system which is located on the inside of the plasma membrane of mammalian cells.

Enzymatically, fragment A1 catalyzes the transfer of the ADP-ribosyl moiety of NAD to a component of the adenylate cyclase system. The process is complex. Adenylate cyclase (AC) is activated normally by a regulatory protein (GS) and GTP; however activation is normally brief because another regulatory protein (Gi), hydrolyzes GTP. The normal situation is described as follows.

The A1 fragment catalyzes the attachment of ADP-Ribose (ADPR) to the regulatory protein forming Gs-ADPR from which GTP cannot be hydrolyzed. Since GTP hydrolysis is the event that inactivates the adenylate cyclase, the enzyme remains continually activated. This situation can be illustrated

Thus, the net effect of the toxin is to cause cAMP to be produced at an abnormally high rate which stimulates mucosal cells to pump large amounts of Cl- into the intestinal contents. H2O, Na+ 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. 


Mechanism of action of cholera enterotoxin according to Finkelstein in Baron, Chapter 24. Cholera toxin approaches target cell surface. B subunits bind to oligosaccharide of GM1 ganglioside. Conformational alteration of holotoxin occurs, allowing the presentation of the A subunit to cell surface. The A subunit enters the cell. The disulfide bond of the A subunit is reduced by intracellular glutathione, freeing A1 and A2. NAD is hydrolyzed by A1, yielding ADP-ribose and nicotinamide. One of the G proteins of adenylate cyclase is ADP-ribosylated, inhibiting the action of GTPase and locking adenylate cyclase in the "on" mode.

Colonization of the Small Intestine

There are several characteristics of pathogenic V. cholerae that are important determinants of the colonization process. These include adhesins, neuraminidase, motility, chemotaxis and toxin production. If the bacteria are able to survive the gastric secretions and low pH of the stomach, they are well adapted to survival in the small intestine. V. cholerae is resistant to bile salts and can penetrate the mucus layer of the small intestine, possibly aided by secretion of neuraminidase and proteases (mucinases). They withstand propulsive gut motility by their own swimming ability and chemotaxis directed against the gut mucosa.

Specific adherence of V. cholerae to the intestinal mucosa is probably mediated by long filamentous fimbriae that form bundles at the poles of the cells. These fimbriae have been termed Tcp pili (for toxin coregulated pili), because expression of these pili genes is coregulated with expression of the cholera toxin genes. Not much is known about the interaction of Tcp pili with host cells, and the host cell receptor for these fimbriae has not been identified. Tcp pili share amino acid sequence similarity with N-methylphenylalanine pili of Pseudomonas and Neisseria.

Two other possible adhesins in V. cholerae are a surface protein that agglutinates red blood cells (hemagglutinin) and a group of outer membrane proteins which are products of the acf (accessory colonization factor) genes. acf mutants have been shown to have reduced ability to colonize the intestinal tract. It has been suggested that V. cholerae might use these nonfimbrial adhesins to mediate a tighter binding to host cells than is attainable with fimbriae alone.

V. cholerae produces a protease originally called mucinase that degrades different types of protein including fibronectin, lactoferrin and cholera toxin itself. Its role in virulence is not known but it probably is not involved in colonization since mutations in the mucinase gene (designated hap for hemagglutinin protease) do not exhibit reduced virulence. It has been suggested that the mucinase might contribute to detachment rather than attachment. Possibly the vibrios would need to detach from cells that are being sloughed off of the mucosa in order to reattach to newly formed mucosal cells.

Genetic Organization and Regulation of Virulence Factors in Vibrio cholerae

In Vibrio cholerae, the production of virulence factors is regulated at several levels. Regulation of genes at the transcriptional level, especially the genes for toxin production and fimbrial synthesis, has been studied in the greatest detail.

V. cholerae enterotoxin is a product of ctx genes. ctxA encodes the A subunit of the toxin, and ctxB encodes the B subunit. The genes are part of the same operon. The transcript (mRNA) of the ctx operon has two ribosome binding sites (rbs), one upstream of the A coding region and another upstream of the B coding region. The rbs upstream of the B coding region is at least seven-times stronger than the rbs of the A coding region. In this way the organism is able to translate more B proteins than A proteins, which is required to assemble the toxin in the appropriate 1A: 5B proportion. The components are assembled in the periplasm after translation. Any extra B subunits can be excreted by the cell, but A must be attached to 5B in order to exit the cell. Intact A subunit is not enzymatically active, but must be nicked to produce fragments A1 and A2 which are linked by a disulfide bond. Once the cholera toxin has bound to the GM1 receptor on host cells, the A1 subunit is released from the toxin by reduction of the disulfide bond that links it to A2, and enters the cell by an unknown translocation mechanism. One hypothesis is that the 5 B subunits form a pore in the host cell membrane through which the A1 unit passes.

Transcription of the ctxAB operon is regulated by a number of environmental signals, including temperature, pH, osmolarity, and certain amino acids. Several other V. cholerae genes are coregulated in the same manner including the tcp operon, which is concerned with fimbrial synthesis and assembly. Thus the ctx operon and the tcp operon are part of a regulon, the expression of which is controlled by the same environmental signals.

The proteins involved in control of this regulon expression have been identified as ToxR, ToxS and ToxT. ToxR is a transmembranous protein with about two-thirds of its amino terminal part exposed to the cytoplasm. ToxR dimers, but not ToxR monomers, will bind to the operator region of ctxAB operon and activate its transcription. ToxS is a periplasmic protein. It is thought that ToxS can respond to environmental signals, change conformation, and somehow influence dimerization of ToxR which activities transcription of the operon.  ToxR and ToxS appear to form a standard two-component regulatory system with ToxS functioning as a sensor protein that phosphorylates and thus converts ToxR to its active DNA binding form. ToxT is  a cytoplasmic protein that is a transcriptional activator of the tcp operon. Expression of ToxT is activated by ToxR, while ToxT, in turn, activates transcription of tcp genes for synthesis of tcp pili.

Thus, the ToxR protein is a regulatory protein which functions as an inducer in a system of positive control. Tox R is thought to interact with ToxS in order to sense some change in the environment and transmit a molecular signal to the chromosome which induces the transcription of genes for attachment (pili formation) and toxin production. It is reasonable to expect that the environmental conditions that exist in the GI tract (i.e., 37o temperature, low pH, high osmolarity, etc.), as opposed to conditions in the extraintestinal (aquatic) environment of the vibrios, are those that are necessary to induce formation of the virulence factors necessary to infect. However, there is conflicting experimental evidence in this regard, which leads to speculation of the ecological function of the toxin during human infection.

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Kenneth Todar is an emeritus lecturer at University of Wisconsin-Madison. He has taught microbiology to undergraduate students at The University of Texas, University of Alaska and University of Wisconsin since 1969.

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